Bright is a B-cell specific transcription factor that also localizes to lipid rafts in resting B cells. Upon B cell receptor (BCR) stimulation, Bright dissociates from lipid rafts and its localization changes. Bright regulates the threshold of BCR signaling by accumulating in lipid rafts of resting B cells, where it interacts with BCR signaling components. Increased levels of raft-localized Bright result in decreased sensitivity of B cells to BCR stimulation.
1) Human pulmonary artery endothelial cells were treated with normal TGF-Ī² or nano-encapsulated TGF-Ī² to analyze gene expression related to TGF-Ī² and NF-ĪŗB signaling pathways.
2) The nano-encapsulated TGF-Ī² decreased expression of genes in the NF-ĪŗB pathway and fibronectin, while increasing expression of Smad3 in the TGF-Ī² pathway, compared to normal TGF-Ī².
3) This suggests that nano-encapsulated TGF-Ī² may attenuate the NF-ĪŗB activation pathway and improve TGF-Ī²ās therapeutic potential for treating pulmonary hypertension and sickle cell disease.
This document discusses signal transduction and how it relates to cancer. It describes how growth factors and receptors contribute to normal signal transduction and how this process is deregulated in cancer. It explains that growth factors regulate growth, proliferation and survival, which are all altered in cancer. Several growth factors and receptors that can contribute to oncogenesis are identified. It also summarizes several key intracellular signaling pathways, like MAPK pathways, that are activated by growth factors and can result in the cancer phenotype if altered.
TiF1-gamma Plays an Essential Role in Murine Hematopoiesis and Regulates Tran...Joe Lee
Ā
This document summarizes a study examining the role of the transcriptional co-factor TIF1-gamma in murine hematopoiesis and erythropoiesis. The study finds that deletion of TIF1-gamma in mice leads to:
1) A block in erythroid maturation in the bone marrow, compensated by enhanced spleen erythropoiesis.
2) Defects in other blood lineages, including a loss of B cells and expansion of granulocytes.
3) Decreased hematopoietic stem cell function.
The deletion of TIF1-gamma also results in reduced transcription elongation of erythroid genes in bone marrow cells. This establishes T
1) The study found that pancreatic cancer cell lines with constitutive activity of the RelA transcription factor overexpressed the anti-apoptotic protein Bcl-xl. 2) The bcl-xl promoter contains two NF-kB binding sites (kB/A and kB/B) that interact with different NF-kB complexes - kB/A binds RelA/p50 heterodimers while kB/B binds p50/p50 homodimers. 3) Inhibition of RelA activity through expression of a dominant negative IkBa mutant reduced expression of Bcl-xl, indicating Bcl-xl is a target gene regulated by RelA/p50 complexes.
This paper identifies genes required for accurate chromosome segregation through systematic yeast screens. The researchers performed genome-wide synthetic lethal and synthetic dosage lethal screens using kinetochore mutants as starting points. They identified 211 nonessential gene deletions that were unable to tolerate defects in kinetochore function. A secondary screen then assessed defects in chromosome segregation. Genes identified were enriched for those with known roles in chromosome segregation. They also uncovered genes with diverse functions, like RCS1, which encodes an iron transcription factor. RCS1 was identified in all three screens and was confirmed to play a role in chromosome stability. The screens revealed genes important for chromosome maintenance that may not have been found through other approaches.
1) GRK5 regulates prostate cancer cell migration and invasion in vitro and tumor growth and metastasis in vivo.
2) GRK5 phosphorylates the cytoskeletal protein moesin, regulating its subcellular distribution and localization to the cell periphery.
3) Phosphorylation of moesin at threonine 66 by GRK5 is important for cell spreading, and mutation of this site reduces cell spreading.
1) NF-kB is constitutively activated in pancreatic cancer cells but not normal pancreatic cells, suggesting it plays a role in pancreatic tumorigenesis.
2) Expressing a mutant IkBa (IkBaM) that inhibits NF-kB suppressed the tumorigenicity of pancreatic cancer cells in a mouse model.
3) Inhibiting NF-kB reduced expression of pro-survival genes like Bcl-xL and Bcl-2, and reduced VEGF and IL-8 expression which are involved in angiogenesis. This suggests NF-kB inhibition can suppress tumorigenesis by reducing pro-survival and angiogenic factors.
The document outlines six hallmarks of cancer cells:
1. Self-sufficiency in growth signals through autocrine or paracrine signaling and expression of growth-promoting extracellular matrix receptors.
2. Insensitivity to antigrowth signals through mutations that block cell cycle inhibitors like Rb or induce differentiation.
3. Evading apoptosis by mutations in genes like p53 that derail cell suicide pathways and activate pro-survival signals.
4. Limitless replicative potential enabled by mechanisms like telomerase that maintain telomere length indefinitely.
5. Sustained angiogenesis driven by factors like VEGF that promote growth of blood vessels.
6. Tissue invasion and
1) Human pulmonary artery endothelial cells were treated with normal TGF-Ī² or nano-encapsulated TGF-Ī² to analyze gene expression related to TGF-Ī² and NF-ĪŗB signaling pathways.
2) The nano-encapsulated TGF-Ī² decreased expression of genes in the NF-ĪŗB pathway and fibronectin, while increasing expression of Smad3 in the TGF-Ī² pathway, compared to normal TGF-Ī².
3) This suggests that nano-encapsulated TGF-Ī² may attenuate the NF-ĪŗB activation pathway and improve TGF-Ī²ās therapeutic potential for treating pulmonary hypertension and sickle cell disease.
This document discusses signal transduction and how it relates to cancer. It describes how growth factors and receptors contribute to normal signal transduction and how this process is deregulated in cancer. It explains that growth factors regulate growth, proliferation and survival, which are all altered in cancer. Several growth factors and receptors that can contribute to oncogenesis are identified. It also summarizes several key intracellular signaling pathways, like MAPK pathways, that are activated by growth factors and can result in the cancer phenotype if altered.
TiF1-gamma Plays an Essential Role in Murine Hematopoiesis and Regulates Tran...Joe Lee
Ā
This document summarizes a study examining the role of the transcriptional co-factor TIF1-gamma in murine hematopoiesis and erythropoiesis. The study finds that deletion of TIF1-gamma in mice leads to:
1) A block in erythroid maturation in the bone marrow, compensated by enhanced spleen erythropoiesis.
2) Defects in other blood lineages, including a loss of B cells and expansion of granulocytes.
3) Decreased hematopoietic stem cell function.
The deletion of TIF1-gamma also results in reduced transcription elongation of erythroid genes in bone marrow cells. This establishes T
1) The study found that pancreatic cancer cell lines with constitutive activity of the RelA transcription factor overexpressed the anti-apoptotic protein Bcl-xl. 2) The bcl-xl promoter contains two NF-kB binding sites (kB/A and kB/B) that interact with different NF-kB complexes - kB/A binds RelA/p50 heterodimers while kB/B binds p50/p50 homodimers. 3) Inhibition of RelA activity through expression of a dominant negative IkBa mutant reduced expression of Bcl-xl, indicating Bcl-xl is a target gene regulated by RelA/p50 complexes.
This paper identifies genes required for accurate chromosome segregation through systematic yeast screens. The researchers performed genome-wide synthetic lethal and synthetic dosage lethal screens using kinetochore mutants as starting points. They identified 211 nonessential gene deletions that were unable to tolerate defects in kinetochore function. A secondary screen then assessed defects in chromosome segregation. Genes identified were enriched for those with known roles in chromosome segregation. They also uncovered genes with diverse functions, like RCS1, which encodes an iron transcription factor. RCS1 was identified in all three screens and was confirmed to play a role in chromosome stability. The screens revealed genes important for chromosome maintenance that may not have been found through other approaches.
1) GRK5 regulates prostate cancer cell migration and invasion in vitro and tumor growth and metastasis in vivo.
2) GRK5 phosphorylates the cytoskeletal protein moesin, regulating its subcellular distribution and localization to the cell periphery.
3) Phosphorylation of moesin at threonine 66 by GRK5 is important for cell spreading, and mutation of this site reduces cell spreading.
1) NF-kB is constitutively activated in pancreatic cancer cells but not normal pancreatic cells, suggesting it plays a role in pancreatic tumorigenesis.
2) Expressing a mutant IkBa (IkBaM) that inhibits NF-kB suppressed the tumorigenicity of pancreatic cancer cells in a mouse model.
3) Inhibiting NF-kB reduced expression of pro-survival genes like Bcl-xL and Bcl-2, and reduced VEGF and IL-8 expression which are involved in angiogenesis. This suggests NF-kB inhibition can suppress tumorigenesis by reducing pro-survival and angiogenic factors.
The document outlines six hallmarks of cancer cells:
1. Self-sufficiency in growth signals through autocrine or paracrine signaling and expression of growth-promoting extracellular matrix receptors.
2. Insensitivity to antigrowth signals through mutations that block cell cycle inhibitors like Rb or induce differentiation.
3. Evading apoptosis by mutations in genes like p53 that derail cell suicide pathways and activate pro-survival signals.
4. Limitless replicative potential enabled by mechanisms like telomerase that maintain telomere length indefinitely.
5. Sustained angiogenesis driven by factors like VEGF that promote growth of blood vessels.
6. Tissue invasion and
The cell cycle and molecular basis of cancer can be summarized as follows:
The cell cycle is tightly regulated by cyclins, cyclin-dependent kinases (CDKs), and their inhibitors to ensure proper cell division and growth. Defects in cell cycle regulation can lead to uncontrolled cell growth and cancer. Cancer develops through the accumulation of genetic mutations in oncogenes and tumor suppressor genes, which disrupt the normal cell cycle checkpoints and allow cells to proliferate uncontrollably. Key cellular capabilities acquired during cancer progression include self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis.
This document discusses a study that found constitutive membrane association greatly enhances the ability of Bruton's tyrosine kinase (Btk) to transform cells. The study targeted Btk to the plasma membrane using fusion proteins, which led to high tyrosine phosphorylation of Btk. Cell transformation required membrane localization of Btk, its kinase activity, phosphorylation by Src kinases, and an intact SH2 domain. The results suggest membrane localization is a critical early step in Btk activation.
This document discusses the RAS/MAP kinase pathway and targeted therapies that inhibit proteins in this pathway for cancer treatment. It specifically mentions that mutations in BRAF occur in the kinase domain and that BRAF inhibitors like vemurafenib and dabrafenib block BRAF to treat cancers. MEK inhibitors and multikinase inhibitors like sorafenib that target multiple nodes in the MAPK pathway are also discussed as cancer therapies.
The document describes research showing that treatment with SIN3 interaction domain (SID) decoy peptides inhibits invasion and Wnt/Ī²-catenin signaling in triple-negative breast cancer cells. The SID decoy peptides bind to the PAH2 domain of SIN3A, disrupting its interaction with the transcription factor TGIF1. TGIF1 knockdown also inhibited Wnt target genes and cell invasion. The findings suggest targeting the SIN3A-TGIF1 interaction with SID decoys is a novel strategy to block tumor invasion and metastasis by reversing the epithelial-to-mesenchymal transition program and inhibiting Wnt/Ī²-catenin signaling in triple-negative breast cancer.
Herstatin is an autoinhibitor of the epidermal growth factor receptor 2 (ErbB2/HER2) that blocks receptor interactions and signaling. This study investigated how herstatin expression affects early epidermal growth factor (EGF) and transforming growth factor beta (TGF-Ī²) signaling pathways and the downstream effects on cell proliferation in mouse fibroblasts. The results showed that herstatin decreased EGF-induced EGFR phosphorylation and delayed receptor downregulation. It also blocked EGF and TGF-Ī² stimulation of the Akt pathway but not the MAPK pathway. While MAPK was fully activated, herstatin prevented TGF-Ī²-induced DNA synthesis and EGF-induced proliferation. These
This document summarizes a study investigating how herstatin, an autoinhibitor of the epidermal growth factor receptor 2 (ErbB2/HER2) tyrosine kinase, modulates epidermal growth factor (EGF) and transforming growth factor beta (TGF-Ī²) signaling pathways. The study found that herstatin expression in NIH3T3 cells decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation. Herstatin almost completely blocked Akt stimulation by EGF and TGF-Ī² but did not affect mitogen-activated protein kinase (MAPK) activation. While MAPK was fully activated, herstatin prevented TGF-Ī²-induced DNA synthesis and
This document discusses the role of Smad signaling pathways in regulating hematopoiesis. It summarizes that BMP signaling regulates hematopoietic stem cell specification during development, while TGF-Ī²1, 2, and 3 are not essential for hematopoietic stem cell generation. TGF-Ī² acts as a negative regulator of hematopoietic progenitor and stem cells in vitro, but TGF-Ī² signaling deficiency in vivo does not affect hematopoietic stem cell proliferation or lineage choice. Smad signaling regulates hematopoiesis through crosstalk with other regulatory signals in the bone marrow microenvironment. Future research will further define how various pathways interact to regulate hematopoietic stem and progenitor cells.
This document reports on a study examining the role of platelet-derived growth factor (PDGF) autocrine loops in human astrocytoma cells. The study shows that dominant-negative mutants of PDGF that disrupt PDGF ligand formation are able to revert the transformed phenotype of PDGF-transformed BALB/c 3T3 cells and two independent human astrocytoma cell lines. In contrast, the mutants did not alter the growth of cell lines transformed by other oncogenes or of other human cancer cell lines. These results support the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas.
1. The document summarizes a study that analyzed DNA copy number alterations in lung adenocarcinoma patients with and without EGFR mutations.
2. The results showed that chromosome 7p had the highest rate of DNA gain, including the EGFR gene. Six genes on chromosome 7p were identified that could predict survival outcomes in patients with EGFR mutations.
3. A "copy number-based risk score" using the six genes was able to identify high-risk and low-risk patients and predict survival. Higher copy numbers of the six genes were also associated with less favorable responses to EGFR-TKI targeted therapy.
The RET proto-oncogene encodes a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor family of extracellular signalling molecules. RET loss of function mutations are associated with the development of Hirschsprung's disease, while gain of function mutations are associated with the development of various types of human cancer, including medullary thyroid carcinoma, multiple endocrine neoplasias type 2A and 2B, pheochromocytoma and parathyroid hyperplasia.
RET is an abbreviation for "rearranged during transfection", as the DNA sequence of this gene was originally found to be rearranged within a 3T3 fibroblast cell line following its transfection with DNA taken from human lymphoma cells. The human gene RET is localized to chromosome 10 (10q11.2) and contains 21 exons.
The natural alternative splicing of the RET gene results in the production of 3 different isoforms of the protein RET. RET51, RET43 and RET9 contain 51, 43 and 9 amino acids in their C-terminal tail respectively. The biological roles of isoforms RET51 and RET9 are the most well studied in-vivo as these are the most common isoforms in which RET occurs.
Common to each isoform is a domain structure. Each protein is divided into three domains: an N-terminal extracellular domain with four cadherin-like repeats and a cysteine-rich region, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain, which is split by an insertion of 27 amino acids. Within the cytoplasmic tyrosine kinase domain, there are 16 tyrosines (Tyrs) in RET9 and 18 in RET51. Tyr1090 and Tyr1096 are present only in the RET51 isoform.
The extracellular domain of RET contains nine N-glycosylation sites. The fully glycosylated RET protein is reported to have a molecular weight of 170 kDa although it is not clear to which isoform this molecular weight relates.
This document discusses oncogenes and their role in cancer development. It notes that oncogenes are activated versions of normal cellular genes that regulate cell proliferation, growth, survival and other processes. Oncogenes become activated through genetic mutations, chromosomal translocations, or gene amplification. Some key oncogenes mentioned include Ras, MYC, and BCL2. The document also explains how certain oncogenes contribute to cancer properties like unlimited replication, evading cell death, and stimulating angiogenesis.
This document discusses oncogenes, which are genes that can cause cancer. It defines oncogenes as genes that were originally proto-oncogenes, which are normal genes involved in cell growth that become mutated and cause uncontrolled cell division. The document covers several topics related to oncogenes, including the classes of genes involved in cancer development (proto-oncogenes, tumor suppressor genes, apoptosis genes), the mechanisms by which proto-oncogenes become activated, the properties of cancer cells, and the role of carcinogens and oncogenic retroviruses in cancer development. It also provides examples of specific oncogenes and tumor suppressor genes.
overexpression of mrps18 2 in cancer cell lines resultsAnimatedWorld
Ā
Human mitochondrial ribosomal protein MRPS18-2 (S18-2) is encoded by a cellular gene that is located on the human chromosome 6p21.3.
They observed the overexpression of the S18-2 protein led to immortalization and de-differentiation of primary rat embryonic fibroblasts and then Cells showed anchorage-independent growth pattern.
There observation is not limited to overexpression but they also see the evidence of s18-2 somehow involves in cell cycle regulation and cause disturbances.
S18-2 protein when over expressed it also cause appearance of multinucleated cells in the selected clones.
The document describes a new method called the mutagenic chain reaction (MCR) that uses the CRISPR/Cas9 system to efficiently generate homozygous loss-of-function mutations from an initial heterozygous mutation. The researchers developed an MCR construct containing Cas9, a guide RNA targeting a genomic sequence of interest, and flanking homology arms. This construct inserts into the target site and expresses Cas9 and guide RNA to cleave the opposite chromosome, converting it to homozygosity via homology-directed repair. Testing this method in Drosophila, they found it efficiently spread mutations from the initial chromosome to the homologous one in somatic and germline cells, demonstrating the potential of MCR for
Cellular Signaling Pathways have direct implications on our understanding of tumor cell behavior. A general overview is presented here followed by a brief discussion of some of the major pathways currently implicated in cancer progression : Ras/RAF/MAP kinase pathway and PI3K/AKT/mTOR pathway s
2015-11-26
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"Leukocyte Trafficking in Heath and Diseases" Motomu Shimaoka, MD, PhD. Professor (Mie University Medical School)
Cancer cells exhibit six hallmarks that allow tumor growth and metastasis. They are: self-sufficiency in growth signals, insensitivity to anti-growth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Cancer cells achieve these hallmarks through genetic and epigenetic alterations that disrupt normal cell signaling pathways.
TIF1-gamma Controls Erythroid Cell Fate by Regulating Transcription ElongationJoe Lee
Ā
1) A genetic suppressor screen in zebrafish identified a mutation in the cdc73 gene that rescued the erythroid defect in tif1g (moonshine) mutants.
2) cdc73 encodes a subunit of the PAF elongation complex. Knockdown of other PAF subunits also rescued tif1g mutants, indicating TIF1g antagonizes the PAF complex.
3) Biochemical studies showed TIF1g interacts with erythroid transcription factors and elongation factors, coupling them to promote transcription elongation of erythroid genes by counteracting polymerase II pausing.
The Ras pathway allows cells to respond to external signals by controlling processes like proliferation, survival, and apoptosis. When growth factors bind to receptor tyrosine kinases, it activates Ras which can then activate the MAPK, PI3K, and other pathways to regulate gene expression and cell behavior. Mutations that cause Ras to be constantly active are implicated in many cancers. Inhibiting Ras function through drugs like farnesyltransferase inhibitors may block its ability to drive uncontrolled cell growth.
This document discusses a school's use of the STaR Chart, a rubric used in Texas to assess schools' technology integration. It received Advanced Tech ratings in Teaching/Learning and Educator Development, and Target Tech in Leadership/Support. To improve, it will focus on technology trainings and becoming Target Tech across all areas. The STaR Chart helps schools develop students' technology skills and monitor effective integration to meet state and federal requirements.
The cell cycle and molecular basis of cancer can be summarized as follows:
The cell cycle is tightly regulated by cyclins, cyclin-dependent kinases (CDKs), and their inhibitors to ensure proper cell division and growth. Defects in cell cycle regulation can lead to uncontrolled cell growth and cancer. Cancer develops through the accumulation of genetic mutations in oncogenes and tumor suppressor genes, which disrupt the normal cell cycle checkpoints and allow cells to proliferate uncontrollably. Key cellular capabilities acquired during cancer progression include self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis.
This document discusses a study that found constitutive membrane association greatly enhances the ability of Bruton's tyrosine kinase (Btk) to transform cells. The study targeted Btk to the plasma membrane using fusion proteins, which led to high tyrosine phosphorylation of Btk. Cell transformation required membrane localization of Btk, its kinase activity, phosphorylation by Src kinases, and an intact SH2 domain. The results suggest membrane localization is a critical early step in Btk activation.
This document discusses the RAS/MAP kinase pathway and targeted therapies that inhibit proteins in this pathway for cancer treatment. It specifically mentions that mutations in BRAF occur in the kinase domain and that BRAF inhibitors like vemurafenib and dabrafenib block BRAF to treat cancers. MEK inhibitors and multikinase inhibitors like sorafenib that target multiple nodes in the MAPK pathway are also discussed as cancer therapies.
The document describes research showing that treatment with SIN3 interaction domain (SID) decoy peptides inhibits invasion and Wnt/Ī²-catenin signaling in triple-negative breast cancer cells. The SID decoy peptides bind to the PAH2 domain of SIN3A, disrupting its interaction with the transcription factor TGIF1. TGIF1 knockdown also inhibited Wnt target genes and cell invasion. The findings suggest targeting the SIN3A-TGIF1 interaction with SID decoys is a novel strategy to block tumor invasion and metastasis by reversing the epithelial-to-mesenchymal transition program and inhibiting Wnt/Ī²-catenin signaling in triple-negative breast cancer.
Herstatin is an autoinhibitor of the epidermal growth factor receptor 2 (ErbB2/HER2) that blocks receptor interactions and signaling. This study investigated how herstatin expression affects early epidermal growth factor (EGF) and transforming growth factor beta (TGF-Ī²) signaling pathways and the downstream effects on cell proliferation in mouse fibroblasts. The results showed that herstatin decreased EGF-induced EGFR phosphorylation and delayed receptor downregulation. It also blocked EGF and TGF-Ī² stimulation of the Akt pathway but not the MAPK pathway. While MAPK was fully activated, herstatin prevented TGF-Ī²-induced DNA synthesis and EGF-induced proliferation. These
This document summarizes a study investigating how herstatin, an autoinhibitor of the epidermal growth factor receptor 2 (ErbB2/HER2) tyrosine kinase, modulates epidermal growth factor (EGF) and transforming growth factor beta (TGF-Ī²) signaling pathways. The study found that herstatin expression in NIH3T3 cells decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation. Herstatin almost completely blocked Akt stimulation by EGF and TGF-Ī² but did not affect mitogen-activated protein kinase (MAPK) activation. While MAPK was fully activated, herstatin prevented TGF-Ī²-induced DNA synthesis and
This document discusses the role of Smad signaling pathways in regulating hematopoiesis. It summarizes that BMP signaling regulates hematopoietic stem cell specification during development, while TGF-Ī²1, 2, and 3 are not essential for hematopoietic stem cell generation. TGF-Ī² acts as a negative regulator of hematopoietic progenitor and stem cells in vitro, but TGF-Ī² signaling deficiency in vivo does not affect hematopoietic stem cell proliferation or lineage choice. Smad signaling regulates hematopoiesis through crosstalk with other regulatory signals in the bone marrow microenvironment. Future research will further define how various pathways interact to regulate hematopoietic stem and progenitor cells.
This document reports on a study examining the role of platelet-derived growth factor (PDGF) autocrine loops in human astrocytoma cells. The study shows that dominant-negative mutants of PDGF that disrupt PDGF ligand formation are able to revert the transformed phenotype of PDGF-transformed BALB/c 3T3 cells and two independent human astrocytoma cell lines. In contrast, the mutants did not alter the growth of cell lines transformed by other oncogenes or of other human cancer cell lines. These results support the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas.
1. The document summarizes a study that analyzed DNA copy number alterations in lung adenocarcinoma patients with and without EGFR mutations.
2. The results showed that chromosome 7p had the highest rate of DNA gain, including the EGFR gene. Six genes on chromosome 7p were identified that could predict survival outcomes in patients with EGFR mutations.
3. A "copy number-based risk score" using the six genes was able to identify high-risk and low-risk patients and predict survival. Higher copy numbers of the six genes were also associated with less favorable responses to EGFR-TKI targeted therapy.
The RET proto-oncogene encodes a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor family of extracellular signalling molecules. RET loss of function mutations are associated with the development of Hirschsprung's disease, while gain of function mutations are associated with the development of various types of human cancer, including medullary thyroid carcinoma, multiple endocrine neoplasias type 2A and 2B, pheochromocytoma and parathyroid hyperplasia.
RET is an abbreviation for "rearranged during transfection", as the DNA sequence of this gene was originally found to be rearranged within a 3T3 fibroblast cell line following its transfection with DNA taken from human lymphoma cells. The human gene RET is localized to chromosome 10 (10q11.2) and contains 21 exons.
The natural alternative splicing of the RET gene results in the production of 3 different isoforms of the protein RET. RET51, RET43 and RET9 contain 51, 43 and 9 amino acids in their C-terminal tail respectively. The biological roles of isoforms RET51 and RET9 are the most well studied in-vivo as these are the most common isoforms in which RET occurs.
Common to each isoform is a domain structure. Each protein is divided into three domains: an N-terminal extracellular domain with four cadherin-like repeats and a cysteine-rich region, a hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain, which is split by an insertion of 27 amino acids. Within the cytoplasmic tyrosine kinase domain, there are 16 tyrosines (Tyrs) in RET9 and 18 in RET51. Tyr1090 and Tyr1096 are present only in the RET51 isoform.
The extracellular domain of RET contains nine N-glycosylation sites. The fully glycosylated RET protein is reported to have a molecular weight of 170 kDa although it is not clear to which isoform this molecular weight relates.
This document discusses oncogenes and their role in cancer development. It notes that oncogenes are activated versions of normal cellular genes that regulate cell proliferation, growth, survival and other processes. Oncogenes become activated through genetic mutations, chromosomal translocations, or gene amplification. Some key oncogenes mentioned include Ras, MYC, and BCL2. The document also explains how certain oncogenes contribute to cancer properties like unlimited replication, evading cell death, and stimulating angiogenesis.
This document discusses oncogenes, which are genes that can cause cancer. It defines oncogenes as genes that were originally proto-oncogenes, which are normal genes involved in cell growth that become mutated and cause uncontrolled cell division. The document covers several topics related to oncogenes, including the classes of genes involved in cancer development (proto-oncogenes, tumor suppressor genes, apoptosis genes), the mechanisms by which proto-oncogenes become activated, the properties of cancer cells, and the role of carcinogens and oncogenic retroviruses in cancer development. It also provides examples of specific oncogenes and tumor suppressor genes.
overexpression of mrps18 2 in cancer cell lines resultsAnimatedWorld
Ā
Human mitochondrial ribosomal protein MRPS18-2 (S18-2) is encoded by a cellular gene that is located on the human chromosome 6p21.3.
They observed the overexpression of the S18-2 protein led to immortalization and de-differentiation of primary rat embryonic fibroblasts and then Cells showed anchorage-independent growth pattern.
There observation is not limited to overexpression but they also see the evidence of s18-2 somehow involves in cell cycle regulation and cause disturbances.
S18-2 protein when over expressed it also cause appearance of multinucleated cells in the selected clones.
The document describes a new method called the mutagenic chain reaction (MCR) that uses the CRISPR/Cas9 system to efficiently generate homozygous loss-of-function mutations from an initial heterozygous mutation. The researchers developed an MCR construct containing Cas9, a guide RNA targeting a genomic sequence of interest, and flanking homology arms. This construct inserts into the target site and expresses Cas9 and guide RNA to cleave the opposite chromosome, converting it to homozygosity via homology-directed repair. Testing this method in Drosophila, they found it efficiently spread mutations from the initial chromosome to the homologous one in somatic and germline cells, demonstrating the potential of MCR for
Cellular Signaling Pathways have direct implications on our understanding of tumor cell behavior. A general overview is presented here followed by a brief discussion of some of the major pathways currently implicated in cancer progression : Ras/RAF/MAP kinase pathway and PI3K/AKT/mTOR pathway s
2015-11-26
ćę„ēååćØē ę°ć島岔ćč¦ćęęļ¼ååē ę ļ¼
"Leukocyte Trafficking in Heath and Diseases" Motomu Shimaoka, MD, PhD. Professor (Mie University Medical School)
Cancer cells exhibit six hallmarks that allow tumor growth and metastasis. They are: self-sufficiency in growth signals, insensitivity to anti-growth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Cancer cells achieve these hallmarks through genetic and epigenetic alterations that disrupt normal cell signaling pathways.
TIF1-gamma Controls Erythroid Cell Fate by Regulating Transcription ElongationJoe Lee
Ā
1) A genetic suppressor screen in zebrafish identified a mutation in the cdc73 gene that rescued the erythroid defect in tif1g (moonshine) mutants.
2) cdc73 encodes a subunit of the PAF elongation complex. Knockdown of other PAF subunits also rescued tif1g mutants, indicating TIF1g antagonizes the PAF complex.
3) Biochemical studies showed TIF1g interacts with erythroid transcription factors and elongation factors, coupling them to promote transcription elongation of erythroid genes by counteracting polymerase II pausing.
The Ras pathway allows cells to respond to external signals by controlling processes like proliferation, survival, and apoptosis. When growth factors bind to receptor tyrosine kinases, it activates Ras which can then activate the MAPK, PI3K, and other pathways to regulate gene expression and cell behavior. Mutations that cause Ras to be constantly active are implicated in many cancers. Inhibiting Ras function through drugs like farnesyltransferase inhibitors may block its ability to drive uncontrolled cell growth.
This document discusses a school's use of the STaR Chart, a rubric used in Texas to assess schools' technology integration. It received Advanced Tech ratings in Teaching/Learning and Educator Development, and Target Tech in Leadership/Support. To improve, it will focus on technology trainings and becoming Target Tech across all areas. The STaR Chart helps schools develop students' technology skills and monitor effective integration to meet state and federal requirements.
The song advocates for collective action to address environmental issues like pollution, global warming, and acid rain. It argues that if people work together on solutions, future generations will benefit from cleaner air, water, and a healthier planet. The lyrics call all people around the world to unite and take steps like reducing pollution and planting trees to help mother nature.
The document provides an overview of using Facebook for business purposes. It discusses the different types of Facebook profiles and pages, how to set up and customize a business page, best practices for posting engaging content on a page, using Facebook ads to promote a page, and analyzing page insights to understand audience engagement. The document also provides examples of businesses that use Facebook effectively and emphasizes promoting social media channels on other marketing materials.
The document is an email notification from Socialware containing details of a Facebook event that was archived. It provides information such as the event name ("Event 21"), details about the user who archived it (lailakhan366@gmail.com), and metadata related to the archiving including the event ID, time, and platform (Facebook).
BW Penman Energy is an international recruitment specialist focused on the energy sector. They operate within the upstream oil and gas sector, covering exploration and production, drilling contracting, oilfield services, engineered products, and engineering procurement and construction. They work with energy companies globally to provide mid-to-senior level talent through their extensive network and specialist knowledge of the industry. Their services include contingency and retained recruitment to match candidates with clients' needs.
The document discusses the National Institutes of Health's (NIH) new policy requiring open access to research publications resulting from NIH-funded research. Key points:
1) The NIH policy aims to make taxpayer-funded research openly accessible to the public without barriers. It will apply to a major portion of biomedical literature.
2) Open access publishing is growing, with successful models like BioMed Central and the Public Library of Science providing immediate access. This benefits researchers through easier dissemination and citation of articles.
3) While some traditional publishers oppose open access, alternatives like open access journals are gaining momentum as major science funders support publishing costs. The NIH estimates it already pays over $30 million annually towards publishing
Exe-eLearning permite el diseƱo rĆ”pido de sitios web de aprendizaje electrĆ³nico que incluyen una herramienta de autor, herramientas de autoevaluaciĆ³n y recursos multimedia, todo en un solo paquete.
Vicki Adcock Oldfield has over 15 years of experience in pharmaceutical sales, currently working at Astellas Pharmaceuticals where she has exceeded sales goals and launched multiple products. She has a track record of growing market share, developing key relationships, and implementing successful sales strategies. Her experience also includes positions at Takeda Pharmaceuticals and AstraZeneca Pharmaceuticals, where she consistently met or exceeded sales targets and objectives.
Biplab Kar has an M.Tech from IIT Bombay in Computer Science and Engineering. He has over 5 years of experience as a database systems developer at Oracle India Pvt. Ltd. and as a JAVA-J2EE web developer at Wipro Technologies. His skills include Java, C, C++, Oracle, PostgreSQL, JSP, and Linux scripting. He led a team of 5 members for a website development project at Wipro.
Regulatory b cells and tolerance in transplantation from animal models to hum...Elsa von Licy
Ā
Regulatory B cells and tolerance in transplantation: from animal models to human provides a review of studies demonstrating the role of regulatory B cells in transplantation tolerance. In animal models, administration of donor B cells combined with blocking of CD40L was shown to increase allograft survival. B cells were also found to induce tolerance more efficiently than T cells. Tolerance induced by anti-CD45RB therapy required host B cells and interactions between co-stimulatory molecules on B and T cells. Short-term immunosuppression in rats led to accumulation of inhibited B cells overexpressing inhibitory receptors. Combined anti-CD45RB and anti-TIM-1 therapy induced B cell-dependent allograft survival associated with upregulation of TIM
Bright is a transcription factor required for both hematopoietic stem cell development and B cell lineage development. Mice lacking Bright die by mid-gestation due to failed hematopoiesis. Rare Bright-deficient mice that survive have deficits in hematopoietic stem cells and impaired B cell development and function, including reduced natural antibody and IgG1 class switching responses. Bright is thus necessary for both the formation of hematopoietic stem cells and their differentiation into specific blood lineages such as B cells.
Huang et al. Cell Death Discovery (2020) 6:70
https://doi.org/10.1038/s41420-020-00301-2 Cell Death Discovery
A R T I C L E Op e n A c c e s s
BECN1 promotes radiation-induced G2/M arrest
through regulation CDK1 activity: a potential role
for autophagy in G2/M checkpoint
Ruixue Huang1, Shanshan Gao2, Yanqin Han2, Huacheng Ning1,2, Yao Zhou1,2, Hua Guan2, Xiaodan Liu2,
Shuang Yan2 and Ping-Kun Zhou2,3
Abstract
Authophagy and G2/M arrest are two important mechanistic responses of cells to ionizing radiation (IR), in particular
the IR-induced fibrosis. However, what interplayer and how it links the autophagy and the G2/M arrest remains elusive.
Here, we demonstrate that the autophagy-related protein BECN1 plays a critical role in ionizing radiation-induced G2/
M arrest. The treatment of cells with autophagy inhibitor 3-methyladenine (3-MA) at 0ā12 h but not 12 h
postirradiation significantly sensitized them to IR, indicating a radio-protective role of autophagy in the early response
of cells to radiation. 3-MA and BECN1 disruption inactivated the G2/M checkpoint following IR by abrogating the IR-
induced phosphorylation of phosphatase CDC25C and its target CDK1, a key mediator of the G2/M transition in
coordination with CCNB1. Irradiation increased the nuclear translocation of BECN1, and this process was inhibited by
3-MA. We confirmed that BECN1 interacts with CDC25C and CHK2, and which is mediated the amino acids 89ā155 and
151ā224 of BECN1, respectively. Importantly, BECN1 deficiency disrupted the interaction of CHK2 with CDC25C and the
dissociation of CDC25C from CDK1 in response to irradiation, resulting in the dephosphorylation of CDK1 and
overexpression of CDK1. In summary, IR induces the translocation of BECN1 to the nucleus, where it mediates the
interaction between CDC25C and CHK2, resulting in the phosphorylation of CDC25C and its dissociation from CDK1.
Consequently, the mitosis-promoting complex CDK1/CCNB1 is inactivated, resulting in the arrest of cells at the G2/M
transition. Our findings demonstrated that BECN1 plays a role in promotion of radiation-induced G2/M arrest through
regulation of CDK1 activity. Whether such functions of BECN1 in G2/M arrest is dependent or independent on its
autophagy-related roles is necessary to further identify.
Introduction
Radiotherapy is a widely used strategy for the treatment of
cancer patients. However, despite major advances in radio-
therapy, the radioresistance of tumors remains the leading
obstacle to their clinical treatment because it results in
radiotherapy failure or tumor recurrence1. Approximately
10ā45% of cancers are resistant to radiation, which greatly
influences the outcomes of radiotherapy.
Autophagy is a process of cellular self-degradation that
plays a critical role in maintaining the balance between
cell survival and cell death2. Recent studies have indicated
that the two roles of autophagy in cancer cells are asso-
ciated with the initiation of a cascade ...
1. The document describes using a deep mutational scanning technique to identify mutations in Bruton's tyrosine kinase (BTK) that confer resistance to ibrutinib, a BTK inhibitor used to treat cancers.
2. A yeast 3-hybrid system will be used to express BTK variants and select for those that maintain kinase activity in the presence of ibrutinib by enabling yeast growth.
3. High-throughput sequencing will identify variants enriched after selection and their resistance levels can be quantified by comparing variant frequencies before and after selection. This will generate a complete resistance map of all BTK mutations.
This document summarizes key signaling pathways in muscle-invasive bladder carcinoma. It discusses molecular markers that indicate basal or luminal subtypes of bladder cancer, which differ in response to treatment. Basal cancers often overexpress EGFR and respond to chemotherapy, while luminal cancers involve alterations in genes like FGFR3, ERBB2/3 and are generally less aggressive. The document also reviews markers for cancer stem cells, receptor tyrosine kinase signaling pathways, cytoskeleton proteins, the PI3K-Akt-mTOR pathway, and VEGF/VEGFR pathways that are clinically significant for modeling and optimizing treatment of muscle-invasive bladder cancer.
A 43-Year-Old Male with PCM1-JAK2 Gene Fusion Experienced T-Lymphoblastic Lym...daranisaha
Ā
Myeloid/lymphoid neoplasms associated with eosinophilia and PCM1-JAK2 is a provisional entity in WHO 2016. Prior case reports have shown quite a few clinical presentations in different patients with this chromosome translocation,characterized by eosinophilia in combination with myelodysplastic/ myeloproliferative neoplasms, acute myeloid leukemia(AML) and rarely,
A 43-Year-Old Male with PCM1-JAK2 Gene Fusion Experienced T-Lymphoblastic Lym...semualkaira
Ā
Myeloid/lymphoid neoplasms associated with eosinophilia and PCM1-JAK2 is a provisional entity in WHO 2016. Prior case reports have shown quite a few clinical presentations in different patients with this chromosome translocation,characterized by eosinophilia in combination with myelodysplastic/ myeloproliferative neoplasms, acute myeloid leukemia(AML) and rarely, T-lymphoblastic lymphoma(T-LBL) or B-acute...
A 43-Year-Old Male with PCM1-JAK2 Gene Fusion Experienced T-Lymphoblastic Lym...semualkaira
Ā
Myeloid/lymphoid neoplasms associated with eosinophilia and PCM1-JAK2 is a provisional entity in WHO 2016. Prior case reports have shown quite a few clinical presentations in different patients with this chromosome translocation,characterized by eosinophilia in combination with myelodysplastic/ myeloproliferative neoplasms, acute myeloid leukemia(AML) and rarely, T-lymphoblastic lymphoma(T-LBL) or B-acute...
A 43-Year-Old Male with PCM1-JAK2 Gene Fusion Experienced T-Lymphoblastic Lym...AnonIshanvi
Ā
Myeloid/lymphoid neoplasms associated with eosinophilia and PCM1-JAK2 is a provisional entity in WHO 2016. Prior case reports have shown quite a few clinical presentations in different patients with this chromosome translocation,characterized by eosinophilia in combination with myelodysplastic/ myeloproliferative neoplasms, acute myeloid leukemia(AML) and rarely, T-lymphoblastic lymphoma(T-LBL) or B-acute
A 43-Year-Old Male with PCM1-JAK2 Gene Fusion Experienced T-Lymphoblastic Lym...NainaAnon
Ā
Myeloid/lymphoid neoplasms associated with eosinophilia and PCM1-JAK2 is a provisional entity in WHO 2016. Prior case reports have shown quite a few clinical presentations in different patients with this chromosome translocation,characterized by eosinophilia in combination with myelodysplastic/ myeloproliferative neoplasms, acute myeloid leukemia(AML) and rarely, T-lymphoblastic lymphoma(T-LBL) or B-acute...
The document provides an overview of apoptosis, or programmed cell death, discussing its molecular mechanisms and role in development and disease. It summarizes that apoptosis occurs through intrinsic and extrinsic pathways, is regulated by Bcl-2 family proteins like Bax and Bcl-2, and involves caspase activation leading to DNA fragmentation and phagocytosis of cell fragments. The document also discusses the importance of apoptosis in immune system development and its relevance to cancer.
Pugacheva et al. COMPLETE GB_16.1_p.161_publ.online_08_14_2015 2Victor Lobanenkov
Ā
CTCF and BORIS are paralogous proteins that bind to DNA through their nearly identical zinc finger domains. The authors performed ChIP-seq experiments in three cancer cell lines to compare genomic binding patterns of CTCF and BORIS. They found that BORIS selectively occupies a subset (~29-38%) of CTCF binding sites that contain clustered CTCF motifs, termed 2xCTSes. In contrast, the majority of CTCF binding sites contain a single CTCF motif (1xCTSes) and are not occupied by BORIS. 2xCTSes are preferentially located at active promoters and enhancers in cancer cells, and are also enriched in regions retaining histones in sperm. The results suggest there are two
Hepatitis B virus X protein (HBx) plays a critical role in mediating cell growth and apoptosis. This study investigated the role of HBx in disrupting stress fiber formation and triggering apoptosis. The results showed that HBx expression increased phosphorylated myosin light chain and myosin light chain kinase levels. HBx disrupted stress fiber formation and regulated focal adhesion kinase and integrin-linked kinase through phosphatase and tensin homolog. Inhibiting myosin light chain kinase or phosphatase and tensin homolog restored effects caused by HBx, suggesting HBx triggers apoptosis through a myosin light chain kinase and phosphatase and tensin homolog dependent pathway by disrupting stress fiber formation.
B lymphocytes, Receptors, Maturation and ActivationBhanu Krishan
Ā
There are two types of lymphocytes namely B-cells and T-cells, which are critical for the immune system.
In addition, several accessory cells and effector cells also participate.
The site of development and maturation of B-cells occurs in bursa fabricius in birds, and bone marrow in mammals. During the course of immune response. B-cells mature into plasma cells and secrete antibodies (immunoglobulins).
The B-cells possess the capability to specifically recognize each antigen and produce antibodies (i.e. immunoglobulins) against it.
Chronic myeloid leukemia (CML), also known as chronic myelogenous leukemia, is a type of
cancer that starts in the blood-forming cells of the bone marrow and invades the blood.
Each human cell contains 23 pairs of chromosomes. Most cases of CML start when a "swapping"
of chromosomal material (DNA) occurs between chromosomes 9 and 22 during cell division due
to attack of DNA by radiation or other damage. Part of chromosome 9 goes to 22 and part of 22
goes to 9. This is known as a translocation and gives rise to a chromosome 22 that is shorter than
normal. This new abnormal chromosome is known as the Philadelphia chromosome.
An allelic variant of mTOR leads to decreased DNA damage response in mouse embryonic fibroblasts. The variant allele, R628C, is found in BALB/c mice and results in a single amino acid substitution in the mTOR protein. Mice with the variant allele (KI mice) have decreased survival after radiation exposure and their embryonic fibroblasts show greater DNA damage, increased proliferation, and lower levels of the cell cycle inhibitor p27 compared to wild type mice. The results suggest the mTOR variant renders cells more susceptible to DNA damage and less able to repair it or arrest the cell cycle after radiation.
Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit ...Ayush Jain
Ā
This document summarizes research on generating stable monoclonal cell lines from a chronic lymphocytic leukemia (CLL) mouse model. The cell lines, called EMC lines, exhibit constant Btk and Akt signaling that is dependent on B-cell receptor signaling, mimicking human CLL. The EMC lines provide a tool to study CLL cell biology and test novel targeted therapies in vitro and in vivo, such as the Btk inhibitor ibrutinib. When engrafted into mice, the EMC lines allow rapid evaluation of drug treatment on CLL progression within reasonable timeframes.
CoH Summer Academy 2016 Poster (Lauren)Lauren T. Hui
Ā
This study examined how two patient-derived glioblastoma cell lines - one proneural (PBT003) and one mesenchymal (PBT030) - responded to tumor necrosis factor (TNF) under different growth conditions. The cell lines were cultured in stem-like or differentiating conditions with and without TNF, then analyzed using a dot migration assay and immunofluorescence staining. The results showed that the cell lines' expression of proteins like VCAM1 and CD44, and binding of chlorotoxin-Cy5.5, varied depending on molecular subtype and exposure to TNF. In particular, PBT030 cells grown in stem-like conditions with TNF had higher expression of the mesenchymal marker CD44.
Multilineage potential and self renewal define an epithelial progenitor cell ...Kahlia Wong
Ā
1) The study identifies a subset of immature thymic epithelial cells (TECs) in the adult thymus that express high levels of the stem cell markers a6-integrin and Sca-1 (a6hiSca-1hi).
2) When assessed in fetal thymus reaggregate grafts and 3D culture, these a6hiSca-1hi TECs demonstrate multilineage potential, generating both cortical and medullary TEC lineages including Aire+ mTECs.
3) The a6hiSca-1hi TECs also exhibit significant self-renewal through in vitro colony formation, maintaining their differentiation capacity upon return to the thymic
This editorial discusses the immunological privilege of the eye and factors that regulate corneal vascularization. It summarizes previous research on how the eye balances immune surveillance with organ function. While much has been learned from studies in mice, the response may differ in humans. Further research is still needed to fully explain phenomena like corneal avascularity and how new materials can be developed considering the local immune environment. The document reviews literature on immune cells, growth factors, and molecular pathways involved in corneal vascularization and their implications.
This document discusses the possibility of developing a vaccine against heart attacks. It reviews evidence from previous studies on the relationship between pneumonia vaccines and reduced risk of acute coronary syndrome. However, it finds the evidence inconclusive and notes limitations in comparing populations across different studies. It also examines potential relationships between antibodies against Chlamydia pneumoniae and coronary heart disease, but finds that the role of these pathogens in heart conditions is still unclear based on the published literature. The document concludes more research is needed to understand these relationships and accurately compare patient groups in different studies.
This study aimed to develop more effective drug delivery systems for cancer treatment by correlating the surface characteristics of drug carriers to their efficacy. Doxorubicin was loaded into soybean oil- and Mygliol 812-based liposomal formulations. Synchrotron small-angle X-ray scattering revealed that doxorubicin loading yielded an abraded surface on the soybean oil formulation. In vitro tests found this formulation more effectively reduced survival of carcinoma cells. A dialysis assay also showed a higher initial burst release of doxorubicin from the soybean oil carriers. The results suggest matching the surface geometry of drug carriers to target cells could help refine development of more effective delivery systems with fewer side effects. This is
This editorial introduces The American Journal of Immunology as a new open access journal in the field of immunology. It discusses key topics in immunology like immune cell interactions with the nervous system and developmental stages of cells. The journal operates under an open access model to increase visibility and commitment to scholarly rigor. Debates on ethical issues in clinical trials are best suited for open access journals, where reasoned arguments from both sides can be accessed by the public. The journal aims to be a forum for exchanging ideas and concepts to benefit the life sciences community.
The authors argue that scholarly papers in oncology should provide precise and lengthy discussions to fully present problems and reasoning, and that supplemental lengthy texts should be allowed. They emphasize maintaining verifiable sources and discussions over time, and publishing raw data to avoid bias from selective referencing or willful neglect. Precise referencing of sources is important for rigor in clinical research.
This document discusses improving the bioavailability of pharmacologically active substances in pharmaceutical and cosmetic formulations. Specifically, it examines using latanoprost, which stimulates eyelash growth when applied topically, for treating hair loss. It also looks at incorporating hyaluronic acid, which treats early skin aging, into cosmetic creams. The authors created liposomes containing tocopherol as a model substance and creams with varying viscosities and hyaluronic acid contents. They also established latanoprost-based foam preparations and examined their foam stability. The goal was to develop surfactant-stabilized cosmetic formulations and pharmaceutical preparations using latanoprost and hyaluronic acid to potentially improve their
This editorial discusses the relationship between oncology research and the shifting political climate in the United States over time. It notes that President Nixon's signing of the National Cancer Act in 1971 was considered progressive at the time, though few today recognize the political implications. The authors reflect on how political views in the U.S. tend to operate around a central point and can drive major changes through subtle shifts left or right. Oncology research must be aware of and adapt to changes in societal values and standards of ethical research as influenced by the pendulum of American democracy.
This editorial discusses the rising costs of cancer treatment and the proposed 21st Century Cures Act. It notes that the average cost to develop a new drug in the US surpassed $2.6 billion in 2014. While the 21st Century Cures Act aims to shorten drug development times to improve access and reduce costs, there are concerns it could compromise safety standards. The Act would increase funding for the NIH and FDA but also direct the FDA to use more efficient trial designs and data analysis methods, which some fear could mean fewer patients and less rigorous testing. Overall the editorial argues the final bill will likely strengthen drug development after extensive review, but it remains unclear if it can truly curb rising drug prices.
The document discusses how technology has helped shed light on cancer through research using large facilities like synchrotron radiation and neutron laboratories. Over 100,000 protein structures have been determined using these techniques to better understand biochemical processes and design drugs. Countries are investing in new facilities to advance scientific development and tackle challenges like cancer. Nanotechnology and drug delivery systems combined with characterization techniques can improve cancer treatment methods.
This document discusses the potential for organ printing as an alternative to organ transplantation. It outlines some of the safety questions that need to be addressed, such as the risk of malignant cell transformation when cells are cultured ex vivo. Specifically, it notes that cells removed from their natural environment and placed in artificial conditions like culture dishes can potentially become immortalized or tumorigenic over time due to factors like prolonged culture or serum exposure. Maintaining patient safety throughout the development of technologies like organ printing will require careful consideration of these types of risks.
This article discusses the risks associated with corneal transplantation from donors with a history of cancer. Specifically, it references a study that found one recipient developed prostate cancer 3 years after receiving a cornea from a donor with lymphoma. The article notes the limited understanding of cancer biology and need for further research on screening donors and the effects of chemotherapy. It concludes that while corneal transplantation provides benefits to patients, more information is needed to fully weigh the risks.
This document summarizes a study that investigated how mechanical stressing of integrin receptors affects tyrosine phosphorylation in osteoblastic cells. The key findings were:
1) Mechanical stressing of both the Ī²1 and Ī±2 integrin subunits induced enhanced tyrosine phosphorylation of proteins compared to integrin clustering alone.
2) Applying cyclic forces at 1 Hz was more effective at inducing tyrosine phosphorylation than continuous stress.
3) Mechanically stressed cells showed tyrosine-phosphorylated proteins becoming anchored to the cytoskeleton in a calcium-dependent manner.
4) Mechanical stressing of integrins also increased phosphorylation of MAP kinases, suggesting it can induce downstream signaling events.
This study investigated the mechanism of biphasic NF-ĪŗB activation in response to proinflammatory cytokines. The results show that:
1) MEKK3 is essential for the rapid activation of NF-ĪŗB, whereas MEKK2 controls the delayed activation.
2) MEKK3 is involved in forming the IĪŗBĪ±:NF-ĪŗB/IKK complex that regulates the transient phase, while MEKK2 participates in assembling the IĪŗBĪ²:NF-ĪŗB/IKK complex for the persistent phase.
3) Different MAP3K kinases and IĪŗB isoforms are involved in specific complex formation with IKK and NF-ĪŗB to regulate the biphasic NF-ĪŗB activation induced by cytokines
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1) Scientists now face new challenges as societies undergo significant changes and available resources decline, reducing surplus funds that previously supported a wide range of research projects.
2) Advances in knowledge are now considered strategic assets, and funding for science can be seen as important for maintaining competitiveness and stimulating beneficial commerce.
3) Scientists may need to become more involved in priority-setting debates to prevent funding cuts that could slow progress and harm standards of living.
This editorial discusses the importance of archiving scientific data and the challenges of maintaining legacy data over time. As research costs rise and specialization increases, vast amounts of data are being generated but not properly archived, resulting in valuable information being lost. Maintaining accessible archives that can preserve data in obsolete formats requires significant resources from both individual researchers and society. The piece argues that more effort must be put toward archiving data to prevent "legacy data extinction" and ensure important information is available to future researchers and generations.
1) The document discusses the relationship between governments and citizens in a democratic society. It argues that while governments need some privilege to handle sensitive information, citizens also have an intrinsic right to access and evaluate information from the government.
2) It states that as governments grow larger, their privileges should not necessarily also grow. All personal information, including that of government employees, should be protected by law.
3) The document concludes that for a healthy democracy, citizens need to actively participate in self-governance by accessing and critically evaluating information from the government and elected representatives. A lack of participation could undermine democracy.
1) Molecular Cancer is an open access journal that aims to maximize the exchange of scientific information by making all of its content freely available.
2) Open access has several broad benefits including universal accessibility of articles online, copyright retention by authors, and permanent archiving of articles which can increase citations and dissemination.
3) Molecular Cancer accepts articles through a peer review process and publishes them online along with supporting materials, allowing for fast publication and wider dissemination of research.
This editorial discusses the first year of the journal Molecular Cancer. It summarizes that in its first year, Molecular Cancer published 16 research papers after rejecting 32 submissions, with an average turnaround time of one month. It also discusses the journal's commitment to open access publishing and rapid peer review. The editorial highlights some of the most accessed papers from the first year and says readership and citation will prove the scientific merit of published papers. It concludes by stating the journal will continue publishing manuscripts in all areas of cancer science.
1. Signalling of the BCR is regulated by a lipid
rafts-localised transcription factor, Bright
Christian Schmidt1
, Dongkyoon Kim1
,
Gregory C Ippolito1
, Hassan R Naqvi2
,
Loren Probst1
, Shawn Mathur1
,
German Rosas-Acosta3
, Van G Wilson3
,
Athenia L Oldham4
, Martin Poenie2
,
Carol F Webb4
and Philip W Tucker1,
*
1
Institute for Cellular and Molecular Biology, The University of Texas at
Austin, Austin, TX, USA, 2
Department of Molecular Cell and
Developmental Biology, The University of Texas at Austin, Austin, TX,
USA, 3
Department of Microbial and Molecular Pathogenesis, Texas A&M
Health Science Center, College Station, TX, USA and 4
Immunobiology
and Cancer Program, Oklahoma Medical Research Foundation,
University of Oklahoma Health Sciences Center, Oklahoma City,
OK, USA
Regulation of BCR signalling strength is crucial for B-cell
development and function. Bright is a B-cell-restricted
factor that complexes with Brutonās tyrosine kinase
(Btk) and its substrate, transcription initiation factor-I
(TFII-I), to activate immunoglobulin heavy chain gene
transcription in the nucleus. Here we show that a palmi-
toylated pool of Bright is diverted to lipid rafts of resting B
cells where it associates with signalosome components.
After BCR ligation, Bright transiently interacts with su-
moylation enzymes, blocks calcium ļ¬ux and phosphoryla-
tion of Btk and TFII-I and is then discharged from lipid
rafts as a Sumo-I-modiļ¬ed form. The resulting lipid raft
concentration of Bright contributes to the signalling
threshold of B cells, as their sensitivity to BCR stimulation
decreases as the levels of Bright increase. Bright regulates
signalling independent of its role in IgH transcription, as
shown by speciļ¬c dominant-negative titration of rafts-
speciļ¬c forms. This study identiļ¬es a BCR tuning mechan-
ism in lipid rafts that is regulated by differential post-
translational modiļ¬cation of a transcription factor with
implications for B-cell tolerance and autoimmunity.
The EMBO Journal (2009) 28, 711ā724. doi:10.1038/
emboj.2009.20; Published online 12 February 2009
Subject Categories: membranes & transport; immunology
Keywords: B cell; immunity; signal transduction
Introduction
B-cell development and response to antigen depend on
signalling through the B-cell antigen receptor (BCR) complex
(Gauld et al, 2002; Meyer-Bahlburg et al, 2008). BCR signal-
ling directs positive and negative selection of immature B
cells and their progression through transitional (T) stages
into mature B cells. Surface markers allow the resolution of
three non-proliferative immature B-cell subpopulations: T1,
T2 and T3 (Allman et al, 2001; Sims et al, 2005). The lineage
origins and signalling requirements of these intermediate
stages of B cells are the subject of considerable interest and
debate (Matthias and Rolink, 2005; Teague et al, 2007; Welner
et al, 2008). It is generally agreed that sequential progression
requires an increasingly higher threshold level of BCR signal-
ling; that is, low or ātonicā threshold signals promote T1 to T2,
whereas relatively higher levels of signalling are needed for
T2 to progress to FO or MZB (Petro et al, 2002; Su and
Rawlings, 2002; Hoek et al, 2006). Strong BCR signalling also
is required to direct non-transitional, fetal progenitors to B-1
fate (Loder et al, 1999; Cariappa et al, 2001; Casola et al,
2004). The amplitude of BCR signalling is positively and
negatively regulated by coreceptors (Carter and Fearon,
1992; Cherukuri et al, 2001; Ravetch and Bolland, 2001)
and crosstalk between the antigen receptors and other path-
ways, particularly BAFF (Guo and Rothstein, 2005; Venkatesh
et al, 2006).
A spatially continuous but mobile unit of critical size within
the plasma membrane is required for efļ¬cient initiation of
BCR activation by multivalent antigen (Dintzis et al, 1976).
Engagement of the antigen receptor yields āmicroclustersā that
can be found in highly ordered domains within the plasma
membrane, known as lipid rafts (Dykstra et al, 2003; Saeki
et al, 2003; Harwood and Batista, 2008). Size and composition
of these platforms of BCR signalling are dynamic and respon-
sive to signalling events mediated by the actin cytoskeleton
through plasma membrane linker proteins, such as Ezrin
(Stoddart et al, 2002; Gupta et al, 2006; Sohn et al, 2006).
Bright (B-cell regulator of IgH transcription/Dril1/
ARID3A) is the founder of the AT-rich interaction domain
(ARID) super-family of DNA-binding proteins (Herrscher
et al, 1995; Wilsker et al, 2005). Bright shuttles between the
cytoplasm and the nucleus in a Crm1- and cell cycle-depen-
dent fashion (Kim and Tucker, 2006). Bright transactivates
the IgH intronic enhancer (Em) and certain IgH promoters by
binding as a tetramer to ATC motifs within nuclear matrix
associating regions (Webb et al, 1999; Kim et al, 2007; Lin
et al, 2007). DNA binding and IgH transcriptional activities of
Bright are stimulated by its interaction with Btk and tran-
scription initiation factor-II (TFII-I), a direct substrate of Btk
(Webb et al, 2000; Rajaiya et al, 2005, 2006). TFII-I also
undergoes nucleocytoplasmic shuttling (Hakre et al, 2006),
and, within the cytoplasm, it associates with PLCg to inhibit
Ca2 Ć¾
mobilisation (Caraveo et al, 2006).
Bright is lineage and stage-speciļ¬cally expressed with high
basal levels in immature B cells and in mitogen or cytokine-
induced mature B cells (Webb et al, 1991a, b, 1998; Nixon
et al, 2004a, b). Shankar et al (2007) recently demonstrated
Received: 30 July 2008; accepted: 9 January 2009; published online:
12 February 2009
*Corresponding author. Institute for Cellular and Molecular Biology,
Molecular Genetics and Microbiology, The University of Texas at Austin,
1 University Station A5000, Austin, TX 78712, USA.
Tel.: Ć¾ 1 512 475 7705; Fax: Ć¾ 1 512 475 7707;
E-mail: philtucker@mail.utexas.edu
The EMBO Journal (2009) 28, 711ā724 | & 2009 European Molecular Biology Organization |All Rights Reserved 0261-4189/09
www.embojournal.org
&2009 European Molecular Biology Organization The EMBO Journal VOL 28 | NO 6 | 2009
EMBO
THE
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JOURNAL
THE
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JOURNAL
711
2. the pathological consequences of loss of this tight control.
Transgenic (TG) mice that over-express wild-type (WT)
Bright speciļ¬cally within the B lineage display spontaneous
autoimmunity. This intrinsic B-cell autoreactivity was not
accompanied by global increase in serum Ig. Instead, a
markedly expanded population of T1 and MZB cells was
observed.
These observations, along with the extranuclear expres-
sion of Bright, TFII-I and their functional association with
Btk, prompted us to examine whether Bright is used in BCR
signal transduction. We show here that a pool of Bright acts
within lipid rafts as a ābrakeā to set a signalling threshold on
the BCR.
Results
Association of Bright with mIgM on B-cell membranes
is reduced after antigen receptor stimulation
Immunostaining of murine B splenocytes indicated that a
fraction of the non-nuclear Bright pool colocalised with mIgM,
suggesting cortical and/or membrane-associated localisation
(Figure 1A and readdressed below). This observation was
conļ¬rmed by computerised 3D reconstructions of the immuno-
ļ¬uorescence data (Figure 1A0
and Supplementary Video 1).
To determine whether this colocalisation remains intact
after engagement of the BCR, cells were stimulated for 5 min
with a-m. Only modest colocalisation of Bright and IgM
was retained, as assessed by computerised 3D reconstruc-
tions of the immunoļ¬uorescence data (Figure 1A00
and
Supplementary Video 2). Inspection of these and additional
images (data not shown) indicated that the observed redis-
tribution of mIgM-associated Bright in stimulated B cells was
not accompanied by signiļ¬cant alteration in either its nuclear
or its cytoplasmic levels (data not shown).
Bright accumulates within lipid rafts of resting but not
stimulated B cells
Because lipid rafts serve as platforms for BCR signalling,
we assayed puriļ¬ed plasma membranes and lipid rafts
(Supplementary Figure 1A) for the presence of Bright. A
small pool of Bright resides in lipid rafts puriļ¬ed from
unstimulated CD43Ć
B cells (Figure 1B, upper panel).
Consistent with the imaging results, Bright was not detected
within lipid rafts after BCR engagement that was sufļ¬cient to
elicit a phosphotyrosine (pY) response (Figure 1B, lower
panel). This suggested that the presence or absence of
Bright within lipid rafts might inļ¬uence BCR signalling.
Levels of Bright within lipid rafts determine BCR
signalling threshold
Normal B cells and mature B-cell lines were examined semi-
quantitatively for lipid raft content of Bright using the B cell-
speciļ¬c lipid rafts component, Raļ¬in (Saeki et al, 2003), as an
internal control (Supplementary Figure 1B and data not
shown). We estimated that raft-localised Bright accounted
for 1ā10% of total cellular Bright, consistent with percentages
previously estimated for mIgM concentrations in lipid rafts
(Sproul et al, 2000; Putnam et al, 2003). Lipid rafts of Raji and
Daudi cells contained B10-fold less Bright than those of CL01
or Ramos (Figure 2A). However, no signiļ¬cant differences
were observed in other subcellular fractions among these
lines (Supplementary Figure 1B).
To achieve maximal BCR responses under minimal anti-
body concentrations and minimal receptor internalisation, an
approach using an anti-IgM mAb in the absence of secondary
cross-linking was optimised (Supplementary Figures 2B and
4B; Materials and methods; data not shown). Lipid rafts from
resting and stimulated cell lines were puriļ¬ed on sucrose
A BBright IgM DNA Merge
A'
A''
Bright
IgM
DNA
Bright
IgM
DNA
Bright
IgM
Untreated
Bright
IgM
Ī±-CD19
Ī±-Ī¼ ā +
ā ā
+
+
pY
Ī³-Tubulin
100 -
75 -
50 -
200 -
150 -
Bright
Raftlin
Bright
75 -
100 -
75 -
50 -
WCL
1 2 3
Lipid rafts
Ī±-Ī¼ Ī±-Ī¼
Untreated
Figure 1 Bright accumulates within lipid rafts of resting but not stimulated B cells. (A) Association of Bright with mIgM on B-cell membranes
is reduced after antigen receptor stimulation. CD43Ć
B cells from spleens of BALB/c adult mice were ļ¬xed and stained for Bright (red), mIgM
(green) and DNA (blue). Arrows point to areas (yellow) where Bright colocalises with membrane IgM. (A0
, A00
) Engagement of the antigen
receptor reduces the colocalisation between Bright and mIgM. CD43Ć
B cells (B1 Ć104
) from spleens of BALB/c adult mice were left untreated
(A0
) or stimulated for 5 min (A00
) with 10 pg a-m, followed by immunostaining as described above. Deconvoluted images are shown with arrows
pointing to areas (yellow) where Bright colocalises with mIgM. (B) BCR engagement leads to a discharge of Bright from lipid rafts. CD43Ć
B
cells (B2 Ć106
) were stimulated with either 2 ng a-m or 2 ng a-m Ć¾ 2 ng a-CD19 for 5 min. Lipid rafts or whole cell lysates (WCL) were prepared
from half of each sample. Proteins from each fraction were analysed by SDSāPAGE/western blot using the antibodies indicated.
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
The EMBO Journal VOL 28 | NO 6 | 2009 &2009 European Molecular Biology Organization712
3. gradients, and fractions were analysed for Bright and other
signalosome occupants (Figure 2B). In agreement with pub-
lished reports (Saeki et al, 2003; Depoil et al, 2008), levels of
mIgM, CD19, and the Bright-interacting partner Btk increased
in lipid rafts after a-m stimulation (Figure 2B). As observed
for normal B cells, Bright moved in the opposite manner.
Fraction
Btk
CL-01
Daudi
Raftlin
Untreated
Bright
CD19
Btk
Bright
CD19
Btk
Bright
CD19
Btk
Ramos
Raji
B
Bright
Bright
CD19
Btk
Raftlin
Raftlin
Raftlin
Raftlin
75 -
150 -
75 -
100 -
75 -
150 -
75 -
100 -
75 -
150 -
75 -
100 -
100 -
75 -
150 -
75 -
100 -
100
12 3 4 5 67 8 9101
910
ā ā ā ++ +
+ ā + ā+ ā
ā + ā +ā +
+ + ā ā+ +
ā ā + +ā ā
+ ++ +
+ +ā ā
ā ā+ +
+ ā+ ā
ā +ā +
ā āā ā
+ +ā ā
ā ā+ +
+ ā+ ā
ā +ā +
11 121314
2 3 4 5 67 8 910
1 2 3 4 5 6 7 8910 1 2 3 4 5 6 7 8910
-
75 -
150 -
75 -
100 -
75 -
150 -
75 -
100 -
100 -100 -
75 -
150 -
75 -
100 -
100 -
75 -
150 -
75 -
100 -
100 -
IP Ī± Bright
IP preimmune
Bright
Btk
3 41 2
Daudi
CL-01
75 -
100 -
75 -
IP pre-immune
Bright
Btk
CD19
7 85 6
Daudi
CL-01
150 -
100 -
75 -
75 -
IP Ī±-Ī¼
IP Ī±-Ī¼
IP Ī±-histoneH1
Raji
Ramos
150 -
100 -
75 -
75 -
Raji
RamosDaudiCL-01
Bright
Raftlin
1 2 3 4
Lipid raft 100 -
75 -
-
C
A
250 -
75 -
37 -
25 -
ā 105 60
Raji Ramos
1 2 3 7 8654
ā 105 60
p-Y
F
G
E
Raji Ramos Daudi CL-01
Bright
Sumo-1
Bright
Sumo-1
3 54 1097 861 2 11 12 15 16 17 22211918 2013 14 23 24
3 54 1097 861 2 11 12 15 16 17 22211918 2013 14 23 24
3 54 1097 861 2 11 12 15 16 17 22211918 2013 14 23 24
WCLLipid rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
WCLLipid rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
WCLLipid rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
WCL
Lipid
rafts
Plasma
membrane
Ī±-CD19 + Ī±-Ī¼
Bright
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
Sumo-1
+IP Ī±-Bright
75 -
75 -
-
-
75 -
75
Ī±-CD19
IP Ī±-Bright
IP Ī±-Bright
75
75
-
D
ā 5 ā 5
CL-01 Daudi
250 -
100 -
37 -
25 -
50 -
1211109
p-Y
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
ā
+
+
+
āĪ±-Ī¼
Ī±-Ī¼
Ī±-Ī¼
Ī±-Ī¼
Ī±-Ī¼
Ī±-Ī¼
Ī±-Ī¼ treated
Ī±-Ī¼ treated
CD19
Ī¼
Btk
Raftlin
Bright
CD19
Ī¼
Btk
Bright
CD19
Ī¼
Ī¼
Ī¼
Ī¼
Ī¼
Btk
Btk
Bright
Bright
CD19
CD19
Ī¼
Ī¼
Ī¼
Ī¼
5%5% 40%40%
5% 5%40% 40%
Untreated
Figure 2 Levels of Bright within lipid rafts determine BCR signalling threshold. (A) Bright levels within lipid rafts vary among B-cell lines.
Lipid rafts were prepared from the indicated exponentially growing human B-cell lines (107
cells) and probed for Bright and (as loading control)
Raftlin. (B, C) Antigen receptor engagement results in a discharge of Bright from lipid raft-localised BCR complexes. (B) The indicated cell lines
(B5 Ć108
) were stimulated for 5 min with 500 ng of a-m, followed by preparation of lipid rafts using discontinuous gradient centrifugation.
Aliquots of each fraction were analysed directly by western or (C) were extracted with RIPA buffer and subjected to co-IP/western using the
antibodies indicated. (D) Raji, Daudi, Ramos and CL01 cells respond differentially to BCR stimulation. The indicated cells were stimulated
(100 ng a-m for 108
cells) for 5, 10 and 60 min (Raji and Ramos; left panel) or for 5 min (CL01 and Daudi; right panel), as indicated, and whole
cell extracts were blotted with a-phosphotyrosine (pY). Equal loading was conļ¬rmed by staining of the ļ¬lters with India Ink (data not shown).
(EāG) BCR Ć¾CD19 stimulation leads to Bright discharge from lipid rafts and accumulation of Sumo-I-Bright in plasma membranes. Raji,
Ramos, CL01 and Daudi (B108
cells) were stimulated for 5 min with (E) 100 ng a-m, (F) 100 ng a-CD19 or (G) 100 ng a-m Ć¾ 100 ng a-CD19. Lipid
rafts (Raft), plasma membranes (membrane) and whole cell lysates (WCL) were analysed by IP/western using the antibodies indicated.
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
&2009 European Molecular Biology Organization The EMBO Journal VOL 28 | NO 6 | 2009 713
4. However, its discharge from lipid rafts was complete only in
cell lines in which its starting levels in lipid rafts were low
(Daudi and Raji, Figure 2B, fractions 3 and 4; boxed in red).
These differences in trafļ¬cking could reļ¬ect differences in
composition of raft-localised complexes, or artifacts resulting
from increased resistance to solubilisation, as BCR ligation is
known to induce coalescence of lipid rafts (Gupta et al, 2006).
Therefore, we compared proļ¬les obtained from RIPA-solubi-
lised versus non-soluble lipid rafts immunoprecipitated (IP)
with a-m, a-Raftlin and a-Btk (Supplementary Figure 2E). We
observed that, as previously published (Saeki et al, 2003), a
complex containing Raļ¬in and IgM was seen only in non-
solubilised rafts (Supplementary Figure 2E); this indicated
that our solubilisation conditions were sufļ¬cient. However,
unexpectedly, Raļ¬in did IP with Btk under both conditions,
suggesting that an IP complex containing Btk and Raļ¬in is not
disrupted by RIPA solubilisation of lipid rafts (readdressed
below). Importantly, Bright remained in a complex with
mIgM and Btk in solubilised lipid rafts of all unstimulated
cells (Figure 2C) but was lost only in a-m stimulated cells
(Daudi and Raji) that contained lower starting levels in their
lipid rafts (Figure 2C, lanes 6 and 12; boxed in red).
These results suggested that B cells that contain more lipid
rafts-associated Bright (Ramos and CL01) would be less
sensitive (higher threshold) to BCR ligation. This was con-
ļ¬rmed by the pY responses of these cell lines to a-m stimula-
tion (Figure 2D). Ramos and CL01 also responded less
vigorously to pro-apoptotic signals shown previously (Chen
et al, 1999) to result from long-term stimulation by a-m
(Supplementary Figure 2C).
Ligation of the BCR coreceptor, CD19, is known to
synergistically enhance antigen receptor-mediated signalling
(Carter and Fearon, 1992; Cherukuri et al, 2001; Depoil et al,
2008). Accordingly, all cell lines responded to a-m Ć¾ a-CD19
costimulation with robust responses (Supplementary Figure
2B). BCR costimulation was required to expel Bright from
lipid rafts of the less sensitive (higher threshold) cell lines
Ramos and CL01 (Figure 2EāG). That Bright migrates as a
doublet is apparent in these experiments (addressed below in
the context of the sumoylation observations).
Thus, engagement of the BCR results in a signiļ¬cant and
speciļ¬c reduction of the small pool of lipid rafts-localised
Bright. This pool is lost from lipid rafts, as Btk and other
signalosome components accumulate there, in proportion to
BCR signalling strength.
Entry of Bright into lipid rafts does not require
interaction with Btk but does require palmitoylation
Bright was readily detected in lipid rafts prepared from
retrovirally transduced NIH/3T3 ļ¬broblasts and other non-B
cells (Figure 3B; data not shown). This indicated that even
though Bright associates (at least transiently) with Btk and
other signalsome components in a-m stimulated lipid rafts
(Figure 2C and addressed further below), these B-cell-re-
stricted proteins are not required to designate or retain
Bright in lipid rafts. Bright point mutants (Figure 3A; Kim
and Tucker, 2006) that are retained either within the cyto-
plasm (K466
A) or the nucleus (G532
A) did not localise to
rafts (Figure 3B). Thus, the rafts-localised pool of Bright is
not directly diverted from the cytoplasmic pool, suggesting
that nucleocytoplasmic shuttling (Kim and Tucker, 2006) is
required.
Palmitoylation of cysteine residues is a feature shared by a
number of lipid raft occupants (Simons and Toomre, 2000;
Ashery et al, 2006). Bright contains a single cysteine (C342)
in its ARID DNA-binding domain, which is conserved among
all identiļ¬ed orthologues and paralogues (Figure 3A; Wilsker
et al, 2005). After transfection into ļ¬broblasts, WT Bright, but
not point mutants (C342S and C342D), were palmitoylated
(Figure 3C).
Sumoylation of Bright regulates its discharge from lipid
rafts into membranes after BCR stimulation
Yeast 2-hybrid cDNA library screening and additional ana-
lyses (Supplementary Figure 3A; data not shown) detected
strong and speciļ¬c Bright interactions with Sumo-I conjugat-
ing enzymes Ubc9 and PIAS1. Further investigations indi-
cated that Bright is conjugated to Sumo-I at a consensus motif
(CKxE, Sampson et al, 2001; Gocke et al, 2005; Bossis and
Melchior, 2006) 401
KIKKE (Figure 3A) both in cultured cell
lines and in vitro (Figure 3E; Supplementary Figure 3B).
Sumo-I-Bright was readily detected in a-Bright IPs of whole
cell lysates prepared from B-cell lines and normal B cells
(Figures 2EāG; Supplementary Figures 2D, 3B and C). We
found no Sumo-I-Bright in lipid rafts regardless of cell source
and stimulation regime; only membranes prepared from
stimulated B cells contained Sumo-I-Bright (Figure 2EāG;
Supplementary Figure 2D). Yet Sumo-I-deļ¬cient (401
KIKK/
AIAA) Bright was capable of entering lipid rafts and mem-
branes as efļ¬ciently as WT in transfected ļ¬broblasts
(Supplementary Figure 3D).
These results prompted us to speculate that the Sumo-I-
Bright pool within stimulated plasma membranes might
derive from a sumoylation reaction initiated in B cell rafts
immediately after BCR ligation. If so, a transient sumoylation
initiation complex might be trapped in lipid rafts under much
weaker BCR stimulation conditions. To test this, we isolated
lipid rafts following conditions (30 s; a-m). Consistent with
our hypothesis, Bright was detected in these mildly stimu-
lated rafts in an IP complex with sumolyation E2 and E3
components, Ubc-9 and PIAS-1 (Figure 3F; Schwarz et al,
1998; Kahyo et al, 2001).
Dominant-negative, lipid rafts localisation-defective
mutants modulate BCR signalling
Because Bright exists primarily as a homo-tetramer, we
established the basis for a dominant-negative approach by
pulling down endogenous Sumo-I-Bright using V5 tagged
401
KIKK/AIAA-Bright (Supplementary Figure 4A; Herrscher
et al, 1995; Kim and Tucker, 2006). Thus, we reasoned that
over-expression of palmitoylation-defective C342S or C342D
Bright should titrate the small pool of palmitoylated Bright
tetramers destined for lipid rafts while sparing tetramers
destined for the nucleus. Conversely, the inability to sumoy-
late Bright should trap it in lipid rafts, leading to suppression
of BCR signalling.
As shown in Figure 4A, rafts prepared from WT transduc-
tants contained increased levels (relative to mock controls) of
V5-tagged retroviral Bright, whereas those expressing C342
substitutions were virtually depleted. Over-expression of
401
KIKK/AIAA-Bright in lipid rafts (Figure 4A) led to retention
of both retroviral and endogenous Bright within rafts after
BCR stimulation.
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
The EMBO Journal VOL 28 | NO 6 | 2009 &2009 European Molecular Biology Organization714
5. We next measured the effect of the dominant-negative
titrations on BCR signalling. As shown in Figure 4B and C,
signalling was markedly increased in Bright C342S and
C342D-infected cells. Notably, the signalling threshold of
the Raji BCR was converted from low to high, because
weak stimulation (a-m only) now resulted in signiļ¬cantly
reduced signalling (Figure 4; Supplementary Figure 4B).
Conversely, over-expression of WT Bright and the 401
KIKK/
AIAA dominant-negative mutant form virtually eliminated
BCR-stimulated Ca2 Ć¾
ļ¬ux and pY activity. Yet, unstimulated
401
KIKK/AIAA-transduced Ramos cells appeared to be con-
stitutively āhyperactivatedā with respect to pY signals
(Figure 4C, lane 7). This was a consistent result (please see
Figure 7A and discussion below) that we suspect derived
from Ramos-speciļ¬c, off-target (i.e., non-BCR mediated)
effects of this dominant negative.
We conclude that a palmitoylated pool of Bright is dis-
patched to lipid rafts to dampen BCR signalling, and sumoy-
lation-triggered discharge of Bright is essential for relieving
this inhibition.
Bright regulates signalling and IgH transcription
independently
As the small lipid rafts-localised pool of Bright is diverted
from its nucleocytoplasmatic shuttling pool (Figure 3B; Kim
and Tucker, 2006), we examined the potential nuclear con-
sequences of the dominant-negative-mediated signalling per-
turbations. Neither nuclear-cytoplasmic ratios nor in vitro
DNA binding of Bright to a target VH-associated promoter
were signiļ¬cantly altered (Figure 5B and C).
In vivo, Bright does not modulate basal levels of IgH
transcription, but like several other trans-activators requires
accessory proteins induced during differentiation (reviewed
in Webb et al, 1999). Thus, we assayed Bright-binding-
dependent luciferase reporter activity in LPS stimulated Raji
and Ramos before and after dominant-negative transduction.
ReklesArid
Bright
Accession # AAB03416
1 601
455 561226 360128 165
Acidic
A
B C D
401
KIKK/AIAA
V5 His6
C342S
C342D
K466A
G532A
wt
K466
A
40%5%
3 4 5 10976 81 2Fraction
G532
A
Bright
75 -
75 -
75 -
41 2 3 9 1085 6 7
+ā + ā +ā + ā+ā
C342S
C342D
Bright
Em
pty
vector
14
C-palmitic
acid
VSV-G
Autoradio
graphy
- 75
- 50
- 37
- 150
- 100
- 75
- 68
Bright
VSV-G
IP Bright
IP VSV
++ + + ā+ + ā++
ā+ + ā +ā ā +āā
C
ells
only
C
342S
(V4)
C
342D
(V4)
1 2 3 4 5
w
tBright(V4)
Vectoronly
(V4)
IP/WB Bright
WB Bright
Lipid rafts
Plasma membrane
- 75
- 75
- 75
- 75
E
GFP-Sumo - + - +- +
GFP
W
T
(1ā602)
404
KIKK
/AIAA
GFP-Bright + + + +- -
1 2 3 4 5 6
100 -
150 -
250 -
Bright
F
Ī±-Ī¼
Raji Ramos Daudi CL-01
ā + ā +ā + ā +
+ + + ++ + + +IP Ī±-Bright
41 2 3 85 6 7
Bright
Ubc-9
PIAS-1
Sumo-1
68 -
18 -
78 -
68 -
Lipid rafts
Lipid rafts
Plasma membrane
Figure 3 Entry and exit of Bright from lipid rafts requires nucleocytoplasmic shuttling, alternative post-translational modiļ¬cations, but not
association with Btk. (A) Schematic of Bright indicating domains and positions of substitution mutations. (B) Cytoplasmic-nuclear shuttling is
a requirement for Brightās inclusion into lipid rafts. NIH/3T3 ļ¬broblasts were transfected with constructs encoding wild type and nuclear
export signal-defective (NES, G532A) and nuclear localisation signal-defective (NLS, K466A) Bright. Fractions from discontinuous (5ā40%)
sucrose gradient puriļ¬cation of lipid rafts were analysed by western for Bright. (C) Palmitoylation of Bright requires cysteine 342. Bright
constructs (indicated at the top) were transfected into Cos-7 cells, and after 48 h, were incubated as indicated with 14
C palmitic acid. Whole cell
lysates were subjected to immunoprecipitation using antibodies against Bright and VSV as indicated (left). SDSāPAGE separated proteins were
transferred onto nitrocellulose, and the metabolic incorporation of 14
C palmitic acid was determined by autoradiography. Solvent control is
indicated by Ć. (D) Speciļ¬cation of Bright to lipid rafts requires cysteine 342. NIH/3T3 ļ¬broblasts were transfected with constructs indicated in
the ļ¬gure. Crude plasma membrane and lipid rafts, prepared as described in previous legends, were analysed by anti-Bright western blotting.
(E) Sumo-I modiļ¬cation of Bright is lost after mutation of 401
KIKK. Cos-7 cells were transfected with GFP-SUMO-I and GFP-Bright expression
constructs, as indicated. Whole cell lysates were prepared using RIPA buffer and analysed by western using a-Bright anti-serum. An arrow
points to a GFP-Sumo-I conjugated species of GFP-Bright. (F) Bright forms a transient, stimulation-speciļ¬c complex with Sumo-I-conjugation
enzymes PIAS1 and Ubc9 in lipid rafts. Indicated B cells (B108
) were stimulated mildly (30 s; 100 ng a-m). Lipid rafts were collected on
gradients, subjected to immunoprecipitation with anti-Bright antiserum, and then analysed by western using the antibodies indicated.
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
&2009 European Molecular Biology Organization The EMBO Journal VOL 28 | NO 6 | 2009 715
6. As shown in Figure 5D, LPS-induced increase in reporter
activity above endogenous levels (lanes 5 and 29) was
equally enhanced by over-expression of WT (lanes 11 and
35) or substitution-mutant forms of Bright (lanes 17, 23, 41
and 47). Five minutes of stimulation using a-m sufļ¬cient to
inļ¬uence BCR signalling (Figure 4) failed to elevate reporter
activity above background levels (Figure 5D, lanes 6, 12, 18,
24, 30, 36, 42 and 48). We conclude that the dominant-
negative effects of C342S/D and 401
KIKK/AIAA are limited
to the rafts-destined pool, and that Bright functions indepen-
dently as both an inducible transactivator of IgH and a BCR
signalling regulator.
Over-expression of Bright impairs BCR signalling of
normal B-cell subpopulations
What is the consequence of manipulating Bright levels within
lipid rafts of normal B cells? Splenic B cells puriļ¬ed from
Bright-TG mice express 3ā5-fold higher levels of Bright within
lipid rafts and whole cells lysates (Figure 6A). TG B cells were
markedly reduced relative to WT in Ca2 Ć¾
and pY responses
over a wild range of a-m doses with concomitant depletion
kinetics of Bright from stimulated rafts (Figure 6A;
Supplementary Figure 5A).
MZB and immature B-cell populations are signiļ¬cantly
elevated in Bright TG, leading to development of sponta-
neously autoimmunity during aging (Shankar et al, 2007). TG
and WT B splenocytes were sorted under conditions that
avoid BCR activation into immature (T1 and T2), MZB, and
FO populations (Figure 6B). Elevated levels of Bright (B2ā5-
fold) were observed in whole cell lysates and in lipid rafts
(Figure 6C and D) prepared from all resting TG populations
except FO. We conļ¬rmed that our a-m stimulation conditions
induced no changes in proliferation or differentiation of these
subpopulations, such as that observed by others under
prolonged stimulation (data not shown; Petro et al, 2002;
readdressed in Discussion).
The movement of mIgM, Btk and CD19 into lipid rafts was
unaffected by the starting levels of Bright, as all TG and WT
populations were indistinguishable (Figure 6D). Likewise, all
WT and TG populations responded to strong (a-m Ć¾ a-CD19)
BCR costimulation with a complete discharge of Bright from
lipid rafts (Figure 6D) and a loss of membrane colocalisation
with mIgM (Supplementary Figure 5B and data not shown).
However, the BCR signalling threshold of immature and MZB
populations, as judged by their response to weak (a-m)
stimulation, correlated inversely with their lipid raft content
of Bright. As shown in Figure 6C, global pY responses of T1,
T2 and MZB TG B cells were reduced relative to WT controls,
consistent with the fact that weak stimulation was insufļ¬-
cient to discharge Bright from their lipid rafts (Figure 6D). pY
responses of FO WT B cells were, as expected (Li et al, 2001),
relatively less robust (Figure 6C). Resting FO B cells con-
tained slightly lower levels of total or lipid raft-localised
Bright (Figure 6C and D; Shankar et al, 2007) and displayed
less colocalisation between Bright and mIgM than the other
subpopulations (Supplementary Figure 5B). Nonetheless,
weak TG FO signalling was consistently dampened in
response to a-m stimulation, and Bright was not fully
discharged from their lipid rafts (Figure 6C and D).
We conclude that lipid rafts-localised Bright increases the
signalling threshold of MZB, immature, and, to a lower
extent, FO B cells. We further suggest that, as BCR signalling
41 2 3 9 1085 6 7
Ī±-CD19
Ī±-Ī¼
75 -
100 -
75 -
Raji (low threshold)
41 2 3 9
+
+ā
ā+
+ā
ā+
+ā
ā+
+ā
ā+
+ā
ā+
+ā
ā+
+ā
ā+
+ā
ā+
+ā
ā+
+ā
ā
1085 6 7
Raftlin
Bright
V5
Ramos (high threshold)
A
0
1
1.6
1.8
2
Rajiāempty vector
2 Ī¼M Ionomycin
40 Ī¼M Digitonin
10 mM EGTA
20 mM Tris
100 s
Fluorescenceratio
RajiāC342S Bright
2 Ī¼M Ionomycin
40 Ī¼M Digitonin
10 mM EGTA
20 mM Tris
Rajiāwild-type Bright
2 Ī¼M Ionomycin
40 Ī¼M Digitonin
10mM EGTA
20 mM Tris
Rajiā
401
KIKK/AIAA Bright
2 Ī¼M Ionomycin
40 Ī¼M Digitonin
10mM EGTA
20 mM Tris
B
WCL
100 -
75 -
50 -
37 -
25 -
200 -
41 2 3 121110985 6 7 131415
Ī±āCD19
Ī±āĪ¼
50 -
Bright
41 2 3 121110985 6 7 1314 15
p-Y
Ī³-Tubulin
C
75 -
Raji (low threshold) Ramos (high threshold)
Lipid rafts
C
342D
C
342S
Bright
Bright
Em
pty
vector
Em
pty
vector
C
342D
C
342S
401
KIKK/AIAA
C
342D
C
342S
Bright
Em
pty
vector401
KIKK/AIAA
C
342D
C
342S
Bright
Em
pty
vector401
KIKK/AIAA
401
KIKK/AIAA
+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā
1.4
1.2
0.2
0.4
0.6
0.8
Figure 4 Dominant-negative, lipid rafts localisation-defective mutants modulate BCR signalling. Raji and Ramos cells (5 Ć108
) were infected
with retroviruses encoding wild type and mutant forms of Bright and were then stimulated for 5 min with 500 ng a-m, 500 ng a-CD19 or 500 ng
a-m Ć¾ 500 ng a-CD19. (A) Levels of Bright in lipid rafts are altered by dominant-negative forms. Lipid rafts levels of total Bright
(endogenous Ć¾ ectopic) were measured by anti-Bright western. Levels of ectopic V5-tagged wild-type Bright, a palmitoylation-defective
(C342S/D) form, which is unable to enter lipid rafts, and a Sumo-I-mutant form (401
KIKK/AIAA), unable to be discharged from rafts, were
detected by anti-V5 western. Raftlin was used as a loading control. (B) Intracellular free [Ca2 Ć¾
] is increased by palmitoylation-deļ¬cient and
decreased by wild type or Sumo-I-deļ¬cient titration of endogenous Bright. Transduced Raji B cells (1 Ć106
cells/ml) were loaded with 2 mM
Indo-1 and stimulated with 1 ng or 40 mg of a-m (indicated as low and high concentrations, respectively, by triangles). Fluorescence signal was
plotted against time (scale bar Ā¼ 100 s). Internal calibration was performed as detailed in Materials and methods. Downward arrows indicate
times of reagent addition. (C) Dominant-negative Bright mutants alter global phosphotyrosine responses. Whole cell lysates prepared from cell
lines established and stimulated in (A) were analysed by anti-pY, Bright and tubulin (loading control) western.
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
The EMBO Journal VOL 28 | NO 6 | 2009 &2009 European Molecular Biology Organization716
7. strength contributes to B-cell subset development (Loder
et al, 1999; Cariappa et al, 2001; Niiro and Clark, 2002;
Petro et al, 2002; Su and Rawlings, 2002; Casola et al, 2004;
Su et al, 2004; Hoek et al, 2006), the skewed T1 and MZB
populations in Bright-over-expressing B-cells derived, at least
in part, from impaired signalling.
Phosphorylation of Btk and TFII-I within lipid rafts is
inhibited by Bright
Btk interacts with Bright (Webb et al, 2000; Rajaiya et al,
2005) to modulate its transcriptional activity in the nucleus
(Rajaiya et al, 2006). Thus, it seemed particularly informative
to determine how Bright levels outside the nucleus affect Btk
activation. First, we examined whole cell extracts prepared
from the dominant-negative transduced cell lines (Figure 7A).
When Bright entry into lipid rafts was blocked by over-
expression of the palmitoylation-defective C342S/D mutant,
pY-Btk was robustly detected after BCR stimulation of either
the less sensitive (Ramos) or the more sensitive (Raji) cell
line. Under conditions in which lipid rafts levels of
Bright were increased (by either WT over-expression or
Bright-401
KIKK/AIAA retention), pY of Btk was inhibited
(Figure 7A). Note that the constitutive hyperactivation
phenotype observed for global pTyr (Figure 4C, lane 7) was
conļ¬rmed in this independent set of 401
-KIKK/AIAA Ramos
transductants.
These results suggested that Btk activation by pY in lipid
rafts is inhibited by Bright. Consistent with this hypothesis,
1 2 3 4
C342S
C342D
wild-type
IVT
IVT
IVT
ā
ā
ā
ā
ā
ā
ā
ā
ā
āā
ā
Bright
S107 VH 1-MAR
401
KIKK/AIAA
IVT
A
C342SC342Dwt Bright
9 10 136 7 8 11 1254321 1514 16
CY NP CH NM CY NP CH NM CY NP CH NM CY NP CH NM
ReklesArid
Bright
Accession # AAB03416
1 601
455 561226 360128 165
Acidic
C342S
C342D
Raji
Bright
401
KIKK/AIAA
401
KIKK/AIAA
75 -
C
B
D
0
2
4
6
Firefly/Renilla
8
10
12
14
LPS
Ī±-Ī¼
+ā
ā ā +
ā +ā
ā ā ā
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā +ā
ā ā +
ā
pGL3-btppGL3 pGL3-btppGL3 pGL3-btppGL3 pGL3-btppGL3 pGL3-btppGL3 pGL3-btppGL3 pGL3-btppGL3 pGL3-btppGL3
Empty vector Bright
401
KIKK/AIAA C342S C342DEmpty vector Bright
401
KIKK/AIAA
Raji Ramos
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Figure 5 Wild type and dominant-negative mutant forms of Bright are indistinguishable in DNA-binding, subcellular localisation and
transcriptional activity. (A) Schematic illustration of Bright amino acid substitution mutations. (B) Subcellular fractionation of Bright is
unaltered by dominant-negative transduction. Raji (107
cells), expressing wild type and the indicated mutant forms of Bright, were subjected to
fractionation into cytoplasm (CY), nucleoplasm (NP), chromatin (CH) and nuclear matrix (NM) and analysed by western using a-Bright anti-
serum. (C) Substitution mutants bind indistinguishably to IgH promoter sites. The indicated forms of Bright were prepared by in vitro
transcription/translation (IVT) and subjected to electrophoretic mobility shift assays using 32
P labelled S107 VH1-MAR probe. Speciļ¬city of
binding was demonstrated by a-Bright super-shift and cold probe competition (not shown) as previously described (Herrscher et al, 1995; Zong
et al, 2000; Kaplan et al, 2001). (D) Bright transactivation of inducible IgH promoter activity is not changed by dominant-negative transduction.
Exponentially growing Raji and Ramos cells, stably transduced with retroviruses encoding empty vector (control) or wild type and mutant
forms of Bright (as indicated), were transfected by electroporation with either a Fireļ¬y luciferase reporter (pGL3) or a pGL3-derived Bright-
responsive (Kim and Tucker, 2006) reporter (VH1-MAR-pGCL3) driven by the S107 VH1-MAR-containing promoter plus Renilla luciferase. After
culture for 2 days, cells were either untreated or stimulated for 5 days with 20 mg/ml LPS or stimulated for 5 min with anti-IgM F(ab0
)2 fragment,
as described in the Materials and methods. Dual luciferase activity was then measured as described (Rajaiya et al, 2006) and expressed as the
Fireļ¬y/Renilla ratio.
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
&2009 European Molecular Biology Organization The EMBO Journal VOL 28 | NO 6 | 2009 717
8. pY-Btk was detected in a-Btk IPs prepared from lipid rafts of
all a-m stimulated WT B-cell populations but in none of the
corresponding Bright-over-expressing TG B cells (Figure 7B).
As predicted by the global pY results, all WT and TG B-cells
responded with robust pY-Btk after strong (a-m Ć¾ a-CD19)
BCR ligation (Figure 6C).
Btk pY is required for TFII-I function as a nuclear tran-
scription factor (Rajaiya et al, 2005, 2006) as well as for its
cytoplasmic interaction with PLCg and subsequent inhibition
of PLCg-mediated Ca2 Ć¾
mobilisation (Guo et al, 2004;
Caraveo et al, 2006). As observed for Btk, TFII-I moved into
lipid rafts of TG and WT B-cell subsets after a-m stimulation
C
D
Btk
75 -
50 -
Raftlin
Bright
Ī³-Tubulin
Ī¼
CD-19
TFII-I
Bright
100 -
100 -
150 -
75 -
100 -
75 -
A
p-Y
2ā5x Bright
transgene wt
2ā5x Bright
transgene wt
ā
ā
ā
+ +
+ā
ā
ā
+ +
+ā
ā
ā
+ +
+ā
ā
ā
+ +
+ā
ā
ā
+ +
+ā
ā
ā
+ +
+ā
ā
ā
+ +
+ā
ā
ā
+ +
+
MZ B cells FO B cells
100 -
75 -
150 -
Ī±-CD19
Ī±-Ī¼
2ā5x Bright
transgene wt
2ā5x Bright
transgene wt
T1 B cells T2 B cells
W
C
L
Lipid rafts
0
1
1.8
2
2 Ī¼M Ionomycin
40 Ī¼M Digitonin
10 mM EGTA
20 mM Tris
2 Ī¼M Ionomycin
40 Ī¼M Digitonin
10mM EGTA
20 mM TrisĪ±-IgM F(abā)2 Ī±-IgM F(abā)2
Fluorescenceratio
100 s Bright transgenic CD43ā
splenocytes Wild-type CD43ā
splenocytes
B
FSC
SSC
B220+ fraction
B220
CD93
CD23
CD21
T 2
T 1
F O B
M Z B
31 2 118 9 1074 5 6 12 13 14 15 16 17 18 19 20 21 22 23 24
1.6
1.4
1.2
0.8
0.6
0.4
0.2
Figure 6 Transgenic over-expression of Bright decreases BCR signalling of normal B cells. (A) Mobilisation of intracellular Ca2 Ć¾
is reduced by
Bright over-expression. Wild type and Bright transgenic mice were sacriļ¬ced, splenic CD43Ć
B cells were prepared by negative selection,
loaded with Indo-1 and then subjected to measurements of intracellular Ca2 Ć¾
using 1 ng (low), 500 ng (medium) or 40 mg a-m (high) to
stimulate 106
cells; Ionomycin, Digitonin, EGTA and Tris were used for internal calibration. (B) Puriļ¬cation of T1, T2, FO and MZ B-cell
populations. B cells were prepared from single cell suspensions of transgenic (shown here) and wild-type (not shown) splenocytes. B220Ć¾
B-cell subpopulations were deļ¬ned as T1 (CD93Ć¾
CD23Ć
CD21Ć
), T2 (CD93Ć¾
CD23Ć¾
CD21Ć¾
), FO (CD93Ć
CD23Ć¾
CD21Ć¾
) and MZ (CD93Ć
CD23Ć
CD21Ć¾
). (C) Over-expression of Bright inhibits global phosphotyrosine responses of isolated B-cell populations. Each indicated
subpopulation (B106
cells) was stimulated for 5 min using 1 ng a-m, 1 ng a-CD19 or 1 ng a-m Ć¾ 1 ng a-CD19. Whole cell lysates (WCL) were
prepared from each and then were subjected to SDSāPAGE/western blotting using a-pY, a-Bright and a-Tubulin (loading control) anti-sera.
(D) Trafļ¬cking of BCR signalling components in and out of lipid rafts is not disturbed in transgenic B-cell populations. The indicated
subpopulations (B2 Ć106
cells each) were stimulated for 5 min using 2 ng a-m, 2 ng a-CD19 or 2 ng a-m Ć¾ 2 ng a-CD19. The entire preparation of
each was then used for the isolation of lipid rafts. Fractions from the gradient centrifugation were collected and divided into two equal parts.
One part was analysed directly by SDSāPAGE/western (lipid rafts from B106
cells per lane) using the antibodies indicated to serve as an input
control for the IP performed with the other half of the RIPA extracted samples (shown in Figure 7B). Here, increased levels of coprecipitated m,
Raftlin and CD19 indicated coalescence of lipid rafts upon BCR engagement (Depoil et al, 2008).
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
The EMBO Journal VOL 28 | NO 6 | 2009 &2009 European Molecular Biology Organization718
9. (Figures 6D and 7B). However, while stronger stimulation
(a-m Ć¾ a-CD19) further concentrated Btk and other signalo-
some components there, TFII-I was codischarged with Bright
(Figures 6D and 7B). Notably, phosphorylation of TFII-I and
Btk was inhibited in a-m stimulated rafts of Bright-over-
expressing TG B cells (Figure 7B).
These results indicate that lipid rafts-localised Bright con-
tributes to dampening of BCR responses by decoupling Btk
activation and downstream immediate early events, such as
tyrosine phosphorylation of TFII-I.
Discussion
We have shown that 1ā10% of Bright, a B-cell-speciļ¬c
transcriptional activator of IgH transcription, associates
with the BCR complex in lipid rafts prepared from trans-
formed mature B-cell lines or from primary B-cell populations
puriļ¬ed from mouse splenocytes or from human PBL. We
demonstrated that Bright is palmitoylated at a single cysteine
residue and that this modiļ¬cation is required for its localisa-
tion in lipid rafts. Our data indicate that the lipid raft
concentration of Bright is used for BCR threshold signalling,
as the sensitivity to BCR stimulation, as measured by calcium
ļ¬ux and transmission of global and Btk-mediated pTyr sig-
nals, depends on the amount of Bright discharged from the
rafts. An inducible, rafts-speciļ¬c association between Bright
and Sumo-I E2/Ubc-9 and E3/PIAS-1 enzymes, along with
accumulation of sumoylated Bright in the plasma membrane
only after BCR stimulation, led us to test whether this post-
translational modiļ¬cation triggered Bright discharge.
Signalling alterations observed in TG B cells that over-express
Bright, or in B-cells transduced with sumoylation-insensitive
and rafts localisation-defective dominant-negative Bright ret-
roviruses, support this notion. The data suggest a model in
which Bright acts as a ābrakeā to set a signalling threshold that
is regulated by alternative post-translational modiļ¬cation.
The strength of BCR-derived signals determines the se-
quential development of immature to mature B cells and their
subsequent fates in the spleen (Casola et al, 2004; Gazumyan
et al, 2006; Patterson et al, 2006; Pao et al, 2007). Shankar
et al reported that T1 and MZB cells are statistically increased
relative to other B-cell populations in Bright-over-expressing
TG mice. Although serum Ig levels were increased only
modestly, spontaneous autoimmunity ensued (Shankar
et al, 2007). Petro et al (2002) showed that under conditions
of prolonged (24 h) BCR engagement with high concentra-
tions of a-m (B10 mg/ml/105
cells), normal T1 B cells undergo
apoptosis, whereas T2 B cells proliferate. Under the same
conditions, T2 B cells display a higher threshold for a-m-
elicited signals than T1 cells (Petro et al, 2002; Hoek et al,
Btk
Raftlin
Ī¼
CD-19
TFII-I
Bright
p-Y
75 -
100 -
100 -
150 -
75 -
100 -
75 -
100 -
31 2 118 9 1074 5 6 12 13 14 15 16 17 18 19 20 21 22 23 24
B
IP Ī±-Btk
2ā5x Bright
transgene wt
2ā5x Bright
transgene wt
+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā+
+
+
āā
ā
MZ B cells FO B cells
Ī±-CD19
Ī±-Ī¼
2ā5x Bright
transgene wt
2ā5x Bright
transgene wt
T1 B cells T2 B cells
Ī±-CD19
Ī±-Ī¼
75 -
IP Ī±-Btk
75 - p-Y
Btk
C
242D
C
242S
Bright
Em
pty
vector
401
KIKK/AIAA
++
ā
ā
+
+
++
ā
ā
+
+
++
ā
ā
+
+
++
ā
ā
+
+
++
ā
ā
+
+
Ramos (high threshold)
75 -
41 2 3 9 1085 6 7
75 - p-Y
Btk
Raji (low threshold)
A
W
C
L
Lipid rafts
Figure 7 BCR-mediated activation of Btk and TFII-I depends on the levels of Bright in lipid rafts. (A) Dominant-negative Bright mutants alter
Btk phosphorylation in B-cell lines. Whole cell lysates prepared from retrovirally transduced cell lines established in Figure 4A were stimulated
for 5 min using either 500 ng a-m or 500 ng a-m Ć¾ 500 ng a-CD19 for 5 Ć108
cells as indicated. After anti-Btk immunoprecipitation, westerns were
performed with anti-Btk (loading control) and anti-pYanti-sera. (B) Transgenic Bright over-expression inhibits Btk and TFII-I phosphorylation
in B-cell subpopulations. Lipid raft extracts prepared using RIPA buffer (described in Figure 6D; equivalent of B106
cells per lane) were
subjected to a-Btk IP, followed by western blotting using the antibodies indicated.
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
&2009 European Molecular Biology Organization The EMBO Journal VOL 28 | NO 6 | 2009 719
10. 2006; Meyer-Bahlburg et al, 2008). To focus on early signal-
ling events and to circumvent apoptotic/proliferation com-
plications, we used B103
-fold lower concentrations of F(ab0
)2
in most of our stimulation assays. We found that over-
expression of Bright led to an elevated level of Bright in
lipid raft-associated signalosomes of all B-cell populations
except FO and increased signalling thresholds to our low
concentrations of a-m. In contrast to the imaging results of
Chung et al (2001), we found that this level of stimulation
was adequate to induce translocation of BCR components
(mIgM, CD19, and Btk) into rafts from all subpopulations.
However, our biochemical approach did not allow us to
address their contention that there are fewer lipid rafts in
immature B cells. That T1 and MZB were particularly sensi-
tive suggests that, in Bright TG mice, these populations would
be compromised in their ability to undergo appropriate
apoptotic responses to self-antigens in vivo. This would
account for the elevated numbers of TG T1 and MZBā
established predecessors for autoreactive B cells (Atencio
et al, 2004; Samuels et al, 2005; Yurasov et al, 2005a, b;
Quinn et al, 2006)āand provide a unique mechanism by
which a B-cell-restricted transcription factor could contribute
intrinsically to B-cell tolerance. On the other hand, and not
mutually exclusive, the selective production of antibodies
associated with the autoimmune syndromes of Bright TGs
(Shankar et al, 2007) could be a direct result of as yet
unidentiļ¬ed, non-IgH transcriptional targets of Bright.
Bright interacts with a well-deļ¬ned signalling molecule,
Btk (Webb et al, 2000; Rajaiya et al, 2005, 2006). Previously
we hypothesised (Webb et al, 1999) that their interaction in
the cytoplasm allowed Bright to deliver Btk to nuclear IgH
promoters. There, Btk could phosphorylate and activate TFII-
I, which at the time, was the only deļ¬ned substrate for Btk
(Novina et al, 1999). Subsequent studies by Webb and
colleagues (Webb et al, 2000; Rajaiya et al, 2005, 2006)
support the notion that Bright delivers Btk to places of active
TFII-I transcription in the nucleus.
Extension of the hypothesis would predict that Btk is
required for Brightās inclusion into lipid rafts and, taken to
the extreme, into lipid raft-localised BCR signalosomes.
However, in Btk-deļ¬cient non-B cells, exogenously expressed
Bright accumulated within lipid rafts, indicating that its
localisation is independent of Btk or other B-cell-speciļ¬c
factors. This led us to hypothesise that Bright might function
in lipid raft-localised BCR complexes to limit or increase the
concentration of Btk. BrightāBtk association was, indeed,
observed in lipid raft-localised BCR complexes. BCR engage-
ment resulted in reversed trafļ¬cking patterns in and out of
lipid rafts; that is, Btk accumulated in lipid raft-localised BCR
complexes as Bright was being depleted. Thus, Bright does
not function to limit signalosome-associated Btk.
Alternatively and in contrast to their kinase-dependent
collaboration in the nucleus (Rajaiya et al, 2006), we rea-
soned that BrightāBtk complexes in lipid rafts may be cata-
lytically inactive or compromised, such that Bright has to be
removed from Btk in order for the Tec kinase to achieve full
activity. Consistent with this notion, activation of Btk by
tyrosine phosphorylation is stimulated when Brightās entry
to lipid rafts is blocked by palmitoylation-defective mutants
and inhibited when Bright is trapped in lipid rafts by a
sumoylation-defective mutant. In further support, we found
that loss of Btk activation occurs in lipid rafts, and inactive
Btk (non-phosphorylated) is associated with Bright there.
This suggested that Bright-containing BCR complexes are
signalling-impaired because the presence of Bright in lipid
rafts raises the threshold of Btk-dependent BCR signalling.
Accordingly, Btk phosphorylation of a direct downstream
substrate, TFII-I, was inhibited within Bright-rich lipid rafts.
The lineage relationships between FO and MZB and the
role that Btk plays in this are controversial (Matthias and
Rolink, 2005; Teague et al, 2007; Welner et al, 2008). MZB
cells have been ascribed to develop either directly from T1
cells (Debnath et al, 2007) or from a subpopulation of CD21int
T2 B cells (Meyer-Bahlburg et al, 2008). Others contend that
both MZB and FO derive from a long lived, post-transitional
follicular B-cell subset described as Follicular Type II
(Cariappa et al, 2007; Allman and Pillai, 2008). Although
Xid phenotypic CBA/N mice show signiļ¬cantly greater loss of
FO (Hardy et al, 1982), their MZB numbers are also reduced
(Liu et al, 1988; Cariappa et al, 2001). The enrichment of
certain Ag speciļ¬cities into MZB requires functional Btk
(Martin and Kearney, 2000; Kanayama et al, 2005). Our
ļ¬ndings support a common progenitor model and suggest
that if MZB require an intact Btk signalling pathway, Bright
has a function in this regulation.
Bright is the ļ¬rst transcription factor shown to function in
lipid rafts, but its residence there is not unprecedented. Small
cytoplasmic fractions of Stat1 and Stat3 constitutively localise
to lipid rafts and have been suggested to function there
during early stages of cytokine signalling (Sehgal et al,
2002). An isoform of OCA-B, an IgH transcriptional coacti-
vator, localises as a myristoylated form to the cytoplasm and
to plasma membranes but not to lipid rafts per se (Yu et al,
2001, 2006). Similarly, TFII-I was previously detected in
cytoplasmic complexes with PLCg (Caraveo et al, 2006).
Btk-dependent phosphorylation of TFII-I was required for
its interaction with and concomitant inhibition of PLCg-
catalysed Ca2 Ć¾
mobilisation (Guo et al, 2004). Both TFII-I
and Bright are regulated by nucleocytoplasmic shuttling
(Novina et al, 1999; Nore et al, 2000; Kim and Tucker,
2006). Lipid rafts-designated Bright derives from a palmitoy-
lated pool that requires continual shuttling, as neither NLS
nor NES mutants accumulate in rafts. That nucleocytopla-
smic shuttling is also required for Bright transcriptional
activity (Kim and Tucker, 2006) raises the possibility that its
occupancy in lipid rafts is prerequisite for assembly of and
subsequent transfer of BrightāBtkāTFII-I complexes to the
nucleus. Consistent with this notion, Bright is codischarged
with activated TFII-I from lipid rafts after BCR ligation as a
Sumo-I-modiļ¬ed form. Although sumoylation has been as-
cribed to mediate nuclear import/export and activity of
transcription factors (Liu et al, 2006), the membrane-loca-
lised metabotropic glutamate receptor is targeted by the
sumoylation machinery (Tang et al, 2005). Similarly, the
sumo pathway was shown to control the activity of the
potassium channel K2P1 (Rajan et al, 2005).
Our observations suggest a new avenue for signal propa-
gation between the membrane and the nucleus. They em-
phasise the need to describe the properties of the
compartmentalised pools, their temporal and spatial regula-
tion and the molecular requirement(s) for the signalling
networks involved. Our results extend and enrich the notion
that mice with an artiļ¬cially altered BCR threshold are more
likely to display features of autoimmunity (Grimaldi et al,
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
The EMBO Journal VOL 28 | NO 6 | 2009 &2009 European Molecular Biology Organization720
11. 2005; Goodnow, 2007). We identify the transcription factor
Bright as an unsuspected component of such a network and
implicate it as regulator of an early event in BCR signalling
and immunologic tolerance.
Materials and methods
Cells
Raji (EBV Ć¾; McConnell et al, 1992; Miller et al, 1993; Caldwell
et al, 1998; Cerimele et al, 2005; Bernasconi et al, 2006), Daudi
(EBV Ć¾; Wang et al, 1990), Ramos (EBVĆ; Cerimele et al, 2005) and
CL01 (EBVĆ; Laskov et al, 2006) were obtained from ATCC
(Manassas, Virginia) and maintained as described (Fell et al,
1986). CD43-B cells were prepared by negative selection of whole
human blood (Gulf Coast Regional Blood Center, Houston, Texas) or
from B10-wk-old BALB/c murince splenocytes (Webb et al, 1998).
Preparative sorts were executed according to Webb et al (1998) and
Shankar et al (2007). B cells were stained as described by Kim and
Tucker (2006), and deconvolution was performed according to
Kuhn and Poenie (2002) and Combs et al (2006).
Molecular and cellular biology
Mutant forms of Bright were generated using the site directed
mutagenesis kit (Stratagene, CA) and transferred into the retroviral
construct pVxy (Ngo et al, 2006).
In vitro translation, sumoylation assays and transduction of B
cells were performed as described (Kienker et al, 1998; Rosas-
Acosta et al, 2005a, b; Kim and Tucker, 2006). Speciļ¬city of
sumoylation reactions was conļ¬rmed by cleavage of modiļ¬ed
Bright by Ulp-1 (Li and Hochstrasser (2003). Stabilisation of Sumo-1
modiļ¬ed Bright was achieved by alkylation with iodoacetic acid
sodium salt (Byrd and Hruby, 2005).
To assay for palmitoylation, WT and mutant forms of Bright as
well as VSV-G were transfected into Cos-7 cells and processed as
described (Rose et al, 1984; a-VSV was kindly provided by Dr
Michael G Roth, U.T. Southwestern Medical Center, Dallas; Yu and
Roth, 2002).
Preparation of stable retrovirally transduced
dominant-negative B-cell lines
Stable transductants were established by employment of the
Phoenix-A retroviral system. We plated 3 Ć105
amphitrophic
Phoenix-A packaging cells in 4 ml of DMEM supplemented with
10% fetal bovine serum (FBS) in 60-mm plates. After one day of
culture, cells were transfected using pBabe constructs using
FuGene6, and viral supernatant was harvested 2 days post-
transfection, centrifuged, and ļ¬ltered to remove live cells and
debris. Target cells (3 Ć105
) were plated into 60-mm plates and
growth medium was replaced with viral mixture. Stable cell lines
were established by selection with 2 mg/ml of puromycin from day 2
post-infection.
Transcriptional analysis
The transcriptional activity of WT Bright and mutant forms were
assayed by luciferase assays (Kim and Tucker, 2006; Rajaiya et al,
2006). Raji or Ramos cells (B5 Ć105
) stably transduced with empty
vector, WT or one of the mutant forms of Bright (401
KIKK/AIAA,
C342S or C342D) were mixed with 125 ng of pRL (Renilla) and
750 ng of either pGL3 or pGL3btp in 300 ml of RPMI. Cells were
incubated for 15 min at room temperature, transferred into
electroporation cuvettes and subjected to electroporation at 975 mF
and 260 V. Electroporated cells were left in the cuvette at room
temperature for 15 min and cultured in RPMI complete growth
medium for 5 h. Cells were then treated with LPS (20 mg/ml) for 3
days in complete growth medium to stimulate cofactors required for
Bright transactivation (Nixon et al, 2004a). Anti-IgM stimulation
was performed for 5 min using 500pg a-m for 5 Ć105
cells.
Luciferase activity was then measured and normalised according
to the Dual Luciferase Reporter Assay Kit from Promega.
B-cell stimulation
To measure signalling effects at low doses of anti-IgM where
receptor internalisation is minimised, we used monoclonal anti-IgM
antibodies in the absence of secondary cross-linking (please see
Supplementary Figures 2B, 4B and 5 for optimisation and further
considerations). To stimulate B cells, 500 ng of F(abā)2 fragments of
a-m (clone JDC-15; Dako [a-human]; OB1022; Southern Biotech
[a-mouse]) and a-CD19 (clone HD37; Dako [a-human]; clone SJ25-
C1 [a-mouse]) were added to 5 Ć108
cells for 5 min at 371C (or other
times as indicated in the ļ¬gures). We determined by FACS analysis
(data not shown) and semi-quantitative western of lipid raft-
associated mIgM (Supplementary Figure 5A) that under these
conditions B1ā5% of mIgM in rafts and membranes are engaged.
Measurement of free intracellular calcium
Approximately 1 Ć106
cells/ml were loaded with 2 mM Indo-1
(Molecular Probes) in HEPES buffered RPMI/1% FBS (pH 7.1) for
30 min at 371C and stimulated successively with 1 ng or 40 mg
(indicated in Figures 4B and 6A as low or high a-m, respectively.
Fluorescence was excited at 340 and 380 nm, and the resulting
signal at each excitation wavelength was plotted against time.
Internal calibration was performed using ļ¬nal concentrations of
2 mM Ionomycin, 40 mM Digitonin, 10 mM EGTA/pH 7 and 20 mM
Tris exactly as described (Vorndran et al, 1995). At the end of each
calcium trace, dye calibration to insure comparable loading and dye
responses were performed by treatment with 2 mM ionomycin
followed by 40 mM digitonin to release Indo-1 into the medium that
contains 1 mM calcium. These levels of calcium saturate the Indo-1
to give the apparent Rmax. Subsequently, excess EGTA/pH7 (10 mM)
is added, followed by 20 mM Tris base to convert EGTA from the -2
or -1 form at pH 7 (and below) to the -4 form, greatly increasing its
chelating ability. That this procedure was sufļ¬cient to achieve Rmin
was conļ¬rmed by the fact that, in the presence of a large excess of
EGTA (10 mM), further additions of Tris base (i.e., beyond two
equivalents) did not lower Indo-1 ratios.
Preparation of lipid rafts
Approximately 500 mg of wet cell pellet were washed twice in ice-
cold phosphate buffered solution (PBS) and homogenised in 5 ml of
10 mM Tris/Cl (pH 7.4), 1 mM EDTA, 250 mM sucrose, 1 mM
phenylmethylsulfonyl ļ¬uoride and 1 mg/ml leupeptin (all from
Sigma, St Louis, Montana) in a tightly ļ¬tted Dounce homogeniser
using ļ¬ve strokes (Shelton et al, 1982; Short and Barr, 2000). The
resulting homogenate was centrifuged at 900 g for 10 min at 41C, the
resulting supernatant was then subjected to centrifugation at
110 000 g for 90 min at 41C (Nagamatsu et al, 1992). The resulting
membrane pellet was resuspended in ice cold 500 ml TNE buffer
(10 mM Tris/Cl [pH 7.4], 150 mM NaCl, 5 mM EDTA, 1% Triton
X-100 [Sigma], 10 Ć protease inhibitors [Complete tablets, Roche,
Indianapolis, IN]). Sucrose gradients for the preparation of lipid
rafts were assembled exactly as described in an earlier publication
(Fuentes-PananaĀ“ et al, 2005). Lipid rafts were isolated by ļ¬otation
on discontinuous sucrose gradients. Membrane pellets were
extracted for 30 min on ice in TNE buffer. For the discontinuous
sucrose gradient, 1 ml of cleared supernatant was mixed with 1 ml
of 85% sucrose in TNE and transferred to the bottom of an
ultracentrifugation tube, followed by overlay with 6 ml of 35%
sucrose in TNE and 3.5 ml of 5% sucrose in TNE. Samples were
spun at 200 000 g for 30 h at 41C; fractions were collected from the
top of the gradient and analysed using western blotting and/or
coimmunoprecipitation, as described in an earlier publication (Kim
and Tucker, 2006).
Immunoprecipitation/western analyses
Unabridged BCR complexes have been successfully immunopreci-
pitated using either stringent RIPA-based buffers (Indraccolo et al,
2002; Gazumyan et al, 2006) or mild conditions, such as Digitonin
or NP-40 (Hombach et al, 1988; Batista et al, 1996). We used a
stringent RIPA formulation of 500 mM NaCl; 10 mM TrisāCl pH 8;
0.1% SDS; 5 mM EDTA, pH 8; 10 Ć protease inhibitor (Complete
tablet, Roche) to solubilise lipid rafts for subsequent immunopre-
cipitation experiments. Brieļ¬y, buoyant fractions, taken from the
discontinuous gradient centrifugation, were pooled and incubated
with the same volume of RIPA buffer on ice for 15 min. Resulting
extracts were pre-cleared by rocking with 1 ml of a 5% slurry of
RIPA equilibrated Protein A beads CL-4B (Amersham Pharmacia,
Uppsala) for 4 h at 41C and removal of the precipitate. The resulting
supernatant was then subjected to IP/western assays as described
(Kim and Tucker, 2006). The following antibodies were used:
a-CD19 (clone 6D5, Dako), a-Bright, a-IgM (BD Pharmingen), a-V5
(Sigma), a-Raftlin (Dr Akihiko Yoshimura; Fukuoka, Japan; Saeki
et al, 2003), a-Sumo-1 (Sigma), a-phosphotyrosine (Sigma),
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
&2009 European Molecular Biology Organization The EMBO Journal VOL 28 | NO 6 | 2009 721
12. a-caspase-3 (Cell Signalling) and anti-TFII-I (kindly provided by Dr
Carol Webb; Rajaiya et al, 2005).
Apoptosis
To assay for DNA fragmentation, 1 Ć108
cells were treated with
100 ng a-m for 72 h and processed as described by Duke et al (1983).
In sum, cells were harvested by centrifugation (200 g; 10 min at
room temperature), washed twice with PBS and resuspended in
2 ml lysis buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.5%
NP-40, and 0.5% SDS, pH 7.4) for 10 min at room temperature. DNA
was extracted twice with an equal volume of phenol followed by a
single extraction with an equal volume of chloroform. DNA
fragments were resolved in a 0.75% agarose gel in TBE running
buffer (90 mM Tris, 90 mM boric acid, 1.5 mM EDTA pH 8.4) and
visualised by staining with ethidium bromide and photography
under UV illumination.
Note added in proof
Nixon et al (2008) recently reported that dominant inhibition of
Bright DNA binding activity in vivo inhibits antibody response to
phosphorylcholine of B1 B cells-a subset whose development and
function are highly dependent on Btk signaling. These data provide
additional support for a mechanistic link between Btk and both
nuclear and lipid rafts-localized Bright.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
The authors are indebted to Paul Das for administrative assistance.
We thank Chhaya Das, Maya Ghosh and June V Harriss for expert
technical assistance. We thank Martin P Kracklauer and Dr Mark A
Brown for critically reading the manuscript and all members of the
Tucker lab for comments on the paper. SM is the recipient of an
Undergraduate Research Fellowship from UT Austin. Financial aid
from the NIH is acknowledged by GCI (CA110624), CFW
(AI044215), MP (AA015437) and PWT (Marie Betzner Morrow
Centennial Endowment, NIH grants AI64886 and CA031534).
References
Allman D, Lindsley RC, DeMuth W, Rudd K, Shinton SA, Hardy RR
(2001) Resolution of three nonproliferative immature splenic B
cell subsets reveals multiple selection points during peripheral B
cell maturation. J Immunol 167: 6834ā6840
Allman D, Pillai S (2008) Peripheral B cell subsets. Curr Opin
Immunol 20: 149ā157
Ashery U, Yizhar O, Rotblat B, Kloog Y (2006) Nonconventional
trafļ¬cking of Ras associated with Ras signal organization. Trafļ¬c
7: 119ā126
Atencio S, Amano H, Izui S, Kotzin BL (2004) Separation of the New
Zealand Black genetic contribution to lupus from New Zealand
Black determined expansions of marginal zone B and B1a cells.
J Immunol 172: 4159ā4166
Batista FD, Anand S, Presani G, Efremov DG, Burrone OR (1996)
The two membrane isoforms of human IgE assemble into
functionally distinct B cell antigen receptors. J Exp Med 184:
2197ā2205
Bernasconi M, Berger C, Sigrist JA, Bonanomi A, Sobek J, Niggli FK,
Nadal D (2006) Quantitative proļ¬ling of housekeeping and
Epstein-Barr virus gene transcription in Burkitt lymphoma cell
lines using an oligonucleotide microarray. Virol J 3: 43
Bossis G, Melchior F (2006) Regulation of SUMOylation by
reversible oxidation of SUMO conjugating enzymes. Mol Cell
21: 349ā357
Byrd CM, Hruby DE (2005) Development of an in vitro cleavage
assay system to examine vaccinia virus I7L cysteine proteinase
activity. Virol J 2: 63
Caldwell RG, Wilson JB, Anderson SJ, Longnecker R (1998) Epstein-
Barr virus LMP2A drives B cell development and survival in the
absence of normal B cell receptor signals. Immunity 9: 405ā411
Caraveo G, van Rossum DB, Patterson RL, Snyder SH, Desiderio S
(2006) Action of TFII-I outside the nucleus as an inhibitor of
agonist-induced calcium entry. Science 314: 122ā125
Cariappa A, Boboila C, Moran ST, Liu H, Shi HN, Pillai S (2007) The
recirculating B cell pool contains two functionally distinct, long-
lived, posttransitional, follicular B cell populations. J Immunol
179: 2270ā2281
Cariappa A, Tang M, Parng C, Nebelitskiy E, Carroll M,
Georgopoulos K, Pillai S (2001) The follicular versus marginal
zone B lymphocyte cell fate decision is regulated by Aiolos, Btk,
and CD21. Immunity 14: 603ā615
Carter RH, Fearon DT (1992) CD19: lowering the threshold for
antigen receptor stimulation of B lymphocytes. Science 256:
105ā107
Casola S, Otipoby KL, Alimzhanov M, Humme S, Uyttersprot N,
Kutok JL, Carroll MC, Rajewsky K (2004) B cell receptor signal
strength determines B cell fate. Nat Immunol 5: 317ā327
Cerimele F, Battle T, Lynch R, Frank DA, Murad E, Cohen C,
Macaron N, Sixbey J, Smith K, Watnick RS, Eliopoulos A,
Shehata B, Arbiser JL (2005) Reactive oxygen signaling and
MAPK activation distinguish Epstein-Barr Virus (EBV)-positive
versus EBV-negative Burkittās lymphoma. Proc Natl Acad Sci USA
102: 175ā179
Chen W, Wang HG, Srinivasula SM, Alnemri ES, Cooper NR (1999)
B cell apoptosis triggered by antigen receptor ligation proceeds
via a novel caspase-dependent pathway. J Immunol 163:
2483ā2491
Cherukuri A, Cheng PC, Sohn HW, Pierce SK (2001) The CD19/CD21
complex functions to prolong B cell antigen receptor signaling
from lipid rafts. Immunity 14: 169ā179
Chung JB, Baumeister MA, Monroe JG (2001) Cutting edge: differ-
ential sequestration of plasma membrane-associated B cell anti-
gen receptor in mature and immature B cells into
glycosphingolipid-enriched domains. J Immunol 166: 736ā740
Combs J, Kim SJ, Tan S, Ligon LA, Holzbaur EL, Kuhn J, Poenie M
(2006) Recruitment of dynein to the Jurkat immunological
synapse. Proc Natl Acad Sci USA 103: 14883ā14888
Debnath I, Roundy KM, Weis JJ, Weis JH (2007) Deļ¬ning in vivo
transcription factor complexes of the murine CD21 and CD23
genes. J Immunol 178: 7139ā7150
Depoil D, Fleire S, Treanor BL, Weber M, Harwood NE, Marchbank
KL, Tybulewicz VL, Batista FD (2008) CD19 is essential for B cell
activation by promoting B cell receptor-antigen microcluster
formation in response to membrane-bound ligand. Nat
Immunol 9: 63ā72
Dintzis HM, Dintzis RZ, Vogelstein B (1976) Molecular determi-
nants of immunogenicity: the immunon model of immune
response. Proc Natl Acad Sci USA 73: 3671ā3675
Duke RC, Chervenak R, Cohen JJ (1983) Endogenous endonuclease-
induced DNA fragmentation: an early event in cell-mediated
cytolysis. Proc Natl Acac Sci USA 80: 6361ā6365
Dykstra M, Cherukuri A, Sohn HW, Tzeng SJ, Pierce SK (2003)
Location is everything: lipid rafts and immune cell signaling.
Annu Rev Immunol 21: 457ā481
Fell HP, Smith RG, Tucker PW (1986) Molecular analysis of the
t(2;14) translocation of childhood chronic lymphocytic leukemia.
Science 232: 491ā494
Fuentes-PananaĀ“ EM, Bannish G, van der Voort D, King LB, Monroe
JG (2005) Iga/Igb complexes generate signals for B cell develop-
ment independent of selective plasma membrane compartmenta-
lization. J Immunol 174: 1245ā1252
Gauld SB, Dal Porto JM, Cambier JC (2002) B cell antigen receptor
signaling: roles in cell development and disease. Science 296:
1641ā1642
Gazumyan A, Reichlin A, Nussenzweig MC (2006) Igb tyrosine
residues contribute to the control of B cell receptor signaling by
regulating receptor internalization. J Exp Med 203: 1785ā1794
Gocke CB, Yu H, Kang J (2005) Systematic identiļ¬cation and
analysis of mammalian small ubiquitin-like modiļ¬er substrates.
J Biol Chem 280: 500ā512
Goodnow CC (2007) Multistep pathogenesis of autoimmune
disease. Cell 130: 25ā35
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
The EMBO Journal VOL 28 | NO 6 | 2009 &2009 European Molecular Biology Organization722
13. Grimaldi CM, Hill L, Xu X, Peeva E, Diamond B (2005) Hormonal
modulation of B cell development and repertoire selection. Mol
Immunol 42: 811ā820
Guo B, Rothstein TL (2005) B cell receptor (BCR) cross-talk: IL-4
creates an alternate pathway for BCR-induced ERK activation that
is phosphatidylinositol 3-kinase independent. J Immunol 174:
5375ā5381
Guo S, Ferl GZ, Deora R, Riedinger M, Yin S, Kerwin JL, Loo JA,
Witte ON (2004) A phosphorylation site in Brutonās tyrosine
kinase selectively regulates B cell calcium signaling efļ¬ciency
by altering phospholipase C-g activation. Proc Natl Acad Sci USA
101: 14180ā14185
Gupta N, Wollscheid B, Watts JD, Scheer B, Aebersold R, DeFranco
AL (2006) Quantitative proteomic analysis of B cell lipid rafts
reveals that ezrin regulates antigen receptor-mediated lipid raft
dynamics. Nat Immunol 7: 625ā633
Hakre S, Tussie-Luna MI, Ashworth T, Novina CD, Settleman J,
Sharp PA, Roy AL (2006) Opposing functions of TFII-I spliced
isoforms in growth factor-induced gene expression. Mol Cell 24:
301ā308
Hardy RR, Hayakawa K, Haaijman J, Herzenberg LA (1982) B-cell
subpopulations identiļ¬ed by two-colour ļ¬uorescence analysis.
Nature 297: 589ā591
Harwood NE, Batista FD (2008) New insights into the early
molecular events underlying B cell activation. Immunity 28:
609ā619
Herrscher RF, Kaplan MH, Lelsz DL, Das C, Scheuermann R, Tucker
PW (1995) The immunoglobulin heavy-chain matrix-associating
regions are bound by Bright: a B cell-speciļ¬c trans-activator
that describes a new DNA-binding protein family. Genes Dev 9:
3067ā3082
Hoek KL, Antony P, Lowe J, Shinners N, Sarmah B, Wente SR, Wang
D, Gerstein RM, Khan WN (2006) Transitional B cell fate is
associated with developmental stage-speciļ¬c regulation of dia-
cylglycerol and calcium signaling upon B cell receptor engage-
ment. J Immunol 177: 5405ā5413
Hombach J, Leclercq L, Radbruch A, Rajewsky K, Reth M (1988) A
novel 34kD protein co-isolated with the IgM molecule in surface
IgM-expressing cells. EMBO J 7: 3451ā3456
Indraccolo S, Minuzzo S, Zamarchi R, Calderazzo F, Piovan E,
Amadori A (2002) Alternatively spliced forms of Iga and Igb
prevent B cell receptor expression on the cell surface. Eur J
Immunol 32: 1530ā1540
Kahyo T, Nishida T, Yasuda H (2001) Involvement of PIAS1 in the
sumoylation of tumor suppressor p53. Mol Cell 8: 713ā718
Kanayama N, Cascalho M, Ohmori H (2005) Analysis of marginal
zone B cell development in the mouse with limited B cell
diversity: role of the antigen receptor signals in the recruitment
of B cells to the marginal zone. J Immunol 174: 1438ā1445
Kaplan MH, Zong RT, Herrscher RF, Scheuermann RH, Tucker PW
(2001) Transcriptional activation by a matrix associating region-
binding protein. contextual requirements for the function of
bright. J Biol Chem 276: 21325ā21330
Kienker LJ, Ghosh MR, Tucker PW (1998) Regulatory elements in
the promoter of a murine TCRD V gene segment. J Immunol 161:
791ā804
Kim D, Probst L, Das C, Tucker PW (2007) REKLES is an ARID3-
restricted multifunctional domain. J Biol Chem 282: 15768ā15777
Kim D, Tucker PW (2006) A regulated nucleocytoplasmic shuttle
contributes to Brightās function as a transcriptional activator of
immunoglobulin genes. Mol Cell Biol 26: 2187ā2201
Kuhn JR, Poenie M (2002) Dynamic polarization of the microtubule
cytoskeleton during CTL-mediated killing. Immunity 16:
111ā121
Laskov R, Berger N, Scharff MD, Horwitz MS (2006) Tumor necrosis
factor-a and CD40L modulate cell surface morphology and induce
aggregation in Ramos Burkittās lymphoma cells. Leuk Lymphoma
47: 507ā519
Li SJ, Hochstrasser M (2003) The Ulp1 SUMO isopeptidase: distinct
domains required for viability, nuclear envelope localization, and
substrate speciļ¬city. J Cell Biol 160: 1069ā1081
Li X, Martin F, Oliver AM, Kearney JF, Carter RH (2001) Antigen
receptor proximal signaling in splenic B-2 cell subsets. J Immunol
166: 3122ā3129
Lin D, Ippolito GC, Zong RT, Bryant J, Koslovsky J, Tucker P (2007)
Bright/ARID3A contributes to chromatin accessibility of the
immunoglobulin heavy chain enhancer. Mol Cancer 6: 23
Liu H, Ippolito GC, Wall JK, Niu T, Probst L, Lee BS, Pulford K,
Banham AH, Stockwin L, Shaffer AL, Staudt LM, Das C, Dyer MJ,
Tucker PW (2006) Functional studies of BCL11A: characterization
of the conserved BCL11A-XL splice variant and its interaction
with BCL6 in nuclear paraspeckles of germinal center B cells. Mol
Cancer 5: 18
Liu YJ, Lortan JE, Oldļ¬eld S, MacLennan IC (1988) CBA/N mice
have marginal zone B cells with normal surface immunoglobulin
phenotype. Adv Exp Med Biol 237: 105ā111
Loder F, Mutschler B, Ray RJ, Paige CJ, Sideras P, Torres R, Lamers
MC, Carsetti R (1999) B cell development in the spleen takes
place in discrete steps and is determined by the quality of B cell
receptor-derived signals. J Exp Med 190: 75ā89
Martin F, Kearney JF (2000) Positive selection from newly formed to
marginal zone B cells depends on the rate of clonal production,
CD19, and btk. Immunity 12: 39ā49
Matthias P, Rolink AG (2005) Transcriptional networks in develop-
ing and mature B cells. Nat Rev Immunol 5: 497ā508
McConnell FM, Shears SB, Lane PJ, Scheibel MS, Clark EA
(1992) Relationships between the degree of cross-linking of
surface immunoglobulin and the associated inositol 1,4,5-
trisphosphate and Ca2+
signals in human B cells. Biochem J
284: 447ā455
Meyer-Bahlburg A, Andrews SF, Yu KO, Porcelli SA, Rawlings DJ
(2008) Characterization of a late transitional B cell population
highly sensitive to BAFF-mediated homeostatic proliferation.
J Exp Med 205: 155ā168
Miller CL, Longnecker R, Kieff E (1993) Epstein-Barr virus latent
membrane protein 2A blocks calcium mobilization in B lympho-
cytes. J Virol 67: 3087ā3094
Nagamatsu S, Kornhauser JM, Burant CF, Seino S, Mayo KE, Bell GI
(1992) Glucose transporter expression in brain. cDNA sequence
of mouse GLUT3, the brain facilitative glucose transporter iso-
form, and identiļ¬cation of sites of expression by in situ hybridi-
zation. J Biol Chem 26: 467ā472
Ngo VN, Davis RE, Lamy L, Yu X, Zhao H, Lenz G, Lam LT, Dave S,
Yang L, Powell J, Staudt LM (2006) A loss-of-function RNA
interference screen for molecular targets in cancer. Nature 441:
106ā110
Niiro H, Clark EA (2002) Regulation of B-cell fate by antigen-
receptor signals. Nat Rev Immunol 2: 945ā956
Nixon JC, Rajaiya J, Webb CF (2004a) Mutations in the DNA-
binding domain of the transcription factor Bright act as dominant
negative proteins and interfere with immunoglobulin transacti-
vation. J Biol Chem 279: 52465ā52472
Nixon JC, Ferrell S, Miner C, Oldham AL, Hochgeschwender U,
Webb CF (2008) Transgenic mice expressing dominant-negative
bright exhibit defects in B1 B cells. J Immunol 181: 6913ā6922
Nixon JC, Rajaiya JB, Ayers N, Evetts S, Webb CF (2004b) The
transcription factor, Bright, is not expressed in all human B
lymphocyte subpopulations. Cell Immunol 228: 42ā53
Nore BF, Vargas L, Mohamed AJ, BrandeĀ“n LJ, BaĀØckesjoĀØ CM, Islam
TC, Mattsson PT, Hultenby K, Christensson B, Smith CI (2000)
Redistribution of Brutonās tyrosine kinase by activation of phos-
phatidylinositol 3-kinase and Rho-family GTPases. Eur J Immunol
30: 145ā154
Novina CD, Kumar S, Bajpai U, Cheriyath V, Zhang K, Pillai S,
Wortis HH, Roy AL (1999) Regulation of nuclear localization and
transcriptional activity of TFII-I by Brutonās tyrosine kinase. Mol
Cell Biol 19: 5014ā5024
Pao LI, Lam KP, Henderson JM, Kutok JL, Alimzhanov M, Nitschke
L, Thomas ML, Neel BG, Rajewsky K (2007) B cell-speciļ¬c
deletion of protein-tyrosine phosphatase Shp1 promotes B-1a
cell development and causes systemic autoimmunity. Immunity
27: 35ā48
Patterson HC, Kraus M, Kim YM, Ploegh H, Rajewsky K (2006) The
B cell receptor promotes B cell activation and proliferation
through a non-ITAM tyrosine in the Iga cytoplasmic domain.
Immunity 25: 55ā65
Petro JB, Gerstein RM, Lowe J, Carter RS, Shinners N, Khan WN
(2002) Transitional type 1 and 2 B lymphocyte subsets are
differentially responsive to antigen receptor signaling. J Biol
Chem 277: 48009ā48019
Putnam MA, Moquin AE, Merrihew M, Outcalt C, Sorge E, Caballero
A, GondreĀ“-Lewis TA, Drake JR (2003) Lipid raft-independent B
cell receptor-mediated antigen internalization and intracellular
trafļ¬cking. J Immunol 170: 905ā912
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
&2009 European Molecular Biology Organization The EMBO Journal VOL 28 | NO 6 | 2009 723
14. Quinn WJ, Noorchashm N, Crowley JE, Reed AJ, Noorchashm H,
Naji A, Cancro MP (2006) Cutting edge: impaired transitional B
cell production and selection in the nonobese diabetic mouse.
J Immunol 176: 7159ā7164
Rajaiya J, Hatļ¬eld M, Nixon JC, Rawlings DJ, Webb CF (2005)
Brutonās tyrosine kinase regulates immunoglobulin promoter
activation in association with the transcription factor Bright.
Mol Cell Biol 25: 2073ā2084
Rajaiya J, Nixon JC, Ayers N, Desgranges ZP, Roy AL, Webb CF
(2006) Induction of immunoglobulin heavy-chain transcription
through the transcription factor Bright requires TFII-I. Mol Cell
Biol 26: 4758ā4768
Rajan S, Plant LD, Rabin ML, Butler MH, Goldstein SA (2005)
Sumoylation silences the plasma membrane leak K+
channel
K2P1. Cell 121: 37ā47
Ravetch JV, Bolland S (2001) IgG Fc receptors. Annu Rev Immunol
19: 275ā290
Rosas-Acosta G, Langereis MA, Deyrieux A, Wilson VG (2005a)
Proteins of the PIAS family enhance the sumoylation of the
papillomavirus E1 protein. Virology 331: 190ā203
Rosas-Acosta G, Russell WK, Deyrieux A, Russell DH, Wilson VG
(2005b) A universal strategy for proteomic studies of SUMO and
other ubiquitin-like modiļ¬ers. Mol Cell Proteomics 4: 56ā72
Rose JK, Adams GA, Gallione CJ (1984) The presence of cysteine in
the cytoplasmic domain of the vesicular stomatitis virus glyco-
protein is required for palmitate addition. Proc Natl Acad Sci USA
81: 2050ā2054
Saeki K, Miura Y, Aki D, Kurosaki T, Yoshimura A (2003) The B cell-
speciļ¬c major raft protein, Raftlin, is necessary for the integrity of
lipid raft and BCR signal transduction. EMBO J 22: 3015ā3026
Sampson DA, Wang M, Matunis MJ (2001) The small ubiquitin-like
modiļ¬er-1 (SUMO-1) consensus sequence mediates Ubc9 binding
and is essential for SUMO-1 modiļ¬cation. J Biol Chem 276:
21664ā21669
Samuels J, Ng YS, Coupillaud C, Paget D, Meffre E (2005) Impaired
early B cell tolerance in patients with rheumatoid arthritis. J Exp
Med 201: 1659ā1667
Schwarz SE, Matuschewski K, Liakopoulos D, Scheffner M, Jentsch
S (1998) The ubiquitin-like proteins SMT3 and SUMO-1 are
conjugated by the UBC9 E2 enzyme. Proc Natl Acad Sci USA 95:
560ā564
Sehgal PB, Guo GG, Shah M, Kumar V, Patel K (2002) Cytokine
signaling: STATS in plasma membrane rafts. J Biol Chem 277:
12067ā12074
Shankar M, Nixon JC, Maier S, Workman J, Farris AD, Webb CF
(2007) Antinuclear antibody production and autoimmunity in
transgenic mice that overexpress the transcription factor Bright.
J Immunol 178: 2996ā3006
Shelton KR, Guthrie VH, Cochran DL (1982) Oligomeric structure of
the major nuclear envelope protein lamin B. J Biol Chem 257:
4328ā4332
Short B, Barr FA (2000) The Golgi apparatus. Curr Biol 10:
R583āR585
Simons K, Toomre D (2000) Lipid rafts and signal transduction.
Nat Rev Mol Cell Biol 1: 31ā39
Sims GP, Ettinger R, Shirota Y, Yarboro CH, Illei GG, Lipsky PE
(2005) Identiļ¬cation and characterization of circulating human
transitional B cells. Blood 105: 4390ā4398
Sohn HW, Tolar P, Jin T, Pierce SK (2006) Fluorescence resonance
energy transfer in living cells reveals dynamic membrane changes
in the initiation of B cell signaling. Proc Natl Acad Sci USA 103:
8143ā8148
Sproul TW, Malapati S, Kim J, Pierce SK (2000) Cutting edge: B cell
antigen receptor signaling occurs outside lipid rafts in immature B
cells. J Immunol 165: 6020ā6023
Stoddart A, Dykstra ML, Brown BK, Song W, Pierce SK, Brodsky FM
(2002) Lipid rafts unite signaling cascades with clathrin to
regulate BCR internalization. Immunity 17: 451ā462
Su TT, Guo B, Wei B, Braun J, Rawlings DJ (2004) Signaling in
transitional type 2 B cells is critical for peripheral B-cell devel-
opment. Immunol Rev 197: 161ā178
Su TT, Rawlings DJ (2002) Transitional B lymphocyte subsets
operate as distinct checkpoints in murine splenic B cell develop-
ment. J Immunol 168: 2101ā2110
Tang Z, El Far O, Betz H, Scheschonka A (2005) Pias1 interaction
and sumoylation of metabotropic glutamate receptor 8. J Biol
Chem 280: 38153ā38159
Teague BN, Pan Y, Mudd PA, Nakken B, Zhang Q, Szodoray P,
Kim-Howard X, Wilson PC, Farris AD (2007) Cutting edge:
Transitional T3 B cells do not give rise to mature B cells, have
undergone selection, and are reduced in murine lupus. J Immunol
178: 7511ā7515
Venkatesh J, Peeva E, Xu X, Diamond B (2006) Cutting edge:
hormonal milieu, not antigenic speciļ¬city, determines the mature
phenotype of autoreactive B cells. J Immunol 176: 3311ā3314
Vorndran C, Minta A, Poenie M (1995) New ļ¬uorescent calcium
indicators designed for cytosolic retention or measuring calcium
near membranes. Biophys J 69: 2112ā2124
Wang F, Gregory C, Sample C, Rowe M, Liebowitz D, Murray R,
Rickinson A, Kieff E (1990) Epstein-Barr virus latent membrane
protein (LMP1) and nuclear proteins 2 and 3C are effectors of
phenotypic changes in B lymphocytes: EBNA-2 and LMP1 co-
operatively induce CD23. J Virol 64: 2309ā2318
Webb C, Zong RT, Lin D, Wang Z, Kaplan M, Paulin Y, Smith E,
Probst L, Bryant J, Goldstein A, Scheuermann R, Tucker P (1999)
Differential regulation of immunoglobulin gene transcription via
nuclear matrix-associated regions. Cold Spring Harb Symp Quant
Biol 64: 109ā118
Webb CF, Das C, Eaton S, Calame K, Tucker PW (1991a) Novel
protein-DNA interactions associated with increased immunoglo-
bulin transcription in response to antigen plus interleukin-5. Mol
Cell Biol 11: 5197ā5205
Webb CF, Das C, Eneff KL, Tucker PW (1991b) Identiļ¬cation of a
matrix-associated region 50
of an immunoglobulin heavy chain
variable region gene. Mol Cell Biol 11: 5206ā5211
Webb CF, Smith EA, Medina KL, Buchanan KL, Smithson G, Dou S
(1998) Expression of bright at two distinct stages of B lymphocyte
development. J Immunol 160: 4747ā4754
Webb CF, Yamashita Y, Ayers N, Evetts S, Paulin Y, Conley ME,
Smith EA (2000) The transcription factor Bright associates with
Brutonās tyrosine kinase, the defective protein in immunodeļ¬-
ciency disease. J Immunol 165: 6956ā6965
Welner RS, Pelayo R, Kincade PW (2008) Evolving views on the
genealogy of B cells. Nat Rev Immunol 8: 95ā106
Wilsker D, Probst L, Wain HM, Maltais L, Tucker PW, Moran E
(2005) Nomenclature of the ARID family of DNA-binding pro-
teins. Genomics 86: 242ā251
Yu S, Roth MG (2002) Casein kinase I regulates membrane binding
by ARF GAP1. Mol Biol Cell 13: 2559ā2570
Yu X, Wang L, Luo Y, Roeder RG (2001) Identiļ¬cation and char-
acterization of a novel OCA-B isoform. Implications for a role in B
cell signaling pathways. Immunity 14: 157ā167
Yu X, Siegel R, Roeder RG (2006) Interaction of the B cell-speciļ¬c
transcriptional coactivator OCA-B and Galectin-1 and a possible
role in regulating BCR-mediated B cell proliferation. J Biol Chem
281: 15505ā15516
Yurasov S, Hammersen J, Tiller T, Tsuiji M, Wardemann H (2005a)
B-cell tolerance checkpoints in healthy humans and patients with
systemic lupus erythematosus. Ann NY Acad Sci 1062: 165ā174
Yurasov S, Wardemann H, Hammersen J, Tsuiji M, Meffre E, Pascual
V, Nussenzweig MC (2005b) Defective B cell tolerance check-
points in systemic lupus erythematosus. J Exp Med 201: 703ā711
Zong RT, Das C, Tucker PW (2000) Regulation of matrix attachment
region-dependent, lymphocyte-restricted transcription through
differential localization within promyelocytic leukemia nuclear
bodies. EMBO J 19: 4123ā4133
Rafts-localised Bright regulates BCR signalling
C Schmidt et al
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