Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain. Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 282, NO. 32, pp. 23306–23315, August 10, 2007 目前已知PTEN(Phosphatase and tensin homolog)是腫瘤抑制蛋白，其由403個氨基酸組成，主要分PTPase及C2 domain，C2 domain使PTEN可與細胞膜作用連結。 PTEN之C-tail(aa 350~403)被發現具有調控PTEN自身活性之功能。前人研究指出C-tail有6個可磷酸化之位置(Thr-366、Ser-370、Ser-380、Thr-382、Thr-383及Ser-385)，這些位置可調控PTEN之腫瘤抑制能力、胞內之分佈及穩定性。前人產生以上位置突變之PTEN變異株，發現這些變異株具有更強的腫瘤抑制能力，但穩定性將降低，這可能是因這些變異株具有更開放結構所致。 本報告針對研究PTEN C-tail在連結細胞膜和在其本身催化活性中扮演的功能。作者先產生一系列之PTEN磷酸化位置變異株，發現S385A會促使PTEN之membrane localization in vivo及加強phosphatase活性in vitro，而且此突變會使Ser-380/Thr-382/Thr-383 cluster的磷酸化程度降低，因此知Ser-385可透過被去磷酸化以調控PTEN。而以phosphomimic residues取代Ser-380/Thr-382/Thr-383會使上述S385A所產生之PTEN催化活性反轉。之後利用免疫沉澱方法，發現C-tail之71-amino acid region會與C2 domain上之CBR3 motif作用，暗示C-tail參與連結細胞膜之調控。最後利用合成之PTEN C-tail peptide，發現其可抑制PTEN之催化活性in vitro，而在細胞表現此peptide則會抑制PTEN之membrane localization，磷酸化之Akt量亦上升。以上實驗顯示C-tail在PTEN之membrane recruitment及PTPase活性調控中扮演Autoinhibitory domain角色。

1. Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029 J Biol Chem. 2007 Aug 10;282(32):23306-15. Epub 2007 Jun 12 IF: 5.520 Design by ChauChanLao 2009.12

3. http://www.cellsignal.com/

4. http://www.cellsignal.com/

7. However, whether these phosphorylation sites contribute to the intrinsic catalytic activity of PTEN remained to be determined Cell Cycle 5, 1523–1527

13. Experimental Design To gain insights into the distribution of PTEN in different cellular compartments Subcellular Fractionation To investigate the relative contribution of individual phosphorylation sites to membrane targeting in mammalian cells Gene Transfection To investigate how individual phosphorylation sites contribute to the innate catalytic property of PTEN Phosphatase Activity Assay 1. To investigate if C-tail could mediate intramolecular interactions with PTEN domains implicated in membrane binding 2.To map the residues in the C2 domain involved in the interaction with the C-tail Immunoprecipitation Assay Peptide Inhibition Assay To investigate whether the C-tail could act as an autoinhibitory domain

14. To gain insights into the distribution of PTEN in different cellular compartments Fig.1A MB: Membrane CYT: Cytosolic NIH3T3, MCF10A, Madin-Darby canine kidney cells Hypotonic Subcellular Fractionation 100,000 g, 50min, 4℃ Nuclei Cytosolic fraction 18,000 g, 10min, 4℃ Membrane fraction Western blot Hypotonic buffer; Homogenization; 500 g , 5min, 4℃ Lactate dehydrogenase ( LDH ) and R-Ras were used as cytosolic and membrane markers, respectively NIH3T3: Primary embryonic fibroblast cells, mouse MCF10A: Non-tumorigenic mammary gland epithelial cell line, human MDCK: Kidney cell, canine

15. Saponin Subcellular Fractionation NIH3T3, MCF10A, Madin-Darby canine kidney cells Lysis buffer (0.01% saponin); 18000 g , 30min Cytosolic fraction Resuspend; 18000 g , 30min Membrane fraction Western blot Fig.1B MB: Membrane CYT: Cytosolic Wikipedia.org the use of a weaker nonionic detergent, saponin (0.01%), increased the amount of PTEN in the membrane fraction to a detectable level Cancer Cell 6, 117–127

16. Nuclear-Cytosolic Fractionation NIH3T3, MCF10A, Madin-Darby canine kidney cells Cytoplasmic Extraction Reagent II; Vortex; 16,000 g , 5min cytoplasmic extract Nuclear Extraction Reagent; Vortex; 16,000 g , 10min nuclear extract Western blot Fig.1C CYT: Cytosolic NU: Nuclear Sp1 was used as nuclear markers

17. Fig.1D Sucrose Gradient Caveolin: membrane protein NIH3T3 MCF10A MDCK Extraction buffer; Homogenization; Sucrose(5,10, 15, 20, 25, 30%); 34,000 g, 18h, 4 °C Western blot 1ml fraction Dilute; 14,000 g 30min

20. To investigate the relative contribution of individual phosphorylation sites to membrane targeting in mammalian cells Gene Transfection Fig.2A PC3 (PTEN null cell line) PTEN WT cDNA, pCEFL-KZ-AU5-PTEN mutant cDNAs; Lipofectamine2000, 4h; Incubation, 48h Saponin Subcellular Fractionation Western blot 4X 3.5X 5.5X 2X 2X

21. To ascertain the role of phosphorylation at positions Ser-380 and Ser-385 in membrane recruitment Fig.2B Gene Transfection Saponin Subcellular Fractionation Western blot PC3 cell

24. Fig.3B,C not statistically significant

30. To investigate if C-tail could mediate intramolecular interactions with PTEN domains implicated in membrane binding Fig.5A

31. Gene Transfection 293T cells Fig.5A,B Immuno-precipitation Buffer A; α-FLAG monoclonal antibody; GammaBind G-Sepharose beads Western blot

32. Fig.5C Gene Transfection Immuno-precipitation Western blot Fig.5D Fig.5E 293T cells Fig.5A

33. PTEN C-tail interacts with the C2 Domain

36. To map the residues in the C2 domain involved in the interaction with the C-tail Fig.7 Gene Transfection Immuno-precipitation Western blot 293T cells

41. Fig.8A,B Peptide Inhibition Assay BL21 bacteria PTEN Spectrophotometer diC 8 -PIP 3 ; Cp-23 (0.1, 0.4, 0.6, 0.8, 1, 2mM), Cp-23DE (0.1, 0.2, 0.3, 0.4, 0.5mM), Cp-23DEr (0, 0.07, 0.2, 0.7, 2mM); 10min, 37 °C 3X IC 50 : inhibitory potency

42. To investigate whether the C-tail peptide could inhibit PTEN catalytic activity in cultured cells Gene Transfection Saponin Subcellular Fractionation Western blot GFP, GFP-Cp-23, GFP-Cp-23/385E expression plasmids 2.5X 293T cells Fig.9A,B,C

46. Conversely, the dephosphorylation of Ser-385 triggers a cascade of events that will unmask the catalytic pocket and the C2 domain PTEN attaining this activation state has a high affinity for plasma membrane and can dephosphorylate PIP3 with great efficiency

54. Thanks for your attentions

59. Fig.S1