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Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan From the Department of Oncological Sciences, Mount Sinai...
<ul><li>Phosphatase and tensin homolog </li></ul><ul><li>55kD </li></ul><ul><li>Phosphatidylinositol (3, 4, 5) triphosphat...
http://www.cellsignal.com/
http://www.cellsignal.com/
<ul><li>403 amino acid </li></ul><ul><li>Consists of a phosphatase domain, and a C2 domain </li></ul>Wikipedia.org PTEN bi...
C-Tail <ul><li>There are six phosphorylation sites at Thr-366, Ser-370, Ser-380, Thr-382, Thr-383, and Ser-385 that are im...
However, whether these phosphorylation sites contribute to the intrinsic catalytic activity of PTEN remained to be determi...
Ser-370 and Ser-385 <ul><li>Ser-370 and Ser-385 have been shown to be preferentially phosphorylated by casein kinase  in v...
Ser-362 and Thr-366 <ul><li>Additional phosphorylation sites at Ser-362 and Thr-366 have been reported to be the targets o...
Ser-380/Thr-382/Thr-383 cluster <ul><li>The cluster of Ser-380, Thr-382, and Thr-383 residues in the tail region are minor...
<ul><li>The mechanism by which PTEN targets the plasma membrane is under intense investigation.  </li></ul><ul><li>In addi...
<ul><li>To investigated the role of the PTEN carboxyl-terminal tail domain in regulating its membrane targeting and cataly...
Experimental Design To gain insights into the distribution of PTEN in different cellular compartments Subcellular Fraction...
To gain insights into the distribution of PTEN in different cellular compartments Fig.1A MB: Membrane CYT: Cytosolic NIH3T...
Saponin Subcellular Fractionation NIH3T3, MCF10A,  Madin-Darby canine kidney cells Lysis buffer (0.01% saponin); 18000 g ,...
Nuclear-Cytosolic Fractionation NIH3T3, MCF10A,  Madin-Darby canine kidney cells Cytoplasmic Extraction Reagent II; Vortex...
Fig.1D Sucrose Gradient Caveolin: membrane protein NIH3T3 MCF10A  MDCK Extraction buffer; Homogenization; Sucrose(5,10, 15...
<ul><li>(a) A sucrose density gradient is created in a centrifuge tube by layering solutions of differing densities. </li>...
<ul><li>All of these data were consistent with several previous reports showing the predominant cytosolic localization of ...
To investigate the relative contribution of individual phosphorylation sites to membrane targeting in mammalian cells Gene...
To ascertain the role of phosphorylation at positions Ser-380 and Ser-385 in membrane recruitment Fig.2B Gene Transfection...
<ul><li>J. Biol. Chem.  278, 33617–33620 </li></ul>Fig.3A Gene Transfection 293T cells, (human kidney epithelial cell) PTE...
Fusing an  N -myristoylation consensus sequence to PTEN-NH 2  terminus <ul><li>The MYR-PTEN was constructed by fusing an a...
Fig.3B,C not statistically significant
<ul><li>Ser-380 and Ser-385 involved in PTEN Membrane Targeting  </li></ul><ul><li>Mutating Ser-385 to alanine (S385A) pro...
To investigate how individual phosphorylation sites contribute to the innate catalytic property of PTEN Gene Transfection ...
<ul><li>Phosphorylation at the Ser-380/Thr-382/Thr-383 cluster may be positively regulated by phosphorylation events at po...
<ul><li>Substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutat...
Phosphorylation State of PTEN C-tail Affects Its Phosphatase Activity <ul><li>S385A mutation was associated with a substan...
To investigate if C-tail could mediate intramolecular interactions with PTEN domains implicated in membrane binding Fig.5A
Gene Transfection 293T cells Fig.5A,B Immuno-precipitation Buffer A; α-FLAG monoclonal antibody; GammaBind G-Sepharose bea...
Fig.5C Gene Transfection Immuno-precipitation Western blot Fig.5D Fig.5E 293T cells Fig.5A
PTEN C-tail interacts with the C2 Domain
<ul><li>Interaction between the PTPase domain and the CTD required the presence of the C-tail region </li></ul>Gene Transf...
<ul><li>PTEN C-tail may interface with the catalytic domain through interacting with the C2 domain </li></ul>
To map the residues in the C2 domain involved in the interaction with the C-tail Fig.7 Gene Transfection Immuno-precipitat...
<ul><li>The conserved CBRIII loop possesses five positively charged and two hydrophobic residues that are implicated in ph...
<ul><li>CBR3 mutant: Lys-260, Lys-263, Lys-266, Lys-267, and Lys-269 to alanines </li></ul><ul><li>Cα2mutant: substituting...
The C-tail Interacts Extensively with the CBRIII Motif of the C2 Domain <ul><li>These results indicate that the interactio...
To investigate whether the C-tail could act as an autoinhibitory domain <ul><li>PTEN displayed a high affinity for acidic ...
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...
To investigate whether the C-tail peptide could inhibit PTEN catalytic activity in cultured cells Gene Transfection Saponi...
<ul><li>These results demonstrated that the phosphorylation cluster within the C-tail region plays an inhibitory role in P...
Summary <ul><li>Ser-380 and Ser-385 involved in PTEN Membrane Targeting </li></ul><ul><li>S385A promoted membrane localiza...
<ul><li>The C-tail forms an extensive interaction with the CBRIII region of the C2 domain through nonelectrostatic means <...
Conversely, the dephosphorylation of Ser-385 triggers a cascade of events that will unmask the catalytic pocket and the C2...
Conclusion <ul><li>The COOH-terminal tail of PTEN can act as an autoinhibitory domain to control both PTEN membrane recrui...
<ul><li>It has been reported that the phosphorylation at Ser-370 can prime the phosphorylation of Ser-366 by glycogen synt...
<ul><li>PTEN phosphorylated at Ser-380 constitutes only a minor pool of total PTEN  in vitro </li></ul>Fig.2B Fig.3C Fed. ...
<ul><li>1. Ser-380 most likely plays a structural role in regulating PTEN membrane-binding capacity. For instance, mutatin...
<ul><li>There have been suggestions that phosphorylation at Ser-385 may prime the phosphorylation of Thr-383 by casein kin...
<ul><li>However, we are unable to discern whether the higher phosphatase activity and membrane localization observed with ...
<ul><li>There is still considerable uncertainty in how the COOH terminal domain of PTEN regulates its biochemical function...
Thanks for your attentions
<ul><li>Positively charged residues interfacing the lipid bilayer at Arg-161, Lys-163, and Lys-164 have been shown to cont...
<ul><li>It is speculated that phosphorylation at these sites constitutes a negative feedback mechanism in attenuating sign...
<ul><li>PIP2 fails to stimulate PTEN when a soluble lipid substrate, inositol 1,3,4,5-trisphosphate, was used argues for a...
<ul><li>All of these findings favor an interfacial activation mechanism of PTEN at the lipid bilayer involving interaction...
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Regulation of pten activity by its carboxyl terminal autoinhibitory

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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。

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Transcript of "Regulation of pten activity by its carboxyl terminal autoinhibitory"

  1. 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
  2. 2. <ul><li>Phosphatase and tensin homolog </li></ul><ul><li>55kD </li></ul><ul><li>Phosphatidylinositol (3, 4, 5) triphosphate (PIP 3 ) phosphatase (PTPase) </li></ul><ul><li>Functions as a tumor suppressor by negatively regulating Akt/PKB signaling pathway </li></ul>PTEN
  3. 3. http://www.cellsignal.com/
  4. 4. http://www.cellsignal.com/
  5. 5. <ul><li>403 amino acid </li></ul><ul><li>Consists of a phosphatase domain, and a C2 domain </li></ul>Wikipedia.org PTEN binds the membrane through its C2 domain bringing the active site to the membrane-bound PIP 3 to de-phosphorylate it PTEN structure
  6. 6. C-Tail <ul><li>There are six phosphorylation sites at Thr-366, Ser-370, Ser-380, Thr-382, Thr-383, and Ser-385 that are implicated in modulating PTEN tumor suppressor functions, subcellular distribution, and stability. </li></ul>Mol. Cell. Biol. 20, 5010–5018
  7. 7. However, whether these phosphorylation sites contribute to the intrinsic catalytic activity of PTEN remained to be determined Cell Cycle 5, 1523–1527
  8. 8. Ser-370 and Ser-385 <ul><li>Ser-370 and Ser-385 have been shown to be preferentially phosphorylated by casein kinase in vivo . </li></ul><ul><li>A double mutant of S370A/S385A has been shown to have shorter half life than the wild-type protein </li></ul><ul><li>The biological relevance of phosphorylation at Ser-370 and Ser-385 is not well defined </li></ul>J. Biol. Chem. 280, 35195–35202
  9. 9. Ser-362 and Thr-366 <ul><li>Additional phosphorylation sites at Ser-362 and Thr-366 have been reported to be the targets of glycogen synthase kinase-3 </li></ul>J. Biol. Chem. 280, 35195–35202
  10. 10. Ser-380/Thr-382/Thr-383 cluster <ul><li>The cluster of Ser-380, Thr-382, and Thr-383 residues in the tail region are minor phosphorylation sites . However, leptin has recently been reported to stimulate their phosphorylation, resulting in the inhibition of PTEN catalytic activity </li></ul><ul><li>Furthermore, Thr-383 has been identified as a putative autodephosphorylation site for PTEN, since its dephosphorylation greatly enhances the ability of PTEN to suppress cell migration in human glioblastoma cell lines </li></ul>Science 303, 1179–1181 J. Biol. Chem. 280, 35195–35202 382 383
  11. 11. <ul><li>The mechanism by which PTEN targets the plasma membrane is under intense investigation. </li></ul><ul><li>In addition, how the intrinsic catalytic activity of PTEN is regulated has not been fully delineated. </li></ul>
  12. 12. <ul><li>To investigated the role of the PTEN carboxyl-terminal tail domain in regulating its membrane targeting and catalytic functions </li></ul>The aim in this article
  13. 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. 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. 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. 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. 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
  18. 18. <ul><li>(a) A sucrose density gradient is created in a centrifuge tube by layering solutions of differing densities. </li></ul><ul><li>(b) The sample to be tested is placed on top of the gradient. </li></ul><ul><li>(c) Centrifugation causes the various components (fractions) of the sample to sediment differentially. </li></ul><ul><li>(d) The different fractions appear as bands in the centrifuged gradient. </li></ul><ul><li>(e) The different bands can be collected separately by collecting samples from the bottom of the tube at fixed time intervals. </li></ul>Sucrose Gradient www.ncbi.nlm.nih.gov
  19. 19. <ul><li>All of these data were consistent with several previous reports showing the predominant cytosolic localization of PTEN in mammalian cells </li></ul>
  20. 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. 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
  22. 22. <ul><li>J. Biol. Chem. 278, 33617–33620 </li></ul>Fig.3A Gene Transfection 293T cells, (human kidney epithelial cell) PTEN WT cDNA, MYR-PTEN, △ N16-PTEN; Lipofectamine2000, 4h; Incubation, 48h DAPI Immunofluorescence Analysis 3% paraformaldehyde; anti-AU5, overnight; Alexa Fluor 488 anti-mouse secondary antibody; DAPI; confocal microscopy Wikipedia.org
  23. 23. Fusing an N -myristoylation consensus sequence to PTEN-NH 2 terminus <ul><li>The MYR-PTEN was constructed by fusing an adaptor, agcttctcgaggccgccaccatg gggagtagcaagagcaagcctaaggaccccagccagcg cg, containing the myristoylation signal peptide ( GSSKSKPKDPSQR ) in the HindIII and BamHI sites of the pCEFL-KZ-AU5 -PTEN expression plasmid. </li></ul>www.univ-angers.fr 肉豆蔻酸 Fig.3A
  24. 24. Fig.3B,C not statistically significant
  25. 25. <ul><li>Ser-380 and Ser-385 involved in PTEN Membrane Targeting </li></ul><ul><li>Mutating Ser-385 to alanine (S385A) promoted membrane localization in vivo </li></ul>
  26. 26. To investigate how individual phosphorylation sites contribute to the innate catalytic property of PTEN Gene Transfection Sf9 insect cells (pupal ovarian tissue , Fall armyworm Spodoptera frugiperda) baculoviruses harboring pET15B-His-PTEN, pET15B-His-C-tail mutant, 5~7 days; Purification Lysed in buffer A; 18,000 g ; His Trap HP column; gel filtration Phosphatase Activity Assay diC8-PIP 2 , diC8-PIP 3 ; 10 min; Spectrophotometer ( reading the absorbance at 620 nm) PIP2 failed to further stimulate their catalytic activity Fig.4A <ul><li>The NH 2 -terminal 16-aa polybasic region constitutes a PIP 2 -binding motif that is essential for PTEN tumor suppressor function, PIP 2 binding to this motif greatly stimulates the intrinsic catalytic activity of PTEN </li></ul>J. Biol. Chem. 278, 33617–33620 2–3-fold decrease 2–3-fold increase
  27. 27. <ul><li>Phosphorylation at the Ser-380/Thr-382/Thr-383 cluster may be positively regulated by phosphorylation events at positions Ser-370 and Ser-385 </li></ul>Fig.4C,D
  28. 28. <ul><li>Substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutation </li></ul>Gene Transfection 293T cells Purification Lysis buffer; Anti-AU5 antibody; GammaBind G-Sepharose beads Phosphatase Activity Assay Fig.4B
  29. 29. Phosphorylation State of PTEN C-tail Affects Its Phosphatase Activity <ul><li>S385A mutation was associated with a substantial reduction in the phosphorylation of the Ser-380/Thr-382/Thr-383 cluster. Therefore, Ser-385 could prime additional dephosphorylation events to regulate PTEN catalytic activity </li></ul>
  30. 30. To investigate if C-tail could mediate intramolecular interactions with PTEN domains implicated in membrane binding Fig.5A
  31. 31. Gene Transfection 293T cells Fig.5A,B Immuno-precipitation Buffer A; α-FLAG monoclonal antibody; GammaBind G-Sepharose beads Western blot
  32. 32. Fig.5C Gene Transfection Immuno-precipitation Western blot Fig.5D Fig.5E 293T cells Fig.5A
  33. 33. PTEN C-tail interacts with the C2 Domain
  34. 34. <ul><li>Interaction between the PTPase domain and the CTD required the presence of the C-tail region </li></ul>Gene Transfection Immuno-precipitation Western blot Fig.6 Science 303, 1179–1181 293T cells
  35. 35. <ul><li>PTEN C-tail may interface with the catalytic domain through interacting with the C2 domain </li></ul>
  36. 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
  37. 37. <ul><li>The conserved CBRIII loop possesses five positively charged and two hydrophobic residues that are implicated in phospholipids binding </li></ul>Cell 99, 323–334
  38. 38. <ul><li>CBR3 mutant: Lys-260, Lys-263, Lys-266, Lys-267, and Lys-269 to alanines </li></ul><ul><li>Cα2mutant: substituting additional positively charged, solvent-exposed residues at the Lys-327, Lys-330, Lys-332, and Arg-335 </li></ul>Fig.S2
  39. 39. The C-tail Interacts Extensively with the CBRIII Motif of the C2 Domain <ul><li>These results indicate that the interaction between the C-tail and the C2 domain most likely involved extensive interface on multiple sites. </li></ul><ul><li>Alternatively, the interactions observed are nonelectrostatic in nature. </li></ul>
  40. 40. To investigate whether the C-tail could act as an autoinhibitory domain <ul><li>PTEN displayed a high affinity for acidic substrates and may mediate the autodephosphorylation of Ser-383 in the tail region </li></ul>Science 303, 1179–1181 Fig.8A Cp-23: residues 368~390 Cp-23DE: S370D, S380D, S385D (D: aspartic acid), T382E, T383E (E: glutamic acid)
  41. 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. 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
  43. 43. <ul><li>These results demonstrated that the phosphorylation cluster within the C-tail region plays an inhibitory role in PTEN membrane targeting and catalytic activities </li></ul>
  44. 44. Summary <ul><li>Ser-380 and Ser-385 involved in PTEN Membrane Targeting </li></ul><ul><li>S385A promoted membrane localization in vivo and phosphatase activity in vitro </li></ul><ul><li>S385A mutation was associated with a substantial reduction in the phosphorylation of the Ser-380/Thr-382/Thr-383 cluster </li></ul><ul><li>Substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutation </li></ul><ul><li>The C-tail Interacts Extensively with the CBRIII Motif of the C2 Domain </li></ul><ul><li>The C-tail Domain Inhibits the Catalytic Activity of PTEN in a Phosphorylation-dependent Manner </li></ul>
  45. 45. <ul><li>The C-tail forms an extensive interaction with the CBRIII region of the C2 domain through nonelectrostatic means </li></ul>This in turn orientates the phosphorylation cluster between aa 380 and 385 to mediate direct interaction with the catalytic pocket and inhibits PTEN enzymatic activity As a result, PTEN attains a closed conformation and localizes predominantly in the cytosol
  46. 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
  47. 47. Conclusion <ul><li>The COOH-terminal tail of PTEN can act as an autoinhibitory domain to control both PTEN membrane recruitment and phosphatase activity </li></ul>
  48. 48. <ul><li>It has been reported that the phosphorylation at Ser-370 can prime the phosphorylation of Ser-366 by glycogen synthase kinase-3. </li></ul><ul><li>However, mutating Ser-366 did not result in any detectable changes in PTEN activities (data not shown) </li></ul>Fig.2A J. Biol. Chem. 280, 35195–35202 Thus, the biological relevance of Ser-366 phosphorylation by glycogen synthase kinase-3 is still unclear
  49. 49. <ul><li>PTEN phosphorylated at Ser-380 constitutes only a minor pool of total PTEN in vitro </li></ul>Fig.2B Fig.3C Fed. Eur. Biochem. Soc. 528, 145–153
  50. 50. <ul><li>1. Ser-380 most likely plays a structural role in regulating PTEN membrane-binding capacity. For instance, mutating Ser-380 to either alanine or glutamic acid induces a conformational change in the tail region to unmask membrane-binding motifs </li></ul>2. PTEN with Ser-380 phosphorylated constitutes only a small transient pool of PTEN that undergoes rapid turnover during membrane binding 3. Phosphorylation of Ser-380 could promote PTEN membrane targeting. Once bound, Ser(P)-380 could be dephosphorylated
  51. 51. <ul><li>There have been suggestions that phosphorylation at Ser-385 may prime the phosphorylation of Thr-383 by casein kinase II </li></ul>J. Biol. Chem. 276, 993–998 J. Biol. Chem. 280, 35195–35202 Biochem. Soc. Trans. 32, 343–347 Fig.4D 382 383
  52. 52. <ul><li>However, we are unable to discern whether the higher phosphatase activity and membrane localization observed with these two mutants is solely due to mutations in Ser-385 and Ser-370 or if it is an indirect effect of the lack of phosphorylation at Ser-380/Thr-382/Thr-383 </li></ul>Fig.4B
  53. 53. <ul><li>There is still considerable uncertainty in how the COOH terminal domain of PTEN regulates its biochemical function </li></ul>
  54. 54. Thanks for your attentions
  55. 55. <ul><li>Positively charged residues interfacing the lipid bilayer at Arg-161, Lys-163, and Lys-164 have been shown to contribute to membrane binding through electrostatic interactions </li></ul>Proc. Natl. Acad. Sci. U. S. A. 100, 7491–7496
  56. 56. <ul><li>It is speculated that phosphorylation at these sites constitutes a negative feedback mechanism in attenuating signaling events initiated by insulin-like growth factor </li></ul>J. Biol. Chem. 280, 35195–35202
  57. 57. <ul><li>PIP2 fails to stimulate PTEN when a soluble lipid substrate, inositol 1,3,4,5-trisphosphate, was used argues for a role of a PIP2-rich membrane in orientating the catalytic pocket of PTEN toward the lipid bilayer </li></ul>J. Biol. Chem. 279, 16606–16613 Wikipedia.org
  58. 58. <ul><li>All of these findings favor an interfacial activation mechanism of PTEN at the lipid bilayer involving interactions of multiple membrane binding moieties </li></ul>Biochem. J. 371, 947–955
  59. 59. Fig.S1

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