Limits...
Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases.

Lee SY, Voronov S, Letinic K, Nairn AC, Di Paolo G, De Camilli P - J. Cell Biol. (2005)

Bottom Line: Cell Biol. 163:1339-1349).We find that Y649 phosphorylation does not stimulate directly PIPKI gamma binding to talin, but may do so indirectly by inhibiting S650 phosphorylation.Conversely, S650 phosphorylation inhibits Y649 phosphorylation by Src.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The interaction of talin with phosphatidylinositol(4) phosphate 5 kinase type I gamma (PIPKI gamma) regulates PI(4,5)P2 synthesis at synapses and at focal adhesions. Here, we show that phosphorylation of serine 650 (S650) within the talin-binding sequence of human PIPKI gamma blocks this interaction. At synapses, S650 is phosphorylated by p35/Cdk5 and mitogen-activated protein kinase at rest, and dephosphorylated by calcineurin upon stimulation. S650 is also a substrate for cyclin B1/Cdk1 and its phosphorylation in mitosis correlates with focal adhesion disassembly. Phosphorylation by Src of the tyrosine adjacent to S650 (Y649 in human PIPKI gamma) was shown to enhance PIPKI gamma targeting to focal adhesions (Ling, K., R.L. Doughman, V.V. Iyer, A.J. Firestone, S.F. Bairstow, D.F. Mosher, M.D. Schaller, and R.A. Anderson. 2003. J. Cell Biol. 163:1339-1349). We find that Y649 phosphorylation does not stimulate directly PIPKI gamma binding to talin, but may do so indirectly by inhibiting S650 phosphorylation. Conversely, S650 phosphorylation inhibits Y649 phosphorylation by Src. The opposite effects of the phosphorylation of Y649 and S650 likely play a critical role in regulating synaptic function as well as the balance between cell adhesion and cell motility.

Show MeSH

Related in: MedlinePlus

Phosphomimetic mutation at S650 (S650D) disrupts the interaction of PIPKIγ90 with talin in vitro. (A) GST-F3 overlay assay. Nitrocellulose blots of WT and mutant His6-PIPKIγ90 were overlaid with or without GST-F3 fusion protein, and then overlaid with anti-GST antibody. CBB staining reveals equal load of the lanes. (B) Pull-down assay from rat brain extracts on bead-immobilized WT and mutant His6-PIPKIγ90. Bound talin was revealed by Western blotting, and equal amount of bait proteins was revealed by CBB staining. (C) The intensities of the GST-F3 and talin bands shown in A and B were quantified by an NIH image analysis software. Values from mutant proteins were normalized to that from WT and are represented as mean ± SD (n = 4). (D) Localization of transfected GFP-PIPKIγ90. NIH3T3 cells were transfected with GFP-PIPKIγ90 or its S650D mutant, and then were processed by immunofluorescence for vinculin immunoreactivity. Bar, 10 μm. (E) Pull-down assay from lysates of CHO cells transfected with WT and S650D mutant GFP-PIPKIγ90. Bead-immobilized GST-integrin β1 tail was used as a bait. Talin and PIPKIγ90 in the lysates or bead fractions were detected by Western blotting. (F) NIH3T3 cells transfected with mutant (S650N) GFP-PIPKIγ90 were immunostained with antivinculin antibody. Bar, 10 μm. (G) CHO cells were transfected with WT, S650D, or S650N GFP-PIPKIγ90. After 24 h of transfection, cell lysates were immunoprecipitated with anti-PIPKIγ90 antibody, and the presence of talin and PIPKIγ90 in the immunoprecipitates was analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171813&req=5

fig4: Phosphomimetic mutation at S650 (S650D) disrupts the interaction of PIPKIγ90 with talin in vitro. (A) GST-F3 overlay assay. Nitrocellulose blots of WT and mutant His6-PIPKIγ90 were overlaid with or without GST-F3 fusion protein, and then overlaid with anti-GST antibody. CBB staining reveals equal load of the lanes. (B) Pull-down assay from rat brain extracts on bead-immobilized WT and mutant His6-PIPKIγ90. Bound talin was revealed by Western blotting, and equal amount of bait proteins was revealed by CBB staining. (C) The intensities of the GST-F3 and talin bands shown in A and B were quantified by an NIH image analysis software. Values from mutant proteins were normalized to that from WT and are represented as mean ± SD (n = 4). (D) Localization of transfected GFP-PIPKIγ90. NIH3T3 cells were transfected with GFP-PIPKIγ90 or its S650D mutant, and then were processed by immunofluorescence for vinculin immunoreactivity. Bar, 10 μm. (E) Pull-down assay from lysates of CHO cells transfected with WT and S650D mutant GFP-PIPKIγ90. Bead-immobilized GST-integrin β1 tail was used as a bait. Talin and PIPKIγ90 in the lysates or bead fractions were detected by Western blotting. (F) NIH3T3 cells transfected with mutant (S650N) GFP-PIPKIγ90 were immunostained with antivinculin antibody. Bar, 10 μm. (G) CHO cells were transfected with WT, S650D, or S650N GFP-PIPKIγ90. After 24 h of transfection, cell lysates were immunoprecipitated with anti-PIPKIγ90 antibody, and the presence of talin and PIPKIγ90 in the immunoprecipitates was analyzed by Western blotting.

Mentions: We next determined whether a phosphomimetic mutation of S650 to aspartic acid (S650D) affected the interaction with talin. WT and S650D His6-PIPKIγ90 were overlaid in a “far-Western” assay with a GST fusion protein of the F3 subdomain of the FERM domain of talin (Fig. 4, A and C). The F3 domain clearly bound to the WT protein, as expected (Di Paolo et al., 2002), but no binding was observed for the S650D mutant (Fig. 4 A). Binding of the F3 domain to the S650A mutant was also reduced, although not abolished, and this is in agreement with a potential role of the side chain of serine in the binding (Di Paolo et al., 2002; Liddington et al., 2003). These results were qualitatively confirmed by His6-PIPKIγ90 pull-down assays from rat brain extracts (Fig. 4, B and C). In this case, the loss of binding produced by the S650A mutation was nearly as large as that produced by the S650D mutation.


Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases.

Lee SY, Voronov S, Letinic K, Nairn AC, Di Paolo G, De Camilli P - J. Cell Biol. (2005)

Phosphomimetic mutation at S650 (S650D) disrupts the interaction of PIPKIγ90 with talin in vitro. (A) GST-F3 overlay assay. Nitrocellulose blots of WT and mutant His6-PIPKIγ90 were overlaid with or without GST-F3 fusion protein, and then overlaid with anti-GST antibody. CBB staining reveals equal load of the lanes. (B) Pull-down assay from rat brain extracts on bead-immobilized WT and mutant His6-PIPKIγ90. Bound talin was revealed by Western blotting, and equal amount of bait proteins was revealed by CBB staining. (C) The intensities of the GST-F3 and talin bands shown in A and B were quantified by an NIH image analysis software. Values from mutant proteins were normalized to that from WT and are represented as mean ± SD (n = 4). (D) Localization of transfected GFP-PIPKIγ90. NIH3T3 cells were transfected with GFP-PIPKIγ90 or its S650D mutant, and then were processed by immunofluorescence for vinculin immunoreactivity. Bar, 10 μm. (E) Pull-down assay from lysates of CHO cells transfected with WT and S650D mutant GFP-PIPKIγ90. Bead-immobilized GST-integrin β1 tail was used as a bait. Talin and PIPKIγ90 in the lysates or bead fractions were detected by Western blotting. (F) NIH3T3 cells transfected with mutant (S650N) GFP-PIPKIγ90 were immunostained with antivinculin antibody. Bar, 10 μm. (G) CHO cells were transfected with WT, S650D, or S650N GFP-PIPKIγ90. After 24 h of transfection, cell lysates were immunoprecipitated with anti-PIPKIγ90 antibody, and the presence of talin and PIPKIγ90 in the immunoprecipitates was analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171813&req=5

fig4: Phosphomimetic mutation at S650 (S650D) disrupts the interaction of PIPKIγ90 with talin in vitro. (A) GST-F3 overlay assay. Nitrocellulose blots of WT and mutant His6-PIPKIγ90 were overlaid with or without GST-F3 fusion protein, and then overlaid with anti-GST antibody. CBB staining reveals equal load of the lanes. (B) Pull-down assay from rat brain extracts on bead-immobilized WT and mutant His6-PIPKIγ90. Bound talin was revealed by Western blotting, and equal amount of bait proteins was revealed by CBB staining. (C) The intensities of the GST-F3 and talin bands shown in A and B were quantified by an NIH image analysis software. Values from mutant proteins were normalized to that from WT and are represented as mean ± SD (n = 4). (D) Localization of transfected GFP-PIPKIγ90. NIH3T3 cells were transfected with GFP-PIPKIγ90 or its S650D mutant, and then were processed by immunofluorescence for vinculin immunoreactivity. Bar, 10 μm. (E) Pull-down assay from lysates of CHO cells transfected with WT and S650D mutant GFP-PIPKIγ90. Bead-immobilized GST-integrin β1 tail was used as a bait. Talin and PIPKIγ90 in the lysates or bead fractions were detected by Western blotting. (F) NIH3T3 cells transfected with mutant (S650N) GFP-PIPKIγ90 were immunostained with antivinculin antibody. Bar, 10 μm. (G) CHO cells were transfected with WT, S650D, or S650N GFP-PIPKIγ90. After 24 h of transfection, cell lysates were immunoprecipitated with anti-PIPKIγ90 antibody, and the presence of talin and PIPKIγ90 in the immunoprecipitates was analyzed by Western blotting.
Mentions: We next determined whether a phosphomimetic mutation of S650 to aspartic acid (S650D) affected the interaction with talin. WT and S650D His6-PIPKIγ90 were overlaid in a “far-Western” assay with a GST fusion protein of the F3 subdomain of the FERM domain of talin (Fig. 4, A and C). The F3 domain clearly bound to the WT protein, as expected (Di Paolo et al., 2002), but no binding was observed for the S650D mutant (Fig. 4 A). Binding of the F3 domain to the S650A mutant was also reduced, although not abolished, and this is in agreement with a potential role of the side chain of serine in the binding (Di Paolo et al., 2002; Liddington et al., 2003). These results were qualitatively confirmed by His6-PIPKIγ90 pull-down assays from rat brain extracts (Fig. 4, B and C). In this case, the loss of binding produced by the S650A mutation was nearly as large as that produced by the S650D mutation.

Bottom Line: Cell Biol. 163:1339-1349).We find that Y649 phosphorylation does not stimulate directly PIPKI gamma binding to talin, but may do so indirectly by inhibiting S650 phosphorylation.Conversely, S650 phosphorylation inhibits Y649 phosphorylation by Src.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The interaction of talin with phosphatidylinositol(4) phosphate 5 kinase type I gamma (PIPKI gamma) regulates PI(4,5)P2 synthesis at synapses and at focal adhesions. Here, we show that phosphorylation of serine 650 (S650) within the talin-binding sequence of human PIPKI gamma blocks this interaction. At synapses, S650 is phosphorylated by p35/Cdk5 and mitogen-activated protein kinase at rest, and dephosphorylated by calcineurin upon stimulation. S650 is also a substrate for cyclin B1/Cdk1 and its phosphorylation in mitosis correlates with focal adhesion disassembly. Phosphorylation by Src of the tyrosine adjacent to S650 (Y649 in human PIPKI gamma) was shown to enhance PIPKI gamma targeting to focal adhesions (Ling, K., R.L. Doughman, V.V. Iyer, A.J. Firestone, S.F. Bairstow, D.F. Mosher, M.D. Schaller, and R.A. Anderson. 2003. J. Cell Biol. 163:1339-1349). We find that Y649 phosphorylation does not stimulate directly PIPKI gamma binding to talin, but may do so indirectly by inhibiting S650 phosphorylation. Conversely, S650 phosphorylation inhibits Y649 phosphorylation by Src. The opposite effects of the phosphorylation of Y649 and S650 likely play a critical role in regulating synaptic function as well as the balance between cell adhesion and cell motility.

Show MeSH
Related in: MedlinePlus