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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.

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Regulation of S650 phosphorylation in synaptosomes. In all panels shown in the figure, with the exception of E and F, levels of total PIPKIγ90 and of its pS650 epitope were analyzed by Western blots of anti-PIPKIγ90 immunoprecipitates obtained from synaptosomal lysates. For other proteins, Western blots were performed directly on synaptosomal lysates. (A) Freshly prepared rat brain synaptosomes, or synaptosomes exposed to a 1-h labeling step with 32Pi, were incubated for 1 min with either control buffer or stimulation buffer (high K+) in the absence or presence of Ca2+ (Bauerfeind et al., 1997). Anti-PIPKIγ90 immunoprecipitates prepared from these samples were analyzed by autoradiography (32P-labeled samples) or by Western blotting for pS650 and total PIPKIγ90. Western blotting for amphiphysin 2 revealed the previously described stimulation-dependent mobility shift of the upper band (because of its dephosphorylation), thus confirming the occurrence of Ca2+-dependent stimulation. (B) Synaptosomes were stimulated with high K+ for 1 min in the absence or presence of 2 μM cyclosporin A, and then were analyzed with antibodies directed against pS650 or phospho-sites 4 and 5 (MAPK sites) of synapsin I. (C) Synaptosomes were exposed for 1 min to either control buffer (resting) or high K+ buffer (depolarization). Aliquots of stimulated synaptosomes were then returned for 15 min to control buffer (repolarization) with and without the Cdk5 inhibitor butyrolactone I (10 μM). (D) Synaptosomes were incubated for 1 min with control buffer or high K+ buffer in the absence or presence of 1 μM okadaic acid, and then were analyzed for levels of pS650, PIPKIγ90, phospho-MAPK1/2, and total MAPK1/2. (E) Synaptosomes were stimulated for 1 min in the absence or presence of 1 μM okadaic acid and the MAPK inhibitor PD98059. (F) In vitro phosphorylation of WT and S650A mutant His6-PIPKIγ90 with purified MAPK1. S650 phosphorylation by MAPK1 was detected by the anti-pS650 antibody.
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fig7: Regulation of S650 phosphorylation in synaptosomes. In all panels shown in the figure, with the exception of E and F, levels of total PIPKIγ90 and of its pS650 epitope were analyzed by Western blots of anti-PIPKIγ90 immunoprecipitates obtained from synaptosomal lysates. For other proteins, Western blots were performed directly on synaptosomal lysates. (A) Freshly prepared rat brain synaptosomes, or synaptosomes exposed to a 1-h labeling step with 32Pi, were incubated for 1 min with either control buffer or stimulation buffer (high K+) in the absence or presence of Ca2+ (Bauerfeind et al., 1997). Anti-PIPKIγ90 immunoprecipitates prepared from these samples were analyzed by autoradiography (32P-labeled samples) or by Western blotting for pS650 and total PIPKIγ90. Western blotting for amphiphysin 2 revealed the previously described stimulation-dependent mobility shift of the upper band (because of its dephosphorylation), thus confirming the occurrence of Ca2+-dependent stimulation. (B) Synaptosomes were stimulated with high K+ for 1 min in the absence or presence of 2 μM cyclosporin A, and then were analyzed with antibodies directed against pS650 or phospho-sites 4 and 5 (MAPK sites) of synapsin I. (C) Synaptosomes were exposed for 1 min to either control buffer (resting) or high K+ buffer (depolarization). Aliquots of stimulated synaptosomes were then returned for 15 min to control buffer (repolarization) with and without the Cdk5 inhibitor butyrolactone I (10 μM). (D) Synaptosomes were incubated for 1 min with control buffer or high K+ buffer in the absence or presence of 1 μM okadaic acid, and then were analyzed for levels of pS650, PIPKIγ90, phospho-MAPK1/2, and total MAPK1/2. (E) Synaptosomes were stimulated for 1 min in the absence or presence of 1 μM okadaic acid and the MAPK inhibitor PD98059. (F) In vitro phosphorylation of WT and S650A mutant His6-PIPKIγ90 with purified MAPK1. S650 phosphorylation by MAPK1 was detected by the anti-pS650 antibody.

Mentions: To further confirm the site of phosphorylation and for use in the studies described later, an antibody that specifically recognizes PIPKIγ90 phosphorylated at S650 (anti–phospho-S650 [pS650] antibody) was raised. The pS650 antibody selectively recognized WT PIPKIγ90 phosphorylated in vitro by p35/Cdk5 (Fig. 2 A). Under the same conditions (after 30-min incubation), the S650A mutant was phosphorylated to a much lower extent as revealed by the incorporation of 32P (reflecting one or other phosphorylation sites besides S650), and no signal was detected with the pS650 antibody (Fig. 2 A). WT or S650A HA-PIPKIγ90 was transfected into CHO cells with p35 plus Cdk5. Using the pS650 antibody to analyze immunoprecipitated PIPKIγ90, the WT, but not the S650 mutant, protein was found to be phosphorylated (Fig. 2 B). Omission of p35 and Cdk5, or transfection with catalytically inactive mutant of Cdk5 (mut-Cdk5; Patrick et al., 1999) resulted in a significantly lower phosphorylation of S650 compared with that observed after transfection with p35/Cdk5 (Fig. 2 C). Under these conditions, S650 may be phosphorylated by low levels of endogenous Cdk5 (Dhavan and Tsai, 2001) and/or by other proline-directed protein kinases (see Figs. 7 and 8).


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)

Regulation of S650 phosphorylation in synaptosomes. In all panels shown in the figure, with the exception of E and F, levels of total PIPKIγ90 and of its pS650 epitope were analyzed by Western blots of anti-PIPKIγ90 immunoprecipitates obtained from synaptosomal lysates. For other proteins, Western blots were performed directly on synaptosomal lysates. (A) Freshly prepared rat brain synaptosomes, or synaptosomes exposed to a 1-h labeling step with 32Pi, were incubated for 1 min with either control buffer or stimulation buffer (high K+) in the absence or presence of Ca2+ (Bauerfeind et al., 1997). Anti-PIPKIγ90 immunoprecipitates prepared from these samples were analyzed by autoradiography (32P-labeled samples) or by Western blotting for pS650 and total PIPKIγ90. Western blotting for amphiphysin 2 revealed the previously described stimulation-dependent mobility shift of the upper band (because of its dephosphorylation), thus confirming the occurrence of Ca2+-dependent stimulation. (B) Synaptosomes were stimulated with high K+ for 1 min in the absence or presence of 2 μM cyclosporin A, and then were analyzed with antibodies directed against pS650 or phospho-sites 4 and 5 (MAPK sites) of synapsin I. (C) Synaptosomes were exposed for 1 min to either control buffer (resting) or high K+ buffer (depolarization). Aliquots of stimulated synaptosomes were then returned for 15 min to control buffer (repolarization) with and without the Cdk5 inhibitor butyrolactone I (10 μM). (D) Synaptosomes were incubated for 1 min with control buffer or high K+ buffer in the absence or presence of 1 μM okadaic acid, and then were analyzed for levels of pS650, PIPKIγ90, phospho-MAPK1/2, and total MAPK1/2. (E) Synaptosomes were stimulated for 1 min in the absence or presence of 1 μM okadaic acid and the MAPK inhibitor PD98059. (F) In vitro phosphorylation of WT and S650A mutant His6-PIPKIγ90 with purified MAPK1. S650 phosphorylation by MAPK1 was detected by the anti-pS650 antibody.
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Related In: Results  -  Collection

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fig7: Regulation of S650 phosphorylation in synaptosomes. In all panels shown in the figure, with the exception of E and F, levels of total PIPKIγ90 and of its pS650 epitope were analyzed by Western blots of anti-PIPKIγ90 immunoprecipitates obtained from synaptosomal lysates. For other proteins, Western blots were performed directly on synaptosomal lysates. (A) Freshly prepared rat brain synaptosomes, or synaptosomes exposed to a 1-h labeling step with 32Pi, were incubated for 1 min with either control buffer or stimulation buffer (high K+) in the absence or presence of Ca2+ (Bauerfeind et al., 1997). Anti-PIPKIγ90 immunoprecipitates prepared from these samples were analyzed by autoradiography (32P-labeled samples) or by Western blotting for pS650 and total PIPKIγ90. Western blotting for amphiphysin 2 revealed the previously described stimulation-dependent mobility shift of the upper band (because of its dephosphorylation), thus confirming the occurrence of Ca2+-dependent stimulation. (B) Synaptosomes were stimulated with high K+ for 1 min in the absence or presence of 2 μM cyclosporin A, and then were analyzed with antibodies directed against pS650 or phospho-sites 4 and 5 (MAPK sites) of synapsin I. (C) Synaptosomes were exposed for 1 min to either control buffer (resting) or high K+ buffer (depolarization). Aliquots of stimulated synaptosomes were then returned for 15 min to control buffer (repolarization) with and without the Cdk5 inhibitor butyrolactone I (10 μM). (D) Synaptosomes were incubated for 1 min with control buffer or high K+ buffer in the absence or presence of 1 μM okadaic acid, and then were analyzed for levels of pS650, PIPKIγ90, phospho-MAPK1/2, and total MAPK1/2. (E) Synaptosomes were stimulated for 1 min in the absence or presence of 1 μM okadaic acid and the MAPK inhibitor PD98059. (F) In vitro phosphorylation of WT and S650A mutant His6-PIPKIγ90 with purified MAPK1. S650 phosphorylation by MAPK1 was detected by the anti-pS650 antibody.
Mentions: To further confirm the site of phosphorylation and for use in the studies described later, an antibody that specifically recognizes PIPKIγ90 phosphorylated at S650 (anti–phospho-S650 [pS650] antibody) was raised. The pS650 antibody selectively recognized WT PIPKIγ90 phosphorylated in vitro by p35/Cdk5 (Fig. 2 A). Under the same conditions (after 30-min incubation), the S650A mutant was phosphorylated to a much lower extent as revealed by the incorporation of 32P (reflecting one or other phosphorylation sites besides S650), and no signal was detected with the pS650 antibody (Fig. 2 A). WT or S650A HA-PIPKIγ90 was transfected into CHO cells with p35 plus Cdk5. Using the pS650 antibody to analyze immunoprecipitated PIPKIγ90, the WT, but not the S650 mutant, protein was found to be phosphorylated (Fig. 2 B). Omission of p35 and Cdk5, or transfection with catalytically inactive mutant of Cdk5 (mut-Cdk5; Patrick et al., 1999) resulted in a significantly lower phosphorylation of S650 compared with that observed after transfection with p35/Cdk5 (Fig. 2 C). Under these conditions, S650 may be phosphorylated by low levels of endogenous Cdk5 (Dhavan and Tsai, 2001) and/or by other proline-directed protein kinases (see Figs. 7 and 8).

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