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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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PIPKIγ90 is phosphorylated by p35/Cdk5 at S650 in vivo. (A) Generation and characterization of a polyclonal antibody specific for phospho-S650 (pS650). Purified WT and S650A mutant His6-PIPKIγ90 were incubated with or without p35/Cdk5 and with either nonradioactive ATP (for Western blotting) or [32P]ATP (for autoradiography) for 30 min. Samples were analyzed by autoradiography, and Western blotting with the anti-pS650 antibody was performed. Coomassie brilliant blue (CBB) staining shows loading of equal amounts of substrate proteins. The autoradiographic signal was quantified by a phosphorimager analysis and values were expressed as a percentage of the radioactivity incorporated into the WT sample. Bar graphs represent mean ± SD (n = 3). (B) CHO cells were transfected with WT or S650A mutant HA-PIPKIγ90 together with p35 and Cdk5. PIPKIγ90 immunoprecipitates were obtained from cell lysates 24 h after transfection. S650 phosphorylation and PIPKIγ90 expression were analyzed by Western blotting with anti-pS650 and anti-HA antibodies, respectively. (C) CHO cells were cotransfected with HA-PIPKIγ90, p35, and Cdk5 or mut-Cdk5. PIPKIγ90 immunoprecipitates from transfected and control cells were analyzed by Western blotting as described above. The faint band visible in nontransfected CHO cells may represent phosphorylated hamster PIPKIγ90. The pS650 immunoreactivity was quantified using an NIH image analysis software and shown as mean ± SD (n = 3).
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fig2: PIPKIγ90 is phosphorylated by p35/Cdk5 at S650 in vivo. (A) Generation and characterization of a polyclonal antibody specific for phospho-S650 (pS650). Purified WT and S650A mutant His6-PIPKIγ90 were incubated with or without p35/Cdk5 and with either nonradioactive ATP (for Western blotting) or [32P]ATP (for autoradiography) for 30 min. Samples were analyzed by autoradiography, and Western blotting with the anti-pS650 antibody was performed. Coomassie brilliant blue (CBB) staining shows loading of equal amounts of substrate proteins. The autoradiographic signal was quantified by a phosphorimager analysis and values were expressed as a percentage of the radioactivity incorporated into the WT sample. Bar graphs represent mean ± SD (n = 3). (B) CHO cells were transfected with WT or S650A mutant HA-PIPKIγ90 together with p35 and Cdk5. PIPKIγ90 immunoprecipitates were obtained from cell lysates 24 h after transfection. S650 phosphorylation and PIPKIγ90 expression were analyzed by Western blotting with anti-pS650 and anti-HA antibodies, respectively. (C) CHO cells were cotransfected with HA-PIPKIγ90, p35, and Cdk5 or mut-Cdk5. PIPKIγ90 immunoprecipitates from transfected and control cells were analyzed by Western blotting as described above. The faint band visible in nontransfected CHO cells may represent phosphorylated hamster PIPKIγ90. The pS650 immunoreactivity was quantified using an NIH image analysis software and shown as mean ± SD (n = 3).

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)

PIPKIγ90 is phosphorylated by p35/Cdk5 at S650 in vivo. (A) Generation and characterization of a polyclonal antibody specific for phospho-S650 (pS650). Purified WT and S650A mutant His6-PIPKIγ90 were incubated with or without p35/Cdk5 and with either nonradioactive ATP (for Western blotting) or [32P]ATP (for autoradiography) for 30 min. Samples were analyzed by autoradiography, and Western blotting with the anti-pS650 antibody was performed. Coomassie brilliant blue (CBB) staining shows loading of equal amounts of substrate proteins. The autoradiographic signal was quantified by a phosphorimager analysis and values were expressed as a percentage of the radioactivity incorporated into the WT sample. Bar graphs represent mean ± SD (n = 3). (B) CHO cells were transfected with WT or S650A mutant HA-PIPKIγ90 together with p35 and Cdk5. PIPKIγ90 immunoprecipitates were obtained from cell lysates 24 h after transfection. S650 phosphorylation and PIPKIγ90 expression were analyzed by Western blotting with anti-pS650 and anti-HA antibodies, respectively. (C) CHO cells were cotransfected with HA-PIPKIγ90, p35, and Cdk5 or mut-Cdk5. PIPKIγ90 immunoprecipitates from transfected and control cells were analyzed by Western blotting as described above. The faint band visible in nontransfected CHO cells may represent phosphorylated hamster PIPKIγ90. The pS650 immunoreactivity was quantified using an NIH image analysis software and shown as mean ± SD (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171813&req=5

fig2: PIPKIγ90 is phosphorylated by p35/Cdk5 at S650 in vivo. (A) Generation and characterization of a polyclonal antibody specific for phospho-S650 (pS650). Purified WT and S650A mutant His6-PIPKIγ90 were incubated with or without p35/Cdk5 and with either nonradioactive ATP (for Western blotting) or [32P]ATP (for autoradiography) for 30 min. Samples were analyzed by autoradiography, and Western blotting with the anti-pS650 antibody was performed. Coomassie brilliant blue (CBB) staining shows loading of equal amounts of substrate proteins. The autoradiographic signal was quantified by a phosphorimager analysis and values were expressed as a percentage of the radioactivity incorporated into the WT sample. Bar graphs represent mean ± SD (n = 3). (B) CHO cells were transfected with WT or S650A mutant HA-PIPKIγ90 together with p35 and Cdk5. PIPKIγ90 immunoprecipitates were obtained from cell lysates 24 h after transfection. S650 phosphorylation and PIPKIγ90 expression were analyzed by Western blotting with anti-pS650 and anti-HA antibodies, respectively. (C) CHO cells were cotransfected with HA-PIPKIγ90, p35, and Cdk5 or mut-Cdk5. PIPKIγ90 immunoprecipitates from transfected and control cells were analyzed by Western blotting as described above. The faint band visible in nontransfected CHO cells may represent phosphorylated hamster PIPKIγ90. The pS650 immunoreactivity was quantified using an NIH image analysis software and shown as mean ± SD (n = 3).
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