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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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S650 of PIPKIγ90 undergoes mitotic phosphorylation by cyclin B1/Cdk1. (A) CHO cells transfected with WT or S650A mutant HA-PIPKIγ90 for 24 h were arrested in the mitotic state (M) by nocodazole treatment. G1 interphase cells (G) were further prepared from mitotically synchronized cells after removal of nocodazole. Cell lysates of both M and G cells were analyzed by Western blotting with anti-pS650 and anti-PIPKIγ90 antibodies. (B) U87MG cells were processed as described in A to generate mitotic and interphase cells. PIPKIγ90 was immunoprecipitated from cell lysates for analysis of pS650, talin and total PIPKIγ90. Cell lysates were also analyzed by Western blotting for levels of histone H3 phosphorylation using a phosphospecific antibody. Bar graphs represent normalized talin immunoreactivity (mean ± SD; n = 4) after quantification as shown in Fig. 4 C. (C) WT and S650A mutant His6-PIPKIγ90 were incubated in vitro with or without the cyclin B1–Cdk1 complex in the presence of [32P]ATP. His6-PIPKIγ90 phosphorylation and cyclin B1 autophosphorylation were examined by autoradiography after SDS-PAGE. In parallel, samples phosphorylated under the same conditions with nonradioactive ATP were processed for Western blotting with anti-pS650 antibody. CBB staining demonstrates equal amounts of proteins.
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fig8: S650 of PIPKIγ90 undergoes mitotic phosphorylation by cyclin B1/Cdk1. (A) CHO cells transfected with WT or S650A mutant HA-PIPKIγ90 for 24 h were arrested in the mitotic state (M) by nocodazole treatment. G1 interphase cells (G) were further prepared from mitotically synchronized cells after removal of nocodazole. Cell lysates of both M and G cells were analyzed by Western blotting with anti-pS650 and anti-PIPKIγ90 antibodies. (B) U87MG cells were processed as described in A to generate mitotic and interphase cells. PIPKIγ90 was immunoprecipitated from cell lysates for analysis of pS650, talin and total PIPKIγ90. Cell lysates were also analyzed by Western blotting for levels of histone H3 phosphorylation using a phosphospecific antibody. Bar graphs represent normalized talin immunoreactivity (mean ± SD; n = 4) after quantification as shown in Fig. 4 C. (C) WT and S650A mutant His6-PIPKIγ90 were incubated in vitro with or without the cyclin B1–Cdk1 complex in the presence of [32P]ATP. His6-PIPKIγ90 phosphorylation and cyclin B1 autophosphorylation were examined by autoradiography after SDS-PAGE. In parallel, samples phosphorylated under the same conditions with nonradioactive ATP were processed for Western blotting with anti-pS650 antibody. CBB staining demonstrates equal amounts of proteins.

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)

S650 of PIPKIγ90 undergoes mitotic phosphorylation by cyclin B1/Cdk1. (A) CHO cells transfected with WT or S650A mutant HA-PIPKIγ90 for 24 h were arrested in the mitotic state (M) by nocodazole treatment. G1 interphase cells (G) were further prepared from mitotically synchronized cells after removal of nocodazole. Cell lysates of both M and G cells were analyzed by Western blotting with anti-pS650 and anti-PIPKIγ90 antibodies. (B) U87MG cells were processed as described in A to generate mitotic and interphase cells. PIPKIγ90 was immunoprecipitated from cell lysates for analysis of pS650, talin and total PIPKIγ90. Cell lysates were also analyzed by Western blotting for levels of histone H3 phosphorylation using a phosphospecific antibody. Bar graphs represent normalized talin immunoreactivity (mean ± SD; n = 4) after quantification as shown in Fig. 4 C. (C) WT and S650A mutant His6-PIPKIγ90 were incubated in vitro with or without the cyclin B1–Cdk1 complex in the presence of [32P]ATP. His6-PIPKIγ90 phosphorylation and cyclin B1 autophosphorylation were examined by autoradiography after SDS-PAGE. In parallel, samples phosphorylated under the same conditions with nonradioactive ATP were processed for Western blotting with anti-pS650 antibody. CBB staining demonstrates equal amounts of proteins.
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fig8: S650 of PIPKIγ90 undergoes mitotic phosphorylation by cyclin B1/Cdk1. (A) CHO cells transfected with WT or S650A mutant HA-PIPKIγ90 for 24 h were arrested in the mitotic state (M) by nocodazole treatment. G1 interphase cells (G) were further prepared from mitotically synchronized cells after removal of nocodazole. Cell lysates of both M and G cells were analyzed by Western blotting with anti-pS650 and anti-PIPKIγ90 antibodies. (B) U87MG cells were processed as described in A to generate mitotic and interphase cells. PIPKIγ90 was immunoprecipitated from cell lysates for analysis of pS650, talin and total PIPKIγ90. Cell lysates were also analyzed by Western blotting for levels of histone H3 phosphorylation using a phosphospecific antibody. Bar graphs represent normalized talin immunoreactivity (mean ± SD; n = 4) after quantification as shown in Fig. 4 C. (C) WT and S650A mutant His6-PIPKIγ90 were incubated in vitro with or without the cyclin B1–Cdk1 complex in the presence of [32P]ATP. His6-PIPKIγ90 phosphorylation and cyclin B1 autophosphorylation were examined by autoradiography after SDS-PAGE. In parallel, samples phosphorylated under the same conditions with nonradioactive ATP were processed for Western blotting with anti-pS650 antibody. CBB staining demonstrates equal amounts of proteins.
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