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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 vitro. Recombinant PIPKIγ90 fusion proteins were incubated with p35–Cdk5 complex in the presence of [32P]ATP for 30 min. Protein phosphorylation was analyzed by autoradiography after SDS-PAGE. (A) GST-PIPKIγ90 was phosphorylated by p35/Cdk5 in the absence and presence of the Cdk5 inhibitors, roscovitine or butyrolactone I (20 μM each). (B and C) Phosphorylation of His6-PIPKIγ90 by p35/Cdk5 and time course of the phosphorylation. (D) Stoichiometry of the phosphorylation of His6-PIPKIγ90 by p35/Cdk5. Purified proteins phosphorylated in vitro in the presence of [32P]ATP were separated by SDS-PAGE and stained with Coomassie brilliant blue. PIPKIγ90 bands were excised and incorporation of 32P was measured. (E) Diagram indicating the position of the phosphorylation site S650 in the COOH-terminal 28-aa tail of PIPKIγ90. (F) 2-D phosphopeptide mapping of His6-PIPKIγ87, His6-PIPKIγ90, and S650A mutant His6-PIPKIγ90 phosphorylated by p35/Cdk5 for 30 min in vitro. A circle outlines the spots present only in the protein that contains S650.
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fig1: PIPKIγ90 is phosphorylated by p35/Cdk5 at S650 in vitro. Recombinant PIPKIγ90 fusion proteins were incubated with p35–Cdk5 complex in the presence of [32P]ATP for 30 min. Protein phosphorylation was analyzed by autoradiography after SDS-PAGE. (A) GST-PIPKIγ90 was phosphorylated by p35/Cdk5 in the absence and presence of the Cdk5 inhibitors, roscovitine or butyrolactone I (20 μM each). (B and C) Phosphorylation of His6-PIPKIγ90 by p35/Cdk5 and time course of the phosphorylation. (D) Stoichiometry of the phosphorylation of His6-PIPKIγ90 by p35/Cdk5. Purified proteins phosphorylated in vitro in the presence of [32P]ATP were separated by SDS-PAGE and stained with Coomassie brilliant blue. PIPKIγ90 bands were excised and incorporation of 32P was measured. (E) Diagram indicating the position of the phosphorylation site S650 in the COOH-terminal 28-aa tail of PIPKIγ90. (F) 2-D phosphopeptide mapping of His6-PIPKIγ87, His6-PIPKIγ90, and S650A mutant His6-PIPKIγ90 phosphorylated by p35/Cdk5 for 30 min in vitro. A circle outlines the spots present only in the protein that contains S650.

Mentions: As a first step to determine whether Cdk5 may be responsible, at least in part, for the constitutive phosphorylation of PIPKIγ90 in resting synapses, we tested the ability of PIPKIγ90 to serve as a substrate for Cdk5 in vitro. Incubation of GST fusion protein of human PIPKIγ90 with purified p35/Cdk5 at an ∼1:1 stoichiometric ratio resulted in its efficient phosphorylation (Fig. 1 A). Similar results were obtained with His6-tagged PIPKIγ90 (Fig. 1 B), which was used for all subsequent phosphorylation experiments. p35, a prominent physiological substrate for the kinase activity of Cdk5 (Dhavan and Tsai, 2001), was also efficiently phosphorylated, as expected, and with similar kinetics during a 30-min incubation (Fig. 1 C). Two Cdk5 inhibitors, roscovitine (Tan et al., 2003) and butyrolactone I (Lee et al., 2004), inhibited the phosphorylation of both proteins (Fig. 1 A). A stoichiometric analysis revealed the incorporation of 2 mol phosphate/mol PIPKIγ90, which suggests at least two phosphorylation sites (Fig. 1 D).


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 vitro. Recombinant PIPKIγ90 fusion proteins were incubated with p35–Cdk5 complex in the presence of [32P]ATP for 30 min. Protein phosphorylation was analyzed by autoradiography after SDS-PAGE. (A) GST-PIPKIγ90 was phosphorylated by p35/Cdk5 in the absence and presence of the Cdk5 inhibitors, roscovitine or butyrolactone I (20 μM each). (B and C) Phosphorylation of His6-PIPKIγ90 by p35/Cdk5 and time course of the phosphorylation. (D) Stoichiometry of the phosphorylation of His6-PIPKIγ90 by p35/Cdk5. Purified proteins phosphorylated in vitro in the presence of [32P]ATP were separated by SDS-PAGE and stained with Coomassie brilliant blue. PIPKIγ90 bands were excised and incorporation of 32P was measured. (E) Diagram indicating the position of the phosphorylation site S650 in the COOH-terminal 28-aa tail of PIPKIγ90. (F) 2-D phosphopeptide mapping of His6-PIPKIγ87, His6-PIPKIγ90, and S650A mutant His6-PIPKIγ90 phosphorylated by p35/Cdk5 for 30 min in vitro. A circle outlines the spots present only in the protein that contains S650.
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Related In: Results  -  Collection

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fig1: PIPKIγ90 is phosphorylated by p35/Cdk5 at S650 in vitro. Recombinant PIPKIγ90 fusion proteins were incubated with p35–Cdk5 complex in the presence of [32P]ATP for 30 min. Protein phosphorylation was analyzed by autoradiography after SDS-PAGE. (A) GST-PIPKIγ90 was phosphorylated by p35/Cdk5 in the absence and presence of the Cdk5 inhibitors, roscovitine or butyrolactone I (20 μM each). (B and C) Phosphorylation of His6-PIPKIγ90 by p35/Cdk5 and time course of the phosphorylation. (D) Stoichiometry of the phosphorylation of His6-PIPKIγ90 by p35/Cdk5. Purified proteins phosphorylated in vitro in the presence of [32P]ATP were separated by SDS-PAGE and stained with Coomassie brilliant blue. PIPKIγ90 bands were excised and incorporation of 32P was measured. (E) Diagram indicating the position of the phosphorylation site S650 in the COOH-terminal 28-aa tail of PIPKIγ90. (F) 2-D phosphopeptide mapping of His6-PIPKIγ87, His6-PIPKIγ90, and S650A mutant His6-PIPKIγ90 phosphorylated by p35/Cdk5 for 30 min in vitro. A circle outlines the spots present only in the protein that contains S650.
Mentions: As a first step to determine whether Cdk5 may be responsible, at least in part, for the constitutive phosphorylation of PIPKIγ90 in resting synapses, we tested the ability of PIPKIγ90 to serve as a substrate for Cdk5 in vitro. Incubation of GST fusion protein of human PIPKIγ90 with purified p35/Cdk5 at an ∼1:1 stoichiometric ratio resulted in its efficient phosphorylation (Fig. 1 A). Similar results were obtained with His6-tagged PIPKIγ90 (Fig. 1 B), which was used for all subsequent phosphorylation experiments. p35, a prominent physiological substrate for the kinase activity of Cdk5 (Dhavan and Tsai, 2001), was also efficiently phosphorylated, as expected, and with similar kinetics during a 30-min incubation (Fig. 1 C). Two Cdk5 inhibitors, roscovitine (Tan et al., 2003) and butyrolactone I (Lee et al., 2004), inhibited the phosphorylation of both proteins (Fig. 1 A). A stoichiometric analysis revealed the incorporation of 2 mol phosphate/mol PIPKIγ90, which suggests at least two phosphorylation sites (Fig. 1 D).

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