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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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Phosphorylation of either Y649 or S650 inhibits the phosphorylation of the adjacent site. (A) Binding of PIPKIγ90 peptides to GST-talin head as determined by ITC. Note that both the dissociation constant (Kd) and the enthalpy (ΔH) of the binding are similar for WT and pY649 peptides. The pY654 peptide has lower affinity, but the pS650 peptide did not bind. N/A, not available. (B) In vitro phosphorylation by Cdk5 and c-Src of WT, pY649 and pS650 12-mer PIPKIγ90 peptides. Each peptide was incubated in the presence of [32P]ATP and of either p35/Cdk5 or c-Src for 20 min at 30°C. Peptides were then recovered on phosphocellulose paper, and the associated radioactivity was measured by Cerenkov counting. Incorporation of radioactivity into pY649 and pS650 peptides was normalized to that of the WT peptide and presented as mean ± SEM (n = 4)
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fig6: Phosphorylation of either Y649 or S650 inhibits the phosphorylation of the adjacent site. (A) Binding of PIPKIγ90 peptides to GST-talin head as determined by ITC. Note that both the dissociation constant (Kd) and the enthalpy (ΔH) of the binding are similar for WT and pY649 peptides. The pY654 peptide has lower affinity, but the pS650 peptide did not bind. N/A, not available. (B) In vitro phosphorylation by Cdk5 and c-Src of WT, pY649 and pS650 12-mer PIPKIγ90 peptides. Each peptide was incubated in the presence of [32P]ATP and of either p35/Cdk5 or c-Src for 20 min at 30°C. Peptides were then recovered on phosphocellulose paper, and the associated radioactivity was measured by Cerenkov counting. Incorporation of radioactivity into pY649 and pS650 peptides was normalized to that of the WT peptide and presented as mean ± SEM (n = 4)

Mentions: Ling et al. (2003) reported that the COOH-terminal region of mouse PIPKIγ90 undergoes phosphorylation by Src at tyrosine 644 (Y649 of human PIPKIγ90) and that this phosphorylation enhances its interaction with talin. Y649 is adjacent to S650 in the talin-binding sequence (WVYSPL), which is identical in mice and humans. The juxtaposition of the two sites raised the possibility that phosphorylation of one might regulate phosphorylation of the other. This possibility was tested using synthetic peptides centered around the talin-binding consensus. Both 12-mer peptides, as well as 17-mer peptides identical to those used by Ling et al. (2003), were tested. First, we revisited the effect of Y649 phosphorylation on talin binding. Surprisingly, an ITC assay did not demonstrate a difference between the affinities of the WT peptides and of the pY649 peptides for GST-talin head (Fig. 6 A). Phosphorylation of tyrosine 654, which is a weak Src target site (Ling et al., 2003), also had no positive effect on talin binding, and in fact, it slightly decreased the binding (Fig. 6 A). The pS650 12-mer peptide exhibited negligible binding to talin, as expected (Figs. 6 A and 3 A). In agreement with the results of the ITC assay, the WT peptide (12-mer) and the pY649 peptide (12-mer) equally competed with the tail for talin binding in pull-down assays from rat brain extracts using GST–28-aa tail (unpublished data).


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)

Phosphorylation of either Y649 or S650 inhibits the phosphorylation of the adjacent site. (A) Binding of PIPKIγ90 peptides to GST-talin head as determined by ITC. Note that both the dissociation constant (Kd) and the enthalpy (ΔH) of the binding are similar for WT and pY649 peptides. The pY654 peptide has lower affinity, but the pS650 peptide did not bind. N/A, not available. (B) In vitro phosphorylation by Cdk5 and c-Src of WT, pY649 and pS650 12-mer PIPKIγ90 peptides. Each peptide was incubated in the presence of [32P]ATP and of either p35/Cdk5 or c-Src for 20 min at 30°C. Peptides were then recovered on phosphocellulose paper, and the associated radioactivity was measured by Cerenkov counting. Incorporation of radioactivity into pY649 and pS650 peptides was normalized to that of the WT peptide and presented as mean ± SEM (n = 4)
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Phosphorylation of either Y649 or S650 inhibits the phosphorylation of the adjacent site. (A) Binding of PIPKIγ90 peptides to GST-talin head as determined by ITC. Note that both the dissociation constant (Kd) and the enthalpy (ΔH) of the binding are similar for WT and pY649 peptides. The pY654 peptide has lower affinity, but the pS650 peptide did not bind. N/A, not available. (B) In vitro phosphorylation by Cdk5 and c-Src of WT, pY649 and pS650 12-mer PIPKIγ90 peptides. Each peptide was incubated in the presence of [32P]ATP and of either p35/Cdk5 or c-Src for 20 min at 30°C. Peptides were then recovered on phosphocellulose paper, and the associated radioactivity was measured by Cerenkov counting. Incorporation of radioactivity into pY649 and pS650 peptides was normalized to that of the WT peptide and presented as mean ± SEM (n = 4)
Mentions: Ling et al. (2003) reported that the COOH-terminal region of mouse PIPKIγ90 undergoes phosphorylation by Src at tyrosine 644 (Y649 of human PIPKIγ90) and that this phosphorylation enhances its interaction with talin. Y649 is adjacent to S650 in the talin-binding sequence (WVYSPL), which is identical in mice and humans. The juxtaposition of the two sites raised the possibility that phosphorylation of one might regulate phosphorylation of the other. This possibility was tested using synthetic peptides centered around the talin-binding consensus. Both 12-mer peptides, as well as 17-mer peptides identical to those used by Ling et al. (2003), were tested. First, we revisited the effect of Y649 phosphorylation on talin binding. Surprisingly, an ITC assay did not demonstrate a difference between the affinities of the WT peptides and of the pY649 peptides for GST-talin head (Fig. 6 A). Phosphorylation of tyrosine 654, which is a weak Src target site (Ling et al., 2003), also had no positive effect on talin binding, and in fact, it slightly decreased the binding (Fig. 6 A). The pS650 12-mer peptide exhibited negligible binding to talin, as expected (Figs. 6 A and 3 A). In agreement with the results of the ITC assay, the WT peptide (12-mer) and the pY649 peptide (12-mer) equally competed with the tail for talin binding in pull-down assays from rat brain extracts using GST–28-aa tail (unpublished data).

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