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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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Cdk5 phosphorylation of PIPKIγ90 inhibits its interaction with talin in vivo. (A) ITC analysis of the binding of 12-mer WT and pS650 peptides from the 28-aa tail of PIPKIγ90 to GST-talin head. Raw data as a function of time are shown in the top panels, and plots of the total heat released as a function of the molar ratio of each ligand are shown in the bottom panels. The continuous line in the bottom panels represents the nonlinear, least-squares best fits to the experimental data using a one-site model of binding. Note the roughly 1:1 stoichiometry indicated by the half-height point of the sigmoidal curve (bottom left; Turnbull and Daranas, 2003), and the complete absence of heat release in the case of the pS650 peptide. (B) CHO cells were cotransfected with HA-PIPKIγ90, p35, and Cdk5 or mut-Cdk5. Protein contents of the starting lysates were revealed by Western blotting. PIPKIγ90 and talin were immunoprecipitated from the cell lysates and presence of PIPKIγ90 and talin in each immunoprecipitate was detected by Western blotting.
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fig3: Cdk5 phosphorylation of PIPKIγ90 inhibits its interaction with talin in vivo. (A) ITC analysis of the binding of 12-mer WT and pS650 peptides from the 28-aa tail of PIPKIγ90 to GST-talin head. Raw data as a function of time are shown in the top panels, and plots of the total heat released as a function of the molar ratio of each ligand are shown in the bottom panels. The continuous line in the bottom panels represents the nonlinear, least-squares best fits to the experimental data using a one-site model of binding. Note the roughly 1:1 stoichiometry indicated by the half-height point of the sigmoidal curve (bottom left; Turnbull and Daranas, 2003), and the complete absence of heat release in the case of the pS650 peptide. (B) CHO cells were cotransfected with HA-PIPKIγ90, p35, and Cdk5 or mut-Cdk5. Protein contents of the starting lysates were revealed by Western blotting. PIPKIγ90 and talin were immunoprecipitated from the cell lysates and presence of PIPKIγ90 and talin in each immunoprecipitate was detected by Western blotting.

Mentions: Given the location of S650 in the middle of the talin-binding sequence (WVYSPL; Di Paolo et al., 2002), the possibility that phosphorylation of S650 of PIPKIγ90 inhibits its interaction with talin was next examined. Isothermal titration calorimetry (ITC) was used to analyze the binding of the GST-talin head (Di Paolo et al., 2002), which comprises the FERM domain, to synthetic 12-mer dephospho- and phosphopeptides encompassing the talin-binding sequence of PIPKIγ90. Kd values of 2 μM and >1 mM were observed for the dephosphopeptide and the pS650 peptide, respectively (Fig. 3 A), indicating a strongly negative role of S650 phosphorylation in talin binding. A Kd of 2 μM (see Fig. 6 A) value for the WT peptide is in agreement with the dissociation constant of the same interaction measured with tryptophan fluorescence by Barsukov et al. (2003), but is higher than the dissociation constant observed by Ling et al. (2003) using fluorescence anisotropy measurement. Differences in the assays and the experimental conditions may explain the discrepancy. For example, our values were obtained at 37oC, whereas the value of Ling et al. (2003) was recorded at room temperature.


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)

Cdk5 phosphorylation of PIPKIγ90 inhibits its interaction with talin in vivo. (A) ITC analysis of the binding of 12-mer WT and pS650 peptides from the 28-aa tail of PIPKIγ90 to GST-talin head. Raw data as a function of time are shown in the top panels, and plots of the total heat released as a function of the molar ratio of each ligand are shown in the bottom panels. The continuous line in the bottom panels represents the nonlinear, least-squares best fits to the experimental data using a one-site model of binding. Note the roughly 1:1 stoichiometry indicated by the half-height point of the sigmoidal curve (bottom left; Turnbull and Daranas, 2003), and the complete absence of heat release in the case of the pS650 peptide. (B) CHO cells were cotransfected with HA-PIPKIγ90, p35, and Cdk5 or mut-Cdk5. Protein contents of the starting lysates were revealed by Western blotting. PIPKIγ90 and talin were immunoprecipitated from the cell lysates and presence of PIPKIγ90 and talin in each immunoprecipitate was detected by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171813&req=5

fig3: Cdk5 phosphorylation of PIPKIγ90 inhibits its interaction with talin in vivo. (A) ITC analysis of the binding of 12-mer WT and pS650 peptides from the 28-aa tail of PIPKIγ90 to GST-talin head. Raw data as a function of time are shown in the top panels, and plots of the total heat released as a function of the molar ratio of each ligand are shown in the bottom panels. The continuous line in the bottom panels represents the nonlinear, least-squares best fits to the experimental data using a one-site model of binding. Note the roughly 1:1 stoichiometry indicated by the half-height point of the sigmoidal curve (bottom left; Turnbull and Daranas, 2003), and the complete absence of heat release in the case of the pS650 peptide. (B) CHO cells were cotransfected with HA-PIPKIγ90, p35, and Cdk5 or mut-Cdk5. Protein contents of the starting lysates were revealed by Western blotting. PIPKIγ90 and talin were immunoprecipitated from the cell lysates and presence of PIPKIγ90 and talin in each immunoprecipitate was detected by Western blotting.
Mentions: Given the location of S650 in the middle of the talin-binding sequence (WVYSPL; Di Paolo et al., 2002), the possibility that phosphorylation of S650 of PIPKIγ90 inhibits its interaction with talin was next examined. Isothermal titration calorimetry (ITC) was used to analyze the binding of the GST-talin head (Di Paolo et al., 2002), which comprises the FERM domain, to synthetic 12-mer dephospho- and phosphopeptides encompassing the talin-binding sequence of PIPKIγ90. Kd values of 2 μM and >1 mM were observed for the dephosphopeptide and the pS650 peptide, respectively (Fig. 3 A), indicating a strongly negative role of S650 phosphorylation in talin binding. A Kd of 2 μM (see Fig. 6 A) value for the WT peptide is in agreement with the dissociation constant of the same interaction measured with tryptophan fluorescence by Barsukov et al. (2003), but is higher than the dissociation constant observed by Ling et al. (2003) using fluorescence anisotropy measurement. Differences in the assays and the experimental conditions may explain the discrepancy. For example, our values were obtained at 37oC, whereas the value of Ling et al. (2003) was recorded at room temperature.

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