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Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
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Differential actions of p60c-Src and Lck kinases on the Ras regulators p120-GAP and GDP/GTP exchange factor CDC25Mm

C Giglione1, S Gonfloni, A Parmeggiani

  • 1Groupe de Biophysique-Equipe 2, Ecole Polytechnique, Palaiseau, France. giglione@isv.cnrs-gif.fr

European Journal of Biochemistry
|June 8, 2001
PubMed

Insights

Src kinases p60c-Src and Lck selectively phosphorylate Ras regulators p120-Ras GTPase activating protein (GAP) and CDC25Mm/GDP/GTP exchange factor (GEF). This phosphorylation differentially modulates their activity towards Ras proteins, impacting the Ras signaling pathway.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • The Ras signaling pathway is crucial for cellular functions.
  • Src kinase family members are known to phosphorylate Ras regulators.
  • The functional consequences of these phosphorylations on Ras pathway modulation remain unclear.

Purpose of the Study:

  • To investigate the phosphorylation of p120-Ras GTPase activating protein (GAP) and CDC25Mm/GDP/GTP exchange factor (GEF) by p60c-Src and Lck.
  • To compare the functional effects of phosphorylation on Ras regulators by different Src kinases.

Main Methods:

  • In vitro kinase assays using full-length and truncated forms of GAP and GEF.
  • Analysis of Ha-Ras GTPase activity and GDP/GTP exchange activity of phosphorylated and unphosphorylated Ras regulators.
  • Utilized catalytic domain of p60c-Src to assess the role of SH3/SH2 domains.

Main Results:

  • Both p60c-Src and Lck phosphorylated p120-GAP and CDC25Mm.
  • Phosphorylation of p120-GAP by p60c-Src inhibited its GTPase stimulating activity, while Lck phosphorylation had no effect.
  • Lck phosphorylation of CDC25Mm enhanced its GDP/GTP exchange activity on Ha-Ras, whereas p60c-Src phosphorylation was ineffective.
  • SH3/SH2 domains of p60c-Src are not required for phosphorylation of these regulators.

Conclusions:

  • Phosphorylation by p60c-Src and Lck differentially modulates the activity of p120-GAP and CDC25Mm.
  • This selective phosphorylation provides a mechanism for fine-tuning the Ras signaling pathway.
  • The findings highlight the specificity of kinase-mediated regulation of Ras pathway components.

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