Differential actions of p60c-Src and Lck kinases on the Ras regulators p120-GAP and GDP/GTP exchange factor CDC25Mm

It is known that the human Ras GTPase activating protein (GAP) p120-GAP can be phosphorylated by different members of the Src kinase family and recent ly phosphorylation of the GDP/GTP exchange factor (GEF) CDC25Mm/ GRF1 by pr oteins of the Src kinase family has been revealed in vivo [Kiyono, M., Kazi ro, Y. & Satoh, T. (2000) J. Biol. Chem. 275, 5441-5446]. As it still remai ns unclear how these phosphorylations can influence the Ras pathway we have analyzed the ability of p60c-Src and Lck to phosphorylate these two Ras re gulators and have compared the activity of the phosphorylated and unphospho rylated forms. Both kinases were found to phosphorylate full-length or trun cated forms of GAP and GEE The use of the catalytic domain of p60c-Src show ed that its SH3/SH2 domains are not required for the interaction and the ph osphorylation of both regulators. Remarkably, the phosphorylations by the t wo kinases were accompanied by different functional effects. The phosphoryl ation of p120-GAP by p60c-Src inhibited its ability to stimulate the Ha-Ras -GTPase activity, whereas phosphorylation by Lck did not display any effect . A different picture became evident with CDC25Mm; phosphorylation by Lck i ncreased its capacity to stimulate the GDP/GTP exchange on Ha-Ras, whereas its phosphorylation by p60c-Src was ineffective. Our results suggest that p hosphorylation by p60c-Src and Lck is a selective process that can modulate the activity of p120-GAP and CDC25Mm towards Ras proteins.