FUNCTIONAL ROLES FOR THE PLECKSTRIN AND DBL HOMOLOGY REGIONS IN THE RAS EXCHANGE FACTOR SON-OF-SEVENLESS

Activation of p21(ras) by receptor tyrosine kinases is thought to resu lt from recruitment of guanine nucleotide exchange factors such as Son -of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2. This hypothesis was tested in the present stud ies by evaluating the ability of truncation and deletion mutants of Dr osophila (d)Sos to enhance [P-32]GTP loading of p21(ras) when expresse d in P-32-labeled COS or 293 cells, The dSos catalytic domain (residue s 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding, COOH-terminal (residues 1126-1596) regions, exhibi ts intrinsic exchange activity as evidenced by its rescue of mutant Sa ccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activi ty, Here we show that this dSos catalytic domain fails to affect GTP.p 21(ras) levels when expressed in cultured mammalian cells unless the N H2-terminal domain is also present, Surprisingly, the COOH-terminal, a daptor binding domain of dSos was not sufficient to confer p21(ras) ex change activity to the Sos catalytic domain in these cells in the abse nce of the NH2-terminal domain. This function of promoting catalytic d omain activity could be localized by mutational analysis to the plecks trin and Dbl homology sequences located just NH2-terminal to the catal ytic domain, The results demonstrate a functional role for these pleck strin and Dbl domains within the dsos protein, and suggest the presenc e of unidentified cellular elements that interact with these domains a nd participate in the regulation of p21(ras).