P21RAS ACTIVATION BY THE GUANINE-NUCLEOTIDE EXCHANGE FACTOR SOS, REQUIRES THE SOS GRB2 INTERACTION AND A 2ND LIGAND-DEPENDENT SIGNAL INVOLVING THE SOS N-TERMINUS/

It has been suggested that a key event in growth factor-induced p21Ras activation by the guanine nucleotide exchange factor Sos, is the recr uitment of Sos to the plasma membrane by its interaction with the adap tor protein Grb2. However, other evidence argues that the sub cellular localisation of Sos is independent of Grb2, and that the Sos/Grb2 int eraction can be dispensed with for p21Ras activation. To clarify the r ole of the Sos/Grb2 interaction in ligand-stimulated p21Ras activation , we have utilised the observation that overexpression of the Sos C-te rminal domain can effectively inhibit p21Ras-dependent signalling in t hree different mammalian systems. We have shown that concurrent expres sion of Grb2, but not SH2 or SH3 domain mutants of Grb2, or the altern ative adaptor protein Nck, can rescue this inhibitory effect: of the C -terminus. This shows that the Grb2/Sos interaction is required to med iate growth factor-dependent activation of p21Ras, and requires the pr esence of intact SH2 and SH3 domains of Grb2. This approach was also u sed for a functional analysis of Sos which revealed that growth factor dependent signals are transmitted through both the N-terminal and C-t erminal domains.