We have been investigating the regulatory mechanism of the SOS guanine nucleotide exchange factor, a critical activator of Ras and previously found that the catalytic activity of SOS was activated upon stimulation of receptor or nonreceptor protein tyrosine kinase coupled to the Ras signaling pathway in various biological systems. In order to define the domains of SOS involved in the regulation of its catalytic activity, we have initiated the mutational analysis of the regulatory domain of SOS. We have found that the insertional mutation near the first proline-rich C-terminal region (Grb2-binding site) of mSOS1 causes the constitutive activation of its activity. This mutant form of mSOS1 neither responded to EGF or PDGF stimulation nor transmitted activation signals from these ligands to Ras. These results appear to be consistent with others' findings that the prolin-rich C-terminal domain of mSOS1 could suppress its catalytic activity, and mSOS1 might be liberated from such a repression as a consequence of conformational change caused by the insertional mutation near this domain. We also investigated the role of other domains of mSOS1 - pleckstrin homology (PH) domain and Dbl oncogene homology (DH) domain. To this end, the construction of expression vectors with deletion mutations in PH and DH domains is in progress, and GST-PH and -DH fusion proteins have been prepared to affinity-purify the cellular factors interacting with these domains. These studies will provide a further insight into the regulatory mechanism of mSOS1. In another study, we have investigated the involvement of GTP-binding proteins of the Ras-super family in apoptosis. Especially, we focussed on the possible regulatory role of Rac in programmed cell death induced by NGF withdrawal from PC12 cells, and the analysis of the inhibitory effect of dominant negative Rac on this form of apoptosis is in progress. The contribution of Rac to committment of cell survival or death will be investigated by using this biological system.
