ROLE OF GAS1 DOWN-REGULATION IN MITOGENIC STIMULATION OF QUIESCENT NIH3T3 CELLS BY V-SRC

Quiescent mammalian fibroblasts can be induced to reenter the cell cyc le by growth factors and oncoproteins, We studied the pathway(s) throu gh which v-Src, the oncogenic tyrosine kinase encoded by the v-src onc ogene of Rous sarcoma virus, forces serum-starved NIH3T3 cells to ente r S-phase, To this purpose, we isolated and characterized a polyclonal population of NIH3T3 cells transformed by the MR31 retroviral vector, encoding G418 resistance and the v-src temperature-sensitive allele f rom the mutant ts LA31 PR-A, NIH(MR31! cells displayed a temperature-c onditional transformed phenotype and could be made quiescent by serum deprivation at the restrictive temperature, Serum stimulation or therm olabile v-Src reactivation induced entry into S-phase to a comparable extent, although with different kinetics. The data suggest that v-Src mitogenic activity involves early activation of the Erk1/Erk2 MAP kina ses with very little tyrosine phosphorylation of the Shc adaptor prote ins at least during the early stages of v-Src reactivation and that v- Src-induced S-phase entry was strongly inhibited by drugs affecting ME K or PI 3-kinase, Our results also suggest that down-regulation of gas 1 gene expression plays an important role in regulating the efficiency of entry into S-phase triggered by reactivated v-Src and that Gas1 do wn-regulation does not require PI 3-kinase dependent signals.