MITOGENESIS OF QUIESCENT CHICK FIBROBLASTS BY V-SRC - DEPENDENCE ON EVENTS AT THE MEMBRANE LEADING TO EARLY CHANGES IN AP-1

Activation of rapidly reversible temperature-sensitive (ts) v-Src in q uiescent chicken embryo fibroblasts (CEFs) results in both morphologic al transformation and exit from G0 to G1, resulting in mitosis. This p henomenon permits examination of cellular responses very soon after ac tivating the oncoprotein, and we have used this to study changes in en dogenous AP-1, and the regulation of its major components, in the firs t few hours after activating v-Src. This approach contrasts with a num ber of studies that have demonstrated enhanced activity of exogenously added AP-1 components in cells transformed by v-Src. Reactivation of a membrane-associated tyrosine kinase (tsRCAN-29) results in a several -fold increase in AP-1 DNA binding and a similar increase in the activ ity of an AP-1-responsive reporter soon after temperature shift. c-Jun and c-Fos are regulated at a number of levels in response to both sti muli. In quiescent RCAN-29-infected CEFs stimulated into cycle by shif t to permissive temperature, c-fos transcripts are elevated by 15 min and remain above basal level for at least 4 h. Serum induces much grea ter elevation of c-fos transcripts, although this response is transien t. Despite the difference in magnitude of the transcript responses, th e stimulation of nuclear c-Fos protein is similar in both serum and v- Src-stimulated cultures. No elevation in c-jun transcripts or nuclear c-Jun protein level is evident in v-Src-stimulated quiescent CEFs. How ever, there is an early change in the tryptic phosphopeptide map of p3 9 c-Jun in response to both v-Src and serum. Upon stimulation we obser ved a novel redistribution of phosphate in the carboxy-terminal trypti c phosphopeptide that may be responsible in part for the increase in A P-1 DNA binding. Phosphorylation of amino-terminal serines 63 and 73 o n peptides Y and X, believed to be responsible for regulation of the t ransactivation function of c-Jun, is constitutively high in resting CE F cultures; stimulation with serum or v-Src results in only a modest i ncrease in phosphorylation at these sites. Significantly, reactivation of a non-myristylated, transformation-defective version of the tsRCAN -29 v-Src protein (RCAN-29A2) is unable to induce resting CEFs to reen ter cycle. In addition, this mutant fails to induce early increases in AP-1 activity, implying that these nuclear changes require crucial si gnalling events at the cell periphery, and that these events correlate with the biological consequences of expression of v-Src.