Mitogenesis by v-Src: fluctuations throughout G1 of classical immediate early AP-1 and mitogen-activated protein kinase responses that parallel the need for the oncoprotein.

ABSTRACT

Activation of the tyrosine kinase of a temperature-sensitive mutant v-Src oncoprotein in quiescent Rat-1 cells leads to passage through the cell cycle. Temperature shift experiments show that v-Src is needed to leave G0, to pass a relatively stable G1 "pause" point, and to pass a later G1 point committing cells to S phase. Classic immediate early responses that activate both AP-1 DNA binding and mitogen-activated protein (MAP) kinase are induced at G0 exit, but unexpectedly they rise again in mid-G1 and before the onset of S phase, fluctuations that parallel the need for v-Src. An estrogen-inducible mutant c-Raf-1 renders these cells susceptible to mitogenic stimulation by beta-estradiol, without v-Src activity, but greatly inhibits the ability of v-Src to induce DNA synthesis and MAP kinase, probably because v-Src physically associates with inactive c-Raf-1 at permissive but not restrictive temperature. This implicates c-Raf-1 association with enzymically active v-Src and consequent activation of the MAP kinase pathway in v-Src mitogenesis. Furthermore, temperature shift experiments indicate that the mid-G1 peak of MAP kinase activity is associated with cells reaching the G1 pause point, while the pre-S phase peak is needed for DNA synthesis. In contrast, cell transformation by v-Src does not require enhanced MAP kinase activity at any stage of the cell cycle.