C-MYC COOPERATES WITH ACTIVATED RAS TO INDUCE THE CDC2 PROMOTER

Expression of c-myc with constitutively active mutants of the ras gene results in the cooperative transformation of primary fibroblasts, although the precise mechanism by which these genes cooperate is unknown. Since c-Myc has been shown to Function as a transcriptional activator, we have examined the ability of c-Myc e and activated Ras (H-Ras(V-12)) to cooperatively induce the promoter activity of cdc2, a gene which is critical for cell cycle progression. Microinjection of expression constructs encoding H-Ras(V-12) and c-Myc along with a cdc2 promoter-luciferase reporter plasmid into quiescent cells led to an increase in cdc2 promoter activity approximately 30 h after injection, a period which coincides with the S-to-G(2)/M transition in these cells. Expression of H-Ras(V-12) alone weakly activated the cdc2 promoter, while expression of c-Myc alone had no effect. Mutants of c-Myc lacking either the leucine zipper dimerization domain or the phosphoacceptor site Ser-62 could not cooperate with H-Ras(V-12) to induce the cdc2 promoter. These mutants also lacked the ability to cooperate with H-Ras(V-12) to stimulate DNA synthesis. Deletion analysis identified a distinct region of the cdc2 promoter which was required for c-Myc responsiveness. Taken together, these observations suggest a mechanistic link between the molecular activities of c-Myc and Ras and induction of the cell cycle regulator Cdc2.