v-Jun Overrides the Mitogen Dependence of S-Phase Entry by Deregulating Retinoblastoma Protein Phosphorylation and E2F-Pocket Protein Interactions as a Consequence of Enhanced Cyclin E-cdk2 Catalytic Activity

Abstract

v-Jun accelerates G₁ progression and shares the capacity of the Myc, E2F, and E1A oncoproteins to sustain S-phase entry in the absence of mitogens; however, how it does so is unknown. To gain insight into the mechanism, we investigated how v-Jun affects mitogen-dependent processes which control the G₁/S transition. We show that v-Jun enables cells to express cyclin A and cyclin A-cdk2 kinase activity in the absence of growth factors and that deregulation of cdk2 is required for S-phase entry. Cyclin A expression is repressed in quiescent cells by E2F acting in conjunction with its pocket protein partners Rb, p107, and p130; however, v-Jun overrides this control, causing phosphorylated Rb and proliferation-specific E2F-p107 complexes to persist after mitogen withdrawal. Dephosphorylation of Rb and destruction of cyclin A nevertheless occur normally at mitosis, indicating that v-Jun enables cells to rephosphorylate Rb and reaccumulate cyclin A without exogenous mitogenic stimulation each time the mitotic “clock” is reset. D-cyclin–cdk activity is required for Rb phosphorylation in v-Jun-transformed cells, since ectopic expression of the cdk4- and cdk6-specific inhibitor p16^INK4A inhibits both DNA synthesis and cell proliferation. Despite this, v-Jun does not stimulate D-cyclin–cdk activity but does induce a marked deregulation of cyclin E-cdk2. In particular, hormonal activation of a conditional v-Jun–estrogen receptor fusion protein in quiescent, growth factor-deprived cells stimulates cyclin E-cdk2 activity and triggers Rb phosphorylation and DNA synthesis. Thus, v-Jun overrides the mitogen dependence of S-phase entry by deregulating Rb phosphorylation, E2F-pocket protein interactions, and ultimately cyclin A-cdk2 activity. This is the first report, however, that cyclin E-cdk2, rather than D-cyclin–cdk, is likely to be the critical Rb kinase target of v-Jun.

ACKNOWLEDGMENTS

We thank the following people for generous provision of reagents and technical advice: G. McLean, Sybille Mittnacht, E. Nigg, K. Parkinson, D. Johnson, J. Bartek, and Max Walker. We thank J. A. Wyke and I. Morgan for comments on the manuscript.

This work was supported by the Cancer Research Campaign (CRC) of the United Kingdom.