v-Jun accelerates G 1 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 G 1 /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 p16INK4A 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.The vertebrate cell division cycle is regulated primarily at the transition between the G 1 and S phases of the cell cycle, also known as the restriction point, beyond which cells become committed to mitosis (49, 51). Normal cells require mitogenic signals in the form of soluble growth factors and substrate attachment in order to make this transition, while oncogenic lesions frequently deregulate cell proliferation by mimicking or circumventing the need for such signals (43).The retinoblastoma (Rb) tumor suppressor protein and the related p107 and p130 "pocket proteins" are negative growth regulators which play a pivotal role in controlling the G 1 /S transition through their association with the E2F and DP-1 families of transcription factors (15, 49). E2F and DP-1 proteins form heterodimers which bind to specific DNA recognition sequences either alone as "free" E2F-DP-1 or as complexes with Rb, p107, or p130 (6). Although the functional consequences of E2F-pocket protein interactions are incompletely understood, free E2F has the potential to activate, whereas E2F-pocket protein complexes repress, target gene transcription (6,15).A critical feature of the p...