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 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...

Distinct roles of Jun : Fos and Jun : ATF dimers in oncogenesis

“…These adenovirus-transformed cells resemble CEFs transformed by ATF3 or the ATF2-prefering mutant v-Jun-m1, as they e ciently proliferate in low serum medium, but only ine ciently in soft agar (van der Eb, personal communication). At least one mammalian c-Jun : ATF2-dependent gene that is induced by E1A, cyclin A, has also been found to be induced by v-Jun and v-Jun-m1 in CEFs (Buchou et al, 1993;Zerfass et al, 1996;Clark et al, 2000;Vial, Perez, Herrlich and Castellazzi, unpublished; Figure 1). Like v-Jun-m1, E1A can suppress the levels and/or activities of (Jun : Fos-dependent) secreted proteases (van Dam and van der Eb, 1994;see above).…”

Microarray analysis identifies Autotaxin, a tumour cell motility and angiogenic factor with lysophospholipase D activity, as a specific target of cell transformation by v-Jun

“…The proliferation rate of ATX-expressing CEF, however, was indistinguishable from vector-infected controls and much slower than v-Jun-transformed CEF (Figure 7b). Furthermore, ATX-expressing CEF did not exhibit any obvious morphological changes or other growth alterations associated with cell transformation by v-Jun such as anchorage-independent growth or continued cell cycle progression in low serum-containing medium (Clark et al, 2000). Although these findings obviously do not rule out a role for ATX in growth deregulation in the context of other gene expression changes induced by v-Jun, ectopic expression of ATX alone is insufficient to induce cell transformation in CEF.…”

“…Decreased expression of the DNA damageinducible and p53-regulated cyclin G1 is intriguing in view of recent observations of genetic and functional interactions between c-Jun and p53 (Schreiber et al, 1999), and could also be indicative of altered stress responses. In contrast, diminished expression of gene products involved in DNA replication, such as ribonucleotide reductase, PCNA, and MCM proteins, seems counter-intuitive, since v-Jun is known to promote cell cycle progression (Clark et al, 2000). The DNAreplication process itself is, however, generally considered to be subordinate to other critical rate-limiting biochemical activities, such as cyclin E/cdk2, which act in G1 to control the onset of S phase and which are amplified by v-Jun (Clark et al, 2000).…”

“…Decreases were also observed in the expression of ribonucleotide reductase (0.2-0.24-fold), MCM4 and 6 (0.25),). This was unexpected, since these gene products are required for DNA replication, and previous studies have shown that v-Jun accelerates G1/ S progression and cell proliferation (Clark and Gillespie, 1997;Clark et al, 2000).…”

“…To explore the possible relationship between ATX expression and cell transformation in more detail, we used RCAS, a replication-competent retroviral vector (Hughes et al, 1987), to express human ATX in normal CEF. Two independent CEF cultures infected with RCAS-ATX (Figure 7, 1 and 2) were generated and analysed for ATX expression and evidence of accelerated cell proliferation, a cardinal characteristic of cell transformation by v-Jun (Clark and Gillespie, 1997;Clark et al, 2000). As shown in Figure 7a, Western blotting analysis revealed that both cultures expressed and secreted soluble ATX into the growth medium at levels which were comparable to those released by v-Jun-transformed CEF (Figure3 and data not shown).…”