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 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.

FIG. 1

v-Jun enables cells to sustain cdk2 kinase activity and cyclin A expression after prolonged mitogen deprivation. (a) Immunoprecipitation (IP) kinase assays of total cdk2 and cyclin (cyc) A-cdk2 kinase activity in cultures of control and v-Jun-transformed CEFs growing in GM or after incubation in LS medium for 48 h. Cell extracts were immunoprecipitated with antibodies specific for cdk2 (top) and cyclin A (bottom), and the precipitates were analyzed for kinase activity using histone H1 as a substrate. Subsequently, the precipitates were analyzed for cdk2 or cyclin A protein expression by Western blotting (WB). (b) DNA content cytograms of cultures of v-Jun-transformed CEFs growing in GM or after incubation in LS medium for 48 h. The plots correspond to the samples shown in panel d. (c) Cultures of v-Jun-transformed CEFs growing in GM or after incubation in LS medium for 48 h were labeled with BrdU for 2, 4, 6, or 8 h. The culture medium was changed immediately before BrdU addition to remove from the LS culture apoptotic cells which had died and detached prior to the start of the experiment. After being labeled, both adherent and detached cells were harvested, and the percentage of labeled cells was determined by flow cytometry. (d) Plots of DNA content versus BrdU incorporation for 8-h GM and LS medium samples shown in panel c. Labeled (L) and unlabeled (U) cell populations are indicated, as are the positions of G₁, S, G₂/M, and apoptotic (A) cells.

FIG. 2

Deregulation of cdk2 is required for v-Jun-induced S-phase entry. v-Jun-transformed CEFs cultured in GM or LS medium were microinjected with control (vector) or expression plasmid encoding catalytically inactive cdk2 protein (dn-cdk2) together with a plasmid encoding GFP to identify injected cells. After sufficient time was allowed for expression, the percentage of injected cells which synthesized DNA in 24 h was determined by BrdU labeling and immunocytochemistry. (a) A representative example of this analysis (LS medium). Injected cells staining positive for BrdU incorporation are indicated with arrows, while negative cells are indicated by asterisks. (b) At least 100 surviving injected cells were analyzed for each growth condition. The experiment was repeated three times with similar results.

FIG. 3

v-Jun overrides mitogen regulation of E2F-pocket protein interactions. (a) Cell extracts prepared from control or v-Jun-transformed CEFs growing in GM or after 48 h in LS medium were analyzed for E2F binding activity by band shift assay using an oligonucleotide spanning the E2F site from the E2A gene promoter as a probe. Complexes corresponding to free E2F–DP-1 and E2F-p107 complexes on the basis of antibody addition experiments (b) are indicated. The putative E2F-p130 detected in quiescent CEFs (LS) is indicated with an asterisk. (b) Analysis of E2F complexes in growing v-Jun CEFs using antibodies specific for DP-1, E2F family members, and individual pocket proteins. DNA binding reactions prepared as described for panel a were preincubated with the indicated antisera prior to addition of the oligonucleotide probe. (c) Western blotting analysis of Rb, p107, and p130 expression in the cell extracts shown in panel a.

FIG. 4

v-Jun promotes mitogen-independent Rb phosphorylation and reaccumulation of cyclin A after mitosis. (a) Flow cytometry analysis of mitogen-deprived v-Jun CEFs separated by centrifugal elutriation. The starting population was separated into nine fractions (F1 to -9) of increasing size as determined by FSC. DNA content cytograms and FSC values of the unfractionated population (U) and successive fractions are shown. (b) Western blotting analysis of cyclin (cyc) A, cdk2, and Rb expression in the elutriated fractions shown in panel a.

FIG. 5

p16^INK4A inhibits DNA synthesis and cell proliferation in v-Jun-transformed CEFs. (a) v-Jun-transformed CEFs cultured in GM or LS medium were microinjected with control or expression plasmid encoding human p16^INK4A together with a plasmid encoding GFP to identify injected cells. After time was allowed for expression, the percentage of injected cells which synthesized DNA in 24 h was determined by BrdU labeling and immunocytochemistry. At least 100 surviving injected cells were analyzed in each case. The experiment was repeated three times with similar results. (b) CEFs doubly infected with the indicated combinations of retroviruses were seeded at two dilutions (10⁵ or 10⁶ cells/dish) and selected with G418 for 5 days after being plated. The cultures were fixed in ethanol and stained with Giemsa stain to visualize colony growth.

FIG. 6

v-Jun does not stimulate D-cyclin expression or cyclin D1-cdk kinase activity. (a) Western blotting analysis of cyclin (cyc) D1 and D2 and cdk6 expression in control and v-Jun CEFs growing in GM or after 48 h in LS medium. (b) Determination of cyclin D1-cdk kinase activity in normal and v-Jun-transformed CEFs growing in GM or after 48 h in LS medium using recombinant Rb as a substrate. Cell extracts prepared from the indicated cultures were immunoprecipitated with a matched pair of anti-cyclin D1 monoclonal antibodies: DCS-6, which precipitates only free cyclin D1, and DCS-11, which precipitates cyclin D1-cdk holoenzyme complexes. After quantitation of kinase activity, the DCS-6 value for each sample was subtracted from the DCS-11 value to correct for background.

FIG. 7

v-Jun stimulates cyclin E-cdk2 catalytic activity. (a) Levels of cyclin (cyc) E-cdk2 kinase activity in normal and v-Jun-transformed CEFs growing in GM or after 48 h in LS medium. Extracts prepared from the indicated cell cultures were immunoprecipitated using a cyclin E-specific antiserum, and the immunoprecipitates were assayed for kinase activity using histone H1 as a substrate. Quantitation of these data (bottom) indicated a ninefold amplification of cyclin E-cdk2 kinase activity in growing v-Jun-transformed cells compared to that in the control. (b) Western blotting analysis of cyclin E, cdk2, and p27^KIP1 expression in normal and v-Jun-transformed CEFs maintained in GM and LS medium.

FIG. 8

Activation of Δv-JunER stimulates cyclin E-cdk2 kinase activity, Rb phosphorylation, and S-phase entry in quiescent, growth factor-deprived CEFs. (a) Cultures of CEFs expressing Δv-JunER, or hER as a control, were rendered quiescent by culture in LS medium for 48 h in the absence of estradiol. Newborn calf serum (10%), estradiol (2 μM), or both were then added, and the cultures were labeled with BrdU for 20 h. After being labeled, the cultures were harvested and the percentage of labeled cells was determined by flow cytometry. (b) Cell extracts prepared from replicate cultures of those shown in panel a were analyzed for Rb phosphorylation and p27^KIP1 expression by Western blotting (WB) and cyclin (cyc) E-cdk2 kinase activity by immunoprecipitation (IP) using histone H1 as a substrate.

FIG. 9

Activation of Δv-JunER does not enhance D-cyclin expression. Cultures of CEFs expressing Δv-JunER or hER were rendered quiescent by culture in LS medium for 48 h in the absence of estradiol. Newborn calf serum (10%) or estradiol (2 μM) was then added as indicated, and replicate cultures were assayed for cyclin (cyc) E-cdk2 kinase activity by immunoprecipitation kinase assay (upper panel) and for expression of cyclin E, cdk2, cyclin D1, cyclin D2, cdk6, and p27^KIP1 by Western blotting (WB) (lower panel).

FIG. 10

Model illustrating the proposed point of action of v-Jun on the cell cycle machinery. In normal CEFs, Rb phosphorylation is catalyzed by the sequential action of D-cyclin-cdks and cyclin E-cdk2. Both are mitogen regulated; however, a basal level of cyclin D1-associated kinase activity persists in quiescent cells. v-Jun stimulates cyclin E-cdk2 kinase activity, accelerating G₁ progression under optimal growth conditions and sustaining Rb phosphorylation after mitogen deprivation. p16INK4A inhibits DNA synthesis by preventing prior modification of Rb (pRb∗) by D-cyclin–cdk's, which is required for subsequent phosphorylation by cyclin E-cdk2.