Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage.

Here, we show that activation of the checkpoint effector kinase Chk1 in response to irradiation-induced DNA damage is minimal in G1, maximal during S-phase and diminishes as cells enter G2. In addition, formation of irradiation-induced replication protein A (RPA)-coated single-stranded DNA (RPA-ssDNA), a structure required for ATM and Rad3-related (ATR)-Chk1 activation, occurs in a broadly similar pattern. Cyclin-dependent kinase (Cdk) activity is thought to promote RPA-ssDNA formation by stimulating DNA strand resection at double-strand breaks (DSBs), providing one possible mechanism of imposing cell cycle dependence on DNA damage signaling. However, it has recently been shown that Chk1 itself is also subject to Cdk-mediated phosphorylation at serines 286 and 301 (S286 and 301). We show that Chk1 S301 phosphorylation increases as cells progress through S and G2 and that both Cdk1 and Cdk2 are likely to contribute to this modification in vivo. We also find that substitution of S286 and S301 with non-phosphorylatable alanine residues strongly attenuates DNA damage-induced Chk1 activation and G2 checkpoint proficiency, but does not eliminate the underlying cell cycle dependence of Chk1 regulation. Taken together, these data indicate that Cdk activity regulates multiple steps in the DNA damage response pathway including full activation of Chk1 and checkpoint proficiency.

Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation by de-repression of Chk1 catalytic activity.

Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ATR kinases during checkpoint activation; however, how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell-cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345 and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/M and S/M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, as the mutants are either partially (317A and 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated by a de-repression mechanism.

Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase.

Whether Chk2 contributes to DNA damage-induced arrest in G2 has been controversial. To investigate this issue further, we generated Chk2-deficient DT40 B-lymphoma cells by gene targeting and compared their cell cycle response to ionizing radiation (IR) with wild-type (WT) and isogenic Chk1-deficient counterparts. After moderate doses of IR (4 Gy), we find that Chk2-/- cells which are in G1 or S phase at the time of irradiation arrest efficiently in G2. In contrast, Chk2-/- cells which are in G2 when DNA damage is incurred exhibit an impaired mitotic delay compared to WT, with the result that cells enter mitosis with damaged DNA as judged by the presence of numerous gamma-H2AX foci on condensed chromosomes. Impaired G2 delay as the result of Chk2 deficiency can be detected at very low doses of radiation (0.1 Gy), and may allow division with spontaneous DNA damage, since a higher proportion of mitotic Chk2-/- cells bear spontaneous gamma-H2AX foci and damaged chromosomes during unperturbed growth compared to WT. The contribution of Chk2 to G2/M delay is epistatic to that of Chk1, since Chk1-/- cells exhibit no measurable mitotic delay at any radiation dose tested. We suggest that this function of Chk2 could contribute to tumour suppression, since cell division with low levels of spontaneous damage is likely to promote genetic instability and thus carcinogenesis.

v-Jun stimulates both cdk2 kinase activity and G1/S progression via transcriptional repression of p21 CIP1.

Previous studies have shown that the viral Jun (v-Jun) oncoprotein induces marked alterations in cell cycle control, which are associated with, and may be caused by, increased cdk2 kinase activity. Since p21 CIP1 is an important regulator of cdk2, we investigated whether aberrant expression of this cyclin-dependent kinase inhibitor might contribute to cell cycle deregulation by v-Jun. We find that the basal levels of p21 CIP1 mRNA and protein expression are greatly reduced in chick embryo fibroblasts (CEF) transformed by v-Jun, and that v-Jun blocks the increases in p21 CIP1 expression that normally accompany growth inhibition induced by serum deprivation or confluency in untransformed CEF. Importantly, ectopic expression of p21 CIP1 in v-Jun-transformed CEF inhibits both cdk2 kinase activity and cell cycle progression, indicating that these alterations in p21 CIP1 expression are likely to be functionally significant for growth deregulation. We also investigated the mechanism through which v-Jun disturbs p21 CIP1 expression and the possible involvement of a known p21 CIP1 regulator, p53, as an intermediate in this process. This analysis revealed that repression is mediated primarily at the level of p21 CIP1 gene transcription, however the mechanism is complex; both p53-dependent and -independent mechanisms contribute as judged by analysis of p21 CIP1 promoter mutants and other assays of p53 transcriptional activity.

Transient deactivation of ERK signalling is sufficient for stable entry into G0 in primary avian fibroblasts.

Re-entry into the cell cycle from quiescence requires the activation of mitogen-activated protein (MAP) kinases of the extracellular-signal-regulated kinase (ERK) family [1,2]. The relationship between ERK and cell-cycle control is, however, complex, as ERK activation can also lead to terminal differentiation [3] or a senescence-like growth arrest [4]. Here, we report that reversible cell-cycle exit induced by serum withdrawal in primary avian fibroblasts is associated with rapid deactivation of ERK, but ERK activity is subsequently regenerated and sustained at high levels in fully quiescent (G0) cells. As in proliferating cells, ERK activation during G0 required the MAPkinase kinase MEK and was partially dependent on cell adhesion. Active, phosphorylated ERK was concentrated in the nucleus in cycling cells, but was largely confined to the cytoplasm during G0. This was unexpected, as activatory phosphorylation mediated by MEK is thought to play an important role in promoting nuclear translocation [5,6]. These results indicate that transient deactivation of ERK signalling can be sufficient for stable cell-cycle exit, and that MEK-mediated phosphorylation is not sufficient for nuclear translocation of active ERK in G0. Cytoplasmic sequestration may prevent active ERK from accessing critical nuclear cell-cycle targets, thus allowing quiescent or post-mitotic cells to retain ERK activity for other physiological functions.

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.

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.

Insulin-stimulated expression of c-fos, fra1 and c-jun accompanies the activation of the activator protein-1 (AP-1) transcriptional complex.

The activator protein-1 (AP-1) transcriptional complex is made up of members of the Fos (c-Fos, FosB, Fra1, Fra2) and Jun (c-Jun, JunB, JunD) families and is stimulated by insulin in several cell types. The mechanism by which insulin activates this complex is not well understood but it is dependent on the activation of the Erk1 and Erk2 isoforms of mitogen-activated protein kinases. In the current study we show that the AP-1 complex isolated from insulin-stimulated cells contained c-Fos, Fra1, c-Jun and JunB. The activation of the AP-1 complex by insulin was accompanied by (i) a transient increase in c-fos expression, and the transactivation of the ternary complex factors Elk1 and Sap1a, in an Erk1/Erk2-dependent fashion; (ii) a substantial increase in the expression of Fra1 protein and mRNA, which was preceded by a transient decrease in its electrophoretic mobility upon SDS/PAGE, indicative of phosphorylation; and (iii) a sustained increase in c-jun expression without increasing c-Jun phosphorylation on serines 63 and 73 or activation of the stress-activated kinase JNK/SAPK. In conclusion, insulin appears to stimulate the activity of the AP-1 complex primarily through a change in the abundance of the components of this complex, although there may be an additional role for Fra1 phosphorylation.

An oncogenic mutation uncouples the v-Jun oncoprotein from positive regulation by the SAPK/JNK pathway in vivo.

Stimulation of c-Jun transcriptional activity via phosphorylation mediated by the stress-activated or c-Jun amino-terminal (SAPK/JNK) subgroup of mitogen-activated protein kinases (MAP kinases) is thought to depend on a kinase-docking site (the delta region) within the amino-terminal activation domain, which is deleted from the oncogenic derivative, v-Jun [1] [2] [3]. This mutation markedly enhances v-Jun oncogenicity [4] [5]; however, its transcriptional consequences have not been resolved. In part, this reflects uncertainty as to whether binding of SAPK/JNK inhibits c-Jun function directly [6] [7] or, alternatively, serves to facilitate and maintain the specificity of positive regulatory phosphorylation [8]. Using a two-hybrid approach, we show that SAPK/JNK stimulates c-Jun transactivation in yeast and that this depends on both catalytic activity and physical interaction between the kinase and its substrate. Furthermore, c-Jun is active when tethered to DNA via SAPK/JNK, demonstrating that kinase binding does not preclude transactivation. Taken together, these results suggest that SAPK/JNK acts primarily as a positive regulator of c-Jun transactivation in situ, and that loss of the docking site physically uncouples v-Jun from this control. This loss-of-function model accounts for the deficit of v-Jun regulatory phosphorylation and repression of TPA response element (TRE)-dependent transcription observed in v-Jun-transformed cells and predicts that an important property of the oncoprotein is to antagonise SAPK/JNK-dependent gene expression.

The v-Jun oncoprotein replaces p39 c-Jun as the predominant AP-1 constituent in ASV17-transformed fibroblasts: implications for SAPK/JNK-mediated signal transduction.

We have investigated the expression of Jun family proteins and composition of AP-1 in chicken embryo fibroblasts before and after transformation by the v-Jun oncoprotein of ASV17. We show that p39 c-Jun is the predominant Jun family protein expressed in normal fibroblasts, and that heterodimers of c-Jun and Fos-related partners (Fra's) account for the majority of the AP-1 DNA binding activity. Unexpectedly, because ASV17-transformed fibroblasts do not express p39 c-Jun, v-Jun replaces c-Jun as the predominant AP-1 constituent in association with similar or identical Fra's. This substitution has little effect on the overall level of TRE-specific DNA binding activity, however it results in a profound reduction in TRE-dependent transcriptional activity and a striking defect in signal-regulated phosphorylation of the Jun component of AP-1; whilst agonists of SAPK/JNK kinases trigger transient N-terminal phosphorylation of c-Jun in normal fibroblasts, no corresponding modification of v-Jun occurs in ASV17-transformed cells. Because SAPK/JNK-mediated phosphorylation is thought to regulate c-Jun transcriptional activity and thereby cellular gene expression in response to extracellular signals, we propose that subversion of this signal transduction process by v-Jun is likely to contribute to oncogenesis by ASV17.

Transcriptional activation by the v-Jun oncoprotein is independent of positive regulatory phosphorylation.

Growth factors, phorbol esters, and oncogenes such as ras, src, and sis are believed to stimulate c-Jun transcriptional activation by inducing increased phosphorylation at two serine residues (S63 and S73) within the N-terminal transactivation domain. Although S63 and S73 are conserved in the mutant v-Jun oncoprotein, they are not phosphorylated by two enzymes which target the corresponding residues in c-Jun in vitro; namely a partially purified c-Jun kinase from TPA-stimulated U937 cells and purified p54 mitogen activated protein (MAP) kinase. In addition, v-Jun activates transcription more strongly than c-Jun when fused to the Gal4 DNA-binding domain, and transcriptional activation by Gal4-v-Jun is unaffected when S63, S73, or both, are replaced with non-phosphorylatable alanine residues, amino acid substitutions which severely impair transcriptional activation by Gal4-c-Jun. The novel biochemical and transcriptional properties of v-Jun result from deletion of a 27 amino acid segment, termed delta, which is important for transforming activity. On the basis of these results we propose that unlike c-Jun, v-Jun transcriptional activation is independent of positive regulatory phosphorylation and that this may contribute to oncogenesis by v-Jun.

Protein phosphatase 2A reverses phosphorylation of c-Jun specified by the delta domain in vitro: correlation with oncogenic activation and deregulated transactivation activity of v-Jun.

Chicken c-Jun proteins synthesized in vitro in reticulocyte extract consist of several electrophoretic isoforms resulting from phosphorylation which can be specifically reversed by purified protein phosphatase 2A (PP2A). Using the phosphatase inhibitors okadaic acid and microcystin-LR, we conclude that the isoforms seen in vitro represent a balance between the action of an unidentified kinase(s) which phosphorylates c-Jun and dephosphorylation by an endogenous PP2A-like phosphatase. c-Jun proteins are also subject to phosphorylation in vivo in chick embryo fibroblasts (CEF), which can be reversed by PP2A. In contrast, the viral Jun oncoprotein encoded by ASV17 is not subject to PP2A-sensitive phosphorylation in vitro and is hypophosphorylated in comparison with c-Jun in ASV17-transformed CEF. Hybrids between c-Jun and v-Jun demonstrate that differential phosphorylation in vitro is a consequence of deletion of 27 amino acids in the N-terminal third of v-Jun. The deletion is important for oncogenic activation and lies in a domain, termed delta, which regulates c-Jun transactivation function. PP2A-sensitive phosphorylation in vitro correlates with the differential responsiveness of c-Jun and v-Jun to a recently identified cell type-specific inhibitor of transactivation function.