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To study the mechanisms by which mitogen- and stress-activated protein kinases regulate cell cycle re-entry, we have used a panel of conditional kinases that stimulate defined MAPK or SAPK cascades. Activation of ?MEKK3:ER* during serum restimulation of quiescent cells causes a strong activation of JNK1 and p38a but only a modest potentiation of serum-stimulated ERK1/2 activity. In CCl39 cells this promoted a sustained G1 arrest that correlated with decreased expression of cyclin D1 and Cdc25A, increased expression of p21CIP1 and inhibition of CDK2 activity. In Rat-1 cells, in which p21CIP1 expression is silenced by methylation, ?MEKK3:ER* activation caused only a transient delay in the S phase entry rather than a sustained G1 arrest. Furthermore, p21CIP1-/- 3T3 cells were defective for the ?MEKK3:ER*-induced G1 cell cycle arrest compared to their wild-type counterparts. These results suggest that activated ?MEKK3:ER* inhibits the G1 ? S progression by two kinetically distinct mechanisms, with expression of p21CIP1 being required to ensure a sustained G1 cell cycle arrest. The ERK1/2 and p38aß pathways cooperated to induce p21CIP1 expression and inhibition of p38aß caused a partial reversal of the cell cycle arrest. In contrast, selective activation of ERK1/2 by ?Raf-1:ER* did not inhibit serum stimulated cell cycle re-entry. Finally, selective activation of JNK by ?MEKK1:ER* failed to inhibit cell cycle re-entry, even in cells that retained wild-type p53, arguing against a major role for JNK alone in antagonizing the G1 ? S transition.Oncogene (2004) 23, 3284-3295. doi:10.1038/sj.onc.1207467 Published online 23 February 2004 [ABSTRACT FROM AUTHOR]

CC139 fibroblasts are one of several model systems in which the Raf?MEK?ERK1/2 pathway can inhibit apoptosis independently of the PI3K pathway; however, the precise mechanism for this protective effect is not known. Serum withdrawal from CC139 fibroblasts resulted in the rapid onset of apoptosis, which was prevented by actinomycin D or cycloheximide. Serum withdrawal promoted the rapid, de novo accumulation of BimEL, a proapoptotic 'BH3-only' member of the Bcl-2 protein family. BimEL expression was an early event, occurring several hours prior to caspase activation. In contrast to studies in neurons, activation of the JNK?c-Jun pathway was neither necessary nor sufficient to induce BimEL expression. Selective inhibition of either the ERK pathway (with U0126) or the PI3K pathway (with LY294002) caused an increase in the expression of BimEL. Furthermore, selective activation of the ERK1/2 pathway by ?Raf-1:ER* substantially reduced BimEL expression, abolished conformational changes in Bax and blocked the appearance of apoptotic cells. The ability of ?Raf-1:ER* to repress BimEL expression required the ERK pathway but was independent of the PI3K?PDK?PKB pathway. Thus, serum withdrawal-induced expression of BimEL occurs independently of the JNK?c-Jun pathway and can be repressed by the ERK pathway independently of the PI3K pathway. This may contribute to Raf- and Ras-induced cell survival at low serum concentrations.Oncogene (2003) 22, 1281-1293. doi:10.1038/sj.onc.1206261 [ABSTRACT FROM AUTHOR]

Whilst many studies have examined the role of the MAP Kinases in regulating the G1→S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant ΔMEKK3:ER* in asynchronous hamster (CCl39) and rat (Rat-1) fibroblasts resulted in the strong activation of endogenous JNK and p38 but only a weak activation of ERK. Activation of ΔMEKK3:ER* inhibited cell proliferation through a combination of an initial G1 and G2 cell cycle arrest, followed by a delayed onset of apoptosis. When cells were synchronized in S phase with aphidicolin and then released, activation of ΔMEKK3:ER* resulted in the up-regulation of p21CIP1 and a pronounced inhibition of cyclin A/CDK2 and cyclin B1/CDK1 kinase activity. Analysis of mitotic figures indicated that cells failed to enter mitosis, arresting late in G2. ΔMEKK3:ER*-mediated CDK inhibition and G2 arrest did not absolutely require p21CIP1, since both events were observed in Rat-1 cells in which p21CIP1 is transcriptionally silenced due to promoter methylation. Rather, CDK inhibition was associated with a down-regulation of cyclin A and B1 expression. Finally, application of the p38 inhibitor SB203580 partially restored cyclin B associated kinase activity and allowed cells to proceed through mitosis into the next G1 phase, suggesting that activation of the p38α/β2 pathway can promote a G2 cell cycle arrest.Oncogene (2002) 21, 8089–8104. doi:10.1038/sj.onc.1206000 [ABSTRACT FROM AUTHOR]

In CCL39 cells thrombin is a potent growth factor which requires sustained activation of mitogen activated protein kinases (MAPKs) to promote DNA synthesis. Some of the effects of thrombin can be mimicked by peptides based on the new amino terminus of the cleaved receptor; however, these thrombin receptor peptides (TRPs) fail to induce sustained activation of MAPK or DNA synthesis. We have used thrombin, TRP-7 and other agonists which elicit sustained or transient MAPK activation to identify immediate-early and delayed-early genes which are only expressed under conditions of sustained MAPK activation focusing on cyclin D1, p21Cip1 and the AP-1 transcription factor. Of the stimuli tested only FBS and thrombin were able to stimulate a sustained activation of MAPK, expression of cyclin D1, p21Cip1 and cell cycle re-entry. The expression of cyclin D1 was strongly, though not completely, inhibited by the MEK1 inhibitor PD098059. Thrombin stimulated a rapid but transient accumulation of c-Fos whereas the expression of Fra-1, Fra-2, c-Jun and JunB was sustained throughout the G1 phase of the cell cycle. We focussed our analysis on c-Fos (typical of AP-1 genes which are expressed rapidly and transiently) and Fra-1 and JunB (typical of AP-1 genes expressed after a delay but in a sustained manner). The expression of c-Fos, Fra-1 and JunB was dependent upon the activation of MAPK since these responses were inhibited by PD098059. However, a comparison of responses to FBS, thrombin, TRPs, LPA and EGF revealed that Fra-1 and JunB expression required sustained activation of MAPK whereas c-Fos expression was strongly induced even by non-mitogenic stimuli which elicited only transient MAPK activation. The expression of c-Fos (in response to thrombin, TRP or LPA) or Fra-1, JunB and cyclin D1 (thrombin only) was also inhibited by pertussis toxin. This suggests that both early and late AP-1 gene expression is regulated by the same Gi-mediated, MEK-dependent... [ABSTRACT FROM AUTHOR]

Whilst many studies have examined the role of the MAP Kinases in regulating the G1-->S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant Delta MEKK3:ER* in asynchronous hamster (CCl39) and rat (Rat-1) fibroblasts resulted in the strong activation of endogenous JNK and p38 but only a weak activation of ERK. Activation of Delta MEKK3:ER* inhibited cell proliferation through a combination of an initial G1 and G2 cell cycle arrest, followed by a delayed onset of apoptosis. When cells were synchronized in S phase with aphidicolin and then released, activation of Delta MEKK3:ER* resulted in the up-regulation of p21(CIP1) and a pronounced inhibition of cyclin A/CDK2 and cyclin B1/CDK1 kinase activity. Analysis of mitotic figures indicated that cells failed to enter mitosis, arresting late in G2. Delta MEKK3:ER*-mediated CDK inhibition and G2 arrest did not absolutely require p21(CIP1), since both events were observed in Rat-1 cells in which p21(CIP1) is transcriptionally silenced due to promoter methylation. Rather, CDK inhibition was associated with a down-regulation of cyclin A and B1 expression. Finally, application of the p38 inhibitor SB203580 partially restored cyclin B associated kinase activity and allowed cells to proceed through mitosis into the next G1 phase, suggesting that activation of the p38 alpha/beta 2 pathway can promote a G2 cell cycle arrest.