ΔMEKK3:ER* activation induces a p38α/β2-dependent cell cycle arrest at the G2 checkpoint

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.