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Delta MEKK3:ER* activation induces a p38 alpha/beta 2-dependent cell cycle arrest at the G2 checkpoint
Andrew P Garner1, Claire R Weston, Daniel E Todd
1Inositide Laboratory, Signalling Programme, The Babraham Institute, Babraham Hall, Cambridge CB2 4AT, UK.
Abstract:
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.
Insights
This study reveals that activating MEKK3 signaling inhibits cell proliferation by causing G1/G2 cell cycle arrest and apoptosis. The p38 pathway specifically promotes G2 arrest, independent of p21(CIP1).
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Mitogen-activated protein (MAP) kinase pathways are crucial for cell cycle regulation, particularly the G1 to S phase transition.
- The specific roles of MAP kinases in other cell cycle transitions, such as G2/M, remain less understood.
- MEKK3 is a key activator in MAP kinase signaling cascades.
Purpose of the Study:
- To investigate the function of MEKK3-activated MAP kinase pathways in regulating cell cycle transitions beyond G1.
- To elucidate the mechanisms by which MEKK3 influences cell proliferation, cell cycle arrest, and apoptosis.
- To determine the specific contribution of p38 and JNK pathways to MEKK3-mediated cell cycle effects.
Main Methods:
- Utilized a conditional mutant (Delta MEKK3:ER*) in hamster (CCl39) and rat (Rat-1) fibroblasts.
- Stimulated MEKK3 activation and monitored endogenous JNK, p38, and ERK activation.
- Assessed cell proliferation, cell cycle distribution (G1, G2, S phase arrest), apoptosis, and kinase activity (cyclin A/CDK2, cyclin B1/CDK1) using aphidicolin synchronization and specific inhibitors (SB203580).
Main Results:
- MEKK3 activation strongly induced JNK and p38, with weak ERK activation, leading to G1 and G2 cell cycle arrest and subsequent apoptosis.
- In S-phase synchronized cells, MEKK3 activation upregulated p21(CIP1) and inhibited cyclin A/CDK2 and cyclin B1/CDK1 activity, causing a G2 arrest.
- G2 arrest and CDK inhibition occurred even in p21(CIP1)-silenced cells, linked to reduced cyclin A and B1 expression; p38 inhibition partially rescued kinase activity and mitosis.
Conclusions:
- MEKK3 signaling inhibits cell proliferation through combined G1/G2 arrest and apoptosis.
- The p38 alpha/beta 2 pathway plays a significant role in promoting G2 cell cycle arrest, independently of p21(CIP1).
- MEKK3-induced G2 arrest involves downregulation of cyclin A and B1 expression and inhibition of CDK2 and CDK1 activity.