DMEKK3:ER* activation induces a p38a/b2-dependent cell cycle arrest at the G 2 checkpoint Andrew P Garner 1,2 , Claire R Weston 1,3 , Daniel E Todd 1 , Kathryn Balmanno 1 and Simon J Cook* ,1 1 Inositide Laboratory, Signalling Programme, The Babraham Institute, Babraham Hall, Cambridge CB2 4AT, UK Whilst many studies have examined the role of the MAP Kinases in regulating the G 1 ?S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant DMEKK3: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 DMEKK3:ER* inhibited cell proliferation through a combination of an initial G 1 and G 2 cell cycle arrest, followed by a delayed onset of apoptosis. When cells were synchronized in S phase with aphidicolin and then released, activation of DMEKK3: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 G 2 . DMEKK3:ER*- mediated CDK inhibition and G 2 arrest did not absolutely require p21 CIP1 , since both events were observed in Rat-1 cells in which p21 CIP1 is transcrip- tionally 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 G 1 phase, suggesting that activation of the p38a/b2 pathway can promote a G 2 cell cycle arrest. Oncogene (2002) 21, 8089 – 8104. doi:10.1038/sj.onc. 1206000 Keywords: MEKK3; p38; G 2 ; cyclin; CDK; p21 CIP1 Introduction Cells that are exposed to chemical stresses, including many commonly used chemotherapeutic agents, under- go cell cycle arrest, allowing the cell to repair the lesion or undergo apoptosis. Cells arrest at specific check- points, either late in G 1 or late in G 2 , to prevent replication or segregation of damaged DNA (for reviews see Sherr, 1996; Clarke and Gime´nez-Albia´n, 2000). The p53-dependent up-regulation of p21 CIP1 (El- Deiry et al., 1993; Macleod et al., 1995; Brugarolas et al., 1995; Deng et al., 1995; Waldmann et al., 1995) is critical for both imposing and maintaining the DNA damage-induced G 1 checkpoint (Brugarolas et al., 1995; Deng et al., 1995). p21 CIP1 over-expression results in inhibition of cyclin-dependent kinase 2 (CDK2)-containing complexes (Harper et al., 1995; Poon et al., 1996), hypophosphorylation of the Retinoblastoma protein (pRb) and sequestration of E2F transcription factors (Hiebert et al., 1992; Knudsen and Wang, 1997). The importance of this pathway in growth regulation is exemplified by the frequency of inactivating mutations in p53 or compo- nents of the Rb pathway in human tumours (Hollstein et al., 1994; Hall and Peters, 1996). The passage of cells through S phase and into G 2 is primarily controlled by cyclin A/CDK2, whilst M phase progression is regulated by the activity of the cyclin B/CDK1 complex. During the normal cell cycle, cyclin B1 is expressed during G 2 , whereupon it binds to CDK1 (Labbe et al., 1989; Pines and Hunter, 1989). CDK1 subsequently undergoes an activating phosphor- ylation at T 161 catalysed by CAK (CDK-activating kinase) (Solomon et al., 1992; Fesquet et al., 1993). Once active cyclin B1/CDK1 is formed, Wee1 and Myt1 phosphorylate CDK1 on T 14 and Y 15 , which serves to inhibit its kinase activity (Russell and Nurse, 1987; Gould and Nurse, 1989; Meijer et al., 1991; Mueller et al., 1995). The transition between metaphase and anaphase is then triggered by the Cdc25C- catalysed dephosphorylation of T 14 and Y 15 , resulting in the activation of CDK1 (reviewed in Piwnica- Worms, 1999). Further auto-phosphorylation events lead to the relocation of MPF to the nucleus, where it subsequently phosphorylates its target substrates (Hagting et al., 1999). Following DNA damage, Cdc25C is phosphorylated by Chk1 and Chk2, which are in turn activated downstream of the ATM or ATR kinases (Matsuoka et al., 1998; Brown et al., 1999; Liu et al., 2000). Phosphorylation of Cdc25C promotes its sequestration in the cytoplasm by 14-3-3 proteins, resulting in enhanced Y 15 phosphorylation and inhibi- Received 17 March 2002; revised 27 August 2002; accepted 29 August 2002 *Correspondence: SJ Cook; E-mail: simon.cook@bbsrc.ac.uk Current addresses: 2 Cancer and Infection Bioscience, AstraZeneca, Alderley Park, Macclesfield SK10 4TG, UK; 3 University of Massachusetts Medical School, II Biotech Park, 373 Plantation St., Suite 306, Worcester, MA 01605, USA Oncogene (2002) 21, 8089 – 8104 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc