ERK1/2 and p38 cooperate to induce a p21 CIP1 -dependent G 1 cell cycle arrest Daniel E Todd 1,3 , Ruth M Densham 1 , Sarah A Molton 1,4 , Kathryn Balmanno 1 , Catherine Newson 1 , Claire R Weston 1,5 , Andrew P Garner 1,6 , Linda Scott 2 and Simon J Cook* ,1 1 Signalling Programme, The Babraham Institute, Babraham Hall, Cambridge CB2 4AT, UK; 2 The Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, Scotland, UK 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 DMEK- K3: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 G 1 arrest that correlated with decreased expression of cyclin D1 and Cdc25A, increased expression of p21 CIP1 and inhibition of CDK2 activity. In Rat-1 cells, in which p21 CIP1 expression is silenced by methylation, DMEKK3:ER* activation caused only a transient delay in the S phase entry rather than a sustained G 1 arrest. Furthermore, p21 CIP1/ 3T3 cellsweredefectiveforthe DMEKK3:ER*-induced G 1 cell cycle arrest compared to their wild-type counterparts. These results suggest that activated DMEKK3:ER* inhibits the G 1 -S progression by two kinetically distinct mechanisms, with expression of p21 CIP1 being required to ensure a sustained G 1 cell cycle arrest. The ERK1/2 and p38ab pathways cooperated to induce p21 CIP1 expression andinhibitionofp38ab causedapartialreversalofthecell cycle arrest. In contrast, selective activation of ERK1/2 by DRaf-1:ER* did not inhibit serum stimulated cell cycle re-entry. Finally, selective activation of JNK by DMEK- K1: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 G 1 -S transition. Oncogene (2004) 23, 3284–3295. doi:10.1038/sj.onc.1207467 Published online 23 February 2004 Keywords: ERK; JNK; MEKK; p21 CIP1 ; p38; Raf Introduction The progress of cells through G 1 and into S phase is regulated by the activity of the retinoblastoma tumour suppressor protein (pRb) (Hiebert et al., 1992; Wein- berg, 1995; Hall and Peters, 1996; Kaelin, 1999). Mitogenic stimulation of quiescent cells promotes the expression of D-type cyclins, which assemble with their catalytic partners, CDK4 and CDK6 (Hall and Peters, 1996; Ekholm and Reed, 2000). These cyclin D/CDK complexes initiate the phosphorylation of pRb, which relieves the transcriptional repression of the cyclin E gene (Zhang et al., 2000). Subsequent expression of cyclin E and assembly of cyclin E/CDK2 complexes promotes further phosphorylation of pRb, causing the release of E2F and expression of E2F-regulated genes that are required for the initiation of S phase, including cyclin A (Knudsen and Wang, 1997; Dyson, 1998; Helin, 1998; Knudsen et al., 1998). CDKs are also regulated by phosphorylation (Gu et al., 1992; Clarke, 1995). Activation of CDK4 and CDK2 requires the phosphorylation of a conserved threonine residue within the catalytic loop and depho- sphorylation of Thr14 and Tyr15 in the ATP-binding pocket. Dephosphorylation of CDK2 is catalysed by Cdc25A, a dual specificity phosphatase, that is expressed during the G 1 and S phases (Jinno et al., 1994; Blomberg and Hoffmann, 1999). CDK activity is inhibited by the actions of small molecular weight CDK inhibitors (CDKIs) (Sherr and Roberts, 1999). The INK4 family CDKIs (e.g. p16 INK4a ) specifically inhibit CDK4 and CDK6, whereas the CIP/ KIP family members (e.g. p27 KIP1 and p21 CIP1 ) bind and inhibit cyclin/CDK2 complexes. In quiescent cells, high p27 KIP1 levels restrain cell cycle re-entry by inhibiting cyclin E/CDK2 complexes, whereas mitogenic signals promote ubiquitin-mediated degradation or proteolytic processing of p27 KIP1 (Pagano et al., 1995; Shirane et al., 1999). p21 CIP1 can exert opposing effects on the G 1 -S transition (Sherr and Roberts, 1999). For example, mitogens stimulate a modest induction of p21 CIP1 , which may facilitate the assembly of cyclin D/CDK4 com- plexes and promote the G 1 phase progression (LaBaer et al., 1997; Cheng et al., 1999). However, in response to DNA damage, p53 mediates a strong induction of Received 10 June 2003; revised 24 November 2003; accepted 6 January 2004; Published online 23 February 2004 *Correspondence: SJ Cook; E-mail: simon.cook@bbsrc.ac.uk 3 Current address: BioFocus Discovery, Cambridge Science Park, Cambridge, UK 4 Current address: UCSF Cancer Centre, Box 0128, UCSF, San Francisco, CA 94143, USA 5 Current address: Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester MA01605, USA 6 Current address: Cancer and Infection Bioscience, AstraZeneca, Alderley Park, Macclesfield, SK10 4TG, UK Oncogene (2004) 23, 3284–3295 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc