Specialized Roles of the Two Mitotic Cyclins in Somatic Cells: Cyclin A as an Activator of M Phase–promoting Factor

Abstract

The role of cyclin B-CDC2 as M phase-promoting factor (MPF) is well established, but the precise functions of cyclin A remain a crucial outstanding issue. Here we show that down-regulation of cyclin A induces a G2 phase arrest through a checkpoint-independent inactivation of cyclin B-CDC2 by inhibitory phosphorylation. The phenotype is rescued by expressing cyclin A resistant to the RNA interference. In contrast, down-regulation of cyclin B disrupts mitosis without inactivating cyclin A-CDK, indicating that cyclin A-CDK acts upstream of cyclin B-CDC2. Even when ectopically expressed, cyclin A cannot replace cyclin B in driving mitosis, indicating the specific role of cyclin B as a component of MPF. Deregulation of WEE1, but not the PLK1-CDC25 axis, can override the arrest caused by cyclin A knockdown, suggesting that cyclin A-CDK may tip the balance of the cyclin B-CDC2 bistable system by initiating the inactivation of WEE1. These observations show that cyclin A cannot form MPF independent of cyclin B and underscore a critical role of cyclin A as a trigger for MPF activation. INTRODUCTIONClassic cell cycle studies in the 1980s culminated in the purification of M phase-promoting factor (MPF) from Xenopus eggs and starfish oocytes and the identification of its components as cyclin B and CDC2 (also called cyclin-dependent kinase [CDK]-1; reviewed in Doree and Hunt, 2002). Cyclin B accumulates from S phase and forms a complex with CDC2. The complex is kept inactive by phosphorylation of CDC2 Thr14/Tyr15 by MYT1 and WEE1 until it is abruptly activated by CDC25 during mitosis. As the cell exits mitosis cyclin B is destroyed via an ubiquitin-mediated mechanism that is catalyzed by the APC/C (reviewed in .Cyclin B-CDC2 catalyzes its own activation by an intricate network of feedback loops. WEE1 is phosphorylated by CDKs, facilitating its degradation by the ubiquitin ligases SCF ␤-TrCP and SCF Tome-1 (Ayad et al., 2003;Watanabe et al., 2004Watanabe et al., , 2005Harvey et al., 2005). The CDC25C phosphatase is activated by multiple CDK phosphorylation (reviewed in Hutchins and Clarke, 2004) and inactivated by phosphorylation on Ser216 (enforced by several kinases, including C-TAK1, CHK1, and CHK2). This phosphorylation creates a 14-3-3 binding site, which masks a proximal nuclear localization sequence and excludes CDC25C from the nucleus. Furthermore, phosphorylation of CDC25C Ser214 inhibits the phosphorylation of CDC25C Ser216 (Bulavin et al., 2003). Thus MPF is essentially a bistable system (Ferrell, 2002) that becomes autocatalytic once a critical portion is activated.What triggers the initial activation of MPF has been the subject of immense speculation. PLK1 is a candidate as it can phosphorylate CDC25C at the nuclear-exporting sequence (NES) and promote nuclear accumulation of CDC25C (Toyoshima-Morimoto et al., 2002). PLK1 likewise phosphorylates the NES of cyclin B and increases its nuclear accumulation (Toyoshima-Morimoto et al., 2001;Yuan et al., 2002), coordinating the localization of both cyclin B and CDC25C t...

Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events

“…In agreement with these reports, the present study demonstrates that inhibition of G 2 phase cyclin A/cdk2 delays the cdc25-dependent activation (Mitra and Enders, 2004;Fung et al, 2007), and nuclear translocation (Gong et al, 2007) of cyclin B/cdk1. The accumulated evidence now presents a convincing case that the regulation of the G 2 phase pool of cyclin A/cdk2 is critical for cyclin B/cdk1 activation and G 2 /M progression.…”

“…A number of laboratories have provided evidence that G 2 phase cyclin A/cdk2 has a critical role in the timing of entry into mitosis (Furuno et al, 1999;Goldstone et al, 2001;Hu et al, 2001;Mitra and Enders, 2004;Fung et al, 2007;Gong et al, 2007), which is confirmed by our siRNA and cdk2-specific inhibitor studies. In agreement with these reports, the present study demonstrates that inhibition of G 2 phase cyclin A/cdk2 delays the cdc25-dependent activation (Mitra and Enders, 2004;Fung et al, 2007), and nuclear translocation (Gong et al, 2007) of cyclin B/cdk1.…”

“…The previous studies examining the mechanism by which depletion of G 2 phase cyclin A/cdk2 regulates progression into mitosis have provided evidence for deregulation of Wee1 activity (Fung et al, 2007) and cytoplasmic retention of cyclin B1 (Gong et al, 2007). The mechanism appears independent of ataxia telangiectasia mutated/ATM and Rad3 related (ATM/ATR) checkpoint signalling (Hu et al, 2001;Fung et al, 2007).…”

Generation of an indestructible cyclin B1 by caspase-6-dependent cleavage during mitotic catastrophe

“…FLAG-cyclin B1 in pUHD-P1 was amplified by PCR with vector forward primer and 5 0 -GATCAGCTCCATTTTCTGCATC-3 0 ; the PCR product was cut with NcoI, and ligated into pUHD-P1 cut with NcoI and BamHI (blunt with Klenow fragment) to produce FLAGcyclin B1(CD160) in pUHD-P1. shRNA against cyclin B1 (Fung et al, 2007) and CHK1 (Ho et al, 2005) were constructed as described. Site-directed mutagenesis was carried out with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) with the following oligonucleotides and their antisense: 5 0 -AAGATGGAGCCGACCC GAACCT-3 0 (for shRNA-resistant cyclin B1), 5 0 -ATT TTGGTTAATACTGCCTCT-3 0 (for D123N), and 5 0 -AAA ATGGAGCCGACCCGAACCT-3 0 (for D160N).…”

“…IR was delivered with a caesium 137 source from an MDS Nordion Gammacell 1000 Elite Irradiator (Ottawa, ON, Canada). To enrich the transfected cells, cells were subjected to a transient selection with blasticidin as outlined earlier (Fung et al, 2007). Double thymidine synchronization (Arooz et al, 2000), determination of mitotic index (Chow et al, 2003a), transfection (Chan et al, 2008b) and preparation of cellfree extracts were performed as described.…”