Mcl-1 (myeloid cell leukaemia-1) is a Bcl-2 family member with short-term pro-survival functions but whose other functions, demonstrated by embryonic lethality of knockout mice, do not involve apoptosis. In the present study, we show a cell-cycle-regulatory role of Mcl-1 involving a shortened form of the Mcl-1 polypeptide, primarily localized to the nucleus, which we call snMcl-1. snMcl-1 interacts with the cell-cycle-regulatory protein Cdk1 (cyclin-dependent kinase 1; also known as cdc2) in the nucleus, and Cdk1 bound to snMcl-1 was found to have a lower kinase activity. The interaction with Cdk1 occurs in the absence of its cyclin partners and is enhanced on treatment of cells with G2/M blocking agents, but not by G1/S blocking. The snMcl-1 polypeptide is present during S and G2 phases and is negligible in G1. Overexpression of human Mcl-1 in a murine myeloid progenitor cell line resulted in a lower rate of proliferation. Furthermore, Mcl-1-overexpressing cells had lower total Cdk1 kinase activity compared with parental cells, in both anti-Cdk1 and anti-cyclin B1 immunoprecipitates. The latter results suggest that binding to snMcl-1 alters the ability of Cdk1 to bind its conventional partner, cyclin B1. Given the important role of Cdk1 in progression through G2 and M phases, it is probable that the inhibition of Cdk1 activity accounts for the inhibitory effect of Mcl-1 on cell growth.

INTRODUCTIONMyeloid cell leukemia 1 (Mcl-1) was first identified as a gene induced during myeloid cell differentiation (Kozopas et al., 1993) and is a prosurvival member of the Bcl-2 family. The activity of Bcl-2 family proteins involves several Bcl-2 homology (BH) domains (Danial and Korsmeyer, 2004) that control protein-protein interactions. These proteins play an important role in cancer by regulating cell death and survival. Compared with other prosurvival members such as Bcl-2 or Bcl-xL, Mcl-1 has been shown to have more transient survival effects (Zhou et al., 1997). Unlike other members of the family, the half-life of Mcl-1 is very short and its expression is highly regulated by growth factors and a variety of other agents. Moreover, Mcl-1 has an extended N-terminus that is not conserved in any other Bcl-2 family protein. The N-terminus contains two PEST domains, which are found in proteins that are rapidly turned over, but deletion of the PEST domains does not extend the short half-life of Mcl-1 (Akgul et al., 2000;Clohessy et al., 2004). When the Mcl-1 gene was knocked out in a mouse model, it resulted in peri-implantation lethality (Rinkenberger et al., 2000), but Mcl-1Ϫ/Ϫ embryos showed no alterations in the extent of apoptosis, indicating that Mcl-1 plays a role early in development distinct from its prosurvival functions. We and others reported that Mcl-1 expression can lead to suppression of cell proliferation and may have effects on cell cycle machinery (Fujise et al., 2000;Jamil et al., 2005). We showed that a short nuclear form of Mcl-1 (snMcl-1) was associated primarily with the inactive form of Cdk-1 in the nucleus, most prominently during the G2 phase of the cell cycle, which suggested a potential means by which Mcl-1 mediates its anti-proliferative effects (Jamil et al., 2005).It is now well established that genome surveillance mechanisms function to cause a delay, or arrest, in progression of the cell cycle at the boundary of G1/S or G2/M in response to DNA damage (Zhou and Elledge, 2000;Abraham, 2001;Kastan, 2001;Nyberg et al., 2002;Sancar et al., 2004). These surveillance mechanisms are known to involve a network of interacting proteins that recognize damage and elicit responses including cell cycle delay, DNA repair, and apoptosis (Elledge, 1996;Zhou and Elledge, 2000). Cdk's, which play a central role in cell cycle progression, are key targets of these checkpoints (Iliakis et al., 2003 MATERIALS AND METHODS Cell LinesHeLa, HL-60, and FDC-P1 cells were obtained from ATCC (Manassas, VA). ATM-deficient HT-144 cells were a kind gift from Dr. P. Olive (BC Cancer Agency). ATR-defective fibroblasts, F02-98, were a kind gift from Dr. P. Jeggo. Hs68, which are primary human foreskin fibroblasts (Wheaton and Riabowol, 2004), maintained within 30 -60 mean population doublings, were kindly provided by Dr. K. Riabowol (University of Calgary). HL-60 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM l-glutamine. FDC-P1 cells were grown in RPMI...