Identification of substrates for cyclin dependent kinases

Abstract

Protein phosphorylation is a key regulatory mechanism for cell cycle control in eukaryotes. From yeast to humans, cell cycle progression and cell division require the activation of a group of serine– threonine protein kinases called cyclin dependent kinases (CDKs) (Morgan, 1997), which initiate and coordinate these processes by orderly phosphorylation of their targets. CDKs require association with a cyclin subunit to activate them,whichprovides substrate specificity (Russo,1997; Solomonet al.,1992), and phosphorylation by an activating protein kinase (CAK) at a conserved threonine residue (Krek and Nigg,1992; Lorca et al., 1992; Solomon et al., 1992). The concentration of cyclins oscillate during the cell cycle and their abundance is regulated at several levels: transcriptional, translational and at the level of protein stability. In particular, at the end of mitosis, ubiquitin-mediated proteolysis results in the disappearance of mitotic cyclins, which results in inactivation of CDKs (Zachariae and Nasmyth, 1999). The key cell cycle transitions involving the activity of CDKs occur at the initiation of S-phase and entry into mitosis. Loss of CDK activity at the end of mitosis permits the return to interphase, and is presumably accompanied by dephosphorylation of the majority of target proteins that were modified when the cells entered mitosis. There is rather specific control of the different phases of the cell cycle, so that cells do not enter mitosis with unreplicated or damaged DNA, and do not exit mitosis until all the chromosomes are correctly aligned on the metaphase plate. There is a theoretical conundrum, however, in explaining how it is that activation of S-phase CDKs, such as cyclin E/CDK2 and cyclin A/ CDK2 in vertebrate cells promotes entry into S-phase and not into mitosis, or why activation of the Mphase CDKs does not promote a further round of DNA replication. One hypothesis to explain why cells enter S-phase, rather than mitosis, when the S-phase CDK (CDK2-cyclin E in higher eukaryotes) is activated would be a restricted substrate specificity for this particular CDK, yet there is very little information on this point. Another possibility is that CDK2-cyclin E resides in the nucleus, and entry