Protein phohatase 1 and protein phospha-

Both cyclins A and B associate with and thereby activate cyclin‐dependent protein kinases (cdks). We have investigated which component in the cyclin‐cdk complex determines its substrate specificity. The A‐ and B‐type cyclin‐cdk complexes phosphorylated histone H1 and their cyclin subunits in an indistinguishable manner, irrespective of the catalytic subunit, p33cdk2 or p34cdc2. In contrast, only the cyclin A‐cdk complexes phosphorylated the Rb‐related p107 protein in vitro. Likewise, binding studies revealed that cyclin A‐cdk complexes bound stably to p107 in vitro, whereas cyclin B‐cdk complexes did not detectably associate with p107, under identical assay conditions. Binding to p107 required both cyclin A and a cdk as neither subunit alone bound to p107. These results demonstrate that although the kinase subunit provides a necessary component for binding, it is the cyclin subunit that plays the critical role in targeting the complex to p107. Finally, we show that the cyclin A‐p33cdk2 complex phosphorylated p107 in vitro at most of its sites that are also phosphorylated in human cells, suggesting that the cyclin A‐p33cdk2 complex is a major kinase for p107 in vivo.

Critical cell cycle transitions are controlled by the coordinate actions of the p34wc2 protein kinase and its regulatory subunits, cyclins. Recently we identified another human p34 homolog, cyclin-dependent kinase 2 (CDK2) by complementation of a cdc284 mutation in Saccharomyces cerevisiae using a AYES human cDNA expression library. CDK2 is 66% identical to CDC2Hs and 89% identical to the Xenopus Egi gene, forming a distinct subfamily of CDC2-related protein kinases. We have found that CDK2 encodes a 33-kDa cyclin A-associated protein kinase that contains phosphotyrosine, two characteristics it shares with CDC2Hs. However, we show that the subunit composition of these two protein kinase complexes can vary in different cell types, that they have different in vitro substrate preferences, and that CDK2 mRNA is observed much earlier than CDC2Hs mRNA when lymphocytes are stimulated to enter the cell cycle. We suggest that cells in different developmental or transformed states may have different mechanisms of cell cycle regulation.Critical transitions in the eukaryotic cell cycle are controlled by the coordinate action of a protein kinase(s), p34cdc2, and the associated regulatory subunits, cyclins (1-3). Originally, cyclins were defined as proteins that varied in abundance with periodicity of the cell cycle (4-7); subsequently cyclins were shown to be a family of structurally related proteins that regulate the timing of activation of p34cdc2 (8)(9)(10)(11)(12) A variety of mammalian cyclins have also been identified (5, 22-28), but except for the B-type cyclins their functions are largely unknown. The best-characterized cyclin complex is the mitotic cyclin B/p34cdc2 kinase, the active component of maturating promoting factor (5, 9-10). Cyclin A accumulates before cyclin B in the cell cycle (23) and may be involved in control of S phase (3, 23). In addition, cyclin A has been implicated in cellular transformation (27) (18) for expression in S. cerevesiae. These epitope-tagged kinases are referred to as HA-Cdk2 and HA-Cdc2Hs. Corresponding plasmids for Cdk2 and Cdc2Hs were from a previous study (17).Abbreviations: PBL, peripheral blood lymphocyte; CDK2, cyclindependent kinase 2; HA, hemagglutinin A; a-Cdk2cr, C-terminalspecific Cdk2 antibodies; a-cyclin ANT, N-terminal-specific cyclin A antibodies; a-cyclin ACHLA-1, C-terminal-specific cyclin A antibodies; RF-A, replication factor A; PHA, phytohemagglutinin-P.§To whom reprint requests should be addressed. 2907The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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