ACTIVATION OF CYCLIN-DEPENDENT KINASE-4 (CDK4) BY MOUSE MO15-ASSOCIATED KINASE

The assembly of functional holoenzymes composed of regulatory D-type c yclins and cyclin-dependent kinases (cdks) is rate limiting for progre ssion through the G(1) phase of the mammalian somatic cell cycle. Comp lexes between D-type cyclins and their major catalytic subunit, cdk4, are catalytically inactive until cyclin-bound cdk4 undergoes phosphory lation on a single threonyl residue (Thr-172). This step is catalyzed by a cdk-activating kinase (CAK) functionally analogous to the enzyme which phosphorylates cdc2 and cdk2 at Thr-161/160. Here, we demonstrat e that the catalytic subunit of mouse cdc2/cdk2 CAK (a 39-kDa protein designated p39(MO15)) assemble with a regulatory protein present in ei ther insect or mammalian cells to generate a CAK activity capable of p hosphorylating and enzymatically activating both cdk2 and cdk4 in comp lexes with their respective cyclin partners. A newly identified 37-kDa cyclin-like protein (cyclin H [R. P. Fisher and D. O. Morgan, Cell 78 :713-724, 1994]) can assemble with p39(MO15) to activate both cyclin A -cdk2 and cyclin D-cdk4 in vitro, implying that CAK is structurally re miniscent of cyclin-cdk complexes themselves. Antisera produced to the p39(MO15) subunit can completely deplete mammalian cell lysates of CA K activity for both cyclin A-cdk2 and cyclin D-cdk4, with recovery of activity in the resulting immune complexes. By using an immune complex CAK assay, CAK activity for cyclin A-cdk2 and cyclin D-cdk4 was detec ted both in quiescent cells and invariantly throughout the cell cycle. Therefore, although it is essential for the enzymatic activation of c yclin-cdk complexes, CAK appears to be neither rate limiting for the e mergence of cells from quiescence nor subject to upstream regulatory c ontrol by stimulatory mitogens.