CELL-CYCLE REGULATION OF THE P34(CDC2) P33(CDK2)-ACTIVATING KINASE P40(MO15)/

A key component of Cdc2/Cdk2-activating kinase (CAK) is p40(MO15),prot ein kinase subunit that phosphorylates the T161/160 residues of p34(cd c2)/p33(cdk2). The level and activity of p40(MO15) were essentially co nstant during cleavage of fertilised Xenopus eggs and in growing mouse 3T3 cells, but serum starvation of these cells reduced both the level and activity of p40(MO15). Although the level and activity of endogen ous p40(MO15) did not vary in the cell cycle, we found that bacteriall y expressed p40(MO15) was activated more rapidly by M-phase cell extra cts than by interphase cell extracts. Bacterially expressed p40(MO15) was phosphorylated mainly on serine 170 (a p34(cdc2) phosphorylation s ite) by mitotic cell extracts, but mutation of S170 to alanine did not affect the activation of p40(MO15), whereas mutation of T176 (the equ ivalent site to T161/T160 in p34(cdc2)/p33(cdk2)) abolished the activa tion of p40(MO15). These studies suggest that the level and activity o f p40(MO15) is probably not a major determinant of p34(cdk2)/p33(cdk2) activity in the cell cycle, and that the activation of p40(MO15) may require phosphorylation on T176.