CELL-CYCLE ANALYSIS OF THE ACTIVITY, SUBCELLULAR-LOCALIZATION, AND SUBUNIT COMPOSITION OF HUMAN CAK (CDK-ACTIVATING KINASE)

The activity of cyclin-dependent kinases (cdks) depends on the phospho rylation of a residue corresponding to threonine 161 in human p(34cdc2 ). One enzyme responsible for phosphorylating this critical residue ha s recently been purified from Xenopus and starfish. It was termed CAK (for cdk-activating kinase), and it was shown to contain p40(MO15) as its catalytic subunit. In view of the cardinal role of cdks in cell cy cle control, it is important to learn if and how CAK activity is regul ated during the somatic cell cycle. Here, we report a molecular charac terization of a human p40(MO15) homologue and its associated CAK activ ity. We have cloned and sequenced a cDNA coding for human p40(MO15), a nd raised specific polyclonal and monoclonal antibodies against the co rresponding protein expressed in Escherichia coli. These tools were th en used to demonstrate that p40(MO15) protein expression and CAK activ ity are constant throughout the somatic cell cycle. Gel filtration sug gests that active CAK is a multiprotein complex, and immunoprecipitati on experiments identify two polypeptides of 34 and 32 kD as likely com plex partners of p40(MO15). Th, association of the three proteins is n ear stoichiometric and invariant throughout the cell cycle. Immunocyto chemistry and biochemical enucleation experiments both demonstrate tha t p40(MO15) is nuclear at all stages of the cell cycle (except for mit osis, when the protein redistributes throughout the cell), although th e p34(cdc2)/cyclin B complex, one of the major purported substrates of CAK, occurs in the cytoplasm until shortly before mitosis. The absenc e of obvious changes in CAK activity in exponentially growing cells co nstitutes a surprise. It suggests that the phosphorylation state of th reonine 161 in p34(cdc2) (and the corresponding residue in other cdks) may be regulated primarily by the availability of the cdk/cyclin subs trates, and by phosphatase(s).