Analysis of CAK activities from human cells

The cdk-activating kinase (CAK) activates cyclin-dependent kinases (cdks) t hat control cell-cycle progression by phosphorylating a threonine residue c onserved in cdks. CAK from humans contains p40(MO15) (cdk7), cyclin H and M AT1, which are also subunits of transcription factor IIH where they phospho rylate the C-terminal domain of the large subunit of RNA polymerase II. In contrast, budding yeast Cak1p is a monomeric enzyme without C-terminal doma in kinase activity. Here, we analyze CAK activities in HeLa cells using cdk 2-affinity chromatography. In addition to MO15, a second CAK activity was d etected that runs on gel filtration at 30-40 kDa. This activity phosphoryla ted and activated cdk2 and cdk6. Furthermore, this 'small CAK' activity res embled Cak1p rather than MO15 in terms of substrate specificity, reactivity to antibodies against MO15 and Cak1p, and sensitivity to 5'-fluorosulfonyl benzoyladenosine, an irreversible inhibitory ATP analog. Our findings sugge st the presence of at least two different CAK activities in human cells.