HUMAN AND YEAST CDK-ACTIVATING KINASES (CAKS) DISPLAY DISTINCT SUBSTRATE SPECIFICITIES

Cell cycle progression is controlled by the sequential functions of cy clin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carri ed out by the cdk-activating kinase (CAK). Human CAK has been identifi ed as a p40(MO15)/cyclin H/MAT1 complex that also functions as part of transcription factor III-I (TFIIH) where it phosphorylates multiple t ranscriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yea st consists of a single polypeptide (Cak1p), is not a component of TFI IH, and lacks CTD kinase activity. Here we report that Cak1p and p40(M O15) have strikingly different substrate specificities. Cak1p preferen tially phosphorylated monomeric cdks, whereas p40(MO15) preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40(MO15) only phos phorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk i nhibitors, including p21(CIPI) p27(KIP1), P57(KIP2), p16(INK4a), and p 18(INK4c), could block phosphorylation by p40(MO15) but not phosphoryl ation by Cak1p. Our results demonstrate that although both Cak1p and p 40(MO15) activate cdks by phosphorylating the same residue, the struct ural mechanisms underlying the enzyme-substrate recognition differ gre atly. Structural and physiological implications of these findings will be discussed.