The CDK-activating kinase (Cak1p) from budding yeast has an unusual ATP-binding pocket

Cak1p is an essential protein kinase that phosphorylates and thereby activa tes the major cyclin-dependent kinase in budding yeast, Cdc28p, The sequenc e of Cak1p differs from other members of the protein kinase superfamily in several conserved regions. Cak1p lacks the highly conserved glycine loop mo tif (GXGXXG) that is found in the nucleotide binding fold of virtually all protein kinases and also lacks a number of conserved amino acids found at s ites throughout the protein kinase core sequence. We have used kinetic and mutagenic analyses to investigate whether these sequence differences affect the nucleotide-binding properties of Cak1p, Although Cak1p differs dramati cally from other protein kinases, it binds ATP with a reasonable affinity, with a K-M of 4.8 mu M. Mutations of the putative invariant lysine in Cak1p (Lys-31), homologous to a residue required for activity in virtually all p rotein kinases and that interacts with the ATP phosphates, moderately reduc ed the ability of Cak1p to bind ATP but did not dramatically affect the cat alytic rate of the kinase. Similarly, Cak1p is insensitive to the ATP analo g 5'-fluorosulfonylbenzoyladenosine, which inhibits most protein kinases th rough covalent modification of the invariant lysine, We found that Cak1p is tolerant of mutations within its glycine loop region. Remarkably, Cak1p re mains functional even following truncation of its first 31 amino acids, inc luding the glycine loop region and the invariant lysine. We conclude that t he Cak1p nucleotide-binding pocket differs significantly from those of most other protein kinases and therefore might provide a specific target for an inhibitory drug.