Domain shuffling as a tool for investigation of protein function: substitution of the cysteine-rich region of raf kinase and PKC eta for that of yeast Pkc1p

With the completion of the sequences of entire genomes, the need for functi onal characterisation of proteins and their domains is becoming acute. Cons erved regions within proteins often share overlapping functions but despite this conservation may fulfil quite different tasks in different species. I n this work, we investigated the cysteine-rich motif (C1 domain) of yeast p rotein kinase C (Pkc1p) as a model to establish a test system for domain fu nction. C1 domains activate kinases through binding of either diacylglycero l and/or phosphatidylserine, as in many members of the protein kinase C (PK C) family, or by binding small GTPases, as in Raf kinase. In contrast to ot her members of the protein kinase C superfamily, Pkc1p of Saccharomyces cer evisiae is activated vice binding of the small G-protein Rho1p to its C1 do main. We developed a system for domain shuffling to establish the function of C1 domains from human Raf kinase and rat PKC eta in yeast. Only the C1 d omain from Raf kinase enabled the chimeric enzyme to bind Rho1p when substi tuted for the native yeast domain. Accordingly, a chimeric Pkc1p carrying t he C1 from Raf kinase, but not that from PKC eta, was able to partially com plement the phenotypes of a yeast pkc1 deletion mutant. We interpret these data as further evidence that interaction with a small GTPase is the main r egulatory function of the C1 domain in yeast. (C) 2001 Academic Press.