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 functional characterisation of proteins and their domains is becoming acute. Conserved regions within proteins often share overlapping functions but despite this conservation may fulfil quite different tasks in different species. In this work, we investigated the cysteine-rich motif (C1 domain) of yeast protein kinase C (Pkc1p) as a model to establish a test system for domain function. C1 domains activate kinases through binding of either diacylglycerol and/or phosphatidylserine, as in many members of the protein kinase C (PKC) family, or by binding small GTPases, as in Raf kinase. In contrast to other members of the protein kinase C superfamily, Pkc1p of Saccharomyces cerevisiae is activated via binding of the small G-protein Rho1p to its C1 domain. 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 domain from Raf kinase enabled the chimeric enzyme to bind Rho1p when substituted for the native yeast domain. Accordingly, a chimeric Pkc1p carrying the C1 from Raf kinase, but not that from PKC eta, was able to partially complement the phenotypes of a yeast pkc1 deletion mutant. We interpret these data as further evidence that interaction with a small GTPase is the main regulatory function of the C1 domain in yeast.
Schmitz
H P
HP
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1 Geb.: 26.12, Düsseldorf, D-40225, Germany.
Jöckel
J
J
Block
C
C
Heinisch
J J
JJ
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010766:Phosphorylation; D011493:Protein Kinase C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C097993:Pkc1 protein, Trichoderma reesei; C081975:protein kinase C eta; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins; D003545:Cysteine