Yeast phenotype classifies mammalian protein kinase C cDNA mutants.
The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable to identity rare, randomly altered but active mammalian PKC mutants. It quantifies their catalytic and biological activities in response to PKC activators or inhibitors for a systematic mapping of PKC structure and function or PKC-drug interaction.
Riedel
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Joslin Diabetes Center, Brigham and Women's Hospital, Boston, Massachusetts.
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Hansen
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D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002455:Cell Division; D004247:DNA / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010641:Phenotype; D010703:Phorbol Esters / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D010703:Phorbol Esters; D004247:DNA; D011493:Protein Kinase C; D002118:Calcium