Activation of conventional mammalian protein kinase C isoforms expressed in budding yeast modulates the cell doubling time--a potential in vivo screen for protein kinase C activators.
Conventional mammalian protein kinase C (PKC) isoforms alpha, beta 1, and gamma were expressed in Saccharomyces cerevisiae and resulted in a differential increase in the yeast doubling time in response to distinct classes of PKC activators. Mutants were created in the regulatory domain of PKC alpha to map the interaction with the different activators. The macrocyclic lactone bryostatin 5 preferentially regulated PKC alpha activity through the second cysteine-rich sequence (CYS2) of Cl, while regulation by the diterpene ester mezerein displayed strong preference for the first cysteine-rich sequence (CYS1) of Cl. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA) regulated PKC enzymatic activity equally potently via CYS1 or CYS2 albeit at reduced levels compared with native PKC alpha. For the diterpene ester ingenol-3, 20-dibenzoate and the indol alkaloids (-)-7-octyl-indolactam V and (-)-indolactam V, no responses were observed for mutants lacking either CYS1 or CYS2 whereas native PKC alpha activity was regulated. These in vivo results were complemented by in vitro binding and catalytic assays which showed correlation between PKC enzymatic activity and the cell growth characteristics. The observed phenotype can be exploited to screen natural compounds in vivo for their PKC regulatory potential and to map the underlying interactions.
Shieh
H L
HL
Section on Molecular Biology, Joslin Diabetes Center, Boston, MA, USA.
Hansen
H
H
Zhu
J
J
Riedel
H
H
D000818:Animals; D054713:Bryostatins; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D004224:Diterpenes; D004789:Enzyme Activation / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007769:Lactams / Q000494:pharmacology; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D010703:Phorbol Esters / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D017384:Sequence Deletion; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D054713:Bryostatins; D004224:Diterpenes; D007211:Indoles; D007527:Isoenzymes; D007769:Lactams; D007783:Lactones; D018942:Macrolides; D010703:Phorbol Esters; D013729:Terpenes; C011309:mezerein; C043969:indolactam V; C078062:bryostatin 5; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate