title: Regulatory domain of human protein kinase C alpha dominantly inhibits protein kinase C beta-I-regulated growth and morphology in Saccharomyces cerevisiae.
abstract: This study demonstrates that the isolated regulatory (R) domain (amino acids 1-270) of human protein kinase C alpha (PKC alpha) is a potent inhibitor of PKC beta-I activity in a yeast expression system. The PKC alpha R domain fused to glutathione-S-transferase competitively inhibited the activity of yeast-expressed rat PKC beta-I in vitro (Ki = 0.2 microns) and was 400-fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19-36 from PKC alpha. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMP-dependent protein kinase. The PKC alpha R domain (without glutathione-S-transferase [GST]) also was tested for its ability to inhibit PKC beta-I activity in vivo, in a yeast strain expressing rat PKC beta-I. Upon treatment with a PKC-activating phorbol ester, yeast cells expressing rat PKC beta-I were growth-inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKC beta-I blocked the phorbol ester-induced inhibition of yeast cell growth and the phorbol ester-dependent alterations in yeast cell morphology. These results indicate that the R domain of PKC alpha acts as a dominant inhibitor of PKC activity in vivo and thus provides a useful genetic tool to assess the roles of PKC in various signal transduction processes.
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
authors.forename: A M
authors.initials: AM
authors.lastname: Parissenti
authors.forename: S A
authors.initials: SA
authors.lastname: Kim
authors.forename: C M
authors.initials: CM
authors.lastname: Colantonio
authors.forename: A L
authors.initials: AL
authors.lastname: Snihura
authors.forename: B P
authors.initials: BP
authors.lastname: Schimmer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D012333:RNA, Messenger / Q000032:analysis; D011869:Radioligand Assay; D051381:Rats; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development*
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015240:Phorbol 12,13-Dibutyrate; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; C496628:PRKCA protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha