Regulatory domain of human protein kinase Cα dominantly inhibits protein kinase Cβ-I regulated growth and morphology in Saccharomyces cerevisiae

✍ Scribed by Amadeo M. Parissenti; Sandra A. Kim; Concettina M. Colantonio; Andrea L. Snihura; Bernard P. Schimmer

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 538 KB
• Volume: 166
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/(sici)1097-4652(199603)166:3<609::aid-jcp16>3.0.co;2-1

✦ Synopsis

This study demonstrates that the isolated regulatory (R) domain (amino acids 1 -270) of human protein kinase C a (PKCa) is a potent inhibitor of PKCP-I activity in a yeast expression system. The PKCa R domain fused to glutathione-S-transferase competitively inhibited the activity of yeast-expressed rat PKCP-I in vitro (Ki = 0.2 pM) and was 400-fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19-36 from PKCa. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMPdependent protein kinase. The PKCa R domain (without glutathione-S-transferase [GST]) also was tested for its ability to inhibit PKCP-I activity in vivo, in a yeast strain expressing rat PKCP-I. Upon treatment with a PKC-activating phorbol ester, yeast cells expressing rat PKCP-I were growth-inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKCP-I blocked the phorbol ester-induced inhibition of yeast cell growth and the phorbol esterdependent alterations in yeast cell morphology. These results indicate that the R domain of PKCa 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.