Regulatory domain of human protein kinase C alpha dominantly inhibits protein kinase C beta-I-regulated growth and morphology in Saccharomyces cerevisiae.

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.