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Regulatory domain of human protein kinase Cα dominantly inhibits protein kinase Cβ‐I regulated growth and morphology in Saccharomyces cerevisiae
Author(s) -
Parissenti Amadeo M.,
Kim Sandra A.,
Colantonio Concettina M.,
Snihura Andrea L.,
Schimmer Bernard P.
Publication year - 1996
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199603)166:3<609::aid-jcp16>3.0.co;2-1
Subject(s) - protein kinase c , yeast , biochemistry , biology , phorbol , protein kinase a , saccharomyces cerevisiae , signal transduction , cell growth , kinase , microbiology and biotechnology
This study demonstrates that the isolated regulatory (R) domain (amino acids 1–270) of human protein kinase Cα (PKCα) is a potent inhibitor of PKCβ‐I activity in a yeast expression system. The PKCα R domain fused to glutathione‐S‐transferase competitively inhibited the activity of yeast‐expressed rat PKCβ‐I in vitro (Ki = 0.2 μM) and was 400‐fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19–36 from PKCα. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMP‐dependent protein kinase. The PKCα R domain (without glutathione‐S‐transferase [GST]) also was tested for its ability to inhibit PKCβ‐I activity in vivo, in a yeast strain expressing rat PKCβ‐I. Upon treatment with a PKC‐activating phorbol ester, yeast cells expressing rat PKCβ‐I were growth‐inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKCβ‐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 PKCα 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. © 1996 Wiley‐Liss, Inc.