The pleckstrin homology domain of protein kinase D interacts preferentially with the η isoform of protein kinase C

Abstract

The results presented here demonstrate that protein kinase D (PKD) and PKCη transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCη. The presence of PKCη in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCη was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCε, and did not bind PKCζ. When PKCη was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCη immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino- terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCη. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCη. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs ε and η, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.