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

The results presented here demonstrate that protein kinase D (PKD) and PKC eta transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKC eta. The presence of PKC eta 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 PHD. The association between PKD and PKC eta 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 PKC epsilon, and did not bind PKC zeta. When PKC eta was coexpressed with PHD mutants containing either complete or partial deletions of the PH domain, both PKC eta immunoreactivity and PKC activity in PHD 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 PBD with PKC eta. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKC eta, These results indicate that the PHD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs epsilon and eta, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.