The pleckstrin homology domain of protein kinase D interacts preferentially with the eta isoform of protein kinase C.
The results presented here demonstrate that protein kinase D (PKD) and PKCeta transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCeta. The presence of PKCeta 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 PKCeta 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 PKCepsilon, and did not bind PKCzeta. When PKCeta was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCeta 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 PKCeta. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCeta. These results indicate that the PKD 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.
Waldron
R T
RT
Department of Medicine, School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California 90095-1786, USA.
Iglesias
T
T
Rozengurt
E
E
D000818:Animals; D001798:Blood Proteins / Q000737:chemistry; D019556:COS Cells; D007527:Isoenzymes / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D017386:Sequence Homology, Amino Acid; D000099221:Protein Kinase C zeta
D001798:Blood Proteins; D007527:Isoenzymes; D010750:Phosphoproteins; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D000099221:Protein Kinase C zeta; C039077:platelet protein P47; C081975:protein kinase C eta; C089212:protein kinase D