The Pleckstrin Homology Domain of Protein Kinase D Interacts Preferentially with the η Isoform of Protein Kinase C

Richard T Waldron,Teresa Iglesias,Enrique Rozengurt

doi:10.1074/jbc.274.14.9224

Abstract

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.

Highlights

Protein kinase C (PKC),1 a major cellular target for the potent tumor-promoting phorbol esters [1, 2], has been implicated in the mediation of diverse cellular functions, including short-term regulation of ion fluxes, receptor ligand binding, and signal transduction, and a wide range of longer term ef
Our results demonstrate that the PH domain is critical for stable PKD1⁄7 PKC␩ complex formation, indicating that these domains can mediate highly selective protein-protein interactions
This band did not appear when protein kinase D (PKD) was coexpressed with the active PKC ⑀ or ␨ mutant forms despite their level of overexpression shown by Western blot analysis (Fig. 2), and despite the fact that the PKC⑀ mutant induced PKD activation to essentially the same degree as did the PKC␩ mutant (Fig. 1A)

Summary

Introduction

Protein kinase C (PKC),1 a major cellular target for the potent tumor-promoting phorbol esters [1, 2], has been implicated in the mediation of diverse cellular functions, including short-term regulation of ion fluxes, receptor ligand binding, and signal transduction, and a wide range of longer term ef-. The results presented here demonstrate that protein kinase D (PKD) and PKC␩ transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKC␩.

Results

Conclusion