Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cδ

Robert V. Stahelin, Michelle A. Digman, Martina Medkova, Bharath Ananthanarayanan, John D. Rafter, Heather R. Melowic, Wonhwa Cho

Research output: Contribution to journalArticlepeer-review

114 Scopus citations


The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins. Although recent reports have indicated that C1A and C1B domains of conventional PKCs play different roles in their DAG-mediated membrane binding and activation, the individual roles of C1A and C1B domains in the DAG-mediated activation of novel PKCs have not been fully understood. In this study, we determined the roles of C1A and C1B domains of PKCδ by means of in vitro lipid binding analyses and cellular protein translocation measurements. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCδ have opposite affinities for DAG and phorbol ester, i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCδ mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKCδ. The studies also indicated that an anionic residue, Glu177, in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidyl-serine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKCδ and mutants showed that because of its phosphatidylserine specificity PKCδ preferentially translocated to the plasma membrane under the conditions in which DAG is randomly distributed among intracellular membranes of HEK293 cells. Collectively, these results provide new insight into the differential roles of C1 domains in the DAG-induced membrane activation of PKCδ and the origin of its specific subcellular localization in response to DAG.

Original languageEnglish (US)
Pages (from-to)29501-29512
Number of pages12
JournalJournal of Biological Chemistry
Issue number28
StatePublished - Jul 9 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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