title: Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cdelta.
abstract: 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-1,2-diacylglycerol (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 PKCdelta 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 PKCdelta 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 PKCdelta mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKCdelta. The studies also indicated that an anionic residue, Glu(177), in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidylserine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKCdelta and mutants showed that because of its phosphatidylserine specificity PKCdelta 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 PKCdelta and the origin of its specific subcellular localization in response to DAG.
authors.affiliation: Department of Chemistry, University of Illinois, Chicago, Illinois 60607, USA.
authors.forename: Robert V
authors.initials: RV
authors.lastname: Stahelin
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Digman
authors.forename: Martina
authors.initials: M
authors.lastname: Medkova
authors.forename: Bharath
authors.initials: B
authors.lastname: Ananthanarayanan
authors.forename: John D
authors.initials: JD
authors.lastname: Rafter
authors.forename: Heather R
authors.initials: HR
authors.lastname: Melowic
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
mesh_terms: D000818:Animals; D002151:Calorimetry; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D004075:Diglycerides; D007527:Isoenzymes; D008563:Membrane Lipids; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; C497260:Prkcd protein, rat; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta