title: Role of the Ca2+/phosphatidylserine binding region of the C2 domain in the translocation of protein kinase Calpha to the plasma membrane.
abstract: Signal transduction via protein kinase C (PKC) is closely regulated by its subcellular localization. To map the molecular determinants mediating the C2 domain-dependent translocation of PKCalpha to the plasma membrane, full-length native protein and several point mutants in the Ca(2+)/phosphatidylserine-binding site were tagged with green fluorescent protein and transiently expressed in rat basophilic leukemia cells (RBL-2H3). Substitution of several aspartate residues by asparagine completely abolished Ca(2+)-dependent membrane targeting of PKCalpha. Strikingly, these mutations enabled the mutant proteins to translocate in a diacylglycerol-dependent manner, suggesting that neutralization of charges in the Ca(2+) binding region enables the C1 domain to bind diacylglycerol. In addition, it was demonstrated that the protein residues involved in direct interactions with acidic phospholipids play differential and pivotal roles in the membrane targeting of the enzyme. These findings provide new information on how the C2 domain-dependent membrane targeting of PKCalpha occurs in the presence of physiological stimuli.
authors.affiliation: Department of Physiology, University College London, Gower St., United Kingdom.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Bolsover
authors.forename: Juan C
authors.initials: JC
authors.lastname: Gomez-Fernandez
authors.forename: Senena
authors.initials: S
authors.lastname: Corbalan-Garcia
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D016296:Mutagenesis; D010718:Phosphatidylserines / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051571:Protein Kinase C-alpha; D021381:Protein Transport; D051381:Rats; D014407:Tumor Cells, Cultured
chemical_list: D010718:Phosphatidylserines; D010743:Phospholipids; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium