title: Annexin V inhibits protein kinase C activity via a mechanism of phospholipid sequestration.
abstract: In this study, we assessed the role of annexin V, a Ca2+-dependent phospholipid-binding protein, as a regulator of protein kinase C (PKC) and characterized its mechanism of inhibition. Several mutants obtained by oligonucleotide site-directed mutagenesis were tested in vitro on PKC activity in cytosolic fractions from Jurkat cells and on purified PKCalpha. Annexin V inhibited phosphorylation of annexin II by endogenous PKC and phosphorylation of myelin basic protein by PKCalpha. In both systems, the use of single Ca2+-binding-site mutants of annexin V led to a partial reversal of inhibition, and the Ca2+-binding site located in the first domain of annexin V was found to have the most important role. An increase in the number of mutated Ca2+-binding sites led to a greater loss of inhibition. These results corroborated those showing the progressive loss of binding of these mutants to phospholipid liposomes. In conclusion, we show that PKC inhibition by annexin V is the consequence of a mechanism involving phospholipid sequestration by annexin V, and that the Ca2+-binding site located in domain 1 of annexin V plays a predominant role in this process. In addition, we show that the R122AIK site, which may act analogously to a PKC-inhibitory pseudosubstrate site, is not involved in PKC inhibition, and that a peptide corresponding to the C-terminal tail of annexin V inhibits PKC activity but to a lesser extent than annexin V itself.
authors.affiliation: INSERM U332, Laboratoire de Signalisation, Inflammation et Transformation Cellulaire, ICGM, 22, Rue Mechain, 75014 Paris, France.
authors.forename: T
authors.initials: T
authors.lastname: Dubois
authors.forename: J P
authors.initials: JP
authors.lastname: Mira
authors.forename: D
authors.initials: D
authors.lastname: Feliers
authors.forename: E
authors.initials: E
authors.lastname: Solito
authors.forename: F
authors.initials: F
authors.lastname: Russo-Marie
authors.forename: J P
authors.initials: JP
authors.lastname: Oudinet
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D017306:Annexin A2 / Q000378:metabolism*; D017304:Annexin A5 / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D004676:Myelin Basic Protein / Q000378:metabolism; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D017306:Annexin A2; D017304:Annexin A5; D007527:Isoenzymes; D004676:Myelin Basic Protein; D010446:Peptide Fragments; D010455:Peptides; D010743:Phospholipids; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium