title: Retinoic acid binds to the C2-domain of protein kinase C(alpha).
abstract: Protein kinase C(alpha) (PKC(alpha)) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKC(alpha), a regulatory module in the protein that binds Ca(2+) and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 A resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca(2+)-binding pocket, where Ca(2+) ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in beta-strands three and four. These observations are strongly supported by [(3)H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca(2+) coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKC(alpha) activity.
authors.affiliation: Instituto de Biología Molecular de Barcelona (CSIC), Jordi Girona Salgado 18-26, E-08034 Barcelona, Spain.
authors.forename: Wendy F
authors.initials: WF
authors.lastname: Ochoa
authors.forename: Alejandro
authors.initials: A
authors.lastname: Torrecillas
authors.forename: Ignacio
authors.initials: I
authors.lastname: Fita
authors.forename: Nuria
authors.initials: N
authors.lastname: Verdaguer
authors.forename: Senena
authors.initials: S
authors.lastname: Corbalán-García
authors.forename: Juan C
authors.initials: JC
authors.lastname: Gomez-Fernandez
mesh_terms: D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D014212:Tretinoin / Q000737:chemistry*
chemical_list: D010743:Phospholipids; D011994:Recombinant Proteins; D014212:Tretinoin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D008239:Lysine