A new phosphatidylinositol 4,5-bisphosphate-binding site located in the C2 domain of protein kinase Calpha.
In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) as a second messenger, we studied the activation of protein kinase Calpha by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase Calpha, we have determined and characterized the PtdIns(4,5)P(2)-binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysine-rich cluster located in the beta3- and beta4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P(2); and the second, the already well described site formed by the Ca(2+)-binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase Calpha needs Ca(2+) before the PtdIns(4,5)P(2)-dependent activation of the enzyme can occur.
Corbalán-García
Senena
S
Department de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apdo 4021, E-30100 Murcia, Spain.
García-García
Josefa
J
Rodríguez-Alfaro
José A
JA
Gómez-Fernández
Juan C
JC
D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D051571:Protein Kinase C-alpha; D051381:Rats; D013379:Substrate Specificity
D019269:Phosphatidylinositol 4,5-Diphosphate; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha