Molecular mechanisms of PKCalpha localization and activation by arachidonic acid. The C2 domain also plays a role.
Arachidonic acid, one of the major unsaturated fatty acids released during cell stimulation, participates in the signaling necessary for activation of different enzymes, including protein kinase C (PKC). Here, we demonstrate that arachidonic acid is a direct activator of PKCalpha, but needs the cooperation of Ca(2+) to exert its function. By using several mutants of the C2 and C1 domains, we were able to determine the molecular mechanism of this activation. More specifically, site-directed mutagenesis in key residues found in the C2 domain showed that the Ca(2+)-binding region was essential for the arachidonic acid-dependent localization and activation of PKCalpha. However, the lysine-rich cluster, also located in the C2 domain, played no relevant role in either the membrane localization or activation of the enzyme. Moreover, site-directed mutagenesis in key residues placed in the C1A and C1B subdomains, which are responsible for the diacylglycerol/phorbil ester interaction, demonstrated that the C1A subdomain was involved in the membrane localization and activation mechanism. Taken together, these data suggest a very precise mechanism for PKCalpha activation by arachidonic acid, involving a sequential model of activation in which an increase in intracytosolic Ca(2+) leads to the interaction of arachidonic acid with the Ca(2+)-binding region; only after this step, does the C1A subdomain interact with arachidonic acid, leading to full activation of the enzyme.
Departemento. de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apdo 4021, E-30100 Murcia, Spain.
Rubén
R
López-Nicolás
M José
MJ
López-Andreo
Consuelo
C
Marín-Vicente
Juan C
JC
Gómez-Fernández
Senena
S
Corbalán-García
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008958:Models, Molecular; D051571:Protein Kinase C-alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats
D016718:Arachidonic Acid; D051571:Protein Kinase C-alpha; D002118:Calcium