Molecular mechanisms of PKCα 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 PKC alpha, but needs the cooperation of Ca2+ to exert its function. By using several mutants of the C2 and C I 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 Ca2+-binding region was essential for the arachidonic acid-dependent localization and activation of PKC alpha. 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 PKC alpha activation by arachidonic acid, involving a sequential model of activation in which an increase in intracytosolic Ca2+ leads to the interaction of arachidonic acid with the Ca2+-binding region; only after this step, does the C1A subdomain interact with arachidonic acid, leading to full activation of the enzyme. (c) 2006 Elsevier Ltd. All rights reserved.