The C2 domain of protein kinase C alpha is directly involved in the diacylglycerol-dependent binding of the C1 domain to the membrane

Protein kinase Ca (PKC alpha), which is known to be critical for the contro l of many cellular processes, was submitted to site-directed mutagenesis in order to test the functionality of several amino acidic residues. Thus, D1 87, D246 and D248, all of which are located at the Ca2+ binding site of the C2 domain, were substituted by N. Subcellular fractionation experiments de monstrated that these mutations are important for both Ca2+-dependent and d iacylglycerol-dependent membrane binding. The mutants are not able to phosp horylate typical PKC substrates, such as histone and myelin basic protein. Furthermore, using increasing concentrations of dioleylglycerol, one of the mutants (D246/248N) was able to recover total activity although the amount s of dioleylglycerol it required were larger than those required by wild ty pe protein. On the other hand, the other mutants (D187N and D187/246/248) o nly recovered 50% of their activity. These data suggest that there is a rel ationship between the C1 domain, where dioleylglycerol binds, and the C2 do main, and that this relationship is very important for enzyme activation. T hese findings led us to propose a mechanism for PKC alpha activation, where C1 and C2 domains cannot be considered independent membrane binding module s. (C) 2000 Elsevier Science B.V. All rights reserved.