The C2 domain of protein kinase calpha is directly involved in the diacylglycerol-dependent binding of the C1 domain to the membrane.
Protein kinase Calpha (PKCalpha), which is known to be critical for the control of many cellular processes, was submitted to site-directed mutagenesis in order to test the functionality of several amino acidic residues. Thus, D187, D246 and D248, all of which are located at the Ca(2+) binding site of the C2 domain, were substituted by N. Subcellular fractionation experiments demonstrated that these mutations are important for both Ca(2+)-dependent and diacylglycerol-dependent membrane binding. The mutants are not able to phosphorylate 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 amounts of dioleylglycerol it required were larger than those required by wild type protein. On the other hand, the other mutants (D187N and D187/246/248) only recovered 50% of their activity. These data suggest that there is a relationship between the C1 domain, where dioleylglycerol binds, and the C2 domain, and that this relationship is very important for enzyme activation. These findings led us to propose a mechanism for PKCalpha activation, where C1 and C2 domains cannot be considered independent membrane binding modules.
Conesa-Zamora
P
P
Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apartado de Correos 4021, E-30080, Murcia, Spain.
Gómez-Fernández
J C
JC
Corbalán-García
S
S
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D014162:Transfection
D004075:Diglycerides; D007527:Isoenzymes; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium