MECHANISM OF INHIBITION OF PROTEIN-KINASE-C BY 14-3-3-ISOFORMS - 14-3-3-ISOFORMS DO NOT HAVE PHOSPHOLIPASE A(2) ACTIVITY

The ability of individual members of the 14-3-3 protein family to inhibit protein kinase C (PKC) has been studied by using a synthetic peptide based on the specific 80 kDa substrate for PKC (MARCKS protein) in two different assay systems. Recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse-phase h.p.l.c. were studied. The detailed effects of diacylglycerol and the phorbol ester phorbol 12-myristate 13-acetate on the inhibition were also investigated. This suggests that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC. Since a region in secreted phospholipase A, (PLA(2))shares similarity with this domain, the ability of 14-3-3 to interact with mammalian PLA(2) was studied. Cytosolic PLA(2) has some similarity to the C2 region of PKC, and the effect of 14-3-3 on this class of PLA(2) was also analysed. In contrast with a previous report, no PLA(2) activity was found in brain 143-3-3 nor in any of the recombinant proteins tested. These include zeta 14-3-3 isoform, on which the original observation was made.