REGULATED BINDING OF THE PROTEIN-KINASE-C SUBSTRATE GAP-43 TO THE VO C2 REGION OF PROTEIN-KINASE C-DELTA/

The interaction between protein kinase C-delta and its neuronal substr ate, GAP-43, was studied. Two forms of protein kinase C-delta were iso lated from COS cells and characterized by differences in gel mobility, GAP-43 binding, and specific GAP-43 and histone kinase activities. A slow migrating, low specific activity form of protein kinase C-delta b ound directly to immobilized GAP-43. Binding was abolished in the pres ence of EGTA, suggesting Ca2+ dependence of the interaction. The free catalytic domain of protein kinase C-delta did not bind GAP-43, sugges ting the existence of a binding site in the regulatory domain. Glutath ione S-transferase-protein kinase C-delta regulatory domain fusion pro teins were generated and tested for binding to GAP-43. The V0/C2-like aminoterminal domain was defined as the GAP-43-binding site. GAP-43 bi nding to this region is inhibited by EGTA and regulated at Ca2+ levels between 10(-7) and 10(-6) M. The interaction between protein kinase C -delta and GAP-43 was studied in intact cells by coexpression of the t wo proteins in human embryonic kidney cells followed by immunoprecipit ation. Complex formation occurred only after treatment of the cells wi th the Ca2+ ionophore ionomycin, indicating that elevation of intracel lular Ca2+ is required for interaction in vivo. It is concluded that p rotein kinase C-S interacts with GAP-43 through the V0/C2-like domain, outside the catalytic site, and that this interaction is modulated by intracellular Ca2+.