Regulated binding of the protein kinase C substrate GAP-43 to the V0/C2 region of protein kinase C-delta.
The interaction between protein kinase C-delta and its neuronal substrate, GAP-43, was studied. Two forms of protein kinase C-delta were isolated 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 bound directly to immobilized GAP-43. Binding was abolished in the presence of EGTA, suggesting Ca2+ dependence of the interaction. The free catalytic domain of protein kinase C-delta did not bind GAP-43, suggesting the existence of a binding site in the regulatory domain. Glutathione S-transferase-protein kinase C-delta regulatory domain fusion proteins were generated and tested for binding to GAP-43. The V0/C2-like amino-terminal domain was defined as the GAP-43-binding site. GAP-43 binding 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 two proteins in human embryonic kidney cells followed by immunoprecipitation. Complex formation occurred only after treatment of the cells with the Ca2+ ionophore ionomycin, indicating that elevation of intracellular Ca2+ is required for interaction in vivo. It is concluded that protein kinase C-delta interacts with GAP-43 through the V0/C2-like domain, outside the catalytic site, and that this interaction is modulated by intracellular Ca2+.
Dekker
L V
LV
Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Parker
P J
PJ
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta
D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D006133:Growth Substances; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D010750:Phosphoproteins; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D002118:Calcium