MONOCLONAL-ANTIBODY NM2 RECOGNIZES THE PROTEIN-KINASE-C PHOSPHORYLATION SITE IN B-50 (GAP-43) AND IN NEUROGRANIN (BICKS)

Mouse monoclonal B-50 antibodies (Mabs) were screened to select a Mab that may interfere with suggested functions of B-50 (GAP-43), such as involvement in neurotransmitter release. Because the Mab NM2 reacted w ith peptide fragments of rat B-50 containing the unique protein kinase C (PKC) phosphorylation site at serine-41, it was selected and charac terized in comparison with another Mab NM6 unreactive with these fragm ents. NM2, but not NM6, recognized neurogranin (BICKS), another PKC su bstrate, containing a homologous sequence to rat B-50 (34-52). To narr ow down the epitope domain, synthetic B-50 peptides were tested in ELI SAs. In contrast to NM6, NM2 immunoreacted with B-50 (39-51) peptide, but not with B-50 (43-51) peptide or a C-terminal B-50 peptide. Preabs orption by B-50 (39-51) peptide of NM2 inhibited the binding of NM2 to rat B-50 in contrast to NM6. NM2 selectively inhibited phosphorylatio n of B-50 during endogenous phosphorylation of synaptosomal plasma mem brane proteins. Preabsorption of NM2 by B-50 (39-51) peptide abolished this inhibition. In conclusion, NM2 recognizes the QASFR peptide in B -50 and neurogranin. Therefore, NM2 may be a useful tool in physiologi cal studies of the role of PKC-mediated phosphorylation and calmodulin binding of B-50 and neurogranin.