MonoclonalAntibodyNM2 RecognizestheProteinKinaseC PhosphorylationSiteinB-50 (GAP-43) and inNeurogranin(BILKS)

Abstract : Mouse,monoclonal,B-50 antibodies,(Mabs) were,screened,to select,a Mab,that,may,interferewith suggested functions of B-50 (GAP-43), such as involve- ment,in neurotransmitter,release.Because,the Mab,NM2 reacted,with peptide,fragments,of ratB-50 containing,the unique,protein,kinase,C (PKC) phosphorylation,siteatser- ine-41, itwas selected and characterized in comparison with another,Mab,NM6 unreactive,with these,fragments,. NM2, but not NM6, recognized neurogranin (BILKS), an- other PKC substrate, containing a homologous sequence to ratB-50 (34-52) .To narrow down the epitope domain, syntheticB-50 peptides,were,tested,inELISAs .Incontrast 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,.Preabsorption,by B-50 (39-51) peptide,of NM2 inhibitedthe,binding,of NM2 to rat B-50 in contrast,to NM6 . NM2 selectivelyinhibitedphosphorylation,of B-50 during,endogenous,phosphorylation,of,synaptosomal plasma,membrane,proteins. Preabsorption,of NM2 by B-50 (39-51) peptide,abolished,this inhibition. In con- clusion, NM2 recognizes the QASFR peptide in B-50 and neurogranin .Therefore, NM2 may be a useful toolin physiological,studies,of the,role of PKC-mediated,phos- phorylation,and,calmodulin,binding,of B-50 and,neuro-