Monoclonal Antibody NM2 Recognizes the Protein Kinase C Phosphorylation Site in B-50 (GAP-43) and in Neurogranin (BICKS) |
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Authors: | A. Beate Oestreicher,Jacques J. H. Hens,Arnoud Marquart,Marc Mercken,Pierre N. E. De,Graan ,Henk Zwiers&dagger ,Willem Hendrik Gispen |
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Affiliation: | Division of Molecular Neurobiology, Rudolf Magnus Institute, University of Utrecht, Utrecht, The Netherlands;Innogenetics, Ghent, Laboratory of Neurobiology, Born Bunge Foundation, University of Antwerp, Antwerp-Wilrijk, Belgium;Departments of Medical Physiology/Medical Biochemistry, University of Calgary, Health Science Centre, Calgary, Alberta, Canada |
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Abstract: | Abstract: 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 with peptide fragments of rat B-50 containing the unique protein kinase C (PKC) phosphorylation site at serine-41, it was selected and characterized in comparison with another Mab NM6 unreactive with these fragments. NM2, but not NM6, recognized neurogranin (BICKS), another PKC substrate, containing a homologous sequence to rat B-50 (34–52). To narrow down the epitope domain, synthetic B-50 peptides were tested in ELISAs. 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. Preabsorption by B-50 (39–51) peptide of NM2 inhibited the binding of NM2 to rat B-50 in contrast to NM6. NM2 selectively inhibited phosphorylation of B-50 during endogenous phosphorylation of synaptosomal plasma membrane 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 physiological studies of the role of PKC-mediated phosphorylation and calmodulin binding of B-50 and neurogranin. |
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Keywords: | Epitope mapping GAP-43 Monoclonal antibody Protein kinase C Phosphorylation site of rat B-50 |
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