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 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.
Oestreicher
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AB
Division of Molecular Neurobiology, Rudolf Magnus Institute, University of Utrecht, The Netherlands.
Hens
J J
JJ
Marquart
A
A
Mercken
M
M
De Graan
P N
PN
Zwiers
H
H
Gipsen
W H
WH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002148:Calmodulin-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D051579:Neurogranin; D010446:Peptide Fragments / Q000276:immunology; D010447:Peptide Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013574:Synaptosomes / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C496651:Nrgn protein, rat; D010446:Peptide Fragments; D051579:Neurogranin; D011493:Protein Kinase C; D010447:Peptide Hydrolases