Cellular phosphorylation of neurofilament heavy-chain by cyclin-dependent kinase-5 masks the epitope for monoclonal antibody N52.
N52 is a widely used monoclonal antibody reported to recognise both phosphorylated and non-phosphorylated forms of neurofilament (NF)-H. N52 is therefore classified as a phosphorylation-independent-type antibody. N52 is strongly reactive with NF-H in COS cells transfected with NF-H alone but co-transfection of NF-H with the neurofilament kinase cdk-5 and one of its activators p35, induced phosphorylation of NF-H that abolished this reactivity. Treatment of the cdk-5 phosphorylated NF-H with alkaline phosphatase so as to remove phosphate restored N52 reactivity. A fragment of NF-H containing the consensus cdk-5 sites was reactive with N52 but following co-transfection with cdk-5/p35 a slower migrating fragment species generated by cdk-5 was not labelled by N52. These results demonstrate that N52 is not a truly phosphorylation-independent-type NF-H antibody and suggest that the N52 epitope contains sites targeted for phosphorylation by cdk-5.
Department of Neurology, Institute of Psychiatry, Denmark Hill, London, UK.
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Guidato
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Bajaj
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Miller
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016900:Neurofilament Proteins; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; C495905:CDK5 protein, human; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases