Neurofibrillary tangle-associated collapsin response mediator protein-2 (CRMP-2) is highly phosphorylated on Thr-509, Ser-518, and Ser-522.
3F4, a monoclonal antibody raised against partially purified paired helical filaments (PHFs), strongly labeled neurofibrillary tangles and some plaque neurites but barely labeled neuropil threads. The levels of the 65-kDa antigen were significantly increased in the soluble fraction of the brains affected by Alzheimer's disease (AD), as compared with that in the case of control brains. The antigen was previously identified as human collapsin response mediator protein-2 (hCRMP-2) by sequencing the immunoaffinity-purified 65-kDa antigen [Yoshida, H., Watanabe, A., and Ihara, Y. (1998) J. Biol. Chem. 273, 9761-9768]. Here, we show that the 3F4 antigen represents a highly phosphorylated form of CRMP-2. The 3F4-reactive phosphoepitope was localized to the carboxyl-terminal portion of hCRMP-2, and was created by a novel 45-50-kDa protein kinase in rat brain extract. Site-directed mutagenesis of this portion showed that multiple sites of CRMP-2 are differentially phosphorylated within residues 507-522, and that phosphorylation of three sites, Thr-509, Ser-518, and Ser-522, is required for full 3F4 binding. The phosphorylation of this particular portion carboxyl-terminal to the basic region of CRMP-2 may play an important role in regulating its activity, and may be involved in the formation of degenerating neurites in AD brain.
Gu
Y
Y
Department of Neuropathology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
Hamajima
N
N
Ihara
Y
Y
D000544:Alzheimer Disease / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001923:Brain Chemistry; D019556:COS Cells; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000276:immunology / Q000378:metabolism*; D010769:Phosphothreonine / Q000276:immunology / Q000378:metabolism*; D058225:Plaque, Amyloid / Q000737:chemistry*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D040121:Semaphorin-3A; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D040121:Semaphorin-3A; C095114:collapsin response mediator protein-2; D010769:Phosphothreonine; D010768:Phosphoserine; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases