Neurofibrillary Tangle-Associated Collapsin Response Mediator Protein-2 (CRMP-2) Is Highly Phosphorylated on Thr-509, Ser-518, and Ser-522

Abstract

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.

Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation

“…To confirm that crosstalk primarily occurs when phosphorylation is present at P−3, a peptide from CRMP2 whose O-GlcNAcylation acceptor site is well established (19), was synthesized so that residues upstream and downstream of the O-GlcNAcylation site were either left unmodified or phosphorylated (SI Appendix, Table S2). The locations of the phosphorylation sites were chosen based on their previous detection in vivo (20). Interestingly, comparable O-GlcNAcylation levels were observed on Ser517 for the unmodified peptide and the peptide in which Ser522 had been phosphorylated (at P+5 relative to the O-GlcNAc site) (SI Appendix, Figs.…”

Collapsin Response Mediator Proteins (CRMPs) Are a New Class of Microtubule-associated Protein (MAP) That Selectively Interacts with Assembled Microtubules via a Taxol-sensitive Binding Interaction

“…Hyper-phosphorylated CRMP2 is found in disease states such as Alzheimer disease, where the C-terminal GSK3␤ sites show increased phosphorylation (74,75), and the role of GSK3␤ in promoting neurofibrillary tangles of Alzheimer disease involving Tau has been suggested (47). Tau and CRMP2 share several similarities; both drive MT stabilization, both are substrates of Cdk5 (for priming) and GSK3␤, and both are hyper-phosphorylated in Alzheimer disease (47).…”

Evidence That Collapsin Response Mediator Protein-2 Is Involved in the Dynamics of Microtubules

“…Cell Lines with Inducible CRMP-2 Expression-A pTARGET-hCRMP-2 construct expressing hCRMP-2 was prepared by polymerase chain reaction as described before (10). The cDNA fragment encoding the complete sequence for hCRMP-2 was then cut with EcoRI and NotI from the plasmid and inserted into the pIND(SP1) vector.…”

“…The lysates were centrifuged at 10,000 ϫ g for 15 min, and the supernatants were saved. The protein concentration in each sample was determined by bicinchoninic acid protein assay (Pierce), and equal amounts of protein (2.5 g/lane) were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by Western blotting using a mixture of monoclonal antibodies to CRMP-2 (C4G and N3E) as described previously (10). The relative expression levels of CRMP-2 were quantitated using a GS700 Imaging Densitometer (Bio-Rad).…”

“…Highly phosphorylated forms of CRMP-2 were shown to be associated with neurofibrillary tangles in Alzheimer's disease brains (9,10). Semaphorin III/D and CRMP-2 have also been reported to mediate neuronal death in a dopamine-induced apoptotic cell model (11).…”