Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease.
Collapsin response mediating protein-2 (CRMP2) has been identified as an intracellular protein mediating Semaphorin3A (Sema3A), a repulsive guidance molecule. In this study, we demonstrate that cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3beta (GSK3beta) plays a critical role in Sema3A signalling. In In vitro kinase assay, Cdk5 phosphorylated CRMP2 at Ser522, while GSK3beta did not induce any phosphorylation of CRMP2. Phosphorylation by GSK3beta was exclusively observed in Cdk5-phosphorylated CRMP2, but barely in CRMP2T509A. These results indicate that Cdk5 primarily phosphorylates CRMP2 at Ser522 and GSK3beta secondarily phosphorylates at Thr509. The dual-phosphorylated CRMP2, but not non-phosphorylated or single-phosphorylated CRMP2, is recognized with the antibody 3F4, which is highly reactive with the neurofibrillary tangles of Alzheimer's disease. 3F4 recognized the CRMP2 in the wild-type but not cdk5-/- mouse embryonic brain lysates. The phosphorylation of CRMP2 at Ser522 caused reduction of its affinity to tubulin. In dorsal root ganglion neurones, Sema3A stimulation enhanced the levels of the phosphorylated form of CRMP2 detected by 3F4. Over-expression of CRMP2 mutant substituting either Ser522 or Thr509 to Ala attenuates Sema3A-induced growth cone collapse response. These results suggest that the sequential phosphorylation of CRMP is an important process of Sema3A signalling and the same mechanism may have some relevance to the pathological aggregation of the microtubule-associated proteins.
Uchida
Yutaka
Y
Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.
Ohshima
Toshio
T
Sasaki
Yukio
Y
Suzuki
Hiromi
H
Yanai
Shigeki
S
Yamashita
Naoya
N
Nakamura
Fumio
F
Takei
Kohtaro
K
Ihara
Yasuo
Y
Mikoshiba
Katsuhiko
K
Kolattukudy
Papachan
P
Honnorat
Jerome
J
Goshima
Yoshio
Y
D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology; D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000473:pathology; D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5 / Q000235:genetics / Q000378:metabolism*; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D020439:Growth Cones / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D040121:Semaphorin-3A / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D014404:Tubulin / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D040121:Semaphorin-3A; D014404:Tubulin; C095114:collapsin response mediator protein-2; D051360:Cyclin-Dependent Kinase 5; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C495906:Cdk5 protein, mouse; D038362:Glycogen Synthase Kinase 3