title: Collapsin response-mediator protein 5 (CRMP5) phosphorylation at threonine 516 regulates neurite outgrowth inhibition.
abstract: The collapsin response-mediator proteins (CRMPs) are multifunctional proteins highly expressed during brain development but down-regulated in the adult brain. They are involved in axon guidance and neurite outgrowth signalling. Among these, the intensively studied CRMP2 has been identified as an important actor in axon outgrowth, this activity being correlated with the reorganisation of cytoskeletal proteins via the phosphorylation state of CRMP2. Another member, CRMP5, restricts the growth-promotional effects of CRMP2 by inhibiting dendrite outgrowth at early developmental stages. This inhibition occurs when CRMP5 binds to tubulin and the microtubule-associated protein MAP2, but the role of CRMP5 phosphorylation is still unknown. Here, we have studied the role of CRMP5 phosphorylation by mutational analysis. Using non-phosphorylatable truncated constructs of CRMP5 we have demonstrated that, among the four previously identified CRMP5 phosphorylation sites (T509, T514, T516 and S534), only the phosphorylation at T516 residue was needed for neurite outgrowth inhibition in PC12 cells and in cultured C57BL/6J mouse hippocampal neurons. Indeed, the expression of the CRMP5 non-phosphorylated form induced a loss of function of CRMP5 and the mutant mimicking the phosphorylated form induced the growth inhibition function seen in wildtype CRMP5. The T516 phosphorylation was achieved by the glycogen synthase kinase-3β (GSK-3β), which can phosphorylate the wildtype protein but not the non-phosphorylatable mutant. Furthermore, we have shown that T516 phosphorylation is essential for the tubulin-binding property of CRMP5. Therefore, CRMP5-induced growth inhibition is dependent on T516 phosphorylation through the GSK-3β pathway. The findings provide new insights into the mechanisms underlying neurite outgrowth.
authors.affiliation: Lyon Neuroscience Research Center, INSERM, UMR-S1028, CNRS UMR5292, Neuro-Oncology & Neuro-Inflammation Team, Lyon, F-69372, France; Université de Lyon, Université Claude Bernard Lyon 1, Lyon, F-69000, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Brot
authors.forename: Hinda
authors.initials: H
authors.lastname: Smaoune
authors.forename: Mina
authors.initials: M
authors.lastname: Youssef-Issa
authors.forename: Céline
authors.initials: C
authors.lastname: Malleval
authors.forename: Claire
authors.initials: C
authors.lastname: Benetollo
authors.forename: Roger
authors.initials: R
authors.lastname: Besançon
authors.forename: Carole
authors.initials: C
authors.lastname: Auger
authors.forename: Mahnaz
authors.initials: M
authors.lastname: Moradi-Améli
authors.forename: Jérôme
authors.initials: J
authors.lastname: Honnorat
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003712:Dendrites / Q000378:metabolism* / Q000502:physiology; D006624:Hippocampus / Q000166:cytology; D006867:Hydrolases; D051379:Mice; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism* / Q000502:physiology; D009474:Neurons / Q000378:metabolism* / Q000502:physiology; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D013912:Threonine / Q000737:chemistry; D014404:Tubulin / Q000378:metabolism
chemical_list: D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D014404:Tubulin; D013912:Threonine; C415087:DPYSL5 protein, human; D006867:Hydrolases
keywords: CRMP5; Collapsing response mediator protein; GSK-3β; neurite outgrowth; phosphorylation