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Collapsin response‐mediator protein 5 (CRMP5) phosphorylation at threonine 516 regulates neurite outgrowth inhibition
Author(s) -
Brot Sébastien,
Smaoune Hinda,
YoussefIssa Mina,
Malleval Céline,
Benetollo Claire,
Besançon Roger,
Auger Carole,
MoradiAméli Mahnaz,
Honnorat Jérôme
Publication year - 2014
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1111/ejn.12674
Subject(s) - phosphorylation , neurite , microbiology and biotechnology , mutant , phosphorylation cascade , biology , threonine , protein phosphorylation , serine , tubulin , microtubule , biochemistry , protein kinase a , in vitro , gene
The collapsin response‐mediator proteins ( CRMP s) 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 CRMP 2 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 CRMP 2. Another member, CRMP 5, restricts the growth‐promotional effects of CRMP 2 by inhibiting dendrite outgrowth at early developmental stages. This inhibition occurs when CRMP 5 binds to tubulin and the microtubule‐associated protein MAP 2, but the role of CRMP 5 phosphorylation is still unknown. Here, we have studied the role of CRMP 5 phosphorylation by mutational analysis. Using non‐phosphorylatable truncated constructs of CRMP 5 we have demonstrated that, among the four previously identified CRMP 5 phosphorylation sites (T509, T514, T516 and S534), only the phosphorylation at T516 residue was needed for neurite outgrowth inhibition in PC 12 cells and in cultured C57 BL /6J mouse hippocampal neurons. Indeed, the expression of the CRMP 5 non‐phosphorylated form induced a loss of function of CRMP 5 and the mutant mimicking the phosphorylated form induced the growth inhibition function seen in wildtype CRMP 5. 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 CRMP 5. Therefore, CRMP 5‐induced growth inhibition is dependent on T516 phosphorylation through the GSK ‐3β pathway. The findings provide new insights into the mechanisms underlying neurite outgrowth.