KAR-induced delayed neuronal maturation is mediated by increased T555 phosphorylation of CRMP2, whereas the KAR-induced increase in neurite outgrowth is mediated by decreased phosphorylation of T514 of CRMP2. DRG neurons were infected with Sindbis virus encoding GFP-CRMP2 and phospho-null or phospho-mimetic mutants of CRMP2^T514 and CRMP2^T555 (T514A, T514D, T555A, and T555D). Subsequently, neuronal maturation level and neurite length of GFP⁺-DRG neurons cultured in the presence or in the absence of 300 nm KA were analyzed 20 h after infection. A, Effect on neuronal maturation of expression of CRMP2 and phospho-null and phospho-mimetic mutants. Top: Arrow marks a noninfected stage 3 neuron and arrowheads point to two stage 2 neurons. B, The inhibition of neuronal maturation caused by 300 nm KA observed in CRMP2-expressing cells was still observed in DRG neurons expressing either T514A or T514D mutants, but not in those expressing T555A or T555D mutants. C, Neither the GSK3β inhibitor SB216763 (5 μm) nor the Rho kinase inhibitor GSK 429286 (5 μm) prevented the inhibitory effect of 300 nm KA on neuronal maturation. The data are expressed as the percentage of neurons (mean ± SEM) at each stage of maturation quantified from five to six different cultures with a minimum of 100 neurons analyzed per condition and culture. ***p < 0.001, two-way ANOVA followed by Bonferroni's post hoc test. D, Effect on neurite growth of expression of CRMP2, phospho-null, and phospho-mimetic mutants. E, The enhancement in neurite length induced by 300 nm KA either in GFP- or in CRMP2-expressing DRG neurons was unchanged in cells expressing CRMP2^T555A and CRMP2^T555D, whereas it was impaired in DRG neurons expressing CRMP2^T514A or CRMP2^T514D. F, The increase in neurite elongation caused by 300 nm KA was prevented by the GSK3β inhibitor SB216763 (5 μm) but not by the Rho kinase inhibitor GSK429286 (5 μm). The data are expressed as the fold change (mean ± SEM) relative to GFP-expressing cells in D, untreated cells in E, or those treated with inhibitor alone in F, each quantified from four to five different cultures analyzing 25–50 neurons per stage, condition, and culture. Neurons from all stages of maturation were pooled in D, E, and F because similar effects were observed for each group. *p < 0.05, **p < 0.01, and ***p < 0.001, one-sample t test. #p < 0.05 and ##p < 0.01, one-way ANOVA with Dunnet's post hoc test versus the effect of CRMP2.