Numb-mediated neurite outgrowth is isoform-dependent, and requires activation of voltage-dependent calcium channels

Numb is an evolutionarily conserved protein that controls the differentiation of neuronal progenitor cells by unknown mechanisms. Here we report that the neural cells expressing Numb isoforms with short phosphotyrosine-binding (SPTB) domain undergo extensive neurite outgrowth, an effect that can be blocked by voltage-gated Ca²⁺ channel (VGCC) inhibitor or by Ca²⁺ chelator. In contrast, tyrosine kinase inhibitor, genistein, and selective receptor tyrosine kinase (TrkA) inhibitor, K252α did not affect SPTB Numb-mediated neurite outgrowth. MAP kinase inhibitor, PD98059 partially reduced SPTB Numb-mediated neurite outgrowth. Cells expressing SPTB Numbs exhibit increased whole-cell Ca²⁺ current densities (ICa) which can be prevented by preincubation of either nifedipine or PD98095. Cells expressing LPTB Numbs expressed little ICa (density) and were not able to grow neurites. Our results indicate that Ca²⁺ influx through VGCC may be required for SPTB Numb-mediated neurite outgrowth, suggesting that Numb promotes neuronal differentiation by a mechanism involving PTB domain-specific regulation of Ca²⁺ influx and MAP kinase activation.