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, K252alpha 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.
Lu
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Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD 21224, USA. c.b.lu@leeds.ac.uk
Fu
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Xu
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Mattson
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000096:biosynthesis / Q000502:physiology*; D015640:Ion Channel Gating; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D020932:Nerve Growth Factor / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D016501:Neurites / Q000502:physiology*; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000096:biosynthesis; D051381:Rats
D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C113026:NUMB protein, human; D020033:Protein Isoforms; D021122:Protein Subunits; D019000:Phosphotyrosine; D020932:Nerve Growth Factor; D002118:Calcium