Phosphorylation of type III beta-tubulin PC12 cell neurites during NGF-induced process outgrowth.
Nerve growth factor (NGF) produces both rapid and delayed cellular responses that are involved in neuronal differentiation. Neurite formation, a conspicuous delayed response, is accompanied by phosphorylation of beta-tubulin in PC12 cells. The present work provides further characterization of the phospho form of beta-tubulin in this neuronal model system with regard to isotype, cellular localization, and the circumstances that favor its formation. The results indicate that neuron-specific type III beta-tubulin (beta III-tubulin) is selectively affected during neurite formation. This phosphorylation occurs relatively late in the NGF signal transduction cascade and increases progressively with increasing duration of NGF treatment concomitant with more extensive neurite growth. The subcellular distribution of beta III-tubulin is not markedly different from that of total tubulin, but the phosphorylated protein is uniquely associated with microtubules that are calcium and cold labile. Although NGF is capable of inducing phosphorylation of beta III-tubulin, it is not necessarily sufficient. Based on experiments that employ either nonpermissive substrate conditions or microtubule-depolymerizing drugs, this phosphorylation requires neurite outgrowth. Direct measurements of the phospho form in neurites versus cell bodies by means of a microculture system indicate that phosphorylated beta III-tubulin is enriched in neurites. The enrichment of phospho-beta III-tubulin in calcium- and cold-labile polymer within neurites and its near absence in nonneurite bearing, NGF-treated cells suggests a role for this posttranslationally modified protein in the regulation of dynamic microtubules involved in neurite formation.
Aletta
J M
JM
Department of Pharmacology and Toxicology, University at Buffalo, School of Medicine, New York 14214-3000, USA. jaletta@ubmed.buffalo.edu
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D003078:Colchicine / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects / Q000502:physiology; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D016716:PC12 Cells; D017239:Paclitaxel / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D015398:Signal Transduction; D014404:Tubulin / Q000096:biosynthesis / Q000378:metabolism*
D000906:Antibodies; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D014404:Tubulin; D017239:Paclitaxel; D003078:Colchicine