The N-end rule ubiquitin-conjugating enzyme, HR6B, is up-regulated by nerve growth factor and required for neurite outgrowth

Neurite outgrowth is accompanied by increased levels of high molecular weight ubiquitin conjugates and decreased levels of free ubiquitin. The search for enzymes responsible for increased utilization of ubiquitin revealed the ubiquitin-conjugating enzyme, HR6B (yeast UBC2/RAD6), increased on mRNA and protein level in rat pheochromocytoma (PC12) cells after treatment with nerve growth factor (NGF). HR6B participates in 'N-end rule degradation' that is implicated in the cleavage of proteins with destabilizing N-terminal residues (bulky hydrophobic or basic amino acids) and requires UBR1, the ubiquitin ligase binding N-end rule target proteins. Down-regulation of HR6B or UBR1 mRNA by small interfering RNA and treatment with Leu-Ala, a dipeptide-inhibitor of UBR1, inhibit neurite outgrowth of PC12 cells. Furthermore, axonal regeneration of adult sensory neurons, which express prominent nuclear and membrane-associated HR6 immunoreactivity, is reduced by Leu-Ala in vitro. Therefore, N-end rule ubiquitination is required for neuronal differentiation of PC12 cells and may be involved in axonal regeneration of peripheral neurons.