Neurite outgrowth in PC12 cells - Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (ra-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of I-125-labeled Ub similar to E1 (Ub-activating enzyme) thiol esters and I-125-labeled Ub similar to E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize I-125-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of I-125-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3), However, Uh-dependent proteolysis of I-125-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Uh-protein conjugates, Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Uh-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.