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 (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-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 125 I-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, Ub-dependent proteolysis of 125I-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 Ub-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 Ub-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.
Obin
M
M
Laboratory for Nutrition and Vision Research, Jean Mayer United States Department of Agriculture-Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111, USA. Obin_c1@HNRC.tufts.edu
Mesco
E
E
Gong
X
X
Haas
A L
AL
Joseph
J
J
Taylor
A
A
D000818:Animals; D002454:Cell Differentiation; D004151:Dipeptides / Q000378:metabolism; D006868:Hydrolysis; D007782:Lactoglobulins / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology; D016501:Neurites; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D014452:Ubiquitins / Q000378:metabolism*
D004151:Dipeptides; D007782:Lactoglobulins; D009414:Nerve Growth Factors; D014452:Ubiquitins