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J Biol Chem, Vol. 274, Issue 17, 11789-11795, April 23, 1999

Neurite Outgrowth in PC12 Cells
DISTINGUISHING THE ROLES OF UBIQUITYLATION AND UBIQUITIN-DEPENDENT PROTEOLYSIS

Martin Obin, Eugene Mesco^¶, Xin Gong, Arthur L. Haas, James Joseph^**, and Allen Taylor

From the  Laboratory for Nutrition and Vision Research and ^** Neuroscience Laboratory, Jean Mayer United States Department of Agriculture-Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111, ^¶ Department of Biology and Life Science, Savannah State University, Savannah, Georgia 31404, and  Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

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~E1 (Ub-activating enzyme) thiol esters and ¹²⁵I-labeled Ub~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 -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 -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 -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.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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