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  • Nerve growth factor-specific regulation of protein methylation during neuronal differentiation of PC12 cells.

Nerve growth factor-specific regulation of protein methylation during neuronal differentiation of PC12 cells.

The Journal of cell biology (1997-09-08)
T R Cimato, M J Ettinger, X Zhou, J M Aletta
ABSTRACT

Protein methylation is a posttranslational modification that can potentially regulate signal transduction pathways in a similar manner as protein phosphorylation. The role of protein methylation in NGF signaling was examined by metabolic labeling of PC12 cell proteins with L-[methyl-3H]methionine and by in vitro labeling of cell proteins with L-[methyl-3H]S-adenosylmethionine. Effects of NGF were detected within 15 min. Methyl-labeled proteins were resolved by one and two dimensional SDS-PAGE. NGF affected the methylation of several 68-60-kD proteins (pI 5.8-6.4) and 50-kD proteins (isoelectric point pH 6.7-6.8 and 5.8-6.2). Several NGF-induced changes in methylation increased over several hours and through 4 d. Moreover, methyl labeling of several specific proteins was only detected after NGF treatment, but not in nontreated controls. The effects of NGF on protein methylation were NGF specific since they were not observed with EGF or insulin. A requirement for protein methylation for neurite outgrowth was substantiated with either of two methylation inhibitors: dihydroxycyclopentenyl adenine (DHCA) and homocysteine. DHCA, the more potent of the two, markedly inhibits protein methylation and neurite outgrowth without affecting cell growth, NGF-induced survival, cell flattening, or several protein phosphorylations that are associated with early signaling events. Removal of DHCA leads to rapid protein methylation of several proteins and concurrent neurite outgrowth. The results indicate that NGF regulates the methylation of several specific proteins and that protein methylation is involved in neurite outgrowth from PC12 cells.

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Monoclonal Anti-Phosphotyrosine antibody produced in mouse, clone PT-66, ascites fluid