Nerve Growth Factor–specific Regulation of Protein Methylation during Neuronal Differentiation of PC12 Cells

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.

Generation of polyclonal antiserum for the detection of methylarginine proteins

“…Nuclear extracts from PC12 cells exhibit several methylarginine proteins that are regulated by nerve growth factor (NGF) treatment (Cimato et al, 1997;. We compared the reactivity of anti-mRG with regard to PC12 cell nuclear proteins by western blotting.…”

Activation of methionine synthase by insulin-like growth factor-1 and dopamine: a target for neurodevelopmental toxins and thimerosal

“…[37][38][39] Blocking the methionine cycle (eg with inhibitors of SAH hydrolase) interferes with neurotrophic responses, 10,11 indicating an essential role for methylation in growth factor action. Since differences in cellular phenotype reflect varied patterns of methylation-dependent gene silencing, it is reasonable to hypothesize that growth factors might directly or indirectly modulate genomic methylation status during development.…”

“…This suggestion is reinforced by the observation that blocking methylation interferes with growth factor response. 10,11 Methylation reactions, including DNA methylation, are generally controlled by the ratio of the methyl donor S-adenosylmethionine (SAM) to its demethylated product S-adenosylhomocysteine (SAH), since SAH retains considerable affinity for methyltransferase enzymes. 12,13 Methionine synthase (MS) exerts an important influence on the [SAM] to [SAH] ratio by efficiently converting homocysteine to methionine, using 5-methyltetrahydrofolate as the methyl donor.…”

BTG2TIS21/PC3 induces neuronal differentiation and prevents apoptosis of terminally differentiated PC12 cells

“…In the PC12nn25 mutant cell line, characterized by a defect in NGF receptormediated endocytosis, BTG2 TIS21/PC3 response to NGF is completely abolished and cells remain undifferentiated (Altin et al, 1991). Interestingly, the BTG2 TIS21/PC3 protein physically interacts with and activates the PRMT1 protein-arginine-N-methyltransferase (Lin et al, 1996) whose function is required for PC12 differentiation by NGF (Cimato et al, 1997). To directly investigate the role of BTG2 TIS21/PC3 in neuronal differentiation, rat pheochromocytoma PC12 cells were transfected with an inducible BTG2 TIS21/PC3 expression vector.…”