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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.

Copper incorporation (64 Cu(II)) into Cu,Zn-superoxide dismutase (SOD) was studied in human lymphoblasts. Rapid incorporation of copper with a proportionate increase in SOD activity was detected. No copper incorporation or SOD activation was detected when 64 Cu(II) was added to cell cytosols rather than to intact cells. Thus, incorporation of 64 Cu was not due to isotopic exchange. Cycloheximide had no significant effect on copper incorporation and activation of SOD when the data were corrected for total cell copper. Thus, the data were consistent with copper incorporation into a preexisting apoSOD pool rather than newly synthesized SOD, and no new SOD synthesis was detected over a 15-h incubation period. The size of the apoSOD pool was estimated to be Ϸ35% of the total SOD in lymphoblasts. When cells were preincubated for 15 h with excess copper (15 M Cu(II)), the size of the apo pool markedly decreased but was not eliminated, suggesting that the apoSOD was not due to copper deficiency. These experiments also indicated that newly arrived copper was preferentially incorporated into the apoSOD pool, while the function(s) of an apoSOD pool remains unknown. Copper binding to apoSOD may provide a rapid protective response against copper toxicity.Cu,Zn-superoxide dismutase (SOD) 1 is found in the cytosolic fraction of all cells (1, 2) and has also been detected in nuclei and perioxisomes (2-5). However, the cellular site(s) and stage of protein synthesis of copper incorporation into SOD remain unknown. Moreover, an apo form of SOD was detected in a variety of copper-deficient cells or in cells in which the amount of the apoSOD pool appeared to vary with the state of differentiation (6 -15). The total amount of SOD mRNA and protein may also vary with the state of differentiation of some cells types (16). The cellular location of the apo form of SOD is unknown. While some copper incorporation into apoSOD was reported when high (2 mM) concentrations of copper were added to whole cell homogenates (10), no copper incorporation has been reported for copper addition to the cytosolic fractions from cells containing an apoSOD pool. Since copper is incorporated into apoSOD in neutral buffer, this suggests that either there are factors in cell cytosols which inhibit copper incorporation or that the apoSOD pool is not cytosolic.The time and copper concentration dependencies of copper binding to cytosolic copper binding proteins and copper incorporation into SOD were determined during incubations of lymphoblasts with 64 Cu(II). Since SOD copper does not readily exchange isotopic copper, 64 Cu incorporation exclusively into newly synthesized SOD was anticipated. However, little or no de novo synthesis of SOD protein was detected in lymphoblasts over the time course of these experiments. Instead, an apoSOD pool was detected in lymphoblasts, which were neither copperdeficient nor -differentiating. The results in this and a companion study on copper incorporation into SOD in Menkes (17) lymphoblasts (18) suggest a possible role of...

Uptake and efflux of 64Cu were examined to determine whether hepatic parenchymal cells exhibit the kinetic criteria of a specific transport system for copper and related trace metals. Saturation kinetics were clearly indicated by both v versus [Cu] and 1/v versus 1/[Cu] plots (Km = 11 +/- 0.6 microM and Vmax = 2.7 nmol Cu X min-1 X mg prot-1). Identical results were obtained by cold-copper analyses, and contributions from simple diffusion or nonspecific binding were not detected. Virtually all of the accumulated 64Cu was intracellular by 0.5 min (the initial velocity period), with approximately 40% in the cytosolic fraction. Several related trace metals inhibited 64Cu uptake, but Ni(II) at a 10:1 molar excess did not. Zn(II) acted as a simple competitive inhibitor of 64Cu uptake (Ki = 16 microM). Efflux from preloaded cells was biphasic, with an initial rapid phase of approximately 5 min. Approximately 35% of preloaded 64Cu was transported out of the cells by 40 min, and little efflux occurred thereafter. Thus, hepatocytes exhibit saturation kinetics, competition by related substrates, and countertransport criteria of specific facilitated transport. A wide variety of metabolic inhibitors have no effect on 64Cu uptake under the same conditions that inhibit the active transport of bile acids. Specific inhibitor tests for electrogenic coupling were also negative. Because the identical kinetic parameters were obtained for free 64Cu and the 1:1 64Cu-histidine complex, it is inferred that copper is probably transported as the free ion. Cells incubated with greater than or equal to 10 microM 64Cu showed a net loss of copper after 40- to 60-min incubation, which may involve specific hepatic mechanisms in copper homeostasis.