The methylation pathway. SAHH catalyzes the reversible hydrolysis of SAHcy. Inhibition of SAHH by DHCA or homocysteine leads to an accumulation of SAHcy and inhibition of methyltransferases.

The effects of DHCA and homocysteine on neurite outgrowth and cell morphology. PC12 cells were plated on 35-mm collagen-coated tissue culture dishes at a density of 300,000 cells per dish. Cells were cultured for 7 d under control conditions without NGF (A), with 50 ng/ml NGF (B), NGF plus 1 μM DHCA (C), or NGF with 100 nM DHCA and 100 μM homocysteine (D). Bar, 50 μm.

Dose-dependent effect of DHCA on total protein methylation in PC12 cells correlates with the DHCA effect on inhibition of neurite outgrowth. Inhibition of NGF-stimulated neurite outgrowth (▪) by DHCA was compared to the inhibition of total protein methylation (○) at the same doses of DHCA. PC12 cells were treated with DHCA (30 nM to 3 μM) and NGF (50 ng/ ml) for 6 h in the presence of anisomycin to determine total protein methylation as described under Materials and Methods. Inhibition is plotted relative to control cultures treated with NGF only. The neurite outgrowth data are replotted from Fig. 7 A.

NGF-induced changes in protein methylation persist and progress during prolonged NGF treatment. PC12 cells were incubated with or without NGF (50 ng/ml) for 4 d. Cells were labeled with l-[methyl-³H]methionine (100 μCi/ml) in the presence of anisomycin (10 μM) for 6 h. Equal TCA-precipitable cpm (750,000) of cell lysates were subjected to IEF. IEF gels were loaded on 7.5–15% gradient SDS-PAGE gels to separate methylated proteins by molecular mass. The images shown are fluorograms of the dried gels after exposure of x-ray film for 12 d at −70°C with an intensifying screen. The areas of the gels shown are similar to the boxed areas shown in Fig. 1. The pH gradient of the IEF gels is indicated at the top of the figure. Labels are as in Fig. 1.

The time dependence of total methyl group incorporation into proteins in response to NGF. PC12 cell proteins were metabolically labeled with l-[methyl-³H]methionine (100 μCi/ ml) for 6 h in the presence of anisomycin (10 μM). NGF (50 ng/ ml) was added to the cell cultures for the times indicated. Cells were lysed in SDS-PAGE sample buffer. Total methyl labeling of proteins (counts per minute) was measured for each condition after TCA precipitation of an aliquot of the sample. The relative amount of the ³H-labeled methyl groups in lysate proteins at each time point is given with respect to nontreated control cells. The data shown are the means ± SEM for five independent experiments.

Concentration-dependent inhibition of neurite outgrowth by inhibitors of methylation. (A) Effect of DHCA. PC12 cells on 35-mm collagen-coated tissue culture dishes (300,000 cells per dish) were treated with NGF (50 ng/ml), or NGF and DHCA (10 nM to 3 μM) for 7 d. Neurite-bearing cells were scored as described under Materials and Methods. Cultures treated with NGF and no DHCA received the DMSO vehicle only, which had no effect on neurite outgrowth. The line was fit by least squares regression analysis. The data shown are the means ± SD for three independent experiments. (B) Effect of homocysteine alone and in combination with DHCA. All cultures were treated with NGF (50 ng/ml) and scored for the presence of neurites after 7 d. The data shown are the means ± SD for three independent experiments. The white bar represents the NGF control condition without DHCA or homocysteine. Results from cultures treated with 100 nM DHCA are represented by the adjacent striped bar (DHCA), and those from cultures treated with 100 μM homocysteine in the next striped bar (Hcy). The cross-hatched bar indicates results from cultures treated with 100 nM DHCA plus 100 μM homocysteine (DHCA + Hcy).

The effect of prior NGF treatment on the neurite inhibiting action of DHCA. (A) The effect of varying the time interval between NGF treatment and addition of DHCA. PC12 cells were treated with NGF (50 ng/ml) for 7 d. DHCA (1 μM) was added to the cells either at the time NGF was initially added to the cell cultures (time zero), or after a time interval of 1, 2, 3, 4, 5, or 6 d. Cells were scored for the presence of neurites after 7 d of NGF. The data shown are the means ± SD for three independent experiments. (B) The effect of inhibition of methylation on neurite regeneration. PC12 cells were grown in NGF (50 ng/ml) for 7–14 d. NGF was removed from the cultures by an extensive washing procedure described under Materials and Methods. Cells were removed from the culture dishes by trituration and replated without or with DHCA (3 nM to 3 μM). NGF was added to all cultures 1 h after plating. Neurite regeneration was scored 16–24 h later. The line was fit by least squares regression analysis. The data shown are the means ± SD for three independent experiments.

NGF induces specific changes in the pattern of in vivo protein methylation observed by 2D IEF × SDS-PAGE. PC12 cell proteins were metabolically labeled with l-[methyl-³H]methionine (100 μCi/ml) in the presence of anisomycin (10 μM) for 6 h. Whole cell lysates containing equal TCA-precipitable cpm (800,000) were separated by IEF. IEF gels were loaded onto 8.5– 15% gradient gels for the separation of methylated proteins by molecular mass. Fluorographic images generated from the control and 6 h NGF (50 ng/ml) conditions are illustrated in the top two panels. Molecular mass standards (in kD) are indicated at the left of each panel. The boxed area in the top panels is enlarged below for control, 6 h NGF and 6 h EGF (5 nM) samples. Fluorograms were generated by exposing x-ray film to the dried gels with an enhancing screen for 10 d at −70°C. The pH gradient of the isoelectric focusing tubes is shown below the enlarged images (a–c). Labels (A, *, and +) indicate unaffected methylations, while the remaining letters and numbers indicate protein methylations affected by NGF. These results are representative of three independent experiments.

NGF-treatment of intact cells stimulates in vitro protein methylation in cell-free extracts. (A) PC12 cells were incubated in the presence or absence of NGF (50 ng/ml) for 6 or 16 h. Proteins in the nuclear (500 g, pellet), membrane (100,000 g, pellet), or cytosolic (100,000 g, supernatant) fractions were radiolabeled with l-[methyl-³H]SAM for 1 h at 37°C. The labeling reaction was quenched by the addition of 5× SDS-PAGE sample buffer followed by holding the reaction tube in a boiling water bath for 5 min. Labeled proteins were separated by 7.5–15% gradient SDS-PAGE. The image shown is a fluorogram of the dried gel. Asterisks indicate methylated proteins not detectable in extracts from non-NGF-treated control cells. Arrows identify increases of 67% or greater. The arrowheads (membrane fraction) point out decreases of >30%. These results were reproduced in an independent experiment. Specific changes at 16 h were as follows: nuclear proteins at 97 and 64 kD increase 2.4-fold; cytosolic proteins at 50 kD and 35 kD increase 67 and 79%, respectively. (B) Cell proteins were metabolically radiolabeled followed by preparation of cytosol (in vivo) or the cytosol was prepared first, followed by incubation with [methyl-³H]SAM (in vitro). A portion of the 2D IEF × SDS-PAGE fluorograms from control (−NGF) versus 6 h NGF treatment (+NGF) are displayed. There are five individual protein spots migrating at 64 kD, which are most easily discerned in the in vivo +NGF condition. The 64-kD series of spots in each of the other fluorograms (four spots each) are superimposable with those of the in vivo +NGF condition.

Changes in the pattern of protein methylation are NGF specific and time dependent. (A) PC12 cell proteins were metabolically labeled with l-[methyl-³H]methionine (100 μCi/ml) for 6 h in the presence of anisomycin (10 μM) with or without NGF (50 ng/ml) for the times indicated. Whole cell lysates containing equal TCA-precipitable cpm (300,000) were loaded in each lane. Proteins were separated by 7.5–15% gradient SDS-PAGE. The image shown is a fluorogram of the dried gel after exposure to x-ray film for 3 d at −70°C with an intensifying screen. The bracket indicates the migration position of a 64–62-kD complex of proteins that increase in a time-dependent manner. The arrowhead indicates the migration position of an invariant protein band, the density of which does not change by more than a few percent over the time course. By this measure, the 4-h lane is slightly overloaded, and the 6-h lane, underloaded. Nevertheless, the change in the 64–62-kD protein complex is still evident in the latter. The results illustrated here are representative of five independent experiments. (B) Metabolically radiolabeled methylated proteins (300,000 cpm) were obtained as described above and loaded on 8.5–15% polyacrylamide gradient gels to more favorably resolve the area between 29 and 36 kD. The image shown is a fluorogram derived from the dried gel after exposure to x-ray film for 2 d at −70°C. The arrow indicates the migration position of a regulated 34-kD protein. Cultures were treated with NGF (50 ng/ml), EGF (5 nM), or insulin (1 μM). The results shown are representative of three independent experiments.

Reversal of DHCA-induced inhibition of methyltransferases results in rapid neurite outgrowth and a time-dependent increase in protein methylation. (A) PC12 cells were plated on 35-mm collagen-coated tissue culture dishes at a density of 300,000 cells per dish in NGF (50 ng/ ml) plus or minus 1 μM DHCA for 7 d. The medium was then changed repeatedly to remove DHCA, and neurite outgrowth was scored and plotted over a 3-d period. Controls were maintained in DHCA/NGF (▴) and NGF alone (•). The experimental condition was treatment with DHCA and NGF for 7 d, followed by NGF alone for 3 d (▪). The data are normalized to 100% relative to the number of neurite bearing cells present in cultures treated with NGF alone. The data shown are the means ± SD for three independent experiments. The line shown for the reversal data is the best fit to a single exponential function with a t_1/2 of 12 h. After DHCA removal, the protein methylation pattern was also assessed by SDS-PAGE. (B) Cells were harvested after 10 d of DHCA/NGF treatment or after 7 d DHCA/NGF treatment followed by progressively longer times (-h) in the absence of DHCA. Cell lysates were equalized for total TCA-precipitable ³H counts per minute and separated on 7.5–15% gradient SDS-PAGE. The figure shown is a fluorogram of the dried gel. Exposure time was 5 d. The arrows and brackets indicate the positions of proteins that increased in methylation by 25% or more relative to the 10 d DHCA lane. Asterisks mark the position of protein bands not detected in the 10-d DHCA lane. The changes noted during the reversal period were replicated in four independent experiments.

DHCA treatment does not interfere with cell growth, NGF-mediated survival, or rapid onset NGF-stimulated phosphorylations. (A) PC12 cells were plated on 24-well collagen-coated dishes at a density of 100,000 cells per well. Cells were allowed to attach to the dishes overnight, and then were washed with RPMI-1640 medium to remove serum. Cells were then cultured in complete (serum-containing) medium (CM, ○ and •), plus or minus 1 μM DHCA; RPMI-1640 without serum plus 50 ng/ml NGF, plus or minus 1 μM DHCA (NGF, □ and ▪); RPMI-1640 medium without serum or NGF, plus or minus 1 μM DHCA (No serum, ▵ and ▴). Similar results were observed in two other independent experiments. (B) Effect on Trk phosphorylation. PC12 cells were treated with or without NGF (50 ng/ml) for 5 min. Where indicated, 1 μM DHCA was added to cultures for 1 h before treatment with NGF. Cell lysates were equalized for total protein, immunoprecipitated with anti-phosphotyrosine antibody (as described under Materials and Methods), resolved by 7.5% SDS-PAGE and transferred to Immobilon P membranes. The blot was probed with anti-Trk antibody, followed by donkey anti– rabbit ¹²⁵I-IgG. The image shown is an autoradiogram of the Western blot after a 5-d exposure at −80°C. This result was verified in a second independent experiment. (C) PC12 cells were treated with or without NGF (50 ng/ml) for 2 h. DHCA (1 μM) was added 1 h before the addition of NGF where indicated. Cells were radiolabeled with [³²P]orthophosphate for 2 h as described under Materials and Methods. For long term NGF-induced phosphorylations, PC12 cells were grown in the presence of NGF (50 ng/ml) plus or minus 1 μM DHCA for 10 d. Cells were then radiolabeled with [³²P]orthophosphate for 2 h. For both short and long term NGF treatment experiments, cell lysates were equalized for total TCA-precipitable ³²P-labeled protein and separated on 6–12% gradient SDS-PAGE. The image shown is an autoradiogram of the dried gel. Exposure time was 16 h. The arrows at the right indicate, in decreasing M_r, the positions of the previously identified phosphoproteins, 64-kD chartin, 60-kD tyrosine hydroxylase, and 55-kD β-tubulin.