Prenylcysteine carboxymethyltransferase type III activity is decreased in retinoic acid-treated SH-SY5Y neuroblastoma cells.
Prenylcysteine carboxymethyltransferase (pcCMT) is an enzyme that catalyzes the post-translational carboxymethylation of isoprenylated proteins ensuring a more efficient membrane attachment and proper guiding to a specific target membrane. In this paper, we report on modulation of pcCMT activity in retinoic acid (RA)-treated SH-SY5Y neuroblastoma cells using N-acetyl-S-farnesyl-L-cysteine (AFC) as artificial methyl acceptor. In addition, the methylation of endogenous proteins was followed by the vapor phase equilibrium assay and the storage phosphor screen (P-screen) technique with S-adenosyl-[3H-methyl] methionine (AdoMet) as methyl donor. Methylation of AFC was reduced to 75% of that of the control, the most prominent decrease being observed with the post-nuclear membrane fraction as enzyme source. With regard to protein methylation both screening methods yielded analogous results showing the [3H]-labeling of endogenous proteins in the 21-25kDa molecular mass (MM) range to be diminished by nearly 50%. This questions the role of protein carboxymethylation as an essential component of the differentiation process in SH-SY5Y neuroblastoma cells. The P-screen technique revealed that the methylation of other molecular mass proteins was also affected. Both S-adenosylhomocysteine (AdoHcy) and AFC (AdoHcy being the most effective) inhibited endogenous methylation. An interesting feature was that AFC inhibited the protein methylation proportionally more effective in RA-treated cells. Finally, the levels of three small guanosine-5'-triphosphate (GTP) binding proteins were screened upon differentiation showing rab3A to be increased while rhoA and H-ras were decreased.
Van Dessel
Guido A F
GA
RUCA-Laboratory for Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. guvades@ruca.ua.ac.be
De Busser
Hilde M
HM
Lagrou
Albert R
AR
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D002454:Cell Differentiation; D011905:Genes, ras; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008745:Methylation; D009447:Neuroblastoma / Q000201:enzymology* / Q000473:pathology; D009638:Norepinephrine / Q000378:metabolism; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms; D011496:Protein Methyltransferases / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D020826:rab3A GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000970:Antineoplastic Agents; D008565:Membrane Proteins; D020033:Protein Isoforms; D014212:Tretinoin; D012435:S-Adenosylhomocysteine; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase; D020826:rab3A GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; C068268:N-acetyl-S-farnesylcysteine; D000111:Acetylcysteine; D009638:Norepinephrine