The carboxyl methyltransferase modifying G proteins is a metalloenzyme.
The prenylated protein carboxyl methyltransferase (PPMT) catalyzes the posttranslational methylation of isoprenylated C-terminal cysteine residues found in many signaling proteins such as the small monomeric G proteins and the gamma subunits of heterotrimeric G proteins. Here we report that both membrane-bound PPMT from rat kidney and the recombinant bacterially expressed form of the enzyme required divalent cations for catalytic activity. Unlike EDTA and EGTA, the metal chelator 1,10-phenanthroline strongly inhibited the PPMT activity of kidney intracellular membranes in a dose- and time-dependent manner. 1,10-Phenanthroline was found to inhibit the methylation of the prenylcysteine analog N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, a synthetic substrate for PPMT, with an IC(50) of 2.2 mM. Gel electrophoretic analysis demonstrated that 1,10-phenanthroline almost totally abolished the labeling of methylated proteins in kidney intracellular membranes. Immunoblotting analysis showed that one of the two major peaks of (3)H-methylated proteins in intracellular membranes comigrated with the small G proteins Ras, Cdc42, RhoA, and Rab1. In addition, the methylation of immunoprecipitated Ras and RhoA from kidney intracellular membranes was strongly inhibited when 1,10-phenanthroline was present. Treatment of kidney intracellular membranes with 1,10-phenanthroline increased the proteolytic degradation of PPMT by exogenous trypsin, compared to untreated membranes. We conclude from these data that metal ions are essential for the activity and the stabilization of PPMT. The finding that PPMT is a metalloenzyme may provide new insights into the functions played by this methyltransferase in signal transduction processes.
Desrosiers
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Laboratoire de Médecine Moléculaire, Centre de Cancérologie Charles Bruneau, Université du Québec à Montréal, Succursale Centre-ville, Montréal, Québec, H3C 3P8, Canada.
Nguyen
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D000818:Animals; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015151:Immunoblotting; D007668:Kidney / Q000201:enzymology; D008297:Male; D008745:Methylation; D010618:Phenanthrolines / Q000494:pharmacology; D011496:Protein Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins
D002413:Cations, Divalent; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D010618:Phenanthrolines; D011994:Recombinant Proteins; D004533:Egtazic Acid; D004492:Edetic Acid; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase; D019204:GTP-Binding Proteins; C025205:1,10-phenanthroline