Inhibition of GTPgammaS-dependent L-isoaspartyl protein methylation by tyrosine kinase inhibitors in kidney.
Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS), a non-hydrolysable analogue of GTP. GTPgammaS-stimulated methyl ester group incorporation takes place on isoaspartyl residues, as attested by the alkaline sensitivity of the labelling and its competitive inhibition by L-isoaspartyl-containing peptides. GTPgammaS was the most potent nucleotide tested, whereas GDPbetaS and ATPgammaS also stimulated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein L-isoaspartyl methytransferase (PIMT) activity by GTPgammaS was reached at a physiological pH in the presence of 10 mM MgCl2. Other divalent cations, such as Cu2+, Zn2+ and Co2+ (100 microM), totally inhibited GTPgammaS-dependent carboxyl methylation. The phosphotyrosine phosphatase inhibitor vanadate potentiated the GTPgammaS stimulation of PIMT activity in the kidney cytosol at a concentration lower than 40 microM, but increasing the vanadate concentration to more than 40 microM resulted in a dose-dependent inhibition of the GTPgammaS effect. The tyrosine kinase inhibitors genistein (IC50 = 4 microM) and tyrphostin (IC50 = 1 microM) abolished GTPgammaS-dependent PIMT activity by different mechanisms, as was revealed by acidic gel analysis of methylated proteins. Whereas tyrphostin stabilised the methyl ester groups, genistein acted by blocking a crucial step required for the activation of PIMT activity by GTPgammaS. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTPgammaS-stimulated PIMT activity in the kidney cytosol.
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Département de chimie-biochimie, Université du Québec à Montréal et Centre de Cancérologìe Charles Bruneau, Hôpital Ste-Justine, Montréal, Québec, Canada.
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D000818:Animals; D003600:Cytosol / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism* / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D008297:Male; D008745:Methylation; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011505:Protein-Tyrosine Kinases; D010749:Phosphoprotein Phosphatases