Abstract
Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5′- O-[γ-thio]triphosphate (GTPγS), a non-hydrolysable analogue of GTP. GTPγS-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. GTPγS was the most potent nucleotide tested, whereas GDPβS and ATPγS also stimulated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein l-isoaspartyl methytransferase (PIMT) activity by GTPγS was reached at a physiological pH in the presence of 10 mM MgCl 2. Other divalent cations, such as Cu 2+, Zn 2+ and Co 2+ (100 μM), totally inhibited GTPγS-dependent carboxyl methylation. The phosphotyrosine phosphatase inhibitor vanadate potentiated the GTPγS stimulation of PIMT activity in the kidney cytosol at a concentration lower than 40 μM, but increasing the vanadate concentration to more than 40 μM resulted in a dose-dependent inhibition of the GTPγS effect. The tyrosine kinase inhibitors genistein (IC 50= 4 μM) and tyrphostin (IC 50= 1 μM) abolished GTPγS-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 GTPγS. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTPγS-stimulated PIMT activity in the kidney cytosol.
Published Version
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