INHIBITION OF GTP-GAMMA-S-DEPENDENT L-ISOASPARTYL PROTEIN METHYLATIONBY TYROSINE KINASE INHIBITORS IN KIDNEY

Protein carboxyl methylation in rat kidney cytosol is increased by the addition of guanosine 5'-O-[gamma-thio]triphosphate (GTP gamma S), a non hydrolysable analogue of GTP. GTP gamma S-stimulated methyl ester group incorporation takes place on isoaspartyl residues, as attested b y the alkaline sensitivity of the labelling and its competitive inhibi tion by L-isoaspartyl-containing peptides. GTP gamma S was the most po tent nucleotide tested, whereas GDP beta S and ATP gamma S also stimul ated methylation but to a lesser extent. Maximal stimulation (5-fold) of protein L-isoaspartyl methytransferase (PIMT) activity by GTP gamma S was reached at a physiological pH in the presence of 10 mM MgCl2. O ther divalent cations, such as Cu2+, Zn2+ and Co2+ (100 mu M), totally inhibited GTP gamma S-dependent carboxyl methylation. The phosphotyro sine phosphatase inhibitor vanadate potentiated the GTP gamma S stimul ation of PIMT activity in the kidney cytosol at a concentration lower than 40 mu M, but increasing the vanadate concentration to more than 4 0 mu M resulted in a dose dependent inhibition of the GTP gamma S effe ct. The tyrosine kinase inhibitors genistein (IC50 = 4 mu M) and tyrph ostin (IC50 = 1 mu M) abolished GTP gamma S-dependent PIMT activity by different mechanisms, as was revealed by acidic gel analysis of methy lated proteins. Whereas tyrphostin stabilised the methyl ester groups, genistein acted by blocking a crucial step required for the activatio n of PIMT activity by GTP gamma S. The results obtained with vanadate and genistein suggest that tyrosine phosphorylation regulates GTP gamm a S-stimulated PIMT activity in the kidney cytosol. (C) 1998 Elsevier Science Inc.