INHIBITION OF A SRC HOMOLOGY-2 DOMAIN-CONTAINING PROTEIN-TYROSINE-PHOSPHATASE BY VANADATE IN THE PRIMARY CULTURE OF HEPATOCYTES

Inhibition of protein tyrosine phosphatase (PTP) activities by vanadat e was examined in cultured rat hepatocytes. The incubation of hepatocy tes with sodium orthovanadate inhibited PTP activities, measured with labeled polyglutamate tyrosine (4:1) and insulin receptor peptide (114 2-1153), in a dose- and time-dependent manner. The PTP activities in c ytosolic and particulate fractions were inhibited with the IC50 values of 30-50 and 2-20 mu M, respectively. Vanadate-mediated inhibition of protein phosphatase, type 1 (a serine phosphatase) was less pronounce d, requiring 50- to 150-fold higher concentrations, Molybdate and tung state, the other potent inhibitors of PTPs, exerted similar to 70% les s inhibition of enzyme activities compared to vanadate in intact liver cells. The cytosolic and particulate PTPs inhibited by vanadate were further resolved by fast protein liquid chromatography on Mono Q and S uperose-12 columns, Vanadate exerted stable and differential inhibitio n of several PTPs, One of them was identified as SHPTP2 (Syp, SHP-2) i n cytosolic as well as particulate fractions, Immunoprecipitation of t his PTP with Syp-antibody coupled to protein A-agarose confirmed the v anadate-induced decrease in SHPTP2 activity. Vanadate did not alter th e expression of SHPTP2 and its distribution between cytosolic and part iculate fractions as indicated by the immunoblots. The decrease in the activities of PTPs in vanadate-treated hepatocytes in general was fou nd to be reversed by the reducing agent dithioerythreitol, This study shows that vanadate inhibits many PTPs in intact liver cells, one of t hem being SHPTP2/SHP-2. The inhibition is stable after chromatography on ion-exchange and gel filtration chromatography. The enzyme inhibiti on seems to involve the oxidation of the thiol group of PTPs. (C) 1996 Academic Press, Inc.