Bis(N,N-dimethylhydroxamido)hydroxooxovanadate inhibition of protein tyrosine phosphatase activity in intact cells: comparison with vanadate.

We have shown previously that bis(N,N-dimethylhydroxamido)hydroxooxovanadate (DMHV) is an excellent reversible inhibitor of protein tyrosine phosphatase (PTP) in vitro. DMHV does not carry a charge under physiological pH conditions and is anticipated to permeate cell membranes more easily than vanadate. In the present study, the efficacy of DMHV as a PTP inhibitor in intact cells was compared with that of vanadate by measuring phosphotyrosine levels in various cells treated with these compounds. DMHV was more effective in increasing both the phosphotyrosine levels of various proteins in 3T3L1 fibroblasts and the level of insulin-receptor phosphorylation in CHO cells overexpressing the human insulin receptor. DMHV was about 10- to 20-fold more effective than vanadate in increasing glucose transport and glycogen synthesis in 3T3L1 adipocytes. DMHV, unlike vanadate, also inhibited PTP in Jurkat cells. The implications of these observations are discussed.

Vanadate inhibition of protein tyrosine phosphatases in Jurkat cells: modulation by redox state.

Vanadate is a potent reversible inhibitor of protein tyrosine phosphatases (PTP) in vitro. Vanadate has been shown to increase the phosphotyrosine levels in some cell types whereas in others, like the Jurkat T-lymphoma, vanadate has no effect. The reason for the apparent lack of effect of vanadate in Jurkat cells was investigated in this study. Alteration of the redox state of these cells by reducing the glutathione level with 1-chloro-2,4-dinitrobenzene (DnpCl) had no effect on phosphotyrosine levels. However, the cells became sensitive to vanadate, as measured by an increase in phosphotyrosine levels on a wide range of proteins including the MAP kinases. The increase in phosphotyrosine levels most likely results from inhibition of cellular PTP and suggests that protein tyrosine kinases are constitutively active in cells, resulting in a dynamic phosphorylation-dephosphorylation cycle. The mode of inhibition of PTP by vanadate was investigated by measuring the PTP activity of Jurkat membranes isolated after treatment of cells with vanadate and DnpCl. In contrast to the reversible inhibition of PTP in vitro, the effect of vanadate in the presence of DnpCl was irreversible, raising the possibility that it is peroxovanadate formed in situ that is responsible for the inhibition of PTP in intact cells.