EARLY SIGNALING EVENTS TRIGGERED BY PEROXOVANADIUM [BPV(PHEN)] ARE INSULIN-RECEPTOR KINASE (IRK)-DEPENDENT - SPECIFICITY OF INHIBITION OF IRK-ASSOCIATED PROTEIN-TYROSINE PHOSPHATASE(S) BY BPV(PHEN)

Peroxovanadiums (pVs) are potent protein tyrosine phosphatase (PTP) in hibitors with insulin-mimetic properties in vivo and in vitro. We have established the existence of an insulin receptor kinase (IRK)-associa ted PTP whose inhibition by pVs correlates closely with IRK tyrosine p hosphorylation, activation, and downstream signaling. pVs have also be en shown to activate various tyrosine kinases (TKs) that could partici pate in activation of the insulin-signaling pathway. In the present st udy we have sought to determine whether pV-induced IRK tyrosine phosph orylation requires the intrinsic kinase activity of the IRK, and wheth er IRK activation is necessary to realize the early steps in the insul in-signaling cascade. To address this we evaluated the effect of a pur e pV compound, bis peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma cells overexpressing normal (HTC-IR) or kinase-defici ent (HTC-M1030) mutant IRKs. We showed that at a dose of 0.1 mM, but n ot 1 mM, bpV(phen) induced IRK-dependent events. Thus, 0.1 mM bpV(phen ) increased tyrosine phosphorylation and IRK activity in HTC-IR but no t HTC-M1030 cells. Tyrosine phosphorylation of insulin signal-transduc ing molecules was promoted in HTC-IR but not HTC-M1030 cells by bpV(ph en). The association of p185 and p60 with the src homology-2 (SH2) dom ains of Syp and the p85-regulatory subunit of phosphatidylinositol 3'- kinase was induced by bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin receptor substrate-1-associated phosphatidylinositol 3 '-kinase activity. Thus autophosphorylation and activation of the IRK by bpV(phen) is effected by the IRK itself, and the early events in th e insulin-signaling cascade follow from this activation event. This es tablishes a critical role for PTP(s) in the regulation of IRK activity . bpV(phen) could be distinguished from insulin only in its ability to activate ERK1 in HTC-M1030 cells, thus indicating that this event is IRK independent, consistent with our previous hypothesis that bpV(phen ) inhibits a PTP involved in the negative regulation of mitogen-activa ted protein kinases.