Elsevier

Biochemical Pharmacology

Volume 53, Issue 3, 7 February 1997, Pages 287-298
Biochemical Pharmacology

Research paper
Epidermal growth factor receptor signaling cascade as target for tyrphostin (RG 50864) in epithelial cells: Paradoxical effects on mitogen-activated protein kinase kinase and mitogen-activated protein kinase activities

https://doi.org/10.1016/S0006-2952(96)00688-0Get rights and content

Abstract

Tyrphostins are synthetic compounds that have been described as in vitro inhibitors of epidermal growth factor receptor (EGF-R) tyrosine kinase activity. The inhibitory effect of tyrphostins in intact cells has been shown only after prolonged treatment. However, these compounds appear to be readily incorporated, which suggests that tyrphostin acts indirectly on EGF-R. We studied the effects of a tyrphostin derivative, RG 50864, without preincubation in intact epithelial cells. We selected two human cell lines differing in degree of expression of the p185erbB2 protein, which is closely related to EGF-R. We showed that tyrphostin (RG 50864) had no effect on EGF-dependent EGF-R tyrosine phosphorylation in the parental cell line. On the contrary, it prolonged the EGF-dependent EGF-R and p185erbB2(V-E) tyrosine phosphorylation in p185erbB2(V-E)-expressing cells. Because tyrphostin has been shown to be an inhibitor of p185erbB2 and EGF-R in vitro, this finding indicates that the tyrphostin effect on p185erbB2(V-E) and EGF-R was the result of an indirect mechanism in transfected cells. Tyrphostin treatment alone led to the activation of mitogen-activated protein (MAP) kinase kinase or MAP kinase or extracellular signal-regulated kinase kinase (MEK), suggesting that one of the tyrphostin targets was upstream of MEK1. MAP kinase, however, was not activated after tyrphostin treatment. This finding indicates that tyrphostin had another target in intact cells because MEK1 activation by tyrphostin alone did not correlate with MAP kinase activation. In the two cell lines, tyrphostin modified the time course of EGF-dependent MEK and MAP kinase activation. We conclude that whereas tyrphostins were designed to inhibit EGF-R tyrosine kinase activity, under our conditions EGF-R is not a physiological target for tyrphostin, nor is one of its related protein tyrosine kinases, p185erbB2(V-E). On the contrary, our results show that tyrphostin targets are multiple, leading to complex effects on receptor signaling in these epithelial cells.

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      Two chemicals designed to target either tyrosine kinases (tyrphostin 47) or tyrosine phosphatases (NSC95397 and Me-3,4-dephostatin) were also found to inhibit TGM2 but did so less potently than ZM39923, which was designed to target Janus kinase 3, suggesting that TGM2 shares a similar kinase-like binding site (Faaland et al., 1991; Liu, 2003; Peyregne et al., 2005; Prevost et al., 2003; Suzuki et al., 2001; Tamura et al., 2000; Wolbring et al., 1994). Tyrphostin compounds target tyrosine kinases by binding to the kinase substrate binding site (Nowak et al., 1997). The tyrosine kinase inhibitors used to inhibit TGM2 are used at ∼100 μM in most cellular studies (Bain et al., 2003; Davies et al., 2000); however, our findings suggest Ca-TGM2 is a target at lower concentrations.

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    This work was supported by the Association pour la Recherche sur le Cancer (grant 3014), by Institut National de la Santé et de la recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS) and by the Institut de la Formation Supérieure Biomédicale (IFSBM).

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