Termination of tyrphostin AG1478 application results in different recovery of EGF receptor tyrosine residues 1045 and 1173 phosphorylation in A431 cells

Abstract

Tyrphostin AG1478 is known as a specific and reversible inhibitor of TK (tyrosine kinase) activity of the EGFR [EGF (epidermal growth factor) receptor]. It is attractive as an anticancer agent for cancers with elevated EGFR TK levels. However, post-application effects of AG1478 are not well studied. We have analysed EGFR phosphorylation after termination of AG1478 application using human epidermoid carcinoma A431 cells. It was found that AG1478 inhibitory action is fast, but not fully reversible: removal of tyrphostin resulted in incomplete restoration of the overall EGFR phosphorylation. Analysing the state of two individual autophosphorylation sites of internalized EGFR, Tyr1045 and Tyr1173, we demonstrated that phosphorylation of Tyr1173 involved in stimulation of the MAPK (mitogen-activated protein kinase) cascade was restored much more efficiently than that in position 1045, which binds the ubiquitin ligase c-Cbl and is necessary for targeting the receptor for lysosomal degradation. c-Cbl association with EGFR abolished by AG1478 was not reestablished after tyrphostin cessation. As a consequence, ubiquitination-dependent EGFR delivery to lysosomes was blocked, while phosphorylation of ERK1/2 (extracellular-signal-regulated kinase 1/2) was even increased. Thus, after termination of AG1478, the intracellular level of the inhibitor can be reached at which mitogenic signalling will be restored, whereas the EGFR negative regulation due to lysosomal degradation will not.

TRPC1 is a differential regulator of hypoxia-mediated events and Akt signalling in PTEN-deficient breast cancer cells

“…Increases in TRPC1 channel expression were sensitive to the silencing of a master regulator of hypoxia responses, HIF1A (Weidemann and Johnson, 2008), consistent with the prediction of TRPC1 as a HIF1α target gene (OrtizBarahona et al, 2010). Cellular signalling can selectively control different pathways to EMT induction (Kondratov et al, 2010) and the subsequent array of events that include increased migration, resistance to therapeutic agents and the remodelling of cellular adhesion (Tomaskovic-Crook et al, 2009;Ye and Weinberg, 2015). Our studies provide evidence for TRPC1 as a regulator of hypoxiamediated EMT pathways associated with changes in claudin-4 and Snail.…”

“…We assessed EGFR at Y1173 (an important site of EGFR autophosphorylation; Kondratov et al, 2010) and STAT3 phosphorylation at Y705 in MDA-MB-468 cells under hypoxic conditions for 6, 24 and 48 h. Phosphorylation of EGFR was significantly enhanced after 24 and 48 h of hypoxia, with no significant concomitant change in total EGFR protein ( Fig. 2A).…”

The effect of resveratrol and its methylthio-derivatives on EGFR and Stat3 activation in human HaCaT and A431 cells

“…It also cannot be excluded that other EGFR residues which were not evaluated in this study might be affected and contributed to overall EGFR protein reduction. The possible candidates are 1045 and 1173 tyrosine residues which were found to be phosphorylated in A431 cells, but were not evaluated yet in HaCaT cells [27]. …”

Janus kinases and Src family kinases in the regulation of EGF-induced vimentin expression in MDA-MB-468 breast cancer cells

“…To determine whether regulation of vimentin by either inhibitor may occur upstream of STAT3 activation at the level of the EGFR, we also assessed the effect of pharmacological inhibition on EGF-mediated EGFR phosphorylation. JAK Inhibitor I had no effect on EGFR phosphorylation; however, PP2 (at a concentration greater than that required to significantly inhibit STAT3 phosphorylation) resulted in significant inhibition of EGFR phosphorylation at Tyr1173, an important site of EGFR autophosphorylation (Kondratov et al, 2010). Inhibition of EGF-induced vimentin by PP2 could occur through effects on EGFR activity.…”