Ligand-Independent Tyrosine Kinase Signalling in RTH 149 Trout Hepatoma Cells: Comparison Among Heavy Metals and Pro-Oxidants

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Abstract
Tyrosine phosphorylation depends on the activity of receptor and non-receptor tyrosine kinases and promote cell growth, differentiation and apoptosis. Different stressors are known to stimulate tyrosine kinase activities and this could explain a wide spectrum of effects that these agents produce on different organisms. We studied the effects of heavy metals and pro-oxidants on tyrosine kinase signalling in trout hepatoma cells (RTH 149) by Western immunoblotting. Use of antiphosphotyrosine showed that Hg2+ and Cu2+in the µM range, and H2O2 in the mM range, induced tyrosine phosphorylation. The effect of Cu2+was prevented by pre-incubation with genistein, while those of Hg2+and H2O2 were only decreased, probably due to tyrosine kinase stimulation coupled to phosphatase inhibition. Phosphospecific antibodies against the three types of MAPKs showed that ERK is activated by heavy metals only, while p38 and SAPK/JNK are activated by H2O2, Hg2+, and Cu2+ plus low H2O2. Cell pre-incubation with p38 inhibitors indicated that ERK activation by H2O2 is prevented by concomitant activation of p38. Phosphospecific STAT antibodies revealed activation by H2O2 only. In conclusion, fish cell exposure to heavy metals and pro-oxidants produce specific tyrosine kinase responses, involving cross talk and redox modulatory effects.

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