The sulfhydryl reagent N-ethylmaleimide induces hyperphosphorylation on tyrosine residues in the Jurkat T-cell line.
Tyrosine protein kinases have been shown to be functionally involved in regulation of cellular signalling, proliferation and transformation. The activity of tyrosine protein kinases is counterbalanced by phospho tyrosine phosphatases that maintain constitutively low levels of protein phosphotyrosine in most cells. In this study the effect of N-ethylmaleimide on the protein tyrosine phosphorylation was tested in Jurkat T-cells. Treatment of intact cells for 5-10 mins with 50-100 microM N-ethylmaleimide resulted in a dramatic increase in phosphorylation on tyrosine residues. Phosphoaminoacid analysis revealed an up to ten-fold increase in the content of phosphotyrosine. N-ethylmaleimide blocked the phospho tyrosine phosphatases activity of immunoprecipitated CD45 while in a kinase assay N-ethylmaleimide did not affect the 32P-gamma-ATP phosphorylation of substrates. The N-ethylmaleimide-induced hyperphosphorylation was reversed by treatment with 2 mM dithiotreitol. It is concluded that N-ethylmaleimide offers a novel useful tool for identification of substrates for tyrosine protein kinases and for studies on phosphotyrosine-dependent protein interactions.
Ståhls
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Department of Pathology, University of Helsinki, Finland.
D015703:Antigens, CD; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology*; D006649:Histocompatibility Antigens; D006801:Humans; D007163:Immunosorbent Techniques; D015458:Leukemia, T-Cell; D017493:Leukocyte Common Antigens; D010766:Phosphorylation; D019000:Phosphotyrosine; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D015703:Antigens, CD; D006649:Histocompatibility Antigens; D019000:Phosphotyrosine; D014443:Tyrosine; D017493:Leukocyte Common Antigens; D005033:Ethylmaleimide; D004229:Dithiothreitol