Immunofluorescent quantification of tyrosine phosphorylation of cellular proteins in whole cells by flow cytometry

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Abstract
Tyrosine phosphorylation of proteins, a major event in the transduction of mitogenic signals, was analysed by flow cytometry with a fluorescent antiphosphotyrosine monoclonal antibody, on formaldehyde‐fixed, permeabilized cells. We have used this method (PY‐Facs) to study activation of normal human T lymphocytes and cells of a leukemic T‐cell line: Jurkat. In contrast to normal T cells, Jurkat cells as well as three other leukemic cell lines display a higher constitutive level of tyrosine phosphorylation. This level of tyrosine phosphorylation results from an equilibrium that can be up‐regulated by the tyrosine phosphatase inhibitor, vanadate peroxyde and down‐regulated, by the tyrosine kinase inhibitors, genistein and staurosporine. We have also observed an increased tyrosine phosphorylation of proteins after mitogenic stimulation of Jurkat cells via T‐cell receptor triggering. In addition, the entry of normal purified T cells from G0 phase into the cell cycle after co‐stimulation with a phorbol ester and an antireceptor antibody is correlated with a pronounced increase in tyrosine phosphorylation. We thus confirmed that this biochemical event was tightly associated with the activation status of the cells. The rapidity and sensitivity of the method we describe here make it particularly convenient for routine use and processing of a large number of samples, e.g., during analysis of human tumors. Moreover, because it retains sufficiently the integrity of treated cells and does not alter expression of membrane antigens, this method is suitable for multiparametric analysis, particularly for simultaneous studies associating the measure of tyrosine phosphorylation levels with possible modifications of membrane or intracellular structures as well as with cell cycle status. The PY‐Facs method appears to be an interesting approach to the study of signal transduction events leading to cell activation and proliferation.