The role of protein tyrosine kinases and phosphatases in cell signaling

Abstract

Tyrosine phosphorylation is one of the earlier, highly organised events involved in the signal transduction cascade. This study was aimed at seeking more information concerning the role and site of action of the protein tyrosine kinases and phosphatases, and to identify any mm talk between their pathways and other systems. This was achieved with the use of pharmacological inhibitors applied in experiments, which measured physiological cell responsiveness. Various functional responses, namely activation of the N.IDPH oxidase system, arachidonic acid release and changes in intracellular calcium, which were stimulated by chemotactic peptide were assessed in the human neutrophil. Tyrosine kinases were found to have a significant role in the stimulation of the NADPH oxidase system. 7.4~f of the tyrosine kinase inhibitor genistein reduced superoxide anion generation to 20.0% (±7.6% S.E.M) of control V rna.< values (p<O.OS) and to 11.3% (±1.6% S.E.M) of control O.D.M. values (p<0.01). It was additionally established that tyrosine kinases act in concert with PKC: in the generation of superoxicie anions. 5 IlM of genistein alone caused a 37.5% (±1.1 % S.E.M.) reduction in control V rn:u values. When used in combination with 30 ru\f of the PKC inhibitor staurosporine, which alone caused 2•+.0% (±5.12% S.E.M.) inhibition, a resulting 66.1% (1.5% ±S.E.M.) inhibition of superoxide formulation occurred. The effect of the tyrosine phosphatases on fi\fLP-induced neutrophil responses was found to be less significant than that of the tyrosine kinases, suggesting a more complex signalling mechanism, involving different subsets of phosphatases. The promycloid U937 cell line was used to overcome limiting factors when using human neutrophils. Since this cell line develops properties of host-defence cells when stimulated, it has been used as a simplified model for studying the properties of polymorphonuclear cells. In this study, the U937 cell line was used to examine a role for the phosphatases in the tyrosine phosphorylation of the Nck adapter oncoprotein. In this study, the presence of an endogenous anti-phosphatase was hypothesised, since tyrosine phosphorylation of Nck, which is present in both the stimulated and unstimulated form of U937 cells, was reduced upon incubation with the tyrosine phosphatase inhibitor. This indicates that the antitumour effect of dephostatin may be mediated via the indirect inhibition of the tyrosine phosphorylation of this oncoprotein.