IDENTIFICATION OF A NOVEL PROTEIN TYROSINE KINASE, JAK 3

Abstract

Signal transduction in lymphocytes is accomplished by the use of protein tyrosine kinases. These kinases associate with and phosphorylate receptor subunits upon receptor binding by ligand. The predominant protein tyrosine kinase in T lymphocytes is lck, a member of the src family of protein tyrosine kinases. It has been shown, however, that activation of T lymphocytes occurs in the absence of lck. This would indicate that there are other kinases, which may not have been characterized, involved in activation. Natural Killer (NK) cells resemble activated T lymphocytes in exhibiting a high basal phosphorylation level. In addition, they are able to lyse other cells. For these reasons, NK cells were chosen as a source from which to isolate and characterize unique protein tyrosine kinases. RT PCR was performed on RNA from NK cells. Degenerate primers were then generated corresponding to the conserved catalytic domain of src and non-src kinases. These were then used to amplify the RT PCR generated products from NK cells. Products were then digested, subcloned in pBluescript, and sequenced from both ends using vector primers. Jak 3 is one of the identified kinases from the non-src library. It has a predicted MW of 125KD and contains two tandem nonidentical catalytic domains. Interestingly, it lacks the src homology domains characteristic of the src family of kinases. Jak 3 is instead the fourth member of the Janus family of kinases and is expressed predominantly in NK cells and activated T lymphocytes. The previously cloned Janus kinases are expressed ubiquitously. The selective expression of Jak 3 lead to the examination of its relationship to the T lymphocyte activation pathway. One method of T lymphocyte activation (as well as B and NK cell activation) is by the use of IL2, an autocrine growth factor for T lymphocytes. Upon activation by IL2, protein tyrosine phosphorylation is increased by activation of protein tyrosine kinases associated with cellular subunits of the receptor. Data presented shows that Jak 3 is coupled to the IL2 receptor in human peripheral blood T lymphocytes and NK cells. Specific experiments show that upon activation, Jak 3 is tyrosine phosphorylated in YT cells, the NK cell line NK 3.3, and NK cells. In addition, IL2 stimulated enzymatic activity of Jak 3 as shown by and in vitro kinase assay.