M Kawamura, D W McVicar, J A Johnston, T B Blake, Y Q Chen, B K Lal, A R Lloyd, D J Kelvin, J E Staples, J R Ortaldo

Abstract
Protein-tyrosine kinases (PTKs) are critical enzymes for receptor-mediated signaling in lymphocytes. Because natural killer (NK) cells are large granular lymphocytes with specialized effector function, we set out to identify PTKs preferentially expressed in these cells. One such PTK was identified and molecularly cloned. The predicted amino acid sequence shows that this kinase lacks SH2 or SH3 domains typical of src family kinases but has tandem nonidentical catalytic domains, indicating that it is a member of the Janus family of PTKs. Immunoprecipitation using antiserum generated against a peptide corresponding to the deduced amino acid sequence of this gene revealed a kinase with a molecular weight of approximately 125,000. The pattern of expression of this kinase contrasted sharply with that of other Janus kinases, which are ubiquitously expressed. The kinase described in the present study was found to be more limited in its expression; expression was found in NK cells and an NK-like cell line but not in resting T cells or in other tissues. In contrast, stimulated and transformed T cells expressed the gene, suggesting a role in lymphoid activation. Because of its homology and tissue expression, we have tentatively termed this PTK gene L-JAK for leukocyte Janus kinase.

James A. Johnston*, Masaru Kawamura*, Robert A. Kirken†, Yi-Qing Chen‡, Trevor B. Blake‡, Kyoichi Shibuya§, John R. Ortaldo*, Daniel W. McVicar* & John J. O'Shea*

*Leukocyte Cell Biology Section, Laboratory of Experimental Immunology, f Laboratory of Molecular Immunoregulation, Biological Response Modifier Program, and ‡Biological Carcinogenesis and Development Program, PRI/DynCorp, §Advanced BioSciences Laboratories Inc., National Cancer Institute, FCRDC, Frederick, Maryland 21702-1201, USA

INTERLEUKIN-2 is an autocrine growth factor for T cells1,2 which also activates other cells including B cells3 and natural killer cells4. The subunits of the interleukin-2 receptor (IL-2R) lack intrinsic enzymatic activity, but protein tyrosine phosphorylation is a critical event following ligand binding and src family kinases, such as Lck, are known to be activated by IL-2 (refs 5–9). However, IL- 2 signalling can occur in the absence of receptor interaction with Lck, suggesting that other protein tyrosine kinases might be important10. Here we report that a new member of the Janus family of kinases (Jak-3) is coupled to the IL-2R in human peripheral blood T cells and natural killer cells.

Kirken RA, Rui H, Malabarba MG, Howard OM, Kawamura M, O'Shea JJ, Farrar WL.

Department of Cytokine Molecular Mechanisms Section, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.

Abstract
A number of cytokines and growth factors use the JAK-STAT pathway to signal from the cell membrane to the nucleus. While homodimerizing cytokine receptors may transmit signal via a single form of JAK (i.e. growth hormone receptors), several multicomponent cytokine receptors have been shown to require simultaneous activation of pairs of different JAK kinases (i.e. interferon receptors). Recent evidence for a preferential coupling of JAK3 to interleukin-2 receptor-gamma (IL-2R gamma) and JAK1 to IL-2R beta supports the concept of heterotrans-activation of JAK1 and JAK3 caused by IL-2-induced heterodimerization of their receptor partners. The present study verified the ability of IL-2 to cause tyrosine phosphorylation and activation of JAK1 and JAK3, but demonstrated that IL-2 stimulated JAK3 to a significantly larger extent than JAK1 in human T lymphocytes and the YT cell line. This conclusion was based upon several independent criteria, including more vigorous tyrosine phosphorylation of JAK3, more marked enzymatic activation of JAK3 as well as higher abundance of JAK3 in activated IL-2 receptor complexes. Furthermore, when human IL-2R beta was stably expressed in murine BA/F3 cells, robust IL-2-induced proliferation and JAK3 activation occurred without detectable involvement of either JAK1, JAK2 or TYK2. We therefore propose that IL-2 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL-2-induced heterodimerization of IL-2R beta and IL-2R gamma. Nonetheless, a membrane-proximal region of human IL-2R beta (Asn240-Leu335) was critical for JAK3 activation, and the amount of JAK3 present in activated IL-2 receptor complexes increased with time, suggesting that stabilization of JAK3 binding to the receptor complex relies on both IL-2R beta and IL-2R gamma. Moreover, STAT5 was found to be the predominant STAT transcription factor used by IL-2 in human T cells, and specifically required a COOH-terminal region of IL-2R beta (Ser386-Val525), while STAT5 recruitment was not correlated to activation of IL-2R gamma or JAK3.