Cytokines are secreted proteins that regulate cell growth and differentiation. These factors are especially important in regulating immune and inflammatory responses. Furthermore, cytokines are critical for lymphoid development, homeostasis, tolerance, and memory. Thus understanding the molecular basis of this regulation is likely to provide important insights on the pathogenesis of immune-mediated disease as well as offer new therapeutic targets. We cloned a kinase, Jak3 that is responsible for signaling by a class of cytokines that bind the common gamma chain, gc (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21). We showed that mutation of gc or Jak3 results in severe combined immunodeficiency (SCID). Because of the importance of Jak3, we identified and characterized the Jak3 promoter. We showed that the promoter is active in lymphocytes but not ubiquitously expressed and inducible following T cell activation. We also showed that histone acetylation in the region of the promoter correlates with expression of the gene. The major promoter activity can be delimited to a relatively small 267-bp fragment, which contains Sp-1, AP-1, Ets, Stat, and other binding sites. We showed that the Ets sites are of particular importance in regulating this promoter and using chromatin immunoprecipitation assays, we demonstrated that Ets1/2 binds this site in T cells. These findings therefore provide new insights into the regulation of a key lymphocyte kinase. A major objective of the laboratory is the understanding the structure and function of Jak3, in vitro and in vivo. Using these patient derived mutations as a tool, we have begun studying the trafficking of Jak3 and gc in living cell using fluorescent fusion proteins. We found that Jak3-GFP is cytosolic and cannot traffic to the plasma membrane without its cognate receptor, gc. The structural requirements for the proper localization of Jak3 are quite stringent. Though the amino-terminal band four point one, ezrin, radixin, moiesin (FERM) domain is necessary for receptor association, it is not sufficient; indeed the entire Jak3 molecule is required. Kinase activity is not required, but a number of mutants that disrupt kinase activity do affect trafficking. In addition, several additional patient-derived and artificial mutants were found to disrupt trafficking, including mutations in the pseudokinase domain and the putative SH2 domain. This may explain some of the aberrant functions of the patient-derived Jak3 mutants. Developing this system should also allow us to begin to study ligand-induced alterations in receptor/Jak subcellular distribution and hopefully will provide a better understanding of the cell biology of cytokine signal transduction. In related studies, we have also defined the major sites of autophosphorylation within Jak3. We have recently mapped one site as being an important means by which the adapter molecule SH2Bb is bound. Cells from Jak3-SCID patients also provide another opportunity, namely to examine the role of Jak3 in chemokine receptor signaling. This has been a controversial area that has great ramifications as chemokine receptors belong to the largest gene family, seven transmembrane, G-protein-coupled receptors. In contrast to the information in the literature, we showed, using several different approaches, that chemokine signaling is not dependent upon Jak3 or Jak2. These data suggest that Jaks, in fact, have dedicated functions for Type I/II cytokine receptors and do not have broader functions. After the discovery of Jak3, our group established a CRADA with Pfizer, the goal being the generation of a selective, clinically usefully Jak3 antagonist as a new class of immunosuppressant. Such a compound has now been generated and was tested in two transplant models, mouse and primate; in both settings the drug was effective in blocking transplant rejection. Importantly, the drug has an approximately 20-100 fold selectivity for Jak3 compared to other Jaks. This is important because inhibition of Jak1 and especially Jak2 would be expected to be associated with significant clinical toxicities including anemia, thrombocytopenia and leucopenia. Both in vitro and in cell-based assays, Jak2 signaling was relatively spared. In animals, the drug was associated with only mild anemia. The second major area of investigation is the control of helper T (Th) cell differentiation and the regulation of cell-mediated immunity. Several factors regulate this process including the cytokine IL-12, which we showed activates the Janus kinases, Tyk2 and Jak2 and the transcription factor Stat4. The IL-12R comprises two subunits IL-12R beta 1 and IL-12R beta 2 and we previously demonstrated that the latter is essential for Th1 differentiation. We identified the phosphorylation sites of IL-12R beta 2 for recruiting Stat4 to the receptor. In addition to its tyrosine phosphorylation, Stat4 has a conserved MAPK serine phosphorylation site in its C-terminal transcriptional activation domain. By mutating this site, we were able to show that STAT4 serine phosphorylation is critical for IL-12-induced IFN-gamma production and Th1 differentiation but not not for cell proliferation. This modification is likely mediated by the MKK6/p38 pathway and the intermediates GADD45g and Rac2 appear to be upstream activators of this event. In efforts to better understand target genes activated by cytokines, we utilized microarray technology to identify genes selectively induced by IL-12. We have found that a relatively small number of genes is induced by IL-12, compared to other cytokines like IL-2 and Type I interferon, a finding that argues for an instructive model of cytokine action as opposed to the stochastic models proposed by some workers. One gene that we identified in these microarray studies that was of particular interest is the serine/threonine kinase Cot/Tpl2. This kinase is directly inducible by IL-12 and inhibited by IL-4; accordingly, it is preferentially expressed in Th1 cells. Using siRNA to knock down Cot/Tpl2 levels, we showed that Cot/Tpl2 is important in interferon gamma gene regulation. Thus it appears that a positive feedback mechanism is operative in which IL-12 induces upstream activators of the p38 pathway such as Rac2, Gadd5g and Cot/Tpl2, which in turn promote IL-12-induced interferon gamma production. We also identified a second gene in our microarray studies that we term Cybr, for cytohesin binder and regulator. This gene is also inducible by IL-12 and preferentially expressed in Th1 cells. It associates with guanine nucleotide exchange protein, cytohesin and promotes the GTPase activity of ARFs. We have successfully generated Cybr knockout mice which have defective interferon gamma production by naive CD4+ T cells. Presently we are investigating the mechanism of this deficit.
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