Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.     

Specific contribution of Tyk2 JH regions to the binding and the expression of the interferon alpha/beta receptor component IFNAR1

Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the interferon alpha/beta receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein. Using an in vitro assay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22-221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1-591) of Tyk2. Moreover, substitution of the JH7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor interferon alpha signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-3) contribute in a specific manner to the in vivo assembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in interferon alpha/beta receptor signaling.     

CIS3 and JAB have different regulatory roles in interleukin-6 mediated differentiation and STAT3 activation in M1 leukemia cells

We have reported JAK-signaling modulators, CIS1 (cytokine-inducible SH2 protein-1), CIS3 and JAB (JAK2 binding protein), which are structurally related. In M1 myeloid leukemia cells, CIS3 was induced by neither interleukin 6 (IL6) nor interferon gamma (IFNgamma), while JAB was induced strongly by IFNgamma and slightly by IL6 and leukemia inhibitory factor (ILF). Forced expression of CIS3 and JAB in M1 cells prevented IL6- or LIF-induced growth arrest and differentiation, even when their expression levels were comparable to endogenous ones in several cell lines such as HEL, UT-7, IFNgamma-treated M1, and CTLL2 cells. Pretreatment of parental M1 cells with IFNgamma but not IFNbeta resulted in suppression of LIF-induced STAT3 activation and differentiation, further supporting that physiological level of JAB is sufficient to inhibit LIF-signaling. However, unlike JAB, CIS3 did not inhibit IFNgamma-induced growth arrest, suggesting a difference in cytokine specificity between CIS3 and JAB. CIS3 inhibited STAT3 activation with slower kinetics than JAB and allowed rapid c-fos induction and partial FcgammaRI expression in response to IL6. In 293 cells, CIS3 as well as JAB bound to JAK2 tyrosine kinase domain (JH1), and inhibited its kinase activity, however, the effect of CIS3 on tyrosine kinase activity was weaker than that of JAB, indicating that CIS3 possesses lower affinity to JAK kinases than JAB. These findings suggest that CIS3 is a weaker inhibitor than JAB against JAK signaling, and JAB and CIS3 possess different regulatory roles in cytokine signaling.     

Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphorylation

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (hbetac). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hbetac. We report here a comprehensive screen of the entire hbetac molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hbetac that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hbetac mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hbetac activation, dissociate hbetac tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hbetac.     

Virus-specific immunity after gene therapy in a murine model of severe combined immunodeficiency

Human severe combined immunodeficiency (SCID) can be caused by defects in Janus kinase 3 (JAK3)-dependent cytokine signaling pathways. As a result, patients are at high risk of life-threatening infection. A JAK3 -/- SCID mouse model for the human disease has been used to test whether transplant with retrovirally transduced bone marrow (BM) cells (JAK3 BMT) could restore immunity to an influenza A virus. The immune responses also were compared directly with those for mice transplanted with wild-type BM (+/+ BMT). After infection, approximately 90% of the JAK3 BMT or +/+ BMT mice survived, whereas all of the JAK3 -/- mice died within 29 days. Normal levels of influenza-specific IgG were present in plasma from JAK3 BMT mice at 14 days after respiratory challenge, indicating restoration of B cell function. Influenza-specific CD4(+) and CD8(+) T cells were detected in the spleen and lymph nodes, and virus-specific CD8(+) effectors localized to the lungs of the JAK3 BMT mice. The kinetics of the specific host response correlated with complete clearance of the virus within 2 weeks of the initial exposure. By contrast, the JAK3 -/- mice did not show any evidence of viral immunity and were unable to control this viral pneumonia. Retroviral-mediated JAK3 gene transfer thus restores diverse aspects of cellular and humoral immunity and has obvious potential for human autologous BMT.     

Kinase-negative mutants of JAK1 can sustain interferon-gamma-inducible gene expression but not an antiviral state

The receptor-associated protein tyrosine kinases JAK1 and JAK2 are both required for the interferon (IFN)-gamma response. The effects of expressing kinase-negative JAK mutant proteins on signal transduction in response to IFN-gamma in wild-type cells and in mutant cells lacking either JAK1 or JAK2 have been analysed. In cells lacking endogenous JAK1 the expression of a transfected kinase-negative JAK1 can sustain substantial IFN-gamma-inducible gene expression, consistent with a structural as well as an enzymic role for JAK1. Kinase-negative JAK2, expressed in cells lacking endogenous JAK2, cannot sustain IFN-gamma-inducible gene expression, despite low level activation of STAT1 DNA binding activity. When expressed in wild-type cells, kinase-negative JAK2 acts as a dominant-negative inhibitor of the IFN-gamma response. Further analysis of the JAK/STAT pathway suggests a model for the IFN-gamma response in which the initial phosphorylation of JAK1 and JAK2 is mediated by JAK2, whereas phosphorylation of the IFN-gamma receptor is normally carried out by JAK1. The efficient phosphorylation of STAT 1 in the receptor-JAK complex may again depend on JAK2. Interestingly, a JAK1-dependent signal, in addition to STAT1 activation, appears to be required for the expression of the antiviral state.     

Characterization of the role of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit in the activation of JAK2 and STAT5

The high-affinity human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) consists of an alpha (GMRalpha) and a common beta (betac) subunit. The intracellular domain of betac has been extensively characterized and has been shown to be critical for the activation of both the JAK/STAT and MAP kinase pathways. The function of the intracellular domain of GMRalpha, however, is not as well characterized. To determine the role of this domain in GMR signaling, an extensive structure-function analysis was performed. Truncation mutants alpha362, alpha371, and alpha375 were generated, as well as the site-directed mutants alphaVQVQ and alphaVVVV. Although alpha375beta, alphaVQNQbeta, and alphaVVVVbeta stimulated proliferation in response to human GM-CSF, the truncation mutants alpha362beta and alpha371beta were incapable of transducing a proliferative signal. In addition, both alpha371 and alphaVVVV were expressed at markedly reduced levels, indicating the importance of residues 372 to 374 for proper protein expression. More importantly, we show that GMRalpha plays a direct role in the activation of the JAK/STAT pathway, and electrophoretic mobility shift assays (EMSA) indicate that both GMRalpha and betac play a role in determining the STAT5 DNA binding complex activated by the GMR. Thus, the intracellular domain of the human GMRalpha is important for activation of the JAK/STAT pathway and protein stabilization.