Signal transducers and activators of transcription (STATs) are activated by tyrosine phosphorylation in response to cytokines and mediate many of their functional responses. Stat4 was initially cloned as a result of its homology with Stat1 (refs 4, 5) and is widely expressed, although it is only tyrosine-phosphorylated after stimulation of T cells with interleukin (IL)-12 (refs 6,7). IL-12 is required for the T-cell-independent induction of the cytokine interferon (IFN)-gamma, a key step in the initial suppression of bacterial and parasitic infections. IL-12 is also important for the development of a Th1 response, which is critical for effective host defence against intracellular pathogens. To determine the function of Stat4 and its role in IL-12 signalling, we have produced mice that lack Stat4 by gene targeting. The mice were viable and fertile, with no detectable defects in haematopoiesis. However, all IL-12 functions tested were disrupted, including the induction of IFN-gamma, mitogenesis, enhancement of natural killer cytolytic function and Th1 differentiation.

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Hematopoiesis is regulated through the interaction of a variety of growth factors with specific receptors of the cytokine receptor superfamily. Although lacking catalytic domains, all the receptors couple ligand binding to the rapid induction of protein tyrosine phosphorylation. This is mediated through a novel family of protein tyrosine kinases termed the Janus kinases (Jaks) which associate with the receptors and are activated following ligand binding. Depending upon the cytokine/receptor system, one or more of the four known Jaks (Jak1, Jak2, Jak3, Tyk2) is/are involved. The activated Jaks phosphorylate both themselves and the receptor subunits, creating docking sites for SH2-containing proteins including SHC, which couples receptor engagement to activation of the ras pathway, and HCP, a protein tyrosine phosphatase which negatively affects the response. In addition, the Jaks phosphorylate one or more of a family of signal transducers and activators of transcription (Stats). Phosphorylation of Stats induces their nuclear translocation and DNA-binding activity. Activation of Stats is independent of activation of the ras pathway and represents a novel signaling pathway correlated with mitogenesis.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STATI) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates ofJanus kinases and that proteins related to STATi are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STATI. Unlike STATI, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jakl or Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN--y)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat) gene, suggesting that the genes arose by gene duplication. Unlike Statl, neither IFN-cL nor IFN--y activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colony-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.Hematopoiesis is regulated by the interaction of cytokines with receptors of the cytokine receptor superfamily. These effects are mediated, in part, by inducing the expression of genes associated with growth or differentiation. The mechanisms by which cytokines regulate gene expression are largely unknown. Recent studies have shown that Janus (Jak) kinases associate with receptors of the cytokine receptor superfamily and are activated following ligand binding (21,23,28,30,35). The interferons (IFNs) also activate Jak kinases. Signalling through the IFN-a receptor requires Jakl and Tyk2 (20,25,26,31), while signalling through the IFN--y receptor requires Jakl and Jak2 (20, 34). The Jak kinases are required for phosphorylation of proteins of 91 and 113 kDa, components of the IFN-stimulated gene factor 3ao complex, also termed signal transducers and activators of gene transcription (STAT) 1 and 2, respectively (24). On the basis of these observations, it was proposed (35) that hematopoietic growth factors might utilize similar signalling pathways to regulate gene transcription, either by using STAT1, STAT2, or related family members.The potential involvement of STAT-related proteins has been examined. Recent studies have shown that multiple cytokines induce the tyrosine phosphorylation of proteins that can acquire the ability to bind to IFN-y response elements (12). These activities we...

The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates ␤ 1 integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated ␤ 1 integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas U73122, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in a human leukemic cell line, UT-7. These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as through additional pathways most likely involving phospholipase C␥ and strongly implicate Rap1 in regulation of ␤ 1 integrin-mediated hematopoietic cell adhesion.

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IL-12 is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. IL-12 stimulation results in the activation of Janus kinase 2 and tyrosine kinase 2 and, subsequently, STAT4 and STAT3. In addition, mitogen-activated protein kinase kinase 6/p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways have been recently demonstrated to be activated by IL-12 and play an important role in IL-12 signaling. To further elucidate the molecular mechanism underlying IL-12 signaling, we have performed a yeast two-hybrid screening and identified mouse sphingosine kinase 2 (SPHK2) as a molecule associating with the mouse IL-12Rβ1 cytoplasmic region. Analyses of various mutants of each molecule revealed that the region including the proline-rich domain in SPHK2 is probably responsible for the binding to IL-12Rβ1, while the regions including the carboxyl terminus and Box II in the IL-12Rβ1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-γ production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. A similar decrease in IL-12-induced IFN-γ production was observed when dominant-negative SPHK2 was expressed in activated primary T cells using a retroviral expression system. These results suggest that SPHK2 associates with the IL-12Rβ1 cytoplasmic region and probably plays a role in modulating IL-12 signaling.

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