Signal transducers and activators of transcription (Stats) are activated by tyrosine phosphorylation in response to cytokines, and are thought to mediate many of their functional responses. Stat6 is activated in response to interleukin (IL)-4 and may contribute to various functions including mitogenesis, T-helper cell differentiation and immunoglobulin isotype switching. To evaluate the role of Stat6, we generated Stat6-null mice (Stat6 -/-) by gene disruption in embryonic stem cells. The mice were viable, indicating the lack of a non-redundant function in normal development. Although naive lymphoid cell development was normal, Stat6 -/- mice were deficient in IL-4-mediated functions including Th2 helper T-cell differentiation, expression of cell surface markers, and immunoglobulin class switching to IgE. In contrast, IL-4-mediated proliferation was only partly affected.

No abstract

A recently defined family of cytokines, consisting of ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), and interleukin-6 (IL-6), utilize the Jak-Tyk family of cytoplasmic tyrosine kinases. The beta receptor components for this cytokine family, gp130 and LIF receptor beta, constitutively associate with Jak-Tyk kinases. Activation of these kinases occurs as a result of ligand-induced dimerization of the receptor beta components. Unlike other cytokine receptors studied to date, the receptors for the CNTF cytokine family utilize all known members of the Jak-Tyk family, but induce distinct patterns of Jak-Tyk phosphorylation in different cell lines.

No abstract

The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARF's p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique a chain and a Pc subunit that is shared with the receptors for interleukin-3 (IL3) and . Two regions of the Pc chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Hematopoiesis is regulated through the interaction of one or more of a variety of cytokines with their cognate receptors. Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the proliferation and differentiation of cells at various stages of differentiation along the myeloid lineages (1, 17). The functional, high-affinity receptor for GM-CSF consists of two subunits, each of which is a member of the cytokine receptor superfamily (19,20). The binding of GM-CSF occurs through an a subunit of 60 to 80 kDa, which alone binds GM-CSF with low affinity. Association of the a chain with a 140-kDa 1c chain results in the formation of a high-affinity binding site for GM-CSF. The 1c chain also associates with 'a subunits that specifically bind interleukin-3 (IL-3) and IL-5 and similarly contributes to the formation of high-affinity binding sites for these ligands.Stimulation of growth factor-dependent cell lines with GM-CSF has been shown to induce a variety of immediate cellular responses, including the rapid tyrosine phosphorylation of the 1c subunit and a number of cellular substrates (7, 16, 30); induction of transcription of several immediate-early genes (5); and activation of components of the Ras signalling pathway, including SHC phosphorylation, increases in GTP-bound Ras, activation of Raf-1 kinase, and activation of mitogenactivated protein (MAP) kinase (4,8,23,26,27 inducing mitogenesis, while a distal region is required for activation of Ras, Raf-1, MAP kinase, and S6 kinase.Although considerable evidence demonstrates that induction of protein tyrosine phosphorylation is critical to cytokine function, only recently have the JAK family of kinases been implicated in coupling ligand binding to tyrosine phosphorylation. The JAK family consists of JAK1 (15), JAK2 (29), and TYK2 (9) and is characterized by proteins of approximately 130 kDa that contain a carboxyl kinase domain and a second kinase-like domain and lack SH2 or SH3 domains. JAK2 has been shown to be activated in the responses to erythropoietin (Epo) (36), , growth hormone (2), G-CSF (35a), and prolactin (3). In contrast, IL-6, ciliary neurotrophic factor (CNTF), and leukemia-inhibitory factor activate JAK1, JAK2, and to some extent T...

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.

Interferons (IFNs) alpha/beta (type I) and gamma (type II) bind to distinct cell surface receptors, inducing transcription of overlapping sets of genes by intracellular pathways that have recently attracted much attention. Previous studies using cell lines selected for their inability to respond to IFN-alpha (ref. 4) have shown that the protein kinase Tyk2 plays a central role in the IFN alpha/beta response. Here we report the isolation of the cell line gamma 1A, selected for its inability to express IFN-gamma-inducible cell-surface markers, that is deficient in all aspects of the IFN-gamma response tested, but responds normally to IFNs alpha and beta. The mutant cells can be complemented by the expression of another member of the JAK family of protein tyrosine kinases, JAK2 (refs 6-9). Unlike IFNs alpha and beta, IFN-gamma induces rapid tyrosine phosphorylation of JAK2 in wild-type cells, and JAK2 immunoprecipitates from these cells show tyrosine kinase activity. These responses are absent in gamma 1A cells. JAK2 is therefore required for the response to IFN-gamma but not to IFNs alpha and beta.