Bruce A. Witthuhn

JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid cells through interaction with its receptor (EPOR). Although EPOR is a member of the cytokine receptor superfamily and lacks a kinase domain, EPO induces tyrosine phosphorylation, which is correlated with gene transcription and mitogenesis. Here we demonstrate that EPO induces tyrosine phosphorylation of JAK2 kinase and activates its in vitro autophosphorylation. Using EPOR mutants, phosphorylation and activation of kinase activity correlate with the induction of mitogenesis. Furthermore, JAK2 physically associates with a membrane-proximal region of the EPOR cytoplasmic domain that is required for biological activity. The results support the hypothesis that JAK2 is the kinase that couples EPO binding to tyrosine phosphorylation and mitogenesis.

Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase

Growth hormone receptor (GHR) forms a complex with a tyrosine kinase, suggesting involvement of a ligand-activated tyrosine kinase in intracellular signaling by growth hormone (GH). Here we identify JAK2, a nonreceptor tyrosine kinase, as a GHR-associated tyrosine kinase. Immunological approaches were used to establish GH-dependent complex formation between JAK2 and GHR, activation of JAK2 tyrosine kinase activity, and tyrosyl phosphorylation of both JAK2 and GHR. The JAK2-GHR and JAK2-erythropoietin receptor interactions described here and in the accompanying paper provide a molecular basis for involvement of tyrosyl phosphorylation in physiological responses to these ligands and suggest a shared signaling mechanism among members of the cytokine/hematopoietin receptor family.

Defective Lymphoid Development in Mice Lacking Jak3

The Janus tyrosine kinases (Jaks) play a central role in signaling through cytokine receptors. Although Jak1, Jak2, and Tyk2 are widely expressed, Jak3 is predominantly expressed in hematopoietic cells and is known to associate only with the common γ (γc) chain of the interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 receptors. Homozygous mutant mice in which the Jak3 gene had been disrupted were generated by gene targeting. Jak3-deficient mice had profound reductions in thymocytes and severe B cell and T cell lymphopenia similar to severe combined immunodeficiency disease (SCID), and the residual T cells and B cells were functionally deficient. Thus, Jak3 plays a critical role in γc signaling and lymphoid development.

Signaling by the cytokine receptor superfamily: JAKs and STATs.

A variety of cytokines, lymphokines and growth factors function by interacting with receptors that are members of the cytokine receptor superfamily. These receptors share extracellular motifs and have limited similarity in their cytoplasmic domains. Although lacking catalytic domains, this family of receptors couples ligand binding with the induction of tyrosine phosphorylation. Recent studies have shown that this is mediated by members of the Janus kinase (JAK) family of cytoplasmic protein tyrosine kinases. JAKs physically associate with the membrane-proximal region of the ligand-bound receptor, leading to their tyrosine phosphorylation and activation. The activated JAKs phosphorylate the receptors as well as cytoplasmic proteins belonging to a family of transcription factors called the signal transducers and activators of transcription (STATs), providing a novel signaling pathway that is shared by all members of the cytokine receptor superfamily.

JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c 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. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.

A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6.

The protein tyrosine kinases JAK1, JAK2 and Tyk2 and STATs (signal transducers and activators of transcription) 1 and 3 are activated in response to interleukin‐6 (IL‐6) in human fibrosarcoma cells. In mutant cells lacking JAK1, JAK2 or Tyk2, the absence of one kinase does not prevent activation of the others; activation does not, therefore, involve a sequential three‐kinase cascade. In the absence of JAK1, the phosphorylation of the gp130 subunit of the IL‐6 receptor and the activation of STATs 1 and 3 are greatly reduced. JAK1 is also necessary for the induction of IRF1 mRNA, thus establishing a requirement for the JAK/STAT pathway in the IL‐6 response. JAK2 and Tyk2 although activated cannot, in the absence of JAK1, efficiently mediate activation of STATs 1 and 3. A kinase‐negative mutant of JAK2 can, however, inhibit such activation, and ancillary roles for JAK2 and Tyk2 are not excluded. A major role for JAK1 and the nonequivalence of JAK1 and JAK2 in the IL‐6 response pathway are, nevertheless, clearly established for these cells.

Activation of Jak2 catalytic activity requires phosphorylation of Y1007 in the kinase activation loop.

The Janus protein tyrosine kinases (Jaks) play critical roles in transducing growth and differentiation signals emanating from ligand-activated cytokine receptor complexes. The activation of the Jaks is hypothesized to occur as a consequence of auto- or transphosphorylation on tyrosine residues associated with ligand-induced aggregation of the receptor chains and the associated Jaks. In many kinases, regulation of catalytic activity by phosphorylation occurs on residues within the activation loop of the kinase domain. Within the Jak2 kinase domain, there is a region that has considerable sequence homology to the regulatory region of the insulin receptor and contains two tyrosines, Y1007 and Y1008, that are potential regulatory sites. In the studies presented here, we demonstrate that among a variety of sites, Y1007 and Y1008 are sites of trans- or autophosphorylation in vivo and in in vitro kinase reactions. Mutation of Y1007, or both Y1007 and Y1008, to phenylalanine essentially eliminated kinase activity, whereas mutation of Y1008 to phenylalanine had no detectable effect on kinase activity. The mutants were also examined for the ability to reconstitute erythropoietin signaling in gamma2 cells, which lack Jak2. Consistent with the kinase activity, mutation of Y1007 to phenylalanine eliminated the ability to restore signaling. Moreover, phosphorylation of a kinase-inactive mutant (K882E) was not detected, indicating that Jak2 activation during receptor aggregation is dependent on Jak2 and not another receptor-associated kinase. The results demonstrate the critical role of phosphorylation of Y1007 in Jak2 regulation and function.

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.

A JAK1/JAK2 chimera can sustain alpha and gamma interferon responses.

Cell lines that are mutated in interferon (IFN) responses have been critical in establishing an essential role for the JAK family of nonreceptor tyrosine kinases in interferon signalling. Mutant gamma1A cells have previously been shown to be complemented by overexpression of JAK2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant gamma2A cells lack detectable JAK2 mRNA and protein. By using gamma2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-gamma signalling was examined. Individual mutation of six conserved tyrosine residues, mutation of a potential phosphatase binding site, or mutation of the arginine residue in the proposed SH2-like domain had no apparent effect on signalling in response to IFN-gamma. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-gammaR2 subunit requires the amino-terminal region but not the pseudokinase domain. Consistent with this, in chimeras with JAK1, the JAK2 amino-terminal region was required for receptor association and STAT1 activation. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -gamma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different receptor and signalling proteins rather than in the substrate specificity of their kinase domains.

Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes.

The activation of Janus protein tyrosine kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins by interleukin (IL)‐2, the T cell antigen receptor (TCR) and interferon (IFN) alpha was explored in human peripheral blood‐derived T cells and the leukemic T cell line Kit225. An IL‐2‐induced increase in JAK1 and JAK3, but not JAK2 or Tyk2, tyrosine phosphorylation was observed. In contrast, no induction of tyrosine phosphorylation of JAKs was detected upon stimulation of the TCR. IFN alpha induced the tyrosine phosphorylation of JAK1 and Tyk2, but not JAK2 or JAK3. IFN alpha activated STAT1, STAT2 and STAT3 in T cells, but no detectable activation of these STATs was induced by IL‐2. However, IL‐2 regulates the DNA binding and tyrosine phosphorylation of two STAT‐like protein complexes which do not include STAT1, STAT2 or STAT3. STAT4 is not activated by IL‐2. The activation of STAT5 cannot be excluded, so the IL‐2‐activated complexes most probably include at least one novel STAT. No STAT activity was detected in TCR‐stimulated lymphocytes, indicating that the JAK/STAT pathway defined in this study constitutes an IL‐2R‐mediated signaling event which is not shared by the TCR. Finally, in other cell types the correlation between JAK1 activation and the induction of STAT1 has suggested that JAK1 may activate STAT1. The observation that IL‐2 and IFN alpha activate JAK1 to a comparable degree, but only IFN alpha activates STAT1, indicates that JAK1 activation is not the only determining factor for STAT1 activation. Moreover, the data show that JAK1 stimulation is also not sufficient for STAT3 activation.