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.

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.

Receptors for interferons and other cytokines signal through the action of associated protein tyrosine kinases of the JAK family and latent cytoplasmic transcription factors of the STAT family. Genetic and biochemical analysis of interferon signaling indicates that activation of STATs by interferons requires two distinct JAK family kinases. Loss of either of the required JAKs prevents activation of the other JAK and extinguishes STAT activation. These observations suggest that JAKs provide interferon receptors with a critical catalytic signaling function and that at least two JAKs must be incorporated into an active receptor complex. JAK and STAT proteins are also activated by ligands such as platelet-derived growth factor (PDGF), which act through receptors that possess intrinsic protein tyrosine kinase activity, raising questions about the role of JAKs in signal transduction by this class of receptors. Here, we show that all three of the ubiquitously expressed JAKs-JAK1, JAK2, and Tyk2-become phosphorylated on tyrosine in both mouse BALB/c 3T3 cells and human fibroblasts engineered to express the PDGF-␤ receptor. All three proteins are also associated with the activated receptor. Through the use of cell lines each lacking an individual JAK, we find that in contrast to interferon signaling, PDGF-induced JAK phosphorylation and activation of STAT1 and STAT3 is independent of the presence of any other single JAK but does require receptor tyrosine kinase activity. These results suggest that the mechanism of JAK activation and JAK function in signaling differs between receptor tyrosine kinases and interferon receptors.Receptor tyrosine kinases (RTKs), such as the platelet-derived growth factor (PDGF) receptor (PDGFR), possess intrinsic protein tyrosine kinase activity in their cytoplasmic domains. In contrast, receptors for cytokines have no such activity. Despite this difference, recent data suggest that the two types of receptor have common intracellular signal transduction pathways involving members of the JAK and STAT families (for reviews, see references 4, 7, 15, 16, 21, 37, 43, 44, and 56).The JAKs, also known as Janus kinases, are nontransmembrane protein tyrosine kinases. The structure of the JAK family members is characterized by the presence of seven highly conserved regions (13), the most C-terminal of which harbors the tyrosine kinase activity. The first evidence for a role for JAKs in cytokine signaling came from studies of cells defective in their response to interferons (IFNs). The genetic approach to the dissection of IFN-␣ signaling pathway was developed by analyzing mutant cells selected for nonresponsiveness to IFN-␣. These mutants were derived from an HT1080 human fibrosarcoma cell clone (2fTGH) that expressed the bacterial guanosine phosphoribosyltransferase gene under the control of an IFN-␣-inducible promoter. Mutant group U1 cells fail to respond to IFN-␣ and are deficient in the expression of the Tyk2 protein tyrosine kinase (45). Another mutant line, U4A, responds to neither IFN-␣ nor IF...

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.

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 ␥1A 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 ␤-subunit of the IFN-␥ receptor, consistent with the hypothesis that the mutation in these cells affects JAK2-receptor association. In contrast, mutant ␥2A cells lack detectable JAK2 mRNA and protein. By using ␥2A cells, the role of various domains and conserved tyrosine residues of JAK2 in IFN-␥ 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-␥. Results with deletion mutants, however, indicated that association of JAK2 with the IFN-␥R2 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-␣ and -␥ 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.Janus kinase (JAK) nonreceptor tyrosine kinases and signal transducers and activators of transcription (STATs) are essential in signalling by the interferons (IFNs) and other cytokines (19,27,30,31,34,48,49). JAK1, Tyk2, STAT1, and STAT2 are required for the major IFN-␣/␤ response, and JAK1, JAK2, and STAT1 are required for the IFN-␥ response. The IFN-␥ receptor (IFN-␥R) belongs to the class 2 cytokine receptor family (5) and consists of a 90-kDa ligand-binding ␣ subunit (IFN-␥R1) (1) and a ␤ subunit or accessory factor 1 (AF-1; IFN-␥R2) (16, 44), which is required for receptor function (17). JAK1 is constitutively associated with the IFN-␥R1 subunit (18, 21) and JAK2 with the IFN-␥R2 subunit (4, 24, 39). Work from a number of groups has led to the following model for the initial events at the receptor. On ligand-induced dimerization or oligomerization of the preexisting receptor subunit/JAK complexes, JAK2 phosphorylates itself and JAK1, which further activates both kinases. JAK1 is thought to phosphorylate the IFN-␥R1 subunit of the receptor, creating a docking site for STAT1. JAK2 may again be required for the phosphorylation of STAT1. The activated and phosphorylated STAT1 is released and dimerizes, and the homodimers translocate to the nucleus, where they bind to specific DNA elements to activate transcription (6). Signalling through the IFN-␣␤ receptor(s) is less well unders...