Mutation in the Jak kinase JH2 domain hyperactivates Drosophila and mammalian Jak-Stat pathways.

The Jak (Janus) family of nonreceptor tyrosine kinases plays a critical role in cytokine signal transduction pathways. In Drosophila melanogaster, the dominant hop(Tum-l) mutation in the Hop Jak kinase causes leukemia-like and other developmental defects. Previous studies have suggested that the Hop(Tum-l) protein might be a hyperactive kinase. Here, we report on the new dominant mutation hop(T42), which causes abnormalities that are similar to but more extreme than those caused by hop(Tum-l). We determined that Hop(T42) contains a glutamic acid-to-lysine substitution at amino acid residue 695 (E695K). This residue occurs in the JH2 (kinase-like) domain and is conserved among all Jak family members. We determined that Hop(Tum-1) and Hop(T42) both hyperphosphorylated and hyperactivated D-Stat when overexpressed in Drosophila cells. Moreover, we found that the hop(T42) phenotype was partially rescued by a reduction of wild-type D-stat activity. Finally, generation of the corresponding E695K mutation in murine Jak2 resulted in increased autophosphorylation and increased activation of Stat5 in COS cells. These results demonstrate that the mutant Hop proteins do indeed have increased tyrosine kinase activity, that the mutations hyperactivate the Hop-D-Stat pathway, and that Drosophila is a relevant system for the functional dissection of mammalian Jak-Stat pathways. Finally, we propose a model for the role of the Hop-D-Stat pathway in Drosophila hematopoiesis.     

The Hopscotch Jak kinase requires the Raf pathway to promote blood cell activation and differentiation in<Emphasis Type="Italic"> Drosophila</Emphasis>

Cytokines regulate the development and differentiated functions of hematopoietic cells by activating multiple signaling pathways, including the Jak-Stat pathway, the PI3-kinase pathway, and the Ras/Raf pathway. While the Jak-Stat interaction has been extensively studied, the relationship between this pathway and other cytokine-induced signaling pathways is not fully understood. In Drosophila melanogaster, mutations that result in hyperactivity of the Jak kinase Hopscotch (Hop) cause an activation of the larval blood cell encapsulation response, including blood cell aggregation and differentiation of plasmatocytes into apparent lamellocytes. Here, we demonstrate that Hop requires the activity of the Raf pathway to promote the activation response of larval plasmatocytes, and provide evidence to suggest that the Hop and D-Raf proteins physically interact. We also show that basal level activity of the Raf pathway is required for the accumulation of circulating blood cells.     

Nonhematopoietic expression of Janus kinase 3 is required for efficient recruitment of Th2 lymphocytes and eosinophils in OVA-induced airway inflammation

Tyrosine kinases of the Janus kinase (Jak) family transduce signals from the type I and type II cytokine receptors. Jak3 is unique in this family because its expression must be induced and is predominantly limited to cells of the lymphoid and myeloid lineages. Deficient expression of Jak3 interferes with normal development and function of T, B, and NK cells. Using irradiated Jak3-deficient (Jak3-/-) mice reconstituted with normal bone marrow (Jak3-/-chimeric mice), we have investigated possible actions of Jak3 outside of the hematopoietic system. We show that efficient recruitment of inflammatory cells to the airways of OVA-sensitized mice challenged with aerosolized OVA requires the expression of Jak3 in radioresistant nonhematopoietic cells. Failure to develop eosinophil-predominant airway inflammation in Jak3-/- chimeric mice is not due to failure of T cell sensitization, because Jak3-/- chimeric mice showed delayed-type hypersensitivity responses indistinguishable from wild-type chimeric mice. Jak3-/- chimeric mice, however, express less endothelial-associated VCAM-1 after airway Ag challenge. Given the key role of VCAM-1 in recruitment of Th2 cells and eosinophils, our data suggest that Jak3 in airway-associated endothelial cells is required for the expression of eosinophilic airway inflammation. This requirement for nonhematopoietic expression of Jak3 represents the first demonstration of a physiological function of Jak3 outside of the lymphoid lineages.     

Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1.

SHP-1 is an SH2-containing cytoplasmic tyrosine phosphatase that is widely distributed in cells of the hematopoietic system. SHP-1 plays an important role in the signal transduction of many cytokine receptors, including the receptor for erythropoietin, by associating via its SH2 domains to the receptors and dephosphorylating key substrates. Recent studies have suggested that SHP-1 regulates the function of Jak family tyrosine kinases, as shown by its constitutive association with the Tyk2 kinase and the hyperphosphorylation of Jak kinases in the motheaten cells that lack functional SHP-1. We have examined the interactions of SHP-1 with two tyrosine kinases activated during engagement of the erythropoietin receptor, the Janus family kinase Jak-2 and the c-fps/fes kinase. Immunoblotting studies with extracts from mouse hematopoietic cells demonstrated that Jak2, but not c-fes, was present in anti-SHP-1 immunoprecipitates, suggesting that SHP-1 selectively associates with Jak2 in vivo. Consistent with this, when SHP-1 was coexpressed with these kinases in Cos-7 cells, it associated with and dephosphorylated Jak2 but not c-fes. Transient cotransfection of truncated forms of SHP-1 with Jak2 demonstrated that the SHP-1-Jak2 interaction is direct and is mediated by a novel binding activity present in the N terminus of SHP-1, independently of SH2 domain-phosphotyrosine interaction. Such SHP-1-Jak2 interaction resulted in induction of the enzymatic activity of the phosphatase in in vitro protein tyrosine phosphatase assays. Interestingly, association of the SH2n domain of SHP-1 with the tyrosine phosphorylated erythropoietin receptor modestly potentiated but was not essential for SHP-1-mediated dephosphorylation of Jak2 and had no effect on c-fes phosphorylation. These data indicate that the main mechanism for regulation of Jak2 phosphorylation by SHP-1 involves a direct, SH2-independent interaction with Jak2 and suggest the existence of similar mechanisms for other members of the Jak family of kinases. They also suggest that such interactions may provide one of the mechanisms that control SHP-1 substrate specificity.     

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC₅₀ < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.     

Heme controls the regulation of protein tyrosine kinases Jak2 and Src

Protein tyrosine kinases play key roles in many molecular and cellular processes in diverse living organisms. Their proper functioning is crucial for the normal growth, development, and health in humans, whereas their dysfunction can cause serious diseases, including various cancers. As such, intense studies have been performed to understand the molecular mechanisms by which the activities of protein tyrosine kinases are regulated in mammalian cells. Particularly, small molecules that can modulate the activity of tyrosine kinases are of great importance for discovering therapeutic drug candidates for numerous diseases. Notably, heme cannot only serve as a prosthetic group for hemoglobins and enzymes, but it also is a small signaling molecule that can control the activity of diverse signaling and regulatory proteins. Using a computational search, we found that a group of non-membrane spanning tyrosine kinases contains one or more CP motifs that can potentially bind to heme and mediate heme regulation. We then used experimental approaches to determine whether heme can affect the activity of any of these tyrosine kinases. We found that heme indeed affects the phosphorylation of key tyrosine residues in Jak2 and Src, and is therefore able to modulate Jak2 and Src activity. Further experiments showed that Jak2 and Src bind to heme and that the presence of heme alters the sensitivity of Jak2 and Src to trypsin digestion. These results suggest that heme actively interacts with Jak2 and Src and alters their conformation.     

Fes mediates the IL-4 activation of insulin receptor substrate-2 and cellular proliferation

Although Jak kinases are essential for initiating cytokine signaling, the role of other nonreceptor tyrosine kinases in this process remains unclear. We have examined the role of Fes in IL-4 signaling. Examination of Jak1-deficient cell lines demonstrates that Jak1 is required for the activation of Fes by IL-4. Experiments studying signaling molecules activated by IL-4 receptor suggest that IL-4 signaling can be subdivided into Fes-dependent and Fes-independent pathways. Overexpression of kinase-inactive Fes blocks the IL-4 activation of insulin receptor substrate-2, but not STAT6. Fes appears to be a downstream kinase from Jak1/Jak3 in this process. Further examination of downstream signaling demonstrates that kinase-inactive Fes inhibits the recruitment of phosphoinositide 3-kinase to the activated IL-4 receptor complex and decreases the activation of p70(S6k) kinase in response to IL-4. This inhibition correlates with a decrease in IL-4-induced proliferation. In contrast, mutant Fes does not inhibit the activation of Akt by IL-4. These data demonstrate that signaling pathways activated by IL-4 require different tyrosine kinases. This differential requirement predicts that specific kinase inhibitors may permit the disruption of specific IL-4-induced functions.     

Photochemical preparation of a pyridone containing tetracycle: a Jak protein kinase inhibitor

Jak3 is a protein tyrosine kinase that is associated with the shared gamma chain of receptors for cytokines IL2, IL4, IL7, IL9, and IL13. We have discovered that a pyridone-containing tetracycle (6) may be prepared from trisubstituted imidazole (5) in high yield by irradiation with >350 nm light. Compound 6 inhibits Jak3 with K(I)=5 nM; it also inhibits Jak family members Tyk2 and Jak2 with IC(50)=1 nM and murine Jak1with IC(50)=15 nM. Compound 6 was tested as an inhibitor of 21 other protein kinases; it inhibited these kinases with IC(50)s ranging from 130 nM to >10 microM. Compound 6 also blocks IL2 and IL4 dependent proliferation of CTLL cells and inhibits the phosphorylation of STAT5 (an in vivo substrate of the Jak family) as measured by Western blotting.     

Janus kinase (Jak) subcellular localization revisited: the exclusive membrane localization of endogenous Janus kinase 1 by cytokine receptor interaction uncovers the Jak.receptor complex to be equivalent to a receptor tyrosine kinase

The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor.Janus kinase complexes can be regarded as receptor tyrosine kinases.     

Jak3-independent trafficking of the common gamma chain receptor subunit: chaperone function of Jaks revisited

Janus kinases (Jaks) play an essential role in cytokine signaling and have been reported to regulate plasma membrane expression of their cognate receptors. In this study, we examined whether Jak3 and the common gamma chain (gamma(c)) reciprocally regulate their plasma membrane expression. In contrast to interleukin-2Ralpha, gamma(c) localized poorly to the plasma membrane and accumulated in endosomal-lysosomal compartments. However, gamma(c) was expressed at comparable levels on the surface of cells lacking Jak3, and plasma membrane turnover of gamma(c) was independent of Jak3. Nonetheless, overexpression of Jak3 enhanced accumulation of gamma(c) at the plasma membrane. Without gamma(c), Jak3 localized in the cytosol, whereas in the presence of the receptor, it colocalized with gamma(c) in endosomes and at the plasma membrane. Although the Jak FERM domain is necessary and sufficient for receptor binding, the requirement for full-length Jak3 in gamma(c) membrane trafficking was remarkably stringent; using truncation and deletion mutants, we showed that the entire Jak3 molecule was required, although kinase activity was not. Thus, unlike other cytokine receptors, gamma(c) does not require Jak3 for receptor membrane expression. However, full-length Jak3 is required for normal trafficking of this cytokine receptor/Jak pair, a finding that has important structural and clinical implications.     

Accelerated clearance of Escherichia coli in experimental peritonitis of histamine-deficient mice

IL-5 plays a pivotal role in growth and differentiation of eosinophils. The signal transduction mechanism of IL-5Ralpha is largely unknown. We have demonstrated that IL-5 induces tyrosine phosphorylation of IL-5Ralpha in eosinophils. To identify IL-5Ralpha-associated tyrosine kinases, we have examined the expression of Src family tyrosine kinases in eosinophils. Among the Src family members, Lyn, Hck, Fgr, and Lck are present in eosinophils, and, among these four kinases, only Lyn is associated with the IL-5Ralpha under basal conditions. We also confirm the association of Janus kinase (Jak)2 with IL-5Ralpha. Lyn kinase phosphorylates both IL-5Ralpha and betacR in vitro. The importance of Lyn kinase for eosinophil differentiation was studied using antisense oligodeoxynucleotides. Lyn antisense oligodeoxynucleotide blocks eosinophil differentiation from stem cells in a dose-dependent manner. The Jak2 inhibitor tyrphostin AG490 also inhibits eosinophil differentiation. The importance of Lyn for eosinophil differentiation was further studied using Lyn knockout mice. The IL-5-stimulated eosinophil differentiation from bone marrow cells is significantly inhibited in Lyn(-/-) mice as compared with that in control mice. We conclude that both Lyn and Jak2 play an essential role in IL-5Ralpha signaling, leading to eosinophil differentiation. The effect of Lyn appears to be relatively specific for the eosinophilic lineage.     

Reduction of Lobe leads to TORC1 hypoactivation that induces ectopic Jak/STAT signaling to impair Drosophila eye development

The TOR and Jak/STAT signal pathways are highly conserved from Drosophila to mammals, but it is unclear whether they interact during development. The proline-rich Akt substrate of 40 kDa (PRAS40) mediates the TOR signal pathway through regulation of TORC1 activity, but its functions in TORC1 proved in cultured cells are controversial. The Drosophila gene Lobe (L) encodes the PRAS40 ortholog required for eye cell survival. L mutants exhibit apoptosis and eye-reduction phenotypes. It is unknown whether L regulates eye development via regulation of TORC1 activity. We found that reducing the L level, by hypomorphic L mutation or heterozygosity of the null L mutation, resulted in ectopic expression of unpaired (upd), which is known to act through the Jak/STAT signal pathway to promote proliferation during eye development. Unexpectedly, when L was reduced, decreasing Jak/STAT restored the eye size, whereas increasing Jak/STAT prevented eye formation. We found that ectopic Jak/STAT signaling and apoptosis are mutually dependent in L mutants, indicating that L reduction makes Jak/STAT signaling harmful to eye development. In addition, our genetic data suggest that TORC1 signaling is downregulated upon L reduction, supporting the idea that L regulates eye development through regulation of TORC1 activity. Similar to L reduction, decreasing TORC1 signaling by dTOR overexpression results in ectopic upd expression and apoptosis. A novel finding from our data is that dysregulated TORC1 signaling regulates the expression of upd and the function of the Jak/STAT signal pathway in Drosophila eye development.     

Hop cleavage and function in granzyme B-induced apoptosis

Granzyme B (GzmB) is a cytotoxic protease found in the granules of natural killer cells and cytotoxic T lymphocytes. GzmB cleaves multiple intracellular protein substrates, leading to caspase activation, DNA fragmentation, cytoskeletal instability, and rapid induction of target cell apoptosis. However, no known individual substrate is required for GzmB to induce apoptosis. GzmB is therefore thought to initiate multiple cell death pathways simultaneously to ensure the death of target cells. We previously identified Hop (Hsp70/Hsp90-organizing protein) as a GzmB substrate in a proteomic survey (Bredemeyer, A. J., Lewis, R. M., Malone, J. P., Davis, A. E., Gross, J., Townsend, R. R., and Ley, T. J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 11785-11790). Hop is a co-chaperone for Hsp70 and Hsp90, which have been implicated in the negative regulation of apoptosis. We therefore hypothesized that Hop may have an anti-apoptotic function that is abolished upon cleavage, lowering the threshold for GzmB-induced apoptosis. Here, we show that Hop was cleaved directly by GzmB in vitro and in cells undergoing GzmB-induced apoptosis. Expression of the two cleavage fragments of Hop did not induce cell death. Although cleavage of Hop by GzmB destroyed Hop function in vitro, both cells overexpressing GzmB-resistant Hop and cells with a 90-95% reduction in Hop levels exhibited unaltered susceptibility to GzmB-induced death. We conclude that Hop per se does not set the threshold for susceptibility to GzmB-induced apoptosis. Although it is possible that Hop may be cleaved by GzmB as an "innocent bystander" during the induction of apoptosis, it may also act to facilitate apoptosis in concert with other GzmB substrates.