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At least 19 recordsLinked to original sources
PubMed1999

Identification of SH2-bbeta as a potent cytoplasmic activator of the tyrosine kinase Janus kinase 2.

Janus kinases (JAKs) are cytoplasmic tyrosine kinases critical for signaling by growth hormone (GH) and many other ligands that bind to members of the cytokine receptor superfamily. SH2-Bbeta was previously identified as a JAK2-interacting protein that is tyrosyl phosphorylated in response to GH and other cytokines that activate JAK2. In this study, we examined whether SH2-Bbeta alters the activity of JAK2. SH2-Bbeta, when coexpressed with JAK2, significantly increased the tyrosyl phosphorylation of JAK2 and multiple other cellular proteins and stimulated the kinase activity of JAK2 by approximately 20-fold. Coexpression of SH2-Bbeta with JAK2 dramatically increased tyrosyl phosphorylation of signal transducer and activator of transcription (Stat)5B and Stat3, physiological substrates of JAK2. SH2-Bbeta(R555E) with a defective Src homology 2 domain was unable to stimulate JAK2 and JAK2-mediated tyrosyl phosphorylation of Stat5B and Stat3. More importantly, SH2-Bbeta enhanced GH-induced tyrosyl phosphorylation of endogenous JAK2 and ligand-induced tyrosyl phosphorylation of Stat5B by endogenous JAK2. In contrast, SH2-Bbeta did not potentiate the activation of other tyrosine kinases including the receptors for platelet-derived growth factor, epidermal growth factor, or nerve growth factor (TrkA), tyrosine kinases that also bind SH2-Bbeta. These data demonstrate that SH2-Bbeta is a potent cytoplasmic activator of JAK2 and is thereby expected to be an important cellular regulator of signaling by GH and other hormones and cytokines that activate JAK2.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Unexpected effects of FERM domain mutations on catalytic activity of Jak3: structural implication for Janus kinases.

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.

Adenosine Triphosphate↗
PubMed2002

Mechanisms involved in interleukin-15-induced suppression of human neutrophil apoptosis: role of the anti-apoptotic Mcl-1 protein and several kinases including Janus kinase-2, p38 mitogen-activated protein kinase and extracellular signal-regulated kinases-1/2.

Interleukin-15 (IL-15) is a pro-inflammatory cytokine known as a general inhibitor of apoptosis, which possesses potential therapeutic properties. Although IL-15 was previously found to be a human neutrophil agonist, its mode of action remains unknown. Herein, we were interested in elucidating the mechanisms by which it delays neutrophil apoptosis. IL-15 was found to induce tyrosine phosphorylation events and to prevent loss of the anti-apoptotic Mcl-1 protein expression. Using different signal transduction inhibitors, we found that Janus kinase (Jak)-2, Jak-3, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK), but not G proteins, are involved in IL-15-induced suppression of apoptosis. Furthermore, we found that IL-15 activates Jak-2, p38 MAPK and ERK-1/2, but, unlike granulocyte macrophage-colony-stimulating factor (GM-CSF), it does not activate signal transducer and activator of transcription (STAT)-5a/b. We conclude that IL-15 delays neutrophil apoptosis via several pathways, and that Mcl-1 and several kinases contribute to this. We also conclude that, unlike GM-CSF, IL-15 does not activate the Jak-2/STAT-5 pathway found to be important in neutrophil signaling.

Apoptosis↗
PubMed2004

Dual targeting of Bruton's tyrosine kinase and Janus kinase 3 with rationally designed inhibitors prevents graft-versus-host disease (GVHD) in a murine allogeneic bone marrow transplantation model.

The purpose of the present study was to evaluate the effectiveness of targeting Bruton's tyrosine kinase (BTK) with a specific BTK inhibitor, alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13), for prevention of acute fatal graft-versus-host disease (GVHD) in a murine model of allogeneic bone marrow transplantation (BMT). Vehicle-treated control C57BL/6 mice receiving bone marrow/splenocyte grafts from allogeneic BALB/c donors developed severe multi-organ acute GVHD and died after a median survival time (MST) of 40 d. LFM-A13 treatment (25 mg/kg/d) significantly prolonged the MST of the BMT recipients to 47 d. The probability of survival at 2 months after BMT was 2 +/- 2% for vehicle-treated control mice and 22 +/- 6% for mice treated with LFM-A13 (P = 0.0008). Notably, the combination regimen of LFM-A13 plus the standard anti-GVHD drug methotrexate (MTX) (10 mg/m(2)/d) was more effective than LFM-A13 alone, while the combination regimen of LFM-A13 plus the novel anti-GVHD drug JANEX-1 (60 mg/kg/d), targeting Janus kinase 3, was more effective than LFM-A13, JANEX-1 or MTX alone. More than 70% of recipients receiving this most effective GVHD prophylaxis (LFM-A13 + JANEX-1) remained alive throughout the 80-d observation period with an MST of >80 d. Taken together, these results indicate that targeting BTK with the chemical inhibitor LFM-A13 may attenuate the severity of GVHD, especially when it is combined with other anti-GVHD drugs, such as MTX and JANEX-1.

Acute Disease↗
PubMed2004

Characterization of a peptide inhibitor of Janus kinase 2 that mimics suppressor of cytokine signaling 1 function.

Positive and negative regulation of cytokines such as IFN-gamma are key to normal homeostatic function. Negative regulation of IFN-gamma in cells occurs via proteins called suppressors of cytokine signaling (SOCS)1 and -3. SOCS-1 inhibits IFN-gamma function by binding to the autophosphorylation site of the tyrosine kinase Janus kinase (JAK)2. We have developed a short 12-mer peptide, WLVFFVIFYFFR, that binds to the autophosphorylation site of JAK2, resulting in inhibition of its autophosphorylation as well as its phosphorylation of IFN-gamma receptor subunit IFNGR-1. The JAK2 tyrosine kinase inhibitor peptide (Tkip) did not bind to or inhibit tyrosine autophosphorylation of vascular endothelial growth factor receptor or phosphorylation of a substrate peptide by the protooncogene tyrosine kinase c-src. Tkip also inhibited epidermal growth factor receptor autophosphorylation, consistent with the fact that epidermal growth factor receptor is regulated by SOCS-1 and SOCS-3, similar to JAK2. Although Tkip binds to unphosphorylated JAK2 autophosphorylation site peptide, it binds significantly better to tyrosine-1007 phosphorylated JAK2 autophosphorylation site peptide. SOCS-1 only recognizes the JAK2 site in its phosphorylated state. Thus, Tkip recognizes the JAK2 autophosphorylation site similar to SOCS-1, but not precisely the same way. Consistent with inhibition of JAK2, Tkip inhibited the ability of IFN-gamma to induce an antiviral state as well as up-regulate MHC class I molecules on cells at a concentration of approximately 10 microM. This is similar to the K(d) of SOCS-3 for the erythropoietin receptor. These data represent a proof-of-concept demonstration of a peptide mimetic of SOCS-1 that regulates JAK2 tyrosine kinase function.

Animals↗
PubMed2006

Janus kinase 2 influences growth hormone receptor metalloproteolysis.

GH signals through the GH receptor (GHR), a cytokine receptor superfamily member that couples to the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2). In addition to its role in signaling, we recently implicated JAK2 in the regulation of cell surface GHR abundance by modulation of GHR trafficking and mature GHR stability. GHR is a target for constitutive and inducible metalloprotease-mediated cleavage that alters surface GHR levels and can modulate GH signaling. We previously found that metalloprotease cleavage of GHR is dramatically lessened in fibroblasts derived from mice with targeted deletion of the zinc-binding domain of TNF-alpha-cleaving enzyme [TACE; ADAM17 (a disintegrin and metalloprotease)], implicating this transmembrane ectoenzyme as a GHR metalloprotease. In this study we used a human fibrosarcoma reconstitution system to compare the effects of RNA interference-mediated knockdown of TACE vs. a related metalloprotease, ADAM10. We found that TACE knockdown dramatically reduced both the pace and the degree of inducible GHR proteolysis and augmented the abundance of mature GHR, suggesting a role for TACE in constitutive receptor proteolysis in this system as well. Notably, ADAM10 knockdown also reduced inducible GHR proteolysis, although to a lesser degree than TACE knockdown, suggesting a contribution from this metalloprotease also. To determine whether JAK2 affects GHR proteolysis, we compared JAK2-deficient vs. JAK2-replete cells and found that phorbol 12-methyl 13-acetate-induced GHR proteolysis was significantly diminished in cells that lacked JAK2. Reconstitution with a GHR mutant that lacks the box 1 region (which mediates JAK2 association) resulted in phorbol 12-methyl 13-acetate-induced proteolysis similar in degree to that of the wild-type GHR in JAK2-deficient cells. Introduction of JAK2 did not affect the proteolysis of this box 1-deleted GHR, suggesting GHR-JAK2 association is required for JAK2 to affect GHR proteolysis. Additionally, the inhibitory effect of anti-GHRext-mAb, a conformation-sensitive GHR antibody, on receptor proteolysis was lost in cells that lacked JAK2. Our data indicate that the susceptibility of GHR to proteolysis is substantially affected by JAK2, suggesting yet another role for this kinase in determining GH sensitivity.

ADAM Proteins↗
PubMed2001

PRL-induced ERalpha gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase.

In the rat corpus luteum of pregnancy, PRL stimulation of ER expression is a prerequisite for E2 to have any luteotropic effect. Previous work from our laboratory has established that PRL stimulates ERalpha expression at the level of transcription and that the transcription factor Stat5 (signal transducer and activator of transcription 5) mediates this stimulation. Since it is well established that PRL activates Stat5 through the tyrosine kinase, Janus kinase 2 (Jak2), the role of Jak2 in PRL regulation of ERalpha expression was investigated. In primary luteinized granulosa cells, the general tyrosine kinase inhibitors, genistein and AG18, and the Jak2 inhibitor, AG490, prevented PRL stimulation of ERalpha mRNA levels, suggesting that PRL signaling to the ERalpha gene requires Jak2 activity. However, using an antibody that recognizes the tyrosine-phosphorylated forms of both Stat5a and Stat5b (Y694/Y699), it was found that AG490 could inhibit PRL-induced Stat5a phosphorylation only and had little or no effect on Stat5b phosphorylation. These effects of AG490 were confirmed in COS cells overexpressing Stat5b. Also in COS cells, a kinase-negative Jak2 prevented PRL stimulation of ERalpha promoter activity and Stat5b phosphorylation while a constitutively active Jak2 could stimulate both in the absence of PRL. Furthermore, kinase-negative-Jak2, but not AG490, could inhibit Stat5b nuclear translocation and DNA binding. Therefore, it seems that in the presence of AG490, Stat5b remains phosphorylated, is located in the nucleus and capable of binding DNA, but is apparently transcriptionally inactive. These findings suggest that PRL may activate a second tyrosine kinase, other than Jak2, that is capable of phosphorylating Stat5b without inducing transcriptional activity. To investigate whether another signaling pathway is involved, the src kinase inhibitor PP2 and the phosphoinositol-3 kinase inhibitor (PI3K), LY294002, were used. Neither inhibitor alone had any major effect on PRL regulation of ERalpha promoter activity or on PRL-induced Stat5b phosphorylation. However, the combination of AG490 and LY294002 largely prevented PRL-induced Stat5b phosphorylation. These findings indicate that PRL stimulation of ERalpha expression requires Jak2 and also that PRL can induce Stat5b phosphorylation through two tyrosine kinases, Jak2 and one downstream of PI3K. Furthermore, these results suggest that the role of Jak2 in activating Stat5b may be through a mechanism other than simply inducing Stat5b phosphorylation.

Animals↗
PubMed2004

Janus kinase 3 down-regulates lipopolysaccharide-induced IL-1 beta-converting enzyme activation by autocrine IL-10.

ProIL-1 beta processing by IL-1 beta-converting enzyme (ICE) and the subsequent release of mature IL-1 beta are highly regulated events in the monocyte/macrophage response to pathogens. This process occurs in a controlled way through the activation of the constitutively expressed 45-kDa ICE precursor (proICE). To characterize the signaling pathways involved in ICE regulation in human monocytes/macrophages, we analyzed ICE activation in the presence of specific inhibitors of classic signaling pathways. Although LPS-induced ICE activity was not significantly affected by interruption of extracellular signal-regulated kinase, p38 kinase, or phosphoinositol 3-kinase, Janus kinase 3 (JAK3) inhibition produced a significant dose-dependent enhancement of LPS-induced ICE activity. Support for the inhibitory role of JAK3 was shown by the fact that IL-4 (which uses JAK1 and JAK3 signaling) suppressed LPS-induced ICE activity and by the finding that JAK3 knockout macrophages have increased LPS-induced ICE activation. To understand how JAK3 down-regulates LPS-induced ICE activity in monocytes, we hypothesized that JAK3 signaling enhances IL-10 production. In support of this model we show that LPS-induced IL-10 expression was synchronous with ICE deactivation, IL-4 induced the release of IL-10, exogenous IL-10 suppressed LPS-induced ICE activity, a neutralizing IL-10 Ab increased LPS-induced ICE activity, and, finally, JAK3 knockout macrophages displayed significantly reduced LPS-induced IL-10 production. These findings support a model in which JAK3 signaling enhances IL-10 production leading to down-regulation of ICE activation and suppression of IL-1 beta processing and release.

Animals↗
PubMed2003

Janus kinase 2 determinants for growth hormone receptor association, surface assembly, and signaling.

GH signaling depends on functional interaction of the GH receptor (GHR) and the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which possesses a C-terminal kinase domain, a catalytically inactive pseudokinase domain just N-terminal to the kinase domain, and an N-terminal half shown by us and others to harbor elements for GHR association. Computational analyses indicate that JAKs contain in their N termini ( approximately 450 residues) divergent FERM domains. FERM domains (or subdomains within them) in JAKS may be important for associations with cytokine receptors. For some cytokine receptors, JAK interaction may be required for receptor surface expression. We previously demonstrated that a JAK2 mutant devoid of its N-terminal 239 residues (JAK2-Delta1-239) did not associate with GHR and could not mediate GH- induced signaling. In this report we employ a JAK2-deficient cell line to further define N-terminal JAK2 regions required for physical and functional association with the GHR. We also examine whether JAK2 expression affects cell surface expression of the GHR. Our results suggest that FERM motifs play an important role in the interaction of GHR and JAK2. While JAK2 expression is not required for detectable surface GHR expression, an increased JAK2 level increases the fraction of GHRs that achieves resistance to deglycosylation by endoglycosidase H, suggesting that the GHR-JAK2 association may enhance either the receptor's efficiency of maturation or its stability. Further, we report evidence for the existence of a novel GH-inducible functional interaction between JAK2 molecules that may be important in the mechanism of GH-triggered JAK2 signaling.

Animals↗
PubMed2006

Interleukin (IL)-4 and IL-13 up-regulate monocyte chemoattractant protein-1 expression in human bronchial epithelial cells: involvement of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 and Janus kinase-2 but not c-Jun NH2-terminal kinase 1/2 signalling pathways.

The Th2 cytokines interleukin (IL)-4 and IL-13 and chemokine monocyte chemoattractant protein-1 (MCP-1) are significantly involved in bronchial hyperreactivity (BHR) and remodelling in allergic asthma. Although IL-4 and IL-13 can regulate a number of chemokines from bronchial epithelium, their regulatory effect on the expression of MCP-1 is as yet unproved. We aim to investigate the intracellular signalling mechanisms of IL-4 and IL-13 regulating the expression and secretion of MCP-1 from human bronchial epithelial cells. BEAS-2B cells, derived from a human bronchial epithelial cell line, were activated with or without IL-4 and/or IL-13 for different time intervals. MCP-1 gene expression and protein secretion were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Activation of signalling molecules p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and Janus kinase-2 (JAK-2) was accessed by Western blotting. IL-4 and IL-13 were found to up-regulate gene expression and significantly increase the release of MCP-1 from BEAS-2B cells. Both cytokines could activate p38 MAPK, ERK and JAK-2, but not JNK activity. Inhibition of p38 MAPK, ERK and JAK-2 activities by pretreating the cells with their corresponding inhibitors SB203580, PD98059 and AG490, respectively, significantly suppressed IL-4- and IL-13-induced MCP-1 production in BEAS-2B cells. Together, the above results illustrate that the activation of p38 MAPK, ERK and JAK-2 but not JNK is crucial for IL-4- and IL-13-induced MCP-1 release in human bronchial epithelial cells. Our findings may provide insight into the future development of more effective therapeutic agents for treating allergic asthma.

Blotting, Western↗
PubMed2004

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.

Cell Line↗
PubMed2003

Mechanism of Janus kinase 3-catalyzed phosphorylation of a Janus kinase 1 activation loop peptide.

A high-affinity IL-2 receptor requires two Janus protein tyrosine kinases (JAKs) for IL-2 signal transduction: JAK1 and JAK3. Since transphosphorylation of the two kinases is presumed to occur after receptor engagement we examined the phosphorylation by recombinant JAK3 of a peptide substrate corresponding to the JAK1 activation loop (KAIETDKEYYTVKD), which has two adjacent tyrosines. Mass spectral analysis of the enzymatically phosphorylated peptide showed that the second tyrosine was phosphorylated at a 30-fold greater rate than the first tyrosine. Moreover, no doubly phosphorylated peptide was detected by this analysis. Kinetic analysis of the reactions of singly phosphorylated JAK1 activation loop peptides showed that phosphorylating the first or second tyrosine decreased the k(cat)/K(m) for the phosphorylation of the other 115- and 26-fold, respectively. Singly changing each side chain of the KEYYTV portion of the peptide to a methyl group (alanine) yielded substrates comparable to the wild-type sequences in all cases except that of the first or second tyrosine, which showed a 153- or 70-fold drop in k(cat)/K(m), respectively. Using libraries of immobilized peptides with all 20 naturally occurring amino acids substituted for Y9 or T11 showed that the JAK3 tolerated substitution at T11 but prefers large hydrophobic amino acids at Y9. These results show that JAK3 does not act processively on the JAK1 activation loop in vitro and illustrate the role of Y9 in the recognition of the preferred site of phosphorylation which is Y10.

Amino Acid Substitution↗
PubMed2005

The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor.

JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an important intracellular mediator of cytokine signaling. Mutations of the JAK2 gene are associated with hematologic cancers, and aberrant JAK activity is also associated with a number of immune diseases, including rheumatoid arthritis. Accordingly, the development of JAK2-specific inhibitors has tremendous clinical relevance. Critical to the function of JAK2 is its PTK domain. We report the 2.0 A crystal structure of the active conformation of the JAK2 PTK domain in complex with a high-affinity, pan-JAK inhibitor that appears to bind via an induced fit mechanism. This inhibitor, the tetracyclic pyridone 2-tert-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-1, was buried deep within a constricted ATP-binding site, in which extensive interactions, including residues that are unique to JAK2 and the JAK family, are made with the inhibitor. We present a structural basis of high-affinity JAK-specific inhibition that will undoubtedly provide an invaluable tool for the further design of novel, potent, and specific therapeutics against the JAK family.

Adenosine Triphosphate↗
PubMed1994

Identification of interleukin-2 receptor-associated tyrosine kinase p116 as novel leukocyte-specific Janus kinase.

Janus tyrosine kinase (JAK) has recently been linked to signal transduction by cytokine receptors of the hematopoietin family. We have recently described a 116-kDa tyrosine kinase (p116) present in interleukin-2 (IL-2) receptor complexes in human YT cells that showed functional characteristics of a JAK kinase. These included receptor association, rapid and transient tyrosine phosphorylation kinetics in response to ligand, and in vitro autophosphorylating tyrosine kinase activity (Kirken, R. A., Rui, H., Evans, G. A., and Farrar, W. L. (1993) J. Biol. Chem. 268, 22765-22770). Here we extend these observations by demonstrating structural homologies between IL-2-modulated p116 and prolactin-modulated JAK2 in the rat T cell line Nb2. These include similar net charge as determined by nonequilibrium pH gradient electrofocusing and related primary structure based upon phosphopeptide mapping of V8 protease-digested hyperphosphorylated proteins. This putative JAK kinase underwent marked tyrosine phosphorylation in response to IL-2, IL-4, and IL-7, lymphoid growth factors that use the common IL-2 receptor gamma-chain, but not in response to prolactin. Furthermore, polyclonal antisera to JAK1, JAK2, or tyrosine kinase 2 did not recognize either rat or human p116. However, we identified the IL-2-modulated p116 as the recently cloned novel leukocyte Janus kinase, L-JAK, using an antiserum to a peptide corresponding to the COOH terminus of human L-JAK.

Animals↗
PubMed2005

Inhibition of the antigen-induced activation of rodent mast cells by putative Janus kinase 3 inhibitors WHI-P131 and WHI-P154 in a Janus kinase 3-independent manner.

We analyzed the effects of the Janus kinase 3 (Jak3)-specific inhibitor WHI-P131 (4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) and the Jak3/Syk inhibitor WHI-P154 (4-(3'-bromo-4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline) on the antigen-induced activation of mast cells. In the rat mast cell line RBL-2H3, both WHI-P131 and WHI-P154 inhibited the antigen-induced degranulation and phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK and c-Jun N-terminal kinase (JNK). The phosphorylation of Gab2, Akt and Vav was also inhibited by WHI-P131 and WHI-P154, indicating that these inhibitors suppress the activation of phosphatidylinositol 3-kinase (PI3K). In bone marrow-derived mast cells (BMMCs) from Jak3-deficient (Jak3-/-) mice, degranulation and activation of MAPKs were induced by the antigen in almost the same extent as in BMMCs from wild-type mice. In addition, the antigen-induced degranulation and activation of MAPKs were inhibited by WHI-P131 and WHI-P154 in both groups of BMMCs, indicating that these compounds inhibit a certain step except for Jak3. The antigen-induced increase in the activity of Fyn, a probable tyrosine kinase of Gab2, was also inhibited by WHI-P131 and WHI-P154 in RBL-2H3 cells. In BMMCs from Jak3-/- mice, the antigen stimulation induced tyrosine phosphorylation of Fyn, which was inhibited by WHI-P131, as well as in BMMCs from wild-type mice and in RBL-2H3 cells. These findings suggest that Jak3 does not play a significant role in the antigen-induced degranulation and phosphorylation of MAPKs, and that WHI-P131 and WHI-P154 inhibit the PI3K pathway by preventing the antigen-induced activation of Fyn, thus inhibiting the antigen-induced degranulation and phosphorylation of MAPKs in mast cells.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Mapping the kinase domain of Janus Kinase 3.

The utilization and impact of parallel synthesis on lead exploration around initial hit oxindole (1) are described. The emergent SAR, analogue design and functional impact will also be detailed.

Adjuvants, Immunologic↗
PubMed1998

Conditional activation of Janus kinase (JAK) confers factor independence upon interleukin-3-dependent cells. Essential role of Ras in JAK-triggered mitogenesis.

Cytokines play crucial roles in the growth and differentiation of hematopoietic cells. They bind to specific cell membrane receptors that usually do not possess intrinsic protein-tyrosine kinase activity. Janus kinases (JAKs) are cytoplasmic protein-tyrosine kinases that physically interact with intracellular domains of the cytokine receptors and have been implicated in playing important roles in signal transduction triggered by the cytokine-cytokine receptor interaction. However, it is still uncertain whether JAK activation alone suffices to induce cell proliferation. In this work, we modified Tyk2, a member of the JAK family, by adding a membrane localization sequence and a chemical dimerizer (coumermycin)-dependent dimerization sequence. The modified Tyk2 was activated in a coumermycin-dependent manner, and the activated Tyk2 conferred cytokine independence upon interleukin-3-dependent pro-B lymphoid cells. This cytokine-independent proliferation was completely inhibited by dominant-negative Ras. These results indicate that activation of JAK through membrane-proximal dimerization is sufficient to induce cell cycle progression and that Ras is essentially involved in JAK-triggered mitogenesis.

Aminocoumarins↗
PubMed1997

Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways.

We have recently shown that leptin mimicks insulin effects on glucose transport and glycogen synthesis through a phosphatidylinositol-3 (PI) kinase dependent pathway in C2C12 myotubes. The aim of the present study was to identify the signalling path from the leptin receptor to the PI-3 kinase. We stimulated C2C12 myotubes with insulin (100 nmol/l, 5 min) or leptin (0.62 nmol/l, 10 min) and determined PI-3 kinase activity in immunoprecipitates with specific non-crossreacting antibodies against insulin-receptor substrate (IRS 1/IRS 2) and against janus kinase (JAK 1 and JAK 2). While insulin-stimulated PI-3 kinase activity is detected in IRS-1 and IRS-2 immunoprecipitates, leptin-stimulated PI-3 kinase activity is found only in IRS-2 immunoprecipitates, suggesting that the leptin signal to PI-3 kinase occurs via IRS-2 and not IRS-1. Leptin-, but not insulin-stimulated PI-3 kinase activity is also detected in immunoprecipitates with antibodies against JAK-2, but not JAK-1. The data suggest that JAK-2 and IRS-2 couple the leptin signalling pathway to the insulin signalling chain. Since we have also detected leptin-stimulated tyrosine phosphorylation of JAK-2 and IRS-2 in C2C12 myotubes it can be assumed that leptin activates JAK-2 which induces tyrosine phosphorylation of IRS-2 leading to activation of PI-3 kinase. As we could not detect the long leptin receptor isoform in C2C12 myotubes we conclude that this signalling pathway is activated by a short leptin receptor isoform.

Animals↗