Role Of JAK2 Research Articles

Mechanism of apoptosis induced by IFN-α in human myeloma cells: Role of Jak1 and Bim and potentiation by rapamycin

Interferon-α (IFN-α) has been used for the last 20 years in the maintenance therapy of multiple myeloma (MM), though it is only effective in some patients. Congruent with this, IFN-α induces apoptosis in some MM cell lines. Understanding the mechanism of IFN-α-induced apoptosis could be useful in establishing criteria of eligibility for therapy. Here we show that IFN-α-induced apoptosis in the MM cell lines U266 and H929 was completely blocked by a specific inhibitor of Jak1. The mTOR inhibitor rapamycin mitigated apoptosis in U266 but potentiated it in H929 cells. IFN-α induced PS exposure, ΔΨ m loss and pro-apoptotic conformational changes of Bak, but not of Bax, and was fully prevented by Mcl-1 overexpression in U266 cells. IFN-α treatment caused the release of cytochrome c from mitochondria to cytosol and consequently, a limited proteolytic processing of caspases. Apoptosis induced by IFN-α was only slightly prevented by caspase inhibitors. Levels of the BH3-only proteins PUMA and Bim increased during IFN-α treatment. Bim increase and apoptosis was prevented by transfection with the siRNA for Bim. PUMA-siRNA transfection reduced electroporation-induced apoptosis but had no effect on apoptosis triggered by IFN-α. The potentiating effect of rapamycin on apoptosis in H929 cells was associated to an increase in basal and IFN-α-induced Bim levels. Our results indicate that IFN-α causes apoptosis in myeloma cells through a moderate triggering of the mitochondrial route initiated by Bim and that mTOR inhibitors may be useful in IFN-α maintenance therapy of certain MM patients.

Involvement of JAK2 upstream of the PI 3-kinase in cell–cell adhesion regulation by gastrin

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway has been implicated in cell transformation and proliferation. Besides aberrant cell proliferation, loss of cell–cell adhesion during epithelial–mesenchymal transition (EMT) is an important event which occurs during development of epithelial cancers. However, the role of JAK-dependent pathways in this process is not known. We analyzed the involvement of these pathways in the regulation of E-cadherin-dependent cell–cell adhesion by gastrin, a mitogenic factor for gastrointestinal (GI) tract. We identified JAK2/STAT3 as a new pathway in gastrin signaling. We demonstrated that JAK2 functions as an upstream mediator of the phosphatidylinositol 3 (PI 3)-kinase activity in gastrin signaling. Indeed, we observed a coprecipitation of both kinases and an inhibition of gastrin-induced PI 3-kinase activation when JAK2 activity is blocked. We also demonstrated that loss of cell–cell adhesion and the increase in cell motility induced by gastrin required the activation of JAK2 and the PI 3-kinase. Indeed, the modifications in localization of adherens junctions proteins and the migration, observed in gastrin-stimulated cells, were reversed by inhibition of both kinases. These results described the involvement of JAK2 in the modulation of cell–cell adhesion in epithelial cells. They support a possible role of JAK2 in the epithelial–mesenchymal transition which occurs during malignant development.

Prolactin signals through RUSH/SMARCA3 in the absence of a physical association with Stat5a.

Jak/Stat-mediated prolactin signal transduction culminates in the sequence-selective binding of Stat5a. However, in the absence of Stat-binding sites, a RUSH-binding element mediates the prolactin signal in the rabbit uteroglobin promoter. Speculation about the existence of a Jak/RUSH pathway prompted this series of experiments to examine potential interactions between RUSH and Stat5a. Profiles of Jak/Stat pathway-specific genes by RT-PCR showed that mRNA for Jak2 and Stat5a is expressed in the endometrium of estrous, progesterone-treated, and 5-day pseudopregnant rabbits. Interspecies microarrays showed that transcripts for Stat5a were present at equal concentrations in the endometrium regardless of hormone treatment. The absence of a physical interaction between RUSH and individual Stat proteins bound to enhancer sites was demonstrated with transcription factor interaction arrays. These studies confirm that transmission of the prolactin signal through RUSH occurs in the absence of a physical association with Stat5a. Although a strong physical interaction between RUSH and Egr-1 was identified with the same arrays, no Egr-1 consensus sites were found in the region of the uteroglobin promoter (-175/-80) that contains the authentic RUSH site. Because the major transducer molecules (Jak2, Stat5a) are activated by tyrosine phosphorylation, Western analysis of immunoprecipitated samples, and gel shift assays were used to show that tyrosine phosphorylation is required for RUSH-DNA binding. The precise role for Jak2 in this process remains undefined. By comparison, serine-threonine-specific protein phosphorylation had no effect on RUSH-DNA binding.

JAK2 – not just another kinase

Janus family kinases (JAKs) are cytoplasmic tyrosine kinases that transmit signals from receptors such as those for cytokines. The most important of these signaling pathways involves the signal transducers and activators of transcription (STAT) proteins, which are phosphorylated by JAK kinases and translocate to the nucleus to activate gene transcription. The erythropoietin receptor (EpoR) is a cytokine receptor that binds to and activates one member of the JAK family, JAK2. JAK2, upon activation, phosphorylates the EpoR on several tyrosine residues, as well as several other substrates including STAT5.Huang et al. [1xThe N-terminal domain of Janus kinase 2 is required for Golgi processing and cell surface expression of erythropoietin receptor. Huang, L.J. et al. Mol. Cell. 2001; 8: 1327–1338Abstract | Full Text | Full Text PDF | PubMed | Scopus (200)See all References[1] have recently identified another crucial role for JAK2 in Epo signaling – that of promoting cell surface expression of the EpoR. It is known that only a small fraction of the mature, glycosylated form of EpoR is expressed at the cell surface. The authors show that JAK2 binds to the immature form of EpoR in the endoplasmic reticulum and enhances its processing in the Golgi, leading to increased cell surface expression. They demonstrate that this effect is specific to JAK2, and requires residues located within one of its seven JAK homology (JH) domains, JH7. The levels of cellular JAK2 appear to be the limiting factor for EpoR expression because overexpression of JAK2 in cells expressing the endogenous protein further enhanced EpoR surface expression. Interestingly, a highly conserved juxtamembrane hydrophobic motif that was previously identified as being crucial for JAK2 activation [2xThe erythropoietin receptor cytosolic juxtamembrane domain contains an essential, precisely oriented, hydrophobic motif. Constantinescu, S.N. et al. Mol. Cell. 2001; 7: 377–385Abstract | Full Text | Full Text PDF | PubMed | Scopus (123)See all References[2] was not required for JAK2-mediated enhancement of EpoR processing. This indicates that distinct residues could participate in the binding of JAK2 to EpoR and, therefore, in EpoR cell surface expression and activation.JAK kinases, in contrast to most other signaling molecules, are prebound to the unactivated cytokine receptors. The report by Huang et al. provides evidence as to why this might occur. Because the EpoR–JAK2 complex is more efficiently processed, it results in the expression of EpoR prebound to JAK2 at the cell surface. This complex is then ready to signal upon activation by the ligand, leading to JAK2 transphosphorylation and activation of downstream signaling pathways. A recent study [3xSee all References[3] found similar results with the oncostatin M receptor using another JAK family kinase, JAK1, suggesting that this might be a general phenomenon.

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.

Prevention of apoptosis in J2E erythroid cells by erythropoietin: involvement of JAK2 but not MAP kinases

The J2E erythroid cell line, transformed by retroviral v-raf/v-myc oncogenes, proliferates and differentiates in response to erythropoietin. Here we show that J2E cells undergo apoptosis rapidly after serum withdrawal and that only erythropoietin of seven growth factors tested, could protect the cells from death. The role of JAK2 and MAP kinases in transmitting viability signals initiated by erythropoietin was examined in these cells. Despite constitutive raf kinase activity, phosphorylation of MAP kinases fell after serum withdrawal. However, an antisense oligonucleotide strategy revealed that JAK2, but not the MAP kinases, was involved in transmitting signals to maintain the viability of J2E cells. Several cell cycle proteins and transcription factors were also studied; although c-jun rose sharply during apoptosis, erythropoietin could not suppress this increase. It was concluded that erythropoietin-induced protection from apoptosis involved JAK2, but not MAP kinases or c-jun.

JAK2 is constitutively associated with c-Kit and is phosphorylated in response to stem cell factor.

Stem cell factor (SCF) interacts with the receptor tyrosine kinase c-Kit and has potent effects on hematopoiesis. We have examined the role of JAK2 in the SCF signal transduction pathway. JAK2 and c-Kit were constitutively associated, and treatment with SCF resulted in rapid and transient tyrosine phosphorylation of JAK2. Incubation of cells with JAK2 antisense oligonucleotides resulted in significant decreases in SCF-induced proliferation. These data suggest that JAK2 plays a role in SCF-induced proliferation.