JAK2 Kinase Activity Research Articles

Degrasyn-Induced Trafficking of BCR-ABL as a Novel Mechanism of Kinase Inactivation.

Inhibitors that inactivate specific tyrosine kinases have proven to be a very effective form of therapy of many leukemias and hematopoetic disorders. Most inhibitors function by competing for the ATP-binding pocket or by preventing association with protein substrates. However, clinical and molecular studies have shown that small changes in the structure of the target kinase (point mutations, post-translational modification) affect inhibitor binding affinities, resulting in resistance to this class of inhibitor. Therefore, development of agents that reduce the activity of leukemogenic kinases through alternate mechanisms are of great interest. We previously described a novel class of compounds, termed degrasyns, which reduced BCR-ABL (and Jak2) kinase activity through a unique mechanism. Degrasyn treatment of CML or BaF3 cells expressing wild-type or mutant (T315I) BCR-ABL resulted in a reduction of cytoplasmic BCR-ABL protein levels and loss of downstream signaling without a direct effect on BCR-ABL enzymatic activity. Biochemical studies demonstrated that degrasyn induced a rapid translocation of BCR-ABL from the cytosol to the cytoskeletal fraction (complete within 60 min) and this event correlated with loss of BCR-ABL signaling and initiation of apoptosis. A GFP-fusion protein composed of wild-type or T315I mutant BCR-ABL demonstrated that degrasyn induced high density translocation to the cytoskeletal fraction, as determined by direct fluorescence imaging. Translocation was specific for BCR-ABL and not other kinases (except Jak2) or signaling proteins. Translocation correlated with degrasyn-mediated tyrosine phosphorylation of a subset of specific proteins, including the tyrosine kinase Lyn, in the insoluble cellular fraction. CML cells expressing high levels of Lyn were more sensitive to degrasyn-mediated apoptosis suggesting that Lyn plays a role in degrasyn activity. Together these results suggest that degrasyn inactivates BCR-ABL by inducing its translocation to a cellular compartment that prevents its participation in oncogenic signaling. Degrasyn-induced BCR-ABL translocation was not effected by mutations that block dasatinib and imatinib activity and shows greater activity against some forms of imatinib resistance (Lyn overexpression). Animal studies demonstrated that degrasyn has anti-leukemic activity and functions through a unique mechanism of action. Development of inhibitors with this mode of action may be of significance for CML patients that fail therapy with ATP-binding pocket-directed tyrosine kinase inhibitors.

JAK2 Activation Stimulates Homologous Recombination and Genetic Instability.

The JAK2 V617F mutation is a recurrent mutation frequently observed in classical myeloproliferatives syndromes such as polycythemia vera (PV) (90%), essential thrombocythemia (ET) and myelofibrosis (MF) (50%). Even if JAK2 V617F is common to these three pathologies, bi-alllic mutation of JAK2V617F have been observed in PV patients due to a loss of heterozygoty following a mitotic recombination event. Moreover, mutations in the receptor of thrombopoietin (c-mpl) have also been identified in MF and in some cases associated with the JAK2V671F mutation. These observations led us to hypothesize that initial mutation of JAK2 can stimulate others abnormalities, in particular genetic abnormalities, which could strengthen the kinase activity of JAK2. The deregulation of the DNA double-strand breaks repair mechanism, homologous recombination (HR), could be involved in the appearance of these new genetic abnormalities. Indeed, mutations in genes controlling HR lead to genomic instability, such as punctual mutation, deletion, loss of heterozygoty and chromosomal rearrangement. As a consequence, we investigated in this study the impact of JAK2 activation either by mutation (JAK2V617F) or by erythropoietin (EPO) on HR and genetic instability. First, we introduced a specific substrate of HR in the genome of Ba/F3 cell line and we measured HR after infecting cells with virus containing either JAK2wt or JAK2V617F. In the presence of EPO, JAK2V617F and at a lesser extend JAK2wt induces a 20 and 5-fold HR increase, respectively, compared to Ba/F3 control cells. In good agreement, in spontaneous condition, we observed an increase in nuclear foci of the key protein involved in this process: RAD51 and in its partner: BRCA1. A spontaneous RAD51 foci formation was also found in CD34+ cells from PV and MF patients compared with CD34+ cells from cytapheresis suggesting that an HR increase also occurs in patients. Consequently, we observed a 40% increased in sister chromatid exchange in JAK2V617F -expressing cells compared to JAK2wt-expressing cells or Ba/F3 control cells. Moreover, increases in centrosome and ploidy abnormalities were also detected in JAK2V617F-expressing cells. Finally, we found a 100-fold and 10 fold increase in mutagenesis at HPRT and Na/K ATPase loci, respectively. This result suggests that JAK2V617F induces more deletion event than punctual mutation. This work highlights a new molecular mechanism of regulation of HR by activation of JAK2 and especially by JAK2V617F. It might also give key to understand how a single mutation can give rise to different pathologies. Further works are currently under investigation to determine how JAK2V617F -induced an hyper-recombination phenotype.

Targeting the JAK2 Kinase in Hematological Malignancies.

JAK2 is an intracellular protein tyrosine kinase whose dysregulation has been implicated in leukemia, lymphoma, and myeloproliferative disorders (MPD). Increased kinase activity of JAK2, caused by point mutation of the JH2 autoinhibitory region or formation of JAK2 fusion proteins, causes increased activation of downstream signaling pathways affecting cell differentiation, proliferation, migration, and apoptosis. Through the use of CLIMB™, our proprietary drug discovery process, the published JAK2 crystal structure was used to build several models that were then used as a substrate for in silico docking of 2.3 million virtual small molecule compounds to generate a subset of leads based on calculated binding energies. These leads were further screened using a number of in silico physicochemical and ADMET prediction algorithms to determine “druggable” leads which were most likely to be successful in a biological context. Lead JAK2 inhibitor candidates exhibit low nanomolar IC50 activity against the JAK2 and JAK2 V617F mutant enzymes. Cancer cell lines expressing either the wild-type or mutant JAK2 enzyme demonstrate sensitivity to these inhibitors resulting in IC50 values in low micromolar to nanomolar range. Consistent with the inhibition of the JAK2 enzyme, activity of downstream signaling partners are severely decreased. The phosphorylation level of STAT5, a downstream modulator of JAK2 signaling, in treated HEL cell lysates was analyzed by western blot analysis. These results showed that lead JAK2 candidates caused an inhibition of STAT5 phosphorylation at a low nanomolar EC50. This series of compounds are currently being tested in in vivo xenograft models. Evaluation of lead candidates in biochemical assays against the hERG and CYP450 enzymes showed that these compounds have little inhibitory activity against these enzymes. SuperGen's lead selective JAK2 inhibitors exhibit potent inhibition of wild-type and mutant JAK2 kinase activity translating into potent inhibition of cellular signaling pathways and cancer cell proliferation.

The JAK2 Kinase Inhibitor LS104 Induces Growth-Arrest and Apoptosis in JAK2V617F Positive Cells.

The JAK2V617F-mutation (V617F) is a novel, highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, the V617F mutation confers cytokine independent growth of Ba/F3 cells expressing erythropoietin receptor (EpoR) and constitutive activation of the JAK2 kinase and of the JAK-STAT pathway. In a murine bone-marrow transplant model the V617F-mutation alone is sufficient to induce a polycythemia vera-like phenotype. Therefore, mutant JAK2 kinase is a promising target for kinase inhibitor development. In this report, we characterize the small molecule LS104 (previously CR4; Grunberger et al., Blood 2003) as a novel non-ATP-competitive JAK2V617F kinase inhibitor. Ba/F3 cells stably transfected with EpoR and either the V617F-mutant (Ba/F3-EpoR-VF) or wildtype JAK2 (Ba/F3-EpoR-WT) were treated with LS104. Apoptosis assays as well as immunoblotting of JAK2 and of downstream signalling pathways were performed. The effects of LS104 on kinase activity were determined in an in vitro JAK2 kinase assay. A combination of LS104 with JAK-inhibitor I, which acts via the ATP-binding site, was tested in apoptosis assays. Finally, growth of endogenous erythroid colonies (EECs) obtained from patients with V617F-positive MPD was tested upon addition of LS104. LS104 selectively and dose dependently induced apoptosis in Ba/F3-EpoR-VF cells as compared to Ba/F3-EpoR-WT control cells. By immunoblotting we found inhibition of JAK2 autophosphorylation and of downstream targets as STAT5, AKT and ERK upon treatment with LS104. Activation of these targets by JAK2 was confirmed in experiments using JAK2 siRNA. The IC50 for JAK2 in an in vitro kinase assay using LS104 was <5μM, and this effect was not reversible using increased ATP-doses. Combination treatment of Ba/F3-EpoR-VF cells using LS104 plus JAK-inhibitor I lead to significantly increased apoptosis as compared to each substance alone. Furthermore, we observed 89% inhibition of in vitro formation of EECs at 10μM LS104 whereas growth of CFU-GM obtained from normal controls was virtually unaffected. Taken together, our data show that LS104 specifically inhibits JAK2 kinase activity and JAK2 downstream signals and thereby induces apoptosis in V617F-positive cells. Our data suggest, that LS104 either irreversibly binds to the ATP-binding site or acts as a substrate kinase inhibitor and may be combined with ATP-competitive JAK2 inhibitors to enhance treatment efficacy. Growth of EECs from patients with MPD is shown to be significantly suppressed by LS104. Based on these data, a phase I/II clinical trial of LS104 for patients with JAK2V617F-positive MPD has been initiated recently.

Adaptor Protein Lnk Negatively Regulates Mutant MPL and JAK2 Alleles Associated with Myeloproliferative Disorders

Activating mutations in the cytokine receptor/JAK2 signaling axis are found at high frequency in myeloproliferative disorders (MPD). Lnk, an SH2-containing adaptor protein, is a negative regulator of several hematopoietic cytokine receptors including MPL and EpoR. Here, we assessed whether Lnk can attenuate the activity of mutant MPLW515L, JAK2V617F and JAK2K539L found in MPD patients. Lnk overexpression in Ba/F3-MPLW515L cells inhibited cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhanced proliferation. Lnk-mediated growth inhibition was associated with downregulation of JAK/STAT, MAPK and PI3K signaling pathways. Similarly, Lnk inhibited cytokine-independent growth conferred by JAK2V617F and JAK2K539L in Ba/F3-EpoR cells. Following thrombopoietin stimulation, Lnk became tyrosyl-phosphorylated and associated with activated wild-type (WT) MPL and MPLW515L at the plasma membrane of Ba/F3 cells. An SH2 mutant Lnk (R392E) failed to bind and inhibit WT MPL and MPLW515L, demonstrating that the SH2 domain is essential for Lnk down-modulation of the receptors. The Lnk-MPL interaction was also detected with endogenously expressed proteins from cultured bone marrow cells. A series of C-terminally truncated Lnk constructs were used to determine which Lnk regions are required for Lnk inhibition of JAK2. Experiments in 293T cells indicated that Lnk SH2 domain binds to phosphorylated JAK2. In addition, other Lnk regions associated with non-phosphorylated JAK2, and these interactions were critical for Lnk inhibition of JAK2V617F and JAK2K539L constitutive activation. Our data suggest a model wherein Lnk downregulation of the receptor/JAK2 signaling involves two mechanisms; one is inhibition of the cytokine receptor utilizing JAK2, the second is direct suppression of JAK2 kinase activity. Furthermore, while the receptor mediated inhibition requires the SH2 domain, direct inhibition of JAK2 mutants, V617F and K539L, relies on other Lnk domains. Further elucidating the molecular mechanisms underlying Lnk inhibition of signaling pathways abnormally activated by oncogenic alleles, will provide insight into the pathogenesis of MPD and may have therapeutic value.

A Selective and Potent Oral Inhibitor of the JAK2 Tyrosine Kinase Reverses Polycythemia and Leukocytosis Induced by JAK2 V617F in a Mouse Model.

A somatic mutation (V617F) in the pseudokinase domain of the non-receptor tyrosine kinase JAK2 is found in virtually all patients with the myeloproliferative disease (MPD) polycythemia vera (PV), and about half of those with essential thrombocythemia and chronic idiopathic myelofibrosis. When expressed in mice by retroviral bone marrow transduction and transplantation, JAK2 V617F but not JAK2 wild-type induces leukocytosis with neutrophilia and recapitulates the entire erythroid phenotype of PV, with polycythemia, reticulocytosis, low plasma Epo, and endogenous erythroid colonies (Zaleskas et al., PLoS ONE 2006; 1:e18; Wernig et al., Blood 2006; 107:4274; Lacout et al., Blood 2006; 108:1652). By analogy to BCR-ABL in chronic myeloid leukemia, JAK2 V617F is therefore a rational target for therapy in PV and other JAK2 V617F+ MPDs. Previous studies with a nonselective and parenteral tyrphostin JAK2 inhibitor suggested that short-term inhibition of JAK2 could decrease reticulocytosis in recipients of JAK2 V617F-transduced marrow (Zaleskas et al., op cit.). Here, we studied one of a novel series of inhibitors of JAK2, AZ-01, which potently inhibited JAK2 kinase activity (IC50 < 1nM) and suppressed proliferation of cell lines carrying the JAK2 V617F mutation. We treated cohorts of Balb/c mice with JAK2 V617F-induced polycythemia and leukocytosis with AZ-01 at 10 mg/kg by oral gavage twice daily, or with vehicle alone. After 28 days of treatment, AZ-01-treated mice had significant reductions in hematocrit, hemoglobin, reticulocytes, and splenomegaly, as well as normalization of peripheral blood leukocyte counts (Figure 1). These results demonstrate that oral administration of a potent and selective JAK2 inhibitor can reverse the hematopoietic abnormalities of JAK2 V617F-induced MPD in mice, and validate JAK2 as a target for therapy of human JAK2 V617F+ MPD. [Display omitted] An orally available JAK2 inhibitor decreases the leukocyte count, hematocrit, and reticulocyte count in mice with JAK2 V617F-induced MPD. Leukocyte count (left), hematocrit (center) and reticulocyte counts (right) of cohorts of mice with JAK2 V617F-induced MPD treated with vehicle or drug at 10 mg/kg BID. Blood counts were assessed at 15 and 28 days of treatment. Decreases in d28 hematocrit and retic count in the drug-treated cohorts were significant (p=0.008 and p<0.0001, respectively, t-tests).

JAK kinases control IL-5 receptor ubiquitination, degradation, and internalization

IL-5, IL-3, and GM-CSF are related hematopoietic cytokines, which regulate the function of myeloid cells and are mediators of the allergic inflammatory response. These cytokines signal through heteromeric receptors containing a specific alpha chain and a shared signaling chain, betac. Previous studies demonstrated that the ubiquitin (Ub) proteasome degradation pathway was involved in signal termination of the betac-sharing receptors. In this study, the upstream molecular events leading to proteasome degradation of the IL-5 receptor (IL-5R) were examined. By using biochemical and flow cytometric methods, we show that JAK kinase activity is required for betac ubiquitination and proteasome degradation but only partially required for IL-5R internalization. Furthermore, we demonstrate the direct ubiquitination of the betac cytoplasmic domain and identify lysine residues 566 and 603 as sites of betac ubiquitination. Lastly, we show that ubiquitination of the betac cytoplasmic domain begins at the plasma membrane, increases after receptor internalization, and is degraded by the proteasome after IL-5R internalization. We propose an updated working model of IL-5R down-regulation, whereby IL-5 ligation of its receptor activates JAK2/1 kinases, resulting in betac tyrosine phosphorylation, ubiquitination, and IL-5R internalization. Once inside the cell, proteasomes degrade the betac cytoplasmic domain, and the truncated receptor complex is terminally degraded in the lysosomes. These data establish a critical role for JAK kinases and the Ub/proteasome degradation pathway in IL-5R down-regulation.

Activation Mechanisms of STAT5 by Oncogenic Flt3-ITD.

Mutations in the receptor tyrosine kinase Flt3 represent one of the most common known genetic lesions in AML. Internal tandem duplication (ITD) mutations clustered in the juxtamembrane domain are the most frequent and best characterized mutations found in Flt3. We and others have previously reported that oncogenic activation of Flt3 by ITD mutations activate aberrant signaling including activation of STAT5, up-regulation of STAT targets like-Pim and SOCS proteins and repression of the myeloid transcription factors Pu.1 and c/EBP-alpha (Mizuki et al., Blood 96: 3907–14, 2000; Mizuki et al., Blood 101: 3164–73, 2003). Earlier studies have observed activation of Src and Jak kinases by Flt3-ITD but the mechanisms of STAT5 activation by Flt3-ITD and the role of Src or Jak kinases in this process remains unclear. Using the specific small molecule inhibitors PP-1 and PP-2, we analyzed the role of Src kinases in Flt3-ITD mediated STAT5 activation. Treatment of Flt3-ITD expressing 32D cells with PP-1 and 2 had only marginal effect on Flt3-ITD induced STAT5 activation, which was due to a proportional inhibition of Flt3 kinase by these inhibitors. Similar results were obtained with AG490, reportedly a specific inhibitor of Jak2. However, use of cell a line deficient for all Src family kinases revealed a Src-independent activation of STAT5 by Flt3-ITD. Also, using cell lines deficient for Jak2 or Tyk2 we show that Flt3-ITD induced STAT5 activation independent of Jak2 or Tyk2 kinases. To rule out a possible redundant role of different Jak kinases in Flt3-ITD mediated STAT5 activation, we used SOCS1, a target of STAT5 and a potent molecular inhibitor of all Jak kinases. Surprisingly, retroviral transduction of 32D cells with SOCS1 inhibited IL-3 but not Flt3-ITD mediated proliferation of these cells. Similar results were obtained with SOCS3, another inhibitor of Jak kinases. Detailed signaling analyses of stable 32D cells co-expressing Flt3-ITD and SOCS1 revealed that activation of the STAT5 and up-regulation of STAT5 target gene Pim-2 by Flt3-ITD is completely resistant to SOCS1. In contrast, expression of SOCS1 severely inhibited IL-3 mediated STAT5 activation. Furthermore, using purified recombinant STAT5 and Flt3 kinase in in-vitro kinase assays, we show that STAT5 is indeed a direct target of Flt3. Taken together, our data show that Flt3-ITD directly activates STAT5 independent of Src or Jak kinases, providing the mechanistic basis of Flt3-ITD induced STAT5 activation. Furthermore, our finding that Flt3-ITD up-regulates SOCS proteins but activate STAT5 in a SOCS1 resistant manner may have important implications for Flt3-ITD-mediated modification of cytokine signaling.

Absence of JAK-2 (Val617Phe) Point Mutations in Human Plasma Cell Myeloma.

Multiple myeloma (MM) is a terminal differentiated B cell chronic lymphoproliferative disorder characterized by the latent accumulation of plasma cells with a low proliferative index and an extended life span in bone marrow or extramedullary tissues. While studies on neoplastic cells isolated directly from MM patients or on MM-derived cell lines demonstrated that IL-6 is a major growth and survival factor for initiation of signaling in MM cells in an autocrine or paracrine manner, some MM cells independent on extracellular signaling such as IL-6 have been reported, suggesting other mechanisms may be involved in signal transduction pathways in MM cells. Accumulating evidence showed that IL-6 induces intracellular signaling through members of the signal transducers and activators of transcription (STAT) family of proteins by activating the janus kinase(JAK) family of protein tyrosine kinases, which subsequently phosphorylate and activate cytoplasmic Stat proteins. Activated Stat proteins dimerize and translocate to the nucleus, where they bind to specific DNA response elements and induce expression of Stat-regulated genes. Our previous studies demonstrated that one of the Stat family proteins, Stat3, was constitutively activated (phosphorylated) in the majority of MM patients, suggesting constitutive activation of JAK-2 kinase activities. However, other mechanisms might be also responsible JAK2 constitutive activation in MM cells, in particular in IL-6 independent MM cells. A specific point mutation of JAK-2 kinase gene has been identified recently showing its presence in higher frequency of certain types of chronic myeloproliferative disorders. The point mutation yields constitutive activation of JAK-2 kinase activity in the absence of extracellular signaling and plays an important role in the pathogenesis of those disorders. In this study, we try to determine whether JAK-2 (Val617Phe) point mutation was present in MM patients and if so, what kind of role it may play in the pathogenesis of MM. JAK-2 (Val617Phe) point mutation was analyzed by using an allele-specific polymerase chain reaction followed by separation and detection with capillary electrophoresis. The test has a detection sensitivity of up to 200 picogram mutated DNA. Bone marrow aspirates with various degrees of involvement by MM (ranged10–80%) were selected and all the cases have been verified by morphologic examination and demonstrated to have positive immunoglobulin heavy or kappa light gene rearrangements. Bone marrow specimens from total 59 MM patients were tested for JAK2 (Val617Phe) mutation and none of any MM cases with the specific point mutation were identified (0/59). The results indicated that JAK2 (Val617Phe) mutation was very infrequency or absence in MM patients, despite the fact that constitutional activation of JAK2 kinase as well as Stat3 activation was commonly seen in MM patients. The study suggests that other yet unknown mechanisms may also involve JAK2 activation, particularly in IL-6 independent MM cells.

Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.

Human rhinovirus induces robust IP-10 release by monocytic cells, which is independent of viral replication but linked to type I interferon receptor ligation and STAT1 activation

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10), which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however, there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore, the involvement of resident airway immune cells, predominantly bronchoalveolar macrophages, in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard, our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release, which occurred in a time- and dose-dependent manner. Furthermore, HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus, this work supports a model, wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo.

Structural Basis for Phosphotyrosine Recognition by Suppressor of Cytokine Signaling-3

Suppressor of cytokine signaling (SOCS) proteins are indispensable negative regulators of cytokine-stimulated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathways. SOCS proteins (SOCS1-7 and CIS) consist of a variable N-terminal region, a central Src homology-2 (SH2) domain, and a C-terminal SOCS box. The N-terminal region in SOCS1 and SOCS3 includes the so-called kinase inhibitory region that has been shown to inhibit the catalytic activity of JAK2. Here, we present a crystal structure at 2.0 A resolution of the N-terminally extended SH2 domain of SOCS3 in complex with its phosphopeptide target on the cytokine receptor gp130. The structure reveals that major insertions in the EF and BG loops of the SOCS3 SH2 domain are responsible for binding to gp130 with high affinity and specificity. In addition, the structure provides insights into the possible mechanisms by which SOCS3 and SOCS1 inhibit JAK2 kinase activity.

Phosphorylation of JAK2 at Serine 523: a Negative Regulator of JAK2 That Is Stimulated by GrowthHormone and Epidermal Growth Factor

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling for multiple receptor tyrosine kinases and several G-protein-coupled receptors. In this study, phosphopeptide affinity enrichment and mass spectrometry identified serine 523 (Ser523) in JAK2 as a site of phosphorylation. A phosphoserine 523 antibody revealed that Ser523 is rapidly but transiently phosphorylated in response to growth hormone (GH). MEK1 inhibitor UO126 suppresses GH-dependent phosphorylation of Ser523, suggesting that extracellular signal-regulated kinases (ERKs) 1 and/or 2 or another kinase downstream of MEK1 phosphorylate Ser523 in response to GH. Other ERK activators, phorbol 12-myristate 13-acetate and epidermal growth factor, also stimulate phosphorylation of Ser523. When Ser523 in JAK2 was mutated, JAK2 kinase activity as well as GH-dependent tyrosyl phosphorylation of JAK2 and Stat5 was enhanced, suggesting that phosphorylation of Ser523 inhibits JAK2 kinase activity. We hypothesize that phosphorylation of Ser523 in JAK2 by ERKs 1 and/or 2 or other as-yet-unidentified kinases acts in a negative feedback manner to dampen activation of JAK2 in response to GH and provides a mechanism by which prior exposure to environmental factors that regulate Ser523 phosphorylation might modulate the cell's response to GH.

A functional Jak2 tyrosine kinase domain is essential for mouse development

Jak2 is a member of the Janus family of tyrosine kinases and is involved in cytokine signaling. As a part of a study to determine biological functions of Jak2, we used molecular modeling to identify W1038 as a residue that is critical for tyrosine kinase function. Mutation of W1038, in tandem with E1046, generates a dominant-negative form of the Jak2 protein. Mice that were engineered to express two copies of this dominant-negative Jak2 protein died in utero. Additionally, heterozygous mice expressing Jak2 with kinase activity that is moderately reduced when compared to wild-type activity appear phenotypically normal. Collectively, these data suggest that Jak2 kinase activity is essential for normal mammalian development.

Active Conformation of the Erythropoietin Receptor: RANDOM AND CYSTEINE-SCANNING MUTAGENESIS OF THE EXTRACELLULAR JUXTAMEMBRANE AND TRANSMEMBRANE DOMAINS

In the absence of erythropoietin (Epo) cell surface Epo receptors (EpoR) are dimeric; dimerization is mediated mainly by the transmembrane domain. Binding of Epo changes the orientation of the two receptor subunits. This conformational change is transmitted through the juxtamembrane and transmembrane domains, leading to activation of JAK2 kinase and induction of proliferation and survival signals. To define the active EpoR conformation(s) we screened libraries of EpoRs with random mutations in the transmembrane domain and identified several point mutations that activate the EpoR in the absence of ligand, including changes of either of the first two transmembrane domain residues (Leu(226) and Ile(227)) to cysteine. Following this discovery, we performed cysteine-scanning mutagenesis in the EpoR juxtamembrane and transmembrane domains. Many mutants formed disulfide-linked receptor dimers, but only EpoR dimers linked by cysteines at positions 223, 226, or 227 activated EpoR signal transduction pathways and supported proliferation of Ba/F3 cells in the absence of cytokines. These data suggest that activation of dimeric EpoR by Epo binding is achieved by reorienting the EpoR transmembrane and the connected cytosolic domains and that certain disulfide-bonded dimers represent the activated dimeric conformation of the EpoR, constitutively activating downstream signaling. Based on our data and the previously determined structure of Epo bound to a dimer of the EpoR extracellular domain, we present a model of the active and inactive conformations of the Epo receptor.

Nuclear Localization of STAT5A Modified with O-Linked N-Acetylglucosamine and Early Involution in the Mammary Gland of Hirosaki Hairless Rat

Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.

Effects of a Leukemia-Associated Gain-of-Function Mutation of SHP-2 Phosphatase on IL-3 Signaling.

Mutations in SHP-2 phosphatase that cause its hyper-activation have been identified in human leukemias, in particular, juvenile myelomonocytic leukemia (JMML) that is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce leukemia are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in catalytic-dependent and -independent manners and that overexpression (5-to-6 fold) of wild-type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through dephosphorylation of STAT5. This raised the possibility that SHP-2-associated JMML was not solely attributed to the increased catalytic activities of SHP-2 GOF mutants. The SHP-2 mutants must have gained other additional functions. To test this possibility, we investigated effects of a GOF mutation (E76K, the most frequent SHP-2 mutation seen in JMML) of SHP-2 on IL-3 signal transduction in great detail by comparing signaling activities of SHP-2 E76K to WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. E76K mutation in the N-terminal Src homology 2 domain significantly increased binding of the mutant SHP-2 to Grb2 and Gab2, two adaptor proteins coupling downstream Erk and PI3 kinase pathways to the proximity of the IL-3 receptor. As a result, IL-3-induced Erk and PI3 kinase pathways were highly activated by SHP-2 E76K mutation. In addition, Jak2 kinase activation was also markedly enhanced and due to the E76K mutation the substrate specificity of SHP-2 toward STAT5 was changed. Dephosphorylation of STAT5 by SHP-2 E76K was alleviated. These studies suggest that in addition to elevated catalytic activity, profound changes in protein-protein interaction capacities of GOF mutant SHP-2 play an important role in the pathogenesis of SHP-2-related leukemias.

Novel Quantitative Flow Cytometry-Based Signaling Assays Reveal a Potential Role for HSP90 Inhibitors in the Treatment of JAK2 Mutant-Positive Diseases.

The V617F mutation in the JAK2 tyrosine kinase, recently described in a majority of patients with myeloproliferative disorders (MPDs), confers growth factor independence in vitro and oncogenicity in mice. Therefore, targeted inhibition of mutant JAK2 kinase activity may be an effective strategy for treatment of MPD patients with this mutation. The ability to measure the activation status of JAK2 in patient samples will thus be of substantial value for monitoring therapeutic efficacy. We have developed quantitative flow cytometry-based assays for rapid and reproducible measurement of intracellular total and phosphorylated proteins of the canonical JAK/STAT pathway, as well as heat shock proteins (HSPs). In this study we examined the ability of these assays to detect altered levels of total and phosphorylated JAK/STAT signaling pathway components and HSP in a cell line (HEL) that is homozygous for the V617F JAK2 mutant. HEL cell cultures were incubated with 17AAG, a geldanamycin analog with clinical utility in a broad range of diseases. 17AAG exerts its inhibitory effect by binding to heat shock protein 90 (HSP90), preventing its chaperone association with client oncoproteins. AKT is among these client proteins and a component of the JAK/STAT pathway, representing a potential therapeutic target. 17AAG exposure reduced total AKT protein levels by 42%. 17AAG also inhibited mutant JAK2 activity by 66% and had a smaller effect (17%) on total JAK2 levels, suggesting that mutant JAK2 activation may rely on HSP90, either directly or through dependence on other client proteins. Exposure to 17AAG also reduced levels of P-STAT5 (50%) and, to a lesser extent, total STAT5 (27%). 17AAG-treated cells showed a 55% reduction in HSP90 levels and a 14% increase in HSP70 protein levels. JAK Inhibitor I (Calbiochem), a potent pan-JAK Inhibitor that blocks JAK1, JAK2, and JAK3 activity, caused reductions in P-JAK2 and P-STAT5 levels (29% and 26% decreases, respectively). However, the combining of JAK Inhibitor I with 17AAG did not result in an enhanced effect beyond what was observed with 17AAG treatment alone. Similar results were seen with AG490, a potent and selective JAK2 inhibitor. 17AAG caused a 40% decrease in viable cells after 18 hrs of treatment, compared with a 35% reduction for the pan-JAK inhibitor and a 20% decrease for AG490. Combining 17AAG with the pan-JAK inhibitor or AG490 caused only minor enhancement of these cytotoxic effects (46% and 41% reduction in cell viability, respectively). Our data support the potential utility of HSP90 inhibitors such as 17AAG in the development of small-molecule therapy for mutant JAK2 kinase-positive MPD. These results also show that flow cytometry-based assays for JAK/STAT signaling components and HSPs can be used to quantitatively monitor drug efficacy at the protein level in intact cells. These tests are likely to have broad clinical utility given the spectrum of diseases in which a pathogenic role for mutant JAK2 kinase is implicated.

RhoA, Rho kinase, JAK2, and STAT3 may be the intracellular determinants of longevity implicated in the progeric influence of obesity: Insulin, IGF-1, and leptin may all conspire to promote stem cell exhaustion

The aging process in higher mammals is increasingly being shown to feature a potentially substantial contribution from the longitudinal deterioration of normative stem cell dynamics seen with the passage of time. The precise mechanistic sequence producing this phenomenon is not entirely understood, but recent evidence has strongly implicated intracellular downstream effectors of endocrinologic pathways thought to be engaged by the obese state, specifically the insulin, IGF-1, and leptin signaling pathways. Among the intracellular effectors of these signals, a uniquely potent influence on stem cell dynamics may be attributable to Rho/ROCK, JAK kinase activity and STAT3 activity. In particular, it has already been shown that specific tyrosine kinase activities, such as that seen with Rho kinase, are presently thought to be associated with adverse health outcomes in numerous clinical contexts. Furthermore, the Rho GTPase is thought to be contributing to end-stage renal disease. However, in addition to its contribution to organ system dysfunction, the Rho/ROCK pathway has recently been shown to be activated by insulin and IGF-1, providing a tantalizing connection to nutrition and aging science. The JAK-STAT pathway, in contrast, has long been associated with pro-inflammatory cytokines, but has recently been implicated in leptin signaling as well. Importantly, JAK-STAT signaling has, similarly to Rho/ROCK signaling, been implicated as capable of accelerating stem cell proliferation. The implications of these recent determinations, in light of the recent finding of telomere attrition in humans associated with obesity, are that the intracellular determinants of aging may already be known, and the known common influence of these signaling elements on longitudinal stem cell dynamics is a pronounced induction of proliferation, an elevation that has been linked to the pathologic evolution of longitudinal organ-level dysfunction and the organismal-level physiologic decline seen with the inexorable passage of time. Besides the obvious utility for the management for human age-related dysfunction that investigation of pharmacologic inhibitors of these proteins would provide, interventions such as caloric restriction and possibly intermittent fasting may beneficially influence stem cell proliferation dynamics and reduce intracellular correlates of mitogenic drive. Integrating the findings present in the present body of research may reveal endocrinological states that are compatible with longevity, and will also provide novel insight into the specific proteomic determinants of age-related physiologic decline, ushering in a new epoch of medicine that fosters the management of the "pre-etiopathology" of chronic disease and disability of aging, therefore mitigating the suffering widely thought to be inherent in the latter stages of life.

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.