Regulation of the Jak2 Tyrosine Kinase by Its Pseudokinase Domain (original) (raw)
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Journal of Biological Chemistry, 2002
Janus (Jak) tyrosine kinases contain a tyrosine kinase (JH1) domain adjacent to a catalytically inactive pseudokinase domain (JH2). The JH2 domain has been implicated in regulation of Jak activity, but its function remains poorly understood. Here, we found that the JH2 domain negatively regulates the activity of Jak2 and Jak3. Deletion of JH2 resulted in increased tyrosine phosphorylation of the Jak2-and Jak3-JH2 deletion mutants as well as of coexpressed STAT5. In cytokine receptor signaling, the deletion of the Jak2-and Jak3-JH2 domains resulted in interferon-␥ and interleukin-2-independent STAT activation, respectively. However, cytokine stimulations did not further induce the JH2 deletion mutant-mediated STAT activation. The deletion of the Jak2 JH2 domain also abolished interferon-␥-inducible kinase activation, although it did not affect the reciprocal Jak1-Jak2 interaction in 293T cells. Chimeric constructs, where the JH2 domains were swapped between Jak2 and Jak3, retained low basal activity and cytokine inducible signaling, indicating functional conservation between the two JH2 domains. However, the basal activity of Jak2 was significantly lower than that of Jak3, suggesting differences in the regulation of Jak2 and Jak3 activity. In conclusion, we found that the JH2 domain has a conserved function in Jak2 and Jak3. The JH2 domain is required for two distinct functions in cytokine signaling: (i) inhibition of the basal activity of Jak2 and Jak3, and (ii) cytokine-inducible activation of signaling. The Jak-JH2 deletion mutants are catalytically active, activate STAT5, and interact with another Jak kinase, but the JH2 domain is required to connect these signaling events to receptor activation. Thus, we propose that the JH2 domain contributes to both the uninduced and ligand-induced Jak-receptor complex, where it acts as a cytokine-inducible switch to regulate signal transduction. Janus (Jak) 1 tyrosine kinases are essential mediators of cytokine-induced signal transduction (1). The Jak kinases bind to
Nature Structural & Molecular Biology, 2011
JAK2 belongs to the Janus family of cytoplasmic tyrosine kinases (JAK1-JAK3, TYK2) and functions as a crucial mediator of signaling for hematopoietic cytokines and hormones such as erythropoietin (Epo), thrombopoietin (Tpo), interferon-γ (IFN-γ), several interleukins, growth hormone, prolactin, leptin and granulocytemacrophage colony-stimulating factor (GM-CSF) 1,2 . JAK2 serves as a triggering kinase for cytokine receptors, and phosphorylation and activation of downstream signaling proteins and the progression of signal transduction are dependent on its activity. JAK2 associates with the cytoplasmic domains of cytokine or hormone receptors, and ligand-induced receptor rearrangement facilitates JAK2 trans-phosphorylation of activation-loop Tyr1007 and Tyr1008 in JH1 (the tyrosine kinase domain), leading to its activation. Subsequent phosphorylation by JAK2 of tyrosine residues in the receptors creates docking sites for SH2-containing signaling proteins such as the signal transducer and activator of transcription (STAT) proteins 3,4 .
Characterization of JAK1 Pseudokinase Domain in Cytokine Signaling
Cancers
The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) pathway mediates essential biological functions from immune responses to haematopoiesis. Deregulated JAK-STAT signaling causes myeloproliferative neoplasms, leukaemia, and lymphomas, as well as autoimmune diseases. Thereby JAKs have gained significant relevance as therapeutic targets. However, there is still a clinical need for better JAK inhibitors and novel strategies targeting regions outside the conserved kinase domain have gained interest. In-depth knowledge about the molecular details of JAK activation is required. For example, whether the function and regulation between receptors is conserved remains an open question. We used JAK-deficient cell-lines and structure-based mutagenesis to study the function of JAK1 and its pseudokinase domain (JH2) in cytokine signaling pathways that employ JAK1 with different JAK heterodimerization partner. In interleukin-2 (IL-2)-induced STAT5 activation JAK1 w...
Proceedings of the National Academy of Sciences, 2014
Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.
New insights into the structure and function of the pseudokinase domain in JAK2
Biochemical Society Transactions, 2013
JAK (Janus kinase) 2 plays a critical role in signal transduction through several cytokine receptors. JAKs contain a typical tyrosine kinase domain preceded by a pseudokinase [JH2 (JAK homology 2)] domain which has been considered to be catalytically inactive. Identification of activating mutations in the JH2 domain of JAK2 as the major cause for polycythaemia vera and other MPNs (myeloproliferative neoplasms) demonstrate the critical regulatory function for this domain, but the underlying mechanisms have remained elusive. We have performed biochemical and functional analysis on the JH2 domain of JAK2. The results indicate that JH2 functions as an active protein kinase and phosphorylates two residues in JAK2 (Ser 523 and Tyr 570 ) that have been shown previously to be negative regulatory sites for JAK2 activity. The crystal structure of the JAK2 JH2 domain provides an explanation for the functional findings and shows that JH2 adopts a prototypical kinase fold, but binds MgATP through a non-canonical mode. The structure of the most prevalent pathogenic JH2 mutation V617F shows a high level of similarity to wild-type JH2. The most notable structural deviation is observed in the N-lobe αC-helix. The structural and biochemical data together with MD (molecular dynamics) simulations show that the V617F mutation rigidifies the αC-helix, which results in hyperactivation of the JH1 domain through an as yet unidentified mechanism. These results provide structural and functional insights into the normal and pathogenic function of the JH2 domain of JAK2.
The regulation of JAKs in cytokine signaling and its breakdown in disease
Cytokine, 2018
The JAK-STAT signal transduction pathway is responsible for mediating signals of over fifty cytokines, growth factors and hormones. Signaling through the JAK-STAT pathway is regulated on multiple levels, including intramolecular regulation by the JAK pseudokinase domain, and intermolecular regulation by a host of regulatory proteins. The advent of accessible genomic tools have provided a wealth of information on disease-associated mutations in the JAK-STAT pathway and its regulatory components. The vast number of these mutations in diseases ranging from immunodeficiencies and obesity to many cancers highlight the importance of correct regulation of JAK-STAT signaling for biological processes such as hematopoiesis, regulation of the immune system, metabolism, and growth. Simultaneously, JAK inhibitors are gaining traction in clinical use, both for treatment of diseases driven by JAK mutations, and for a host of inflammatory disorders, in which proinflammatory cytokine signaling through the JAK-STAT pathway is an integral part of pathogenesis. The elucidation of molecular mechanisms in the pathogenesis of complex diseases has also, however, brought the limitations of our current understanding on the regulation of cytokine signaling to the foreground. Indeed, deeper understanding of these regulatory mechanisms are a prerequisite for the development of the next generation of pharmacological modulators of the JAK-STAT pathway. In this review we discuss the current state of knowledge of the intra-and intermolecular regulation of the JAK-STAT pathway, with a focus on diseases arising from disruptions in the regulatory apparatus.
Crystal structures of the JAK2 pseudokinase domain and the pathogenic mutant V617F
2012
The protein tyrosine kinase Jak2 mediates signaling through numerous cytokine receptors. Jak2 possesses a pseudokinase domain (JH2) and a tyrosine kinase domain (JH1). Through unknown mechanisms, JH2 regulates the catalytic activity of JH1, and hyperactivating mutations in the JH2 region of human Jak2 are causative for myeloproliferative neoplasms (MPNs). We showed previously that Jak2 JH2 is in fact catalytically active. Here, we present crystal structures of human Jak2 JH2, both wild-type and the most prevalent MPN mutant, V617F. The structures reveal that JH2 adopts the fold of a prototypical protein kinase but binds Mg-ATP noncanonically. The structural and biochemical data indicate that the V617F mutation rigidifies αhelix C in the N lobe of JH2, which facilitates trans-phosphorylation of JH1. The crystal structures of JH2 afford new opportunities for the design of novel Jak2 therapeutics targeting MPNs. Jak2, a member of the Janus family of protein tyrosine kinases (Jak1-3, Tyk2), associates with the cytoplasmic regions of various cytokine receptors, including those for growth hormone, erythropoietin, leptin, interferon-γ and interleukins IL-3 and IL-5 (ref. 1). Jak2 is activated through cytokine-mediated receptor dimerization or rearrangement and signals through the Jak-Stat (signal transducer and activators of transcription) pathway 2 , which is essential for myeloid cell development, proliferation and survival, as well as for the initial stages of the immune response. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Proceedings of the National Academy of Sciences, 1997
Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, ␥c and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980͞981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.
Regulation of Jak2 function by phosphorylation of Tyr317 and Tyr637 during cytokine signaling
Molecular and cellular biology, 2009
Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr(317) in the FERM domain and Tyr(637) in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr(317) promotes increased Jak2 activity, and the phosphorylation of Tyr(317) during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr(317) in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr(317) in the attenuation of Jak2 signaling. In contrast, mutation of Tyr(637) decreased Jak2 signaling and activi...