Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

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 non-canonically. The structural and biochemical data indicate that the V617F mutation rigidifies α-helix 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.

Interleukin-15 shares many biological activities with IL-2 and signals through the IL-2 receptor beta and gamma chains. However, IL-15 and IL-2 differ in their controls of expression and secretion, their range of target cells and their functional activities. These dissimilarities may include differential effects on apoptosis. For example, IL-2 induces or inhibits T-cell apoptosis in vitro, depending on T-cell activation, whereas IL-15 inhibits cytokine deprivation-induced apoptosis in activated T cells. Studying whether and how IL-15 modulates distinct apoptosis pathways, we show here that apoptosis induced by anti-Fas, anti-CD3, dexamethasone, and/or anti-IgM in activated human T and B cells in vitro is inhibited by IL-15 in a manner dependent on RNA synthesis. In vivo, anti-Fas-induced lethal multisystem apoptosis in mice is suppressed by a novel IL-15-IgG2b fusion protein. Only IL-15, but not IL-2, completely protected from lethal hepatic failure. Thus, IL-15 is a potent, general inhibitor of apoptosis in vitro and in vivo with intriguing therapeutic potential.

Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity contributing to myeloproliferative neoplasms (MPNs). Here, we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2, Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Importantly, different MPN mutations were found to abrogate JH2 activity in cells, and in MPN (V617F) patient cells, phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2.

Janus kinase-2 (JAK2) mediates signaling by various cytokines, including erythropoietin and growth hormone. JAK2 possesses tandem pseudokinase and tyrosine kinase domains. Mutations in the pseudokinase domain are causally linked to myeloproliferative neoplasms (MPNs) in humans. The structure of the JAK2 tandem kinase domains is unknown, and therefore the molecular bases for pseudokinase-mediated autoinhibition and pathogenic activation remain obscure. Using unbiased molecular dynamics simulations of protein-protein docking, we produced a structural model for the autoinhibitory interaction between the JAK2 pseudokinase and kinase domains. A striking feature of our model, which is supported by mutagenesis experiments, is that nearly all of the disease mutations map to the domain interface. The simulations indicate that the kinase domain is stabilized in an inactive state by the pseudokinase domain, and they offer a molecular rationale for the hyperactivity of V617F, the predominant JAK2 MPN mutation.

The family of cytoplasmic Janus (Jak) tyrosine kinases plays an essential role in cytokine signal transduction, regulating cell survival and gene expression. Ligand-induced receptor dimerization results in phosphorylation of Jak2 on activation loop tyrosine Y1007 and stimulation of its catalytic activity, which, in turn, results in activation of several downstream signaling cascades. Recently, the catalytic activity of Jak2 has been found to be subject to negative regulation through various mechanisms including association with SOCS proteins. Here we show that the ubiquitin-dependent proteolysis pathway is involved in the regulation of the turnover of activated Jak2. In unstimulated cells Jak2 was monoubiquitinated, and interleukin-3 or gamma interferon stimulation induced polyubiquitination of Jak2. The polyubiquitinated Jak2 was rapidly degraded through proteasomes. By using different Jak2 mutants we show that tyrosine-phosphorylated Jak2 is preferentially polyubiquitinated and degraded. Furthermore, phosphorylation of Y1007 on Jak2 was required for proteasomal degradation and for SOCS-1-mediated downregulation of Jak2. The proteasome inhibitor treatment stabilized the Jak2-SOCS-1 protein complex and inhibited the proteolysis of Jak2. In summary, these results indicate that the ubiquitin-proteasome pathway negatively regulates tyrosine-phosphorylated Jak2 in cytokine receptor signaling, which provides an additional mechanism to control activation of Jak2 and maintain cellular homeostasis.

IntroductionRegulation of signal transducer and activator of transcription 1 (STAT1) involves posttranslational modifications such as phosphorylation of Tyr701 and Ser727 as well as arginine methylation. 1,2 STAT1 signaling is negatively regulated by protein tyrosine phosphatases (PTPs), suppressors of cytokine signaling (SOCS), and protein inhibitor of activated STAT1 (PIAS) proteins. 1-3 The family of PIAS proteins consists of 5 members that have been implicated in the regulation of several nuclear proteins. PIAS1 and PIAS3 were identified as interaction partners for STAT1 and STAT3, respectively, and they were found to inhibit the DNA-binding activity of activated STATs. [3][4][5] The other members, PIASx␣/ARIP3 (androgen receptor-interacting protein 3), PIASx␤/ Miz1, and PIASy, function as transcriptional coregulators for steroid hormone receptors. [6][7][8] Recently, several PIAS proteins have been shown to function as E3-type small ubiquitin-like modifier (SUMO) ligases. 9-12 SUMO-1, -2, and -3 are small modifier proteins that are covalently conjugated to specific lysine residues of target proteins. 13 A number of nuclear proteins, such as transcription factors AR, p53, and c-Jun, become modified by SUMO-1 conjugation, and this reversible posttranslational modification has been implicated in regulation of proteinprotein interactions, protein stability, localization, or activity. 9,13,14 However, it is currently unknown whether STAT factors are modified through sumoylation. Study design ReagentsAntibodies used include anti-SUMO-1 (mouse anti-GMP-1) (Zymed, San Francisco, CA); anti-HA (clone 16B12) (Berkeley-Antibody, Richmond, CA); anti-Flag (anti-Flag M2) (Sigma Aldrich, St Louis, MO); anti-STAT1 (N terminus) (Transduction Laboratories, BD Biosciences); anti-STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA) biotinylated antimouse (Dako, Glustrup, Denmark) and streptavidin-biotin horseradish peroxidase conjugate (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Human interferon-␥ (huIFN-␥) was purchased from Immugenex, Los Angeles, CA. Plasmid constructsSUMO-1-Flag and SUMO-1-Flag-Flag were a kind gift from Dr H. Yasuda. 15 The SUMO-1, Flag-PIAS1, Flag-PIAS1mut (PIAS1⌬310-407), Flag-PIAS3, Flag-ARIP3 Flag-ARIP3mut (ARIP3⌬347-418) plasmids 4,9 as well as STAT1-WT-HA and GAS-luc reporter construct have been previously described. 16 The STAT1 Lys703Arg mutation was created from STAT1-WT-HA using direct polymerase chain reaction (PCR) mutagenesis with the following primers: 5ЈGGAACTGGATATATCAGGACTGAGTTGATTTCTGTGTCTG-3Ј and 5Ј-CAGACACAGAAATCAACTCAGTCCTGATATATCCAGTTCC-3Ј. Transfections, luciferase assay, and immunodetectionCOS-7 cells were electroporated using a Bio-Rad (Hercules, CA) gene pulser at 260 V and 960 microfarads (F) and lysed in Triton X lysis buffer supplemented with 5 mM N-ethylmaleimide (NEM) (Sigma Aldrich). HeLa cells were transfected using a calcium phosphate method. For luciferase assay, U3A cells were transfected using a calcium phosphate method as described. 16 Immunodetection wa...

Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wildtype JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.T he Janus kinases (JAK1-3, TYK2) are a family of nonreceptor tyrosine kinases with essential functions in the regulation of hematopoiesis, the immune system, and cellular metabolism. JAKs interact specifically with various cytokine receptors and couple cytokine binding to cytoplasmic signaling cascades, including the signal transducers and activators of transcription (STAT) pathway. JAKs consist of an N-terminal FERM domain, an SH2-like (Src homology 2) domain, a pseudokinase domain (JAK homology 2, JH2), and the C-terminal tyrosine kinase domain (JH1). JH2 mediates critical regulatory functions in JAKs and primarily serves to inhibit basal JH1 activity. Experimental deletion of JH2 increases JH1 activity in full-length JAK in the absence of stimulation (1-3), and in recombinant systems addition of JH2 suppresses JH1 activity (4-6). JH2 is, however, also required for ligand-induced activation of full-length JAKs in cell (1-3, 6, 7). The regulatory functions of JH2 are corroborated by the multitude of human disease mutations identified in the domain. The most common JAK2 mutation, V617F, leads to cytokine-independent signaling through the exclusively JAK2-dependent homotypic receptors for erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), and thrombopoietin (8). The V617F mutation is found in ∼95% of patients with polycythemia vera (PV) (9...