Analysis of jak2 catalytic function by peptide microarrays: The role of the JH2 domain and V617F mutation

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Kmfor ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Kmfor ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches

Janus kinase 2 regulates Bcr–Abl signaling in chronic myeloid leukemia

Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr–Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr–Abl+ cells reduced levels of the Bcr–Abl protein and also the phosphorylation of Tyr177 of Bcr–Abl, and Jak2 overexpression rescued these knockdown effects. Treatment of Bcr–Abl+ cells with Jak2 inhibitors for 4–6 h but not with IM also reduced Bcr–Abl protein and pTyr177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr177 of Bcr–Abl (a Jak2 consensus site, YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr–Abl in immune complexes but did not reduce levels of Bcr–Abl, suggesting that the reduction of Bcr–Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr–Abl stability and oncogenic signaling in CML cells are under the control of Jak2

A Bipolar Clamp Mechanism for Activation of Jak-Family Protein Tyrosine Kinases

Most cell surface receptors for growth factors and cytokines dimerize in order to mediate signal transduction. For many such receptors, the Janus kinase (Jak) family of non-receptor protein tyrosine kinases are recruited in pairs and juxtaposed by dimerized receptor complexes in order to activate one another by trans-phosphorylation. An alternative mechanism for Jak trans-phosphorylation has been proposed in which the phosphorylated kinase interacts with the Src homology 2 (SH2) domain of SH2-B, a unique adaptor protein with the capacity to homo-dimerize. Building on a rule-based kinetic modeling approach that considers the concerted nature and combinatorial complexity of modular protein domain interactions, we examine these mechanisms in detail, focusing on the growth hormone (GH) receptor/Jak2/SH2-Bβ system. The modeling results suggest that, whereas Jak2-(SH2-Bβ)2-Jak2 heterotetramers are scarcely expected to affect Jak2 phosphorylation, SH2-Bβ and dimerized receptors synergistically promote Jak2 trans-activation in the context of intracellular signaling. Analysis of the results revealed a unique mechanism whereby SH2-B and receptor dimers constitute a bipolar ‘clamp’ that stabilizes the active configuration of two Jak2 molecules in the same macro-complex

The Janus kinases (Jaks)

The Janus kinase (Jak) family is one of ten recognized families of non-receptor tyrosine kinases. Mammals have four members of this family, Jak1, Jak2, Jak3 and Tyrosine kinase 2 (Tyk2). Birds, fish and insects also have Jaks. Each protein has a kinase domain and a catalytically inactive pseudo-kinase domain, and they each bind cytokine receptors through amino-terminal FERM (Band-4.1, ezrin, radixin, moesin) domains. Upon binding of cytokines to their receptors, Jaks are activated and phosphorylate the receptors, creating docking sites for signaling molecules, especially members of the signal transducer and activator of transcription (Stat) family. Mutations of the Drosophila Jak (Hopscotch) have revealed developmental defects, and constitutive activation of Jaks in flies and humans is associated with leukemia-like syndromes. Through the generation of Jak-deficient cell lines and gene-targeted mice, the essential, nonredundant functions of Jaks in cytokine signaling have been established. Importantly, deficiency of Jak3 is the basis of human autosomal recessive severe combined immunodeficiency (SCID); accordingly, a selective Jak3 inhibitor has been developed, forming a new class of immunosuppressive drugs

Tyrosine phosphorylation of the EGF receptor by the kinase Jak2 is induced by growth hormone

When growth hormone binds to its receptor, which belongs to the cytokine receptor superfamily, it activates the Janus kinase Jak2 which has tyrosine- kinase activity and initiates an activation of several key intracellular proteins (for example, mitogen-activated protein (MAP) kinases) that eventually execute the biological actions induced by growth hormone, including the expression of particular genes. In contrast to receptors that themselves have tyrosine kinase activity, the signalling pathways leading to MAP kinase activation that are triggered by growth hormone are poorly understood, but appear to be mediated by the proteins Grb2 and Shc. We now show that growth hormone stimulates tyrosine phosphorylation of the receptor for epidermal growth factor (EGFR) and its association with Grb2 and at the same time stimulates MAP kinase activity in liver, an important target tissue of growth hormone. Expression of EGFR and its mutants revealed that growth- hormone-induced activation of MAP kinase and expression of the transcription factor c-fos requires phosphorylation of tyrosines on EGFR, but not its own intrinsic tyrosine-kinase activity. Moreover, tyrosine at residue 1,068 of the EGFR is proposed to be one of the principal phosphorylation sites and Grb2-binding sites stimulated by growth hormone via Jak2. Our results indicate that the role of EGFR in signalling by growth hormone is to be phosphorylated by Jak2, thereby providing docking sites for Grb2 and activating MAP kinases and gene expression, independently of the intrinsic tyrosine kinase activity of EGFR. This may represent a novel cross-talk pathway between the cytokine receptor superfamily and growth factor receptor