Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-B beta.

Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0662, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 06/2004; 24(10):4557-70.
Source: PubMed


The tyrosine kinase Janus kinase 2 (JAK2) binds to the majority of the known members of the cytokine family of receptors. Ligand-receptor binding leads to activation of the associated JAK2 molecules, resulting in rapid autophosphorylation of multiple tyrosines within JAK2. Phosphotyrosines can then serve as docking sites for downstream JAK2 signaling molecules. Despite the importance of these phosphotyrosines in JAK2 function, only a few sites and binding partners have been identified. Using two-dimensional phosphopeptide mapping and a phosphospecific antibody, we identified tyrosine 813 as a site of JAK2 autophosphorylation of overexpressed JAK2 and endogenous JAK2 activated by growth hormone. Tyrosine 813 is contained within a YXXL sequence motif associated with several other identified JAK2 phosphorylation sites. We show that phosphorylation of tyrosine 813 is required for the SH2 domain-containing adapter protein SH2-B beta to bind JAK2 and to enhance the activity of JAK2 and STAT5B. The homologous tyrosine in JAK3, tyrosine 785, is autophosphorylated in response to interleukin-2 stimulation and is required for SH2-B beta to bind JAK3. Taken together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the SH2-B beta-binding site within JAK2 that is required for SH2-B beta to enhance activation of JAK2.

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Available from: Christin Carter-Su, Jan 03, 2015
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    • "Jude Children's Research Hospital, Memphis, TN) (Silvennoinen et al., 1993). FLAG-JAK2, and the various rat SH2B1b constructs, FLAG-SH2B1b, GFP-SH2B1b WT, GFP-SH2B1b D198– 268 (DNES), GFP-SH2B1b point mutations (9YF, Y439/494F, S161A, S165A, 2SA(S161/165A), 13SA, S161E, S165E, S161/165E, mNLS), myc-SH2B1b WT, and myc-SH2B1b point mutations (Y439/494F, Y439F, Y494F) have been described previously (Chen and Carter-Su, 2004; Kurzer et al., 2004; Maures et al., 2009; Maures et al., 2011; O'Brien et al., 2003). When expressed in 293T and RAW cells, all mutant SH2B1b proteins migrated in SDS-PAGE gels at the predicted size (data not shown). "
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    ABSTRACT: Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1β to the GH-activated, GH receptor-associated tyrosine kinase JAK2, implicate SH2B1β in GH-dependent actin cytoskeleton remodeling, and suggest that phosphorylation at serine 161 and 165 in SH2B1β releases SH2B1β from the plasma membrane. Here, we examined the role of SH2B1β in GH regulation of macrophage migration. We show that GH stimulates migration of cultured RAW264.7 macrophages, and primary cultures of peritoneal and bone marrow-derived macrophages. SH2B1β overexpression enhances, while SH2B1 knockdown inhibits, GH-dependent motility of RAW macrophages. At least two independent mechanisms regulate the SH2B1β-mediated changes in motility. In response to GH, tyrosines 439 and 494 in SH2B1β are phosphorylated. Mutating these tyrosines in SH2B1β decreases both basal and GH-stimulated macrophage migration. In addition, mutating the polybasic nuclear localization sequence (NLS) in SH2B1β or creating the phosphomimetics SH2B1β(S161E) or SH2B1β(S165E), all of which release SH2B1β from the plasma membrane, enhances macrophage motility. Conversely, SH2B1β(S161/165A) exhibits increased localization at the plasma membrane and decreased macrophage migration. Mutating the NLS or the nearby serines does not alter GH-dependent phosphorylation on tyrosines 439 and 494 in SH2B1β. Mutating tyrosines 439 and 494 does not affect localization of SH2B1β at the plasma membrane or movement of SH2B1β into focal adhesions. Taken together, these results suggest SH2B1β enhances GH-stimulated macrophage motility via mechanisms involving phosphorylation of SH2B1β on tyrosines 439 and 494 and movement of SH2B1β out of the plasma membrane (e.g. as a result of phosphorylation of serines 161 and 165).
    Full-text · Article · Feb 2013 · Journal of Cell Science
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    • "Type I and Type II cytokines recruit non-receptor tyrosine kinases such as Janus kinases (JAKs) to initiate signal transduction since they lack kinase domains. Interaction of JAK proteins with receptors induces autophosphorylation and causes JAKs to associate with, and activate members of, the signal-transducers and activators of transcription (STAT) which translocate to the nucleus and up-regulate transcription of a number of inflammatory genes [9]. In the present study, we examined the activation of several signaling pathways and the release of proinflammatory cytokines from co-cultures of astrocytes and microglia isolated from neonatal rats in response to LPS. "
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    ABSTRACT: Compelling evidence has implicated neuroinflammation in the pathogenesis of a number of neurodegenerative conditions. Chronic activation of both astrocytes and microglia leads to excessive secretion of proinflammatory molecules such as TNF α, IL-6 and IL-1 β with potentially deleterious consequences for neuronal viability. Many signaling pathways involving the mitogen-activated protein kinases (MAPKs), nuclear factor κ B (NF κ B) complex and the Janus kinases (JAKs)/signal transducers and activators of transcription (STAT)-1 have been implicated in the secretion of proinflammatory cytokines from glia. We sought to identify signaling kinases responsible for cytokine production and to delineate the complex interactions which govern time-related responses to lipopolysaccharide (LPS). We examined the time-related changes in certain signaling events and the release of proinflammatory cytokines from LPS-stimulated co-cultures of astrocytes and microglia isolated from neonatal rats. TNF α was detected in the supernatant approximately 1 to 2 hours after LPS treatment while IL-1 β and IL-6 were detected after 2 to 3 and 4 to 6 hours, respectively. Interestingly, activation of NF κ B signaling preceded release of all cytokines while phosphorylation of STAT1 was evident only after 2 hours, indicating that activation of JAK/STAT may be important in the up-regulation of IL-6 production. Additionally, incubation of glia with TNF α induced both phosphorylation of JAK2 and STAT1 and the interaction of JAK2 with the TNF α receptor (TNFR1). Co-treatment of glia with LPS and recombinant IL-6 protein attenuated the LPS-induced release of both TNF α and IL-1 β while potentiating the effect of LPS on suppressor of cytokine signaling (SOCS)3 expression and IL-10 release. These data indicate that TNF α may regulate IL-6 production through activation of JAK/STAT signaling and that the subsequent production of IL-6 may impact on the release of TNF α, IL-1 β and IL-10.
    Full-text · Article · Jun 2012 · Journal of Neuroinflammation
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    • "This is in agreement with current knowledge suggesting that most protein tyrosine kinases (PTKs) prefer Asp and Glu at the N-terminus of the tyrosine residue and that strong preferences are found at positions +1 and +3 [34]. In addition, previous studies on the autophosphorylation sites of JAK2 also identified sequences with high similarities with this motif [26], [35], [36], [37]. An additional online search on PhosphoSitePlus [38] rendered several cytoplasmic proteins (like Akt, Crk, Pten, PKC, Ron, SHP, and STAT5) and membrane receptors (like CSF2RB, EPOR and PrlR) involved in JAK2 signaling with phosphorylated tyrosines in sites strongly similar to this motif. "
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    ABSTRACT: 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 K(m) for 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 K(m) for 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.
    Full-text · Article · Apr 2011 · PLoS ONE
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