Isolation of a catalytically competent phosphorylated tyrosine kinase from Rous sarcoma virus-induced rat tumor by immunoadsorption to and hapten elution from phosphotyrosine binding antibodies

A procedure has been developed for the isolation of a catalytically competent phosphorylated tyrosine kinase (RSV Y-kinase) from avian sarcoma virus-induced rat tumors. The procedure involves reaction of partially purified RSV Y-kinase with ATP to effect tyrosyl phosphorylation of catalytically competent RSV Y-kinase. Tyrosyl phosphorylated RSV Y-kinase was isolated from the heterogenous reaction mixture by immunoadsorption on immobilized phosphotyrosyl binding antibodies and elution with the hapten p-nitrophenyl phosphate. Estimation of the phosphate content of the purified phosphorylated RSV Y-kinase indicated that 1-3 tyrosyl groups had been phosphorylated upon reaction with ATP. The specific activity toward histone 2B of the purified phosphorylated RSV Y-kinase was at least 30-fold greater than that estimated for the RSV Y-kinase prepared previously by immunoadsorption on immobilized antiserum from tumor bearing rabbits.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28715/1/0000536.pd

Enhanced Expression of Janus Kinase–Signal Transducer and Activator of Transcription Pathway Members in Human Diabetic Nephropathy

OBJECTIVE—Glomerular mesangial expansion and podocyte loss are important early features of diabetic nephropathy, whereas tubulointerstitial injury and fibrosis are critical for progression of diabetic nephropathy to kidney failure. Therefore, we analyzed the expression of genes in glomeruli and tubulointerstitium in kidney biopsies from diabetic nephropathy patients to identify pathways that may be activated in humans but not in murine models of diabetic nephropathy that fail to progress to glomerulosclerosis, tubulointerstitial fibrosis, and kidney failure

Functional characterization of obesity-associated variants involving the α and β isoforms of human SH2B1.

We have previously reported rare variants in sarcoma (Src) homology 2 (SH2) B adaptor protein 1 (SH2B1) in individuals with obesity, insulin resistance, and maladaptive behavior. Here, we identify 4 additional SH2B1 variants by sequencing 500 individuals with severe early-onset obesity. SH2B1 has 4 alternatively spliced isoforms. One variant (T546A) lies within the N-terminal region common to all isoforms. As shown for past variants in this region, T546A impairs SH2B1β enhancement of nerve growth factor-induced neurite outgrowth, and the individual with the T546A variant exhibits mild developmental delay. The other 3 variants (A663V, V695M, and A723V) lie in the C-terminal tail of SH2B1α. SH2B1α variant carriers were hyperinsulinemic but did not exhibit the behavioral phenotype observed in individuals with SH2B1 variants that disrupt all isoforms. In in vitro assays, SH2B1α, like SH2B1β, enhances insulin- and leptin-induced insulin receptor substrate 2 (IRS2) phosphorylation and GH-induced cell motility. None of the variants affect SH2B1α enhancement of insulin- and leptin-induced IRS2 phosphorylation. However, T546A, A663V, and A723V all impair the ability of SH2B1α to enhance GH-induced cell motility. In contrast to SH2B1β, SH2B1α does not enhance nerve growth factor-induced neurite outgrowth. These studies suggest that genetic variants that disrupt isoforms other than SH2B1β may be functionally significant. Further studies are needed to understand the mechanism by which the individual isoforms regulate energy homeostasis and behavior.This work was supported by the Wellcome Trust (098497/Z/ 12/Z; 077016/Z/05/Z; 096106/Z/11/Z) (to I.S. Farooqi and L.R. Pearce), by the Medical Research Council Metabolic Diseases Unit and NIHR Cambridge Biomedical Research Centre (to I.S. Farooqi, I. Barroso, and S. O’Rahilly) and the Bernard Wolfe Health Neuroscience Fund (I.S. Farooqi); and by NIH grants RO1-DK54222 (to C. Carter-Su), RO1-DK065122 and RO1- DK073601 (to L. Rui), a predoctoral fellowship from the Systems and Integrative Biology Training Grant NIH–T32-GM008322 (to M.E. Doche) and a Rackham Merit Fellowship from the University of Michigan (to R. Joe). Confocal microscopy was performed using the Morphology and Image Analysis Core of the Michigan Diabetes Research Center (NIH grant P60-DK20572).This is the final published version distributed under a Creative Commons Attribution License, which can also be found on the publisher's website at: http://press.endocrine.org/doi/abs/10.1210/en.2014-1264?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubme

Autophosphorylation of JAK2 on Tyrosines 221 and 570 Regulates Its Activity

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 by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2

Tyrosine 813 Is a Site of JAK2 Autophosphorylation Critical for Activation of JAK2 by SH2-Bβ

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-Bβ 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-Bβ to bind JAK3. Taken together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the SH2-Bβ-binding site within JAK2 that is required for SH2-Bβ to enhance activation of JAK2

Phosphorylation of JAK2 at Serine 523: a Negative Regulator of JAK2 That Is Stimulated by Growth Hormone 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

Regulation of Jak2 Function by Phosphorylation of Tyr317 and Tyr637 during Cytokine Signaling▿

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, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 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 Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling

Tyrosines 868, 966, and 972 in the Kinase Domain of JAK2 Are Autophosphorylated and Required for Maximal JAK2 Kinase Activity

Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry analysis of JAK2 activated by an erythropoietin-bound chimeric erythropoietin receptor/leptin receptor. Phosphospecific antibodies suggest that the phosphorylation of all three tyrosines increases in response to GH. Compared with wild-type JAK2, which is constitutively active when overexpressed, JAK2 lacking tyrosine 868, 966, or 972 has substantially reduced activity. Coexpression with GH receptor and protein tyrosine phosphatase1B allowed us to investigate GH-dependent activation of these mutated JAK2s in human embryonic kidney 293T cells. All three mutated JAK2s are activated by GH, although to a lesser extent than wild-type JAK2. The three mutated JAK2s also mediate GH activation of signal transducer and activator of transcription 3 (Stat3), signal transducer and activator of transcription 5b (Stat5b) and ERK1, but at reduced levels. Coexpression with Src-homology 2B1β (SH2B1β), like coexpression with GH-bound GH receptor, partially restores the activity of all three JAK2 mutants. Based on these results and the crystal structure of the JAK2 kinase domain, we hypothesize that small changes in the conformation of the regions of JAK2 surrounding tyrosines 868, 966, and 972 due to e.g. phosphorylation, binding to a ligand-bound cytokine receptor, and/or binding to Src-homology 2B1, may be essential for JAK2 to assume a maximally active conformation