Christin Carter-su | University of Michigan (original) (raw)

Papers by Christin Carter-su

The American journal of physiology

Intestinal epithelial cells isolated from 6-wk-old chickens maintain the capability for Na+-depen... more Intestinal epithelial cells isolated from 6-wk-old chickens maintain the capability for Na+-dependent concentrative accumulation of 3-O-methylglucose (3-OMG). Cells depleted of ATP exhibit a transient accumulation of 3-OMG in response to imposed Na+ gradients ([Na+]o greater than [Na+]i) or when transmembrane ion diffusion potentials (cell interior negative) are established. Phlorizin or lack of extracellular Na+ prevents formation of sugar gradients in every case. A nonconcentrative, non-Na+-dependent sugar transport system is also operative in these cells. The latter system is inhibited to various degrees by phloretin, theophylline, cytochalasin B, and a variety of flavonones and flavones, including apigenin. These agents also act to inhibit efflux of sugar from the cell via this pathway. The concentrative system normally operates against a "leak" of sugar through the nonconcentrative carrier. If the passive system is made inoperative by any of the agents named above, a significant enhancement of steady-state sugar gradients maintained by the cells is observed. With cytochalasin B, gradients as large as 30-fold are established. The energy inherent in cellular Na+ gradients cannot account for sugar gradients of this magnitude unless both chemical electrical driving forces are considered. When the passive leak is maximmally inhibited, more than half of the total energy required must be derived from the membrane potential.

Journal of Biological Chemistry

Glucocorticoids are known to rapidly inhibit glucose transport when added to isolated rat adipocy... more Glucocorticoids are known to rapidly inhibit glucose transport when added to isolated rat adipocytes. To determine whether this inhibition of transport persists following isolation of the plasma membranes, adipocytes were incubated in the absence or presence of a maximally inhibitory concentration of dexamethasone, a synthetic glucocorticoid, and plasma membrane vesicles were prepared. D-Glucose uptake into vesicles from steroid-treated cells was inhibited by an average of 40%. The ability of dexamethasone to inhibit transport depended upon pretreatment of cells with hormone prior to membrane isolation. Furthermore, the decreased rate of transport was prevented by the simultaneous addition to the cell of actinomycin D or cycloheximide with dexamethasone, indicating a requirement for RNA and protein synthesis.

Journal of Biological Chemistry

ABSTRACT

The American journal of physiology

The ability of glucocorticoids to modify the effect of insulin on glucose transport was investiga... more The ability of glucocorticoids to modify the effect of insulin on glucose transport was investigated in both intact isolated rat adipocytes and in membranes isolated from hormone-treated adipocytes. In intact adipocytes, dexamethasone, a potent synthetic glucocorticoid, inhibited insulin-stimulated 3-O-methylglucose transport at all concentrations of insulin tested (0-2,000 microU/ml). Insulin sensitivity, as well as the maximal response to insulin, was decreased by dexamethasone in the absence of a change in insulin binding. The inhibition was observed regardless of which hormone acted first, was blocked by actinomycin D, and resulted from a decrease in Vmax rather than an increase in Kt of transport. In plasma membranes isolated from insulin-treated adipocytes, glucose transport activity and the amount of glucose transporter covalently labeled with [3H]cytochalasin B were increased in parallel in a dose-dependent fashion. The amount of labeled transporter in a low-density microsomal fraction (LDMF) was decreased in a reciprocal fashion. In contrast, addition of dexamethasone to insulin-stimulated cells caused decreases in both transport activity and amount of labeled transporter in the plasma membranes. This was accompanied by a small increase in the amount of [3H]cytochalasin B incorporated into the glucose transporter in the LDMF. These results are consistent with both insulin and glucocorticoids altering the distribution of glucose transporters between the plasma membrane and LDMF, in opposite directions.

The American journal of physiology

The ability of the synthetic glucocorticoid, dexamethasone, to alter 3-O-methylglucose transport ... more The ability of the synthetic glucocorticoid, dexamethasone, to alter 3-O-methylglucose transport was investigated using isolated rat adipocytes. A maximally effective dose of dexamethasone (10(-7) M) inhibited transport up to 80% within 60-90 min. Inhibition of transport was evident as early as 15-30 min after addition of steroid, and was prevented by both actinomycin D and cycloheximide. When added within 45 or 60 min after dexamethasone, actinomycin D interfered with the cells' ability to respond to the steroid but had no effect when added between 60 and 90 min or longer after the steroid. Cycloheximide interfered with steroid-induced inhibition of transport when added at any time before the 15- to 30-min period immediately preceding the transport assay. This interference with hormone action appeared to be independent of the length of time cells were exposed to dexamethasone before addition of cycloheximide. Thus cells that were maximally inhibited by dexamethasone by 90 min became only partially inhibited when cycloheximide was added at 90 or 120 min, and cells were incubated for an additional 60 or 30 min, respectively. These findings are consistent with the following: dexamethasone inhibits glucose oxidation as a result of inhibiting hexose transport; inhibition of transport by dexamethasone requires the synthesis of RNA during the first 45-60 min after steroid addition and requires protein synthesis during the entire incubation period with dexamethasone; and transport is inhibited within minutes after protein synthesis is initiated.

Journal of Biological Chemistry

Cytochalasin B (6.9 Ci/mmol) and Protosol were obtained from New England Nuclear. Cytochalasin B,... more Cytochalasin B (6.9 Ci/mmol) and Protosol were obtained from New England Nuclear. Cytochalasin B, cytochalasin E, and cytochalasin A were purchased from Aldrich. Sigma supplied the D-gluCOSe analogues, 2,3-dimethylmaleic anhydride, bovine serum albumin (Fraction V), and rabbit muscle actin. Aqueous scintillation fluid was an Amersham product. All other reagents were of reagent grade quality.

Journal of Biological Chemistry

Previous observations have led to the speculation that activation of a growth hormone (GH) recept... more Previous observations have led to the speculation that activation of a growth hormone (GH) receptorassociated tyrosine kinase is an early, perhaps initiating, event in transmembrane signaling by GH. To test this hypothesis further, a Western blotting assay employing antibodies to phosphotyrosine was used to determine whether proteins other than the GH receptor might serve as substrates of the GH receptor-associated tyrosine kinase. The ability of inhibitors of the GH receptor-associated kinase to block actions of GH was also investigated. Over a physiologically relevant range of concentrations, GH was found to promote, in 3T3-F442A fibroblasts, rapid changes in the level of tyrosyl phosphorylation of more than 13 proteins. At the highest GH concentration employed (500 ng/ml), increased tyrosyl phosphorylation of two proteins, pp121 and pp97, was clearly visible at 1 min, the earliest time tested. Increased tyrosyl phosphorylation of a number of other proteins (pp250, pps160-180, and pp36) and decreased tyrosyl phosphorylation of a 140-kDa protein were apparent after 5-10 min of incubation with GH. Staurosporine, herbimycin A, and tyrphostin were identified as inhibitors of the GH receptor-associated kinase. When added to anti-GH antibody immunoprecipitates from GH-treated cells, they inhibited incorporation of 32P from [y3'P]ATP into tyrosyl residues in GH receptor complexes. When added to cells, all three inhibitors blocked all GHdependent increases in tyrosyl phosphorylation of cellular proteins. Inhibitors of the GH receptor-associated tyrosine kinase also abolished GH-dependent activation of microtubule-associated protein (MAP) kinase. Consistent with these inhibitors inhibiting the GH receptor-associated tyrosine kinase, they had little or no effect on activation of MAP kinase by epidermal growth factor. In contrast, genistein and hydroxy-(2naphthyl)-methylphosphonic acid, tyrosine kinase inhibitors lacking specificity for the GH receptor-associated kinase, decreased GH-dependent tyrosyl phosphorylation of only a subset of GH-responsive bands and partially blocked GH-dependent activation of MAP kinase. These data show that increased tyrosyl phosphorylation of specific cellular proteins is a very rapid response to the binding of GH by the cell and most * Recipient of a student medical research fellowship from the University of Michigan Medical School. ~~~1 4 0 -1 6 0 , ~~1 3 0 , PP90, ~~7 5 , ~~4 5 , ~~4 2 , ~~3 9 , likely involves multiple tyrosine kinases. Furthermore, inhibition of the GH receptor-associated tyrosine kinase blocks at least two actions of GH, the stimulation of tyrosyl phosphorylation of multiple proteins and MAP kinase activation. These results are consistent with the GH receptor-associated kinase playing an important, perhaps initiating, role in trans-membrane signaling by GH.

Journal of Biological Chemistry

We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase ... more We have previously demonstrated that growth hormone (GH) promotes an increase in tyrosine kinase activity associated with the GH receptor. To gain insight into the role of GH-dependent tyrosine kinase activity in signaling by GH, we investigated the possibility that GH might stimulate MAP kinase, a serine/threonine/tyrosine kinase thought to be a common element in tyrosine kinase-initiated response cascades. Treatment of 3T3-F442A fibroblasts with 100 ng/ml GH results in a 3-6-fold increase in the ability of cell-free extracts to phosphorylate MAP-2 and myelin basic protein. GH-stimulated kinase activity is unaffected by heparin, H7, or cAMP-dependent protein kinase inhibitor peptide, partially reduced by staurosporin and inhibited by fluoride and calcium ions, indicating that the kinase is not protein kinase C or A, casein kinase, or a calcium/calmodulin-dependent protein kinase. Based on gel permeation chromatography, the molecular mass of the GH-stimulated MAP kinase is approxima...

Recent progress in hormone research, 1998

During the past 4 years, significant progress has been made in elucidating the earliest events fo... more During the past 4 years, significant progress has been made in elucidating the earliest events following binding of ligands to members of the cytokine receptor superfamily. This is a rapidly growing family of receptors that currently includes receptors for growth hormone (GH); prolactin; erythropoeitin; granulocyte colony-stimulating factor; granulocyte macrophage colony-stimulating factor; interleukin(IL)s 2-7, 9-13, 15; interferon (IFN)-alpha, beta, and gamma; thrombopoietin; leptin; oncostatin M; leukemia inhibitory factor (LIF); ciliary neurotrophic factor; and cardiotropin-1. Despite their diverse physiological effects in the body, ligands that bind to members of this family share multiple signaling pathways. An early and most likely initiating event for all of them is the activation of one or more members of the Janus (or JAK) family of tyrosine kinases. The activated JAK kinases, which form a complex with the cytokine receptor subunits, phosphorylate themselves as well as the...

The Journal of biological chemistry, Jan 31, 1997

We have previously found that the signal transducer and activator of transcription (Stat) 3 is co... more We have previously found that the signal transducer and activator of transcription (Stat) 3 is constitutively activated in cells stably transformed by the v-Src oncoprotein. While activation of Stat proteins has also been observed following epidermal growth factor or platelet-derived growth factor stimulation, Stat3 activation is more commonly associated with signaling through cytokine receptors and activation of the Janus family tyrosine kinases JAK1 or JAK2. We therefore investigated whether JAK1 or JAK2 were activated in Src-transformed cells. In three v-Src-transformed fibroblast cell lines (NIH3T3, Balb/c, and 3Y1), JAK1 displayed increased tyrosyl phosphorylation compared to non-transformed cells. The level of tyrosyl phosphorylation of JAK1 was significantly greater in NIH3T3 cells transformed by expression of v-Src or high levels of a constitutively active mutant of c-Src (Y527F) than in cells overexpressing the less transforming normal c-Src. Enzymatic activity of JAK1 was ...

The Journal of biological chemistry, Jan 5, 1989

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on... more We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled ba...

Growth Factors and Cytokines in Health and Disease, 1996

... Mutational studies in the EPO receptor (R), IL-2-R P-chain, and PRL-R have shown that the WSX... more ... Mutational studies in the EPO receptor (R), IL-2-R P-chain, and PRL-R have shown that the WSXWS motif is essential for ligand binding and subsequent signal transduction (Miyazaki et al., 1991; Quelle et al., 1992; Watowich et al., 1992; Rozakis Adcock and Kelly, 1992). ...

Trends in Endocrinology and Metabolism, 2001

In recent years, significant progress has been made in elucidating the signaling pathways activat... more In recent years, significant progress has been made in elucidating the signaling pathways activated by the growth hormone (GH) receptor. An initiating event is probably the activation of JAK2 (Janus kinase 2), a GH receptor-associated tyrosine kinase. Identification of the proteins recruited to the GH receptor-JAK2 complex and dissection of the signaling pathways that are subsequently activated will ultimately provide a basis for understanding GH action at the molecular level.

Proceedings of the National Academy of Sciences, 1982

When deprived of D-glucose for 24 hr, chicken embryo fibroblasts exhibit a marked increase in hex... more When deprived of D-glucose for 24 hr, chicken embryo fibroblasts exhibit a marked increase in hexose transport activity compared with that of control cells. Scatchard analysis of [3H]cytochalasin B binding to starved cell plasma membranes (46 pmol/mg) indicated a six-fold increase compared with fed cell plasma membranes (7.5 pmol/mg). Irradiation of starved cell plasma membranes with high-intensity UV light in the presence of0.5 IAM [3H]cytochalasin B resulted in covalent labeling ofpoly-

PLoS ONE, 2012

Leptin exerts its action by binding to and activating the long form of leptin receptors (LEPRb). ... more Leptin exerts its action by binding to and activating the long form of leptin receptors (LEPRb). LEPRb activates JAK2 that subsequently phosphorylates and activates STAT3. The JAK2/STAT3 pathway is required for leptin control of energy balance and body weight. Defects in leptin signaling lead to leptin resistance, a primary risk factor for obesity. Body weight is also regulated by nutrients, including glucose. Defects in glucose sensing also contribute to obesity. Here we report crosstalk between leptin and glucose. Glucose starvation blocked the ability of leptin to stimulate tyrosyl phosphorylation and activation of JAK2 and STAT3 in a variety of cell types. Glucose dose-dependently enhanced leptin signaling. In contrast, glucose did not enhance growth hormone-stimulated phosphorylation of JAK2 and STAT5. Glucose starvation or 2deoxyglucose-induced inhibition of glycolysis activated AMPK and inhibited leptin signaling; pharmacological inhibition of AMPK restored the ability of leptin to stimulate STAT3 phosphorylation. Conversely, pharmacological activation of AMPK was sufficient to inhibit leptin signaling and to block the ability of glucose to enhance leptin signaling. These results suggest that glucose and/or its metabolites play a permissive role in leptin signaling, and that glucose enhances leptin sensitivity at least in part by attenuating the ability of AMPK to inhibit leptin signaling.

Molecular Endocrinology, 1998

We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR)... more We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to test the notion that the protein chaperone heat shock protein-90 (hsp90) is required for steroiddependent translocation of the receptor through the cytoplasm along cytoskeletal tracks. The GFP-GR fusion protein undergoes steroid-mediated translocation from the cytoplasm to the nucleus, where it is transcriptionally active. Treatment of 3T3 cells containing steroid-bound GFP-GR with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, inhibits dexamethasone-dependent translocation from the cytoplasm to the nucleus. The t 1/2 for translocation in the absence of geldanamycin is ϳ5 min, and the t 1/2 in the presence of geldanamycin is ϳ45 min. In cells treated for 1 h with the cytoskeletal disrupting agents colcemid, cytochalasin D, and ␤,␤-iminodipropionitrile to completely disrupt the microtubule, microfilament, and intermediate filament networks, respectively, the GFP-GR still translocates rapidly to the nucleus in a strictly dexamethasone-dependent manner but translocation is no longer affected by geldanamycin. After withdrawal of the cytoskeletal disrupting agents for 3 h, normal cytoskeletal architecture is restored, and geldanamycin inhibition of dexamethasone-dependent GFP-GR translocation is restored. We suggest that in cells without an intact cytoskeletal system, the GFP-GR moves through the cytoplasm by diffusion. However, under physiological conditions in which the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery that is dependent upon hsp90 chaperone activity. In contrast to the GR, GFP-STAT5B, a signaling protein that is not complexed with hsp90, undergoes GH-dependent translocation to the nucleus in a manner that is not dependent upon hsp90 chaperone activity. (Molecular Endocrinology

Molecular Endocrinology, 2000

Binding of GH to GH receptor (GHR) rapidly and transiently activates multiple signal transduction... more Binding of GH to GH receptor (GHR) rapidly and transiently activates multiple signal transduction pathways that contribute to the growth-promoting and metabolic effects of GH. While the events that initiate GH signal transduction, such as activation of the Janus tyrosine kinase JAK2, are beginning to be understood, the signaling events that terminate GH signaling, such as dephosphorylation of tyrosyl-phosphorylated signaling molecules, are poorly understood. In this report, we examine the role of the SH2 (Src homology-2) domain-containing protein tyrosine phosphatase SHP-2 in GH signaling. We demonstrate that the SH2 domains of SHP-2 bind directly to tyrosyl phosphorylated GHR from GHtreated cells. Tyrosine-to-phenylalanine mutation of tyrosine 595 of rat GHR greatly diminishes association of the SH2 domains of SHP-2 with GHR, and tyrosine-to-phenylalanine mutation of tyrosine 487 partially reduces association of the SH2 domains of SHP-2 with GHR. Mutation of tyrosine 595 dramatically prolongs the duration of tyrosyl phosphorylation of the signal transducer and activator of transcription STAT5B in response to GH, while mutation of tyrosine 487 moderately prolongs the duration of STAT5B tyrosyl phosphorylation. Consistent with the effects on STAT5B phosphorylation, tyrosine-tophenylalanine mutation of tyrosine 595 prolongs the duration of tyrosyl phosphorylation of GHR and JAK2. These data suggest that tyrosine 595 is a major site of interaction of GHR with SHP-2, and that GHR-bound SHP-2 negatively regulates GHR/JAK2 and STAT5B signaling. (Molecular Endocrinology

Molecular and Cellular Biology, 2006

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hem... more 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-yetunidentified 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.

Molecular and Cellular Biology, 2004

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hem... more 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.

Journal of Cell Science, 2013

Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1b to the G... more Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1b to the GH-activated, GH receptorassociated tyrosine kinase JAK2, implicating SH2B1b in GH-dependent actin cytoskeleton remodeling, and suggesting that phosphorylation at serines 161 and 165 in SH2B1b releases SH2B1b from the plasma membrane. Here, we examined the role of SH2B1b 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. SH2B1b overexpression enhances, whereas SH2B1 knockdown inhibits, GH-dependent motility of RAW macrophages. At least two independent mechanisms regulate the SH2B1b-mediated changes in motility. In response to GH, tyrosines 439 and 494 in SH2B1b are phosphorylated. Mutating these tyrosines in SH2B1b decreases both basal and GH-stimulated macrophage migration. In addition, mutating the polybasic nuclear localization sequence (NLS) in SH2B1b or creating the phosphomimetics SH2B1b(S161E) or SH2B1b(S165E), all of which release SH2B1b from the plasma membrane, enhances macrophage motility. Conversely, SH2B1b(S161/165A) exhibits increased localization at the plasma membrane and decreased macrophage migration. Mutating the NLS or the nearby serine residues does not alter GH-dependent phosphorylation on tyrosines 439 and 494 in SH2B1b. Mutating tyrosines 439 and 494 does not affect localization of SH2B1b at the plasma membrane or movement of SH2B1b into focal adhesions. Taken together, these results suggest that SH2B1b enhances GH-stimulated macrophage motility via mechanisms involving phosphorylation of SH2B1b on tyrosines 439 and 494 and movement of SH2B1b out of the plasma membrane (e.g. as a result of phosphorylation of serines 161 and 165).

The American journal of physiology

Journal of Biological Chemistry

ABSTRACT

The American journal of physiology

Journal of Biological Chemistry

Recent progress in hormone research, 1998

The Journal of biological chemistry, Jan 31, 1997

The Journal of biological chemistry, Jan 5, 1989

Growth Factors and Cytokines in Health and Disease, 1996

Trends in Endocrinology and Metabolism, 2001

Proceedings of the National Academy of Sciences, 1982

PLoS ONE, 2012

Molecular Endocrinology, 1998

Molecular Endocrinology, 2000

Molecular and Cellular Biology, 2006

Molecular and Cellular Biology, 2004

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hem... more 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.

Journal of Cell Science, 2013