Tyrosine Phosphorylation of Jak2 in the JH2 Domain Inhibits Cytokine Signaling

MOLECULAR AND CELLULAR BIOLOGY, June 2004, p. 4968–4978 Vol. 24, No. 110270-7306/04/$08.00�0 DOI: 10.1128/MCB.24.11.4968–4978.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Tyrosine Phosphorylation of Jak2 in the JH2 Domain InhibitsCytokine Signaling

Edward P. Feener,†* Felicia Rosario,† Sarah L. Dunn,† Zlatina Stancheva,and Martin G. Myers, Jr.*

Harvard Medical School and Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215

Received 4 September 2003/Returned for modification 7 November 2003/Accepted 9 March 2004

Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes.Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokinesignaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopicanalysis of activated Jak2 protein from intact cells, we identified Tyr221 and Tyr570 as novel sites of Jak2phosphorylation. Phosphorylation of both sites was stimulated by cytokine treatment of cultured cells, and thisstimulation required Jak2 kinase activity. While we observed no gross alteration of signaling upon mutationof Tyr221, Tyr570 lies within the inhibitory JH2 domain of Jak2, and mutation of this site (Jak2Y570F) resultsin constitutive Jak2-dependent signaling in the absence of cytokine stimulation and enhances and prolongsJak2 activation during cytokine stimulation. Mutation of Tyr570 does not alter the ability of SOCS3 to bind orinhibit Jak2, however. Thus, the phosphorylation of Tyr570 in vivo inhibits Jak2-dependent signaling indepen-dently of SOCS3-mediated inhibition. This Tyr570-dependent mechanism of Jak2 inhibition likely representsan important mechanism by which cytokine function is regulated.

Type I cytokines mediate a plethora of physiologic pro-cesses, ranging from hematopoietic and immune functions(such as those mediated by erythropoietin [EPO] and the in-terleukins [ILs]) to growth and neuroendocrine responses(such as those mediated by growth hormone and leptin) (12,14, 16, 23). These actions are mediated by the activation ofcytokine receptor proteins found on the surface of target cells.Cytokine receptors each contain an extracellular domain thatrecognizes its specific cytokine ligand, a single transmembranedomain, and an intracellular domain that, although devoid ofenzymatic activity, transmits intracellular signals by means ofan associated Jak family tyrosine kinase. Ligand binding acti-vates the associated intracellular Jak kinase, resulting in thetyrosine phosphorylation of the Jak kinase and the intracellulardomain of the cytokine receptor. These tyrosine phosphoryla-tion events mediate the recruitment of downstream signalingmolecules that contain phosphotyrosine-binding SH2 domains(such as STAT proteins) (12, 16); tyrosine phosphorylationmay also mediate other regulatory events during cytokine sig-naling (8, 31).

The Jak kinase family contains four members: Jak1 to Jak3and Tyk2 (12, 16). Of these, Jak1, Jak2, and Tyk2 are ubiqui-tously expressed, while Jak3 is found predominantly in immuneand hematopoietic tissues. Jak kinases are composed of fourconserved domains. The NH2-terminal FERM domain is re-quired for interaction with cytokine receptors (24, 30), while

the adjacent SH2-like fold has no known function. TheCOOH-terminal portion of Jak kinases contains a kinase-likeJH2 domain that is devoid of enzymatic activity but that inhib-its the activity of the COOH-terminal JH1 tyrosine kinasedomain (10, 19, 22, 28, 29).

Our laboratory studies signaling by the long form of theleptin receptor (LRb), which regulates feeding, neuroendo-crine, and immune function in response to nutritional cues (9,11, 23). Jak2 mediates LRb signaling (as well as signaling byEPO, growth hormone, and numerous other cytokines) (14, 17,21, 23). While the tyrosine-phosphorylated residues on LRbare known, LRb mediates some signals that require Jak2 butnot tyrosine phosphorylation sites on LRb, suggesting thatthese signals are mediated by tyrosine phosphorylation of Jak2itself (2, 25). Although it is clear that Jak2 and other Jak kinasefamily members become tyrosine phosphorylated on numeroussites, except for paired sites in the activation loop of the kinasedomain and one other recently mapped site (of unclear func-tion) (5, 8, 31), the identity and function of Jak2 phosphory-lation sites remain unknown.

In order to gain insight into the function of Jak2 tyrosinephosphorylation in LRb signaling, we purified activated Jak2protein for analysis by liquid chromatography-tandem massspectroscopy (LC-MS/MS) in order to identify tyrosine phos-phorylation sites on Jak2. We report that Tyr221 and Tyr570 aresites of Jak2 tyrosine phosphorylation. Phosphorylation ofTyr570, which lies within the inhibitory JH2 domain, inhibitsJak2-mediated cytokine signaling.

MATERIALS AND METHODS

Antibodies, growth factors, and reagents. Rabbit anti-LRb (�-LRb) has beendescribed previously (2); rabbit �-Jak2(758) and �-STAT3(PY705) were raisedagainst synthetic peptides corresponding to amino acids 758 to 770 of murineJak2 and the phosphopeptide corresponding to amino acids 700 to 710, includingphosphorylated Tyr705, of murine STAT3. Antibody to phosphorylated Tyr1007

and Tyr1008 of Jak2 [�-Jak2(PY1007,8)] was raised against a keyhole limpet

* Corresponding author. Mailing address for Edward P. Feener:Joslin Proteomics Core, Joslin Diabetes Center, 1 Joslin Pl., Boston,MA 02215. Fax: (617) 732-2637. E-mail: [email protected]. Present address for Martin G. Myers, Jr.: Division ofMetabolism, Endocrinology and Diabetes, Department of Medicine,University of Michigan Medical School, 4301 MSRB III, Box 0638,1150 W. Medical Center Dr., Ann Arbor, MI 48109-0638. Phone: (734)647-9515. Fax: (734) 936-6684. E-mail: [email protected].

† E.P.F., F.R., and S.L.D. contributed equally to this work.

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hemocyanin-coupled 12-amino-acid synthetic peptide phosphorylated on bothtyrosine residues. Both �-STAT3(PY705) and �-Jak2(PY1007,8) were purifiedon antigen peptide without subtraction against unphosphorylated peptide orirrelevant phosphopeptides; this preparation of �-Jak2(PY1007,8) is commer-cially available from Upstate Biotechnology (Lake Placid, N.Y.). Antibodiesrecognizing phosphorylated Tyr221 and Tyr570 of Jak2 were raised in rabbits byinjection of a keyhole limpet hemocyanin-coupled synthetic 11-amino-acid phos-phorylated peptide centered on Tyr221 and Tyr570, respectively; antisera wereaffinity purified on the antigen peptide coupled to a mixture of Affigel-10 and -15(Bio-Rad), followed by passage over Affigel coupled to irrelevant tyrosyl phos-phopeptides and nonphosphorylated antigen peptide to remove antibodies di-rected against other sites of tyrosine phosphorylation and to the nonphosphory-lated form of the site. Synthetic peptides were purchased from BostonBiomolecules (Framingham, Mass.). Recombinant murine IL-3 was obtainedfrom Pierce Endogen (Rockford, Ill.); monoclonal 4G10 was used for �-PYimmunoblotting (Upstate Biotechnology). Antibodies directed against the phos-phorylated (activated) form of ERK were purchased from Cell Signaling Tech-nology (Beverly, Mass.), and that against Jak2(CT) was from Santa Cruz Bio-technology (Santa Cruz, Calif.). Recombinant mouse EPO was purchased fromCardinal Health. Bovine serum albumin fraction V was purchased from Sigma.Protein A-Sepharose 6MB, [125I]EPO, and [125I]protein A were from AmershamPharmacia Biotech (Piscataway, N.J.).

Generation of mutant Jak2 cDNAs. pcDNA3Jak2 (17) was used as templatefor mutagenesis using the QuikChange kit (Stratagene) to replace Tyr221, Tyr570,or Tyr1007 and Tyr1008 with Phe individually (to generate pcDNA3Jak2Y221F andpcDNA3Jak2Y570F) or in combination (pcDNA3Jak2Y1007,8F). The presence ofthe desired mutations and the absence of adventitious mutations were confirmedby DNA sequencing.

Cell lines. All cells were maintained in a humidified atmosphere containing5% CO2 and 95% air at 37°C. 32D cells were grown in RPMI 1640 mediumsupplemented with 10% fetal bovine serum and 5% WEHI-3-conditioned me-dium (a source of IL-3) (2). 293 cells were grown in Dulbecco’s modified Eagle’smedium supplemented with 10% fetal bovine serum. ELR constructs in pcDNA3were transiently cotransfected with pcDNA3 alone or with the appropriatepcDNA3Jak2 isoform into subconfluent 293 cells using Lipofectamine (Amer-sham Pharmacia). In most experiments, 15-cm dishes were transfected with 15�g of pcDNA3ELR plus 10 �g of the appropriate Jak2 isoform, except for signalattenuation experiments in which 24 �g of ELR and 1 �g of Jak2 were used per15-cm plate.

Preparation of cell lysates for immunoprecipitation. Prior to each experiment,subconfluent cells were made quiescent by overnight incubation in Dulbecco’smodified Eagle’s medium containing 0.5% bovine serum albumin (32D cells for4 h, 293 cells overnight) before stimulation with EPO or IL-3 at 37°C. Cells werelysed in 20 mM Tris, pH 7.4, containing 137 mM NaCl, 2 mM EDTA, 10%glycerol, 50 mM �-glycerophosphate, 50 mM NaF, 1% Nonidet P-40, 2 mMphenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate (lysis buffer).Insoluble material was removed by centrifugation at 16,000 � g at 4°C for 5 min.Protein concentrations of the resulting lysates were determined, and equivalentamounts of protein were added to the appropriate antibodies for immunopre-cipitation or were denatured in 2� Laemmli buffer for direct resolution bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Forimmunoprecipitates, lysates were incubated with antibody at 4°C overnight fol-lowed by incubation with protein A-Sepharose for 30 min. Immune complexeswere collected by centrifugation and washed three times in lysis buffer beforedenaturation in Laemmli buffer and separation by SDS-PAGE.

LC-MS/MS analysis. For preparation of protein for LC-MS/MS analysis, ma-terial was immunoprecipitated from 5 to 10 15-cm dishes of cells and resolved ona single lane of an SDS–7% PAGE gel. Jak2 protein was visualized by stainingwith Coomassie brilliant blue G-250 stain (Bio-Rad) and destaining overnight in10% methanol–10% glacial acetic acid. Gel slices containing Jak2 were digestedwith 5 ng of sequencing-grade modified trypsin (Promega)/�l in 25 mM ammo-nium bicarbonate containing 0.01% N-octylglucoside for 18 h at 37°C. Peptideswere eluted from the gel slices with 80% acetonitrile–1% formic acid. Trypticdigests were separated by capillary high-performance liquid chromatography(C18, 75-�m-inner-diameter Picofrit column; New Objective) using a flow rate of100 nl/min over a 3-h reverse-phase gradient and analyzed using an LCQ DecaXP Plus ion trap LC/MSn system (ThermoFinnigan). Resultant MS/MS spectrawere matched against mouse JAK2 sequence using TurboSequest (BioWorks3.1) with fragment ion tolerance of �0.5 and amino acid modification variablesincluding phosphorylation (80 Da) of Ser, Thr, and Tyr, oxidation (16 Da) ofMet, and methylation (14 Da) of Lys. MS/MS spectra that matched Jak2 phos-phopeptides were then analyzed using DeNovoX software (Thermofinnigan) to

confirm phosphopeptide sequence assignments. Synthetic phosphopeptides, cor-responding to tryptic phosphopeptide sequences derived from Jak2, were ob-tained (Boston Biomolecules, Inc.) and analyzed using the LC-MS/MS protocoldescribed above. MS/MS spectra from these synthetic peptide controls andJak2-derived tryptic peptides were compared for correlation of fragmentationion m/z and abundance.

Immunoblotting. SDS-PAGE gels were transferred to nitrocellulose mem-branes (Schleicher & Schuell) in Towbin buffer containing 0.02% SDS and 20%methanol. Membranes were blocked for 1 h at room temperature or overnight at4°C in buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, and 0.01% Tween20 (wash buffer) supplemented with 3% bovine serum albumin (block buffer).Membranes were incubated in primary antibody in block buffer for 1 h, rinsedthree times with wash buffer, and incubated for 30 min in block buffer. Detectionwas by incubation for 1 h with 125I-labeled protein A in block buffer (precededby a 1-h incubation with rabbit anti-mouse antisera, followed by washing in thecase of 4G10 immunoblotting) or by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz). Blots were rinsed four times inwash buffer before overnight exposure on Kodak X-Omat AR film or a phos-phorimager (for 125I-labeled protein A) or were subjected to enhanced chemi-luminescence analysis (Western Lightning; Pierce) and exposed to film.

Luciferase reporter assays. Various combinations of expression vectors forELR and Jak2 constructs were cotransfected into HEK 293 cells along withcombinations of the STAT3-responsive gamma interferon-activated sequence(GAS)-Luc and pRL-TK (Promega), encoding Renilla luciferase (4). Followingtransfection, cells were switched to serum-free medium and stimulated for var-ious times with various concentrations of EPO. Cells were lysed and assayed withthe Stop-n-Glo dual luciferase reporter kit (Promega) according to the kit’sinstructions; GAS-Luc firefly luciferase activity was normalized for transfectionefficiency with Renilla luciferase from the constitutive pRL-TK plasmid.

Analysis of SOCS3 function. The SOCS3 expression vector pcDNA3SOCS3has been described previously (4). For the study of SOCS3-mediated inhibition,HEK 293 cells were transiently transfected with ELR or ELRL985 (6 �g), Jak2 orJak2Y570F (2 �g), and pcDNA3 plus pcDNA3SOCS3 (0, 0.2, or 2 �g), totaling 2�g. Cells were made quiescent overnight before stimulating in the absence orpresence of EPO for 15 min. Cells were lysed and analyzed as described above.

RESULTS

Since we are interested in mechanisms by which Jak2 maycontribute to LRb signaling, we undertook to identify sites ofJak2 phosphorylation that are regulated by the activation ofthe intracellular domain of LRb. We prepared Jak2 protein by�-Jak2(758) immunoprecipitation from 293 cells cotransfectedwith Jak2 and an EPO receptor-LRb chimera (ELR) thatplaces the intracellular domain of LRb under the control ofEPO; we employed this ELR in place of native LRb, since theextracellular domain of the EPO receptor confers much higherlevels of receptor expression and facilitates the study of signal-ing by the intracellular domain of LRb in transfected cells.Jak2 protein that was immunoprecipitated from cells followingincubation in the absence or presence of EPO for 15 min wasresolved by SDS-PAGE and visualized by staining with Coo-massie blue (Fig. 1A). This Jak2 protein was subjected totryptic proteolysis and extracted from the gel, and the resultingpeptides were subjected to LC-MS/MS analysis. Analysis ofMS/MS spectra using TurboSequest identified two Jak2 trypticpeptides, IQDY221(P)HILTR and REVGDY570(P)GQLHK,with Xcorr scores of �2.0 for 2� charged precursors (Fig. 1Band C, top panels). Both of these spectra displayed ions con-sistent with the neutral ion loss of 40 m/z resulting from the lossof HPO3 from doubly charged phosphotyrosine-containingpeptides. Analysis of these MS/MS spectra using DeNovoXgenerated peptide sequence tags that confirmed Sequest as-signments (data not shown). MS/MS spectra corresponding topeptides containing phosphorylated Tyr221 and phosphory-lated Tyr570 from Jak2 were detected from numerous indepen-

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dent analyses, suggesting that these residues represent sites ofin vivo phosphorylation. In order to further confirm the iden-tity of these peptides, phosphopeptides corresponding to thecandidate phosphorylated Tyr221 and Tyr570 Jak2 tryptic pep-

tides were synthesized and subjected to LC-MS/MS analysis(Fig. 1B and C, lower panels). The appearance and relativeabundance of y and b ions generated from these syntheticphosphopeptides correlated with fragmentation ions observed

FIG. 1. Purification and analysis of Jak2 protein. (A) HEK 293 cells were left untransfected or were transfected with the cDNAs for ELR andJak2, were made quiescent, and were then incubated a further 15 min in the absence (�) or presence (�) of EPO (10 U/ml). Cells were lysed,and lysates were immunoprecipitated with �-Jak2(758). Immunoprecipitated proteins were resolved by SDS-PAGE and detected by staining withCoomassie blue; the relevant region of a representative gel is shown in the panel. The migration of Jak2 protein is indicated to the right of thegel. (B) MS/MS spectra for the Jak2 tryptic peptide phosphorylated at Tyr221 (m/z 620.43; top) and corresponding synthetic peptide,IQDY(P)HILTR (m/z 620.25; bottom). (C) MS/MS spectra for the Jak2 tryptic peptide phosphorylated at Tyr570 (m/z 691.25; top) andcorresponding synthetic peptide, REVGDY(P)GQLHK (m/z 691.64; bottom). Sequest assignments of y� and b� ions are shown in red and blue,respectively. Fragment ions corresponding to the neutral loss of 40 m/z from these doubly charged precursors are shown in green.



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in corresponding spectra of Jak2-derived peptides, confirmingassignment of Jak2 phosphorylation at Tyr221 and Tyr570.

In order to facilitate the study of the in vivo phosphorylationstatus of these residues and to study their regulation, we gen-erated antibodies specific for the phosphorylated forms ofTyr221 [�-Jak2(PY221)] and Tyr570 [�-Jak2(PY570)] and gen-erated Jak2 cDNAs in which Tyr221 or Tyr570 were replacedwith Phe (Jak2Y221F and Jak2Y570F, respectively) or in whichthe activation loop tyrosine residues (Tyr1007 and Tyr1008) werereplaced with Phe (Jak2Y1007,8F; mutation of Tyr1007 andTyr1008 in the activation loop of the Jak2 kinase domain in thelatter mutant impairs Jak2 kinase activation) (8). In order tounderstand the regulation of Tyr221 and Tyr570 phosphoryla-tion, we transfected 293 cells with ELR in combination withcontrol plasmid or the cDNAs for various Jak2 mutants (Fig.2A). Cells were incubated in the absence or presence of EPOand lysed, and lysates were immunoprecipitated with�-Jak2(758) or �-LRb. Since �-Jak2(758) also recognizes anonphosphoprotein from 293 cells that comigrates with Jak2on SDS-PAGE (Fig. 1A), it is difficult to distinguish Jak2 fromthis protein by immunoblotting with �-Jak2(758); we thus de-tect the expression of Jak2 protein by immunoblotting with�-Jak2(CT). We often note the loss of some �-Jak2(CT) re-activity upon Jak2 phosphorylation, however, consistent withour preliminary data suggesting Ser/Thr phosphorylation ofthe COOH terminus of Jak2 (data not shown).

�-PY immunoblotting of �-Jak2(758)-precipitable materialdemonstrated ligand-stimulated tyrosine phosphorylation ofJak2, Jak2Y221F, and Jak2Y570F; as expected, tyrosine phos-phorylation of Jak2Y1007,8F was virtually undetectable. Whilethe phosphorylation of Jak2 and Jak2Y221F was modest in theabsence of ligand stimulation, we noted the increased totaltyrosine phosphorylation of Jak2Y570F in the absence of ligandstimulation. The modest decrease in total �-PY immunoreac-tivity in Jak2Y221F compared to that with Jak2 when normal-ized for the increase Jak2Y221F protein recovered in this ex-periment could be consistent with alteration in the activity ofJak2Y221F or with the loss of a major site of phosphorylation inJak2Y221F.

Immunoblotting with �-Jak2(PY221) demonstrated thatTyr221 was phosphorylated during ligand stimulation of Jak2and Jak2Y570F (as well as on Jak2Y570F from unstimulatedcells), but not on Jak2Y221F or Jak2Y1007,8F. These data suggestthat Jak2 tyrosine kinase activity is required for the ligand-activated phosphorylation of Tyr221 on Jak2 and that Tyr570 isnot required for the phosphorylation of Tyr221, although theabsence of Tyr570 may increase phosphorylation of Tyr221 andother sites. As expected, phosphorylation of Tyr570 was notdetected on Jak2Y570F; while some phosphorylation of Tyr570

was detected on Jak2 and Jak2Y221F from unstimulated cells,ligand treatment increased the phosphorylation of Tyr570 oneach of these Jak2 isoforms. In contrast to the apparent de-crease in total �-PY reactivity relative to protein observed inJak2Y221F, �-Jak2(PY570) reactivity was increased in approx-imate proportion to the increased amount of Jak2Y221F proteinexpressed, suggesting that the observed decrease in �-PY re-activity in Jak2Y221F may be secondary to the loss of a majorsite of phosphorylation in this mutant. Some phosphorylationof Tyr570 was detectable in Jak2Y1007,8F, but it was not in-creased appreciably by EPO stimulation. Since �-Jak2(PY570)

was antigen affinity purified and additionally purified to re-move binding to the unphosphorylated Tyr570 and nonspecificphosphotyrosine (see Materials and Methods for details),these data suggest that Tyr570 of Jak2 is phosphorylated tosome extent in unstimulated cells, even on a poorly activatedJak2 isoform, but that the cytokine-induced phosphorylation ofTyr570 requires Jak2 kinase activity.

We employed �-PY immunoblotting to detect phosphory-lated Jak2 in �-LRb immunoprecipitates, since �-PY immu-noblotting is very sensitive and does not suffer the variousshortcomings of �-Jak2(758) and �-Jak2(CT). This analysis

FIG. 2. Phosphorylation of Tyr221 and Tyr570 in HEK 293 and 32Dcells. (A and B) HEK 293 cells were transfected with ELR and theindicated Jak2 isoform, made quiescent, and incubated in the absence(�) or presence (�) of EPO (10 U/ml) for an additional 15 min beforebeing lysed. (C) Quiescent 32D cells were incubated in the absence(�) or presence (�) of IL-3 (10 U/ml) for 15 min and lysed. In allpanels, lysates were immunoprecipitated with the indicated antibodies.Lysates or immunoprecipitated proteins (as indicated) were resolvedby SDS-PAGE and transferred to nitrocellulose for immunoblottingwith the indicated antibody. The migration of detected signaling pro-teins is noted to the right of the panels.



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demonstrated the ligand-stimulated tyrosine phosphorylationof ELR and of the ELR-associated Jak2 isoforms in cellsexpressing Jak2, Jak2Y221F, and Jak2Y570F, but notJak2Y1007,8F. In order to control for the possibility that theobserved ELR-associated phosphorylated Jak2 representedmaterial that became nonspecifically associated with the�-LRb immunoprecipitate, we analyzed nonimmune and�-Jak2 immunoprecipitates alongside �-LRb immunoprecipi-tate by �-PY immunoblotting (Fig. 2B). Since no phosphory-lated Jak2 was detected in the nonimmune immunoprecipi-tates while it was detected in �-LRb and �-Jak2immunoprecipitates, this suggests that the Jak2 recovered in�-LRb immunoprecipitates represented ELR-associated Jak2protein. Since essentially all of the tyrosine phosphorylation ofJak2 in each immunoprecipitate occurred secondary to recep-tor stimulation, the smaller amount of tyrosine-phosphorylatedJak2 recovered in the �-LRb immunoprecipitate than the�-Jak2(758) immunoprecipitate likely reflects the instability ofthe receptor-Jak2 complex during immunoprecipitation, notthe predominance of Jak2 that is not associated with receptor.Thus, in total, these data suggest that neither Tyr221 nor Tyr570

of Jak2 is required for interaction with or tyrosine phosphor-ylation of ELR.

In order to determine whether the phosphorylation of Tyr221

and Tyr570 are regulated by multiple cytokines and at endog-enous levels of receptor and Jak2, we examined the ability ofIL-3 to stimulate the phosphorylation of Tyr221 and Tyr570 inuntransfected 32D myeloid progenitor cells (Fig. 2C). Quies-cent 32D cells were incubated for 10 min in the absence orpresence of IL-3 and lysed. Lysates were immunoprecipi-tated with �-Jak2(758), and the immunoprecipitated materialwas immunoblotted with �-PY, �-Jak2(PY221), and �-Jak2(PY570). This analysis revealed the expected IL-3-stimulatedtyrosine phosphorylation of Jak2 and STAT3; IL-3 also stim-ulated �-Jak2(PY221) and �-Jak2(PY570) immunoreactivityin immunoprecipitates of endogenous Jak2 from 32D cells,suggesting that Tyr221 and Tyr570 of Jak2 are phosphorylatedduring activation of multiple cytokine receptors at endogenouslevels of receptor and Jak2.

In order to examine the roles of Tyr221 and Tyr570 in signal-ing by the intracellular domain of LRb, we cotransfected 293cells with the ELR cDNA and empty vector or the cDNAs forJak2, Jak2Y221F, Jak2Y570F, and Jak2Y1007,8F. Transfected cellswere incubated in the absence or presence of EPO for 15 minand lysed. Lysates were immunoprecipitated with �-Jak2(758)for detection of Jak2 protein expression and tyrosine phos-phorylation or were directly resolved by SDS-PAGE for de-tection of STAT3 and ERK activation by immunoblotting withantibodies specific to their phosphorylated (activated) forms(Fig. 3). Expression of the various Jak2 isoforms was confirmedby �-Jak2(CT) immunoblotting of �-Jak2(758) immunopre-cipitates. The analysis of signaling demonstrated a smallamount of ligand-stimulated tyrosine phosphorylation of en-dogenous Jak2 and moderate ligand-stimulated activation ofSTAT3 and ERK in cells transfected with ELR only; similarlevels of ELR signaling in cells expressing ELR andJak2Y1007,8F presumably reflected similar amounts of endoge-nous Jak2 activation in these cells, consistent with the inabilityof Jak2Y1007,8F to mediate tyrosine phosphorylation events thatwould substantially increase the amplitude of signaling (8).

Although basal phosphorylation of Jak2, STAT3, and ERKwere poorly detectable in cells expressing Jak2 and Jak2Y221F,ligand stimulation similarly increased the phosphorylation ofJak2, STAT3, and ERK to a much greater level than observedwithout Jak2 overexpression, suggesting that overexpression ofJak2 enhances ELR signaling and that Jak2Y221F mediatesthese ELR signals normally. The slightly greater signaling me-diated by ELRY221F is again consistent with the modestly in-creased expression of this Jak2 isoform in this experiment. Weagain observed increased tyrosine phosphorylation ofJak2Y570F in the absence of ligand stimulation, with furtherincreased phosphorylation following ligand stimulation. Simi-larly, STAT3 and ERK phosphorylation was increased in theabsence of ligand stimulation in cells expressing Jak2Y570F,consistent with the notion that this Jak2 mutant is active in theabsence of cytokine stimulation.

In order to gain further insight into the increased basalactivity of Jak2Y570F, we examined its activity in the absence orpresence of ELR. 293 cells were transfected with ELR plusvector control, Jak2, Jak2Y221F, Jak2Y570F, or with Jak2 orJak2Y570F in the absence of receptor cDNA (Fig. 4). Cells wereincubated in the absence or presence of EPO for 15 min andlysed, and lysates were immunoprecipitated with �-Jak2(758)or resolved directly by SDS-PAGE for immunoblot analysis.Similar amounts of �-Jak2(CT) reactivity were observed incells expressing ELR plus Jak2 and Jak2Y221F, although theincreased phosphorylation of Jak2Y570F rendered this Jak2mutant difficult to detect by immunoblotting with �-Jak2(CT).As seen previously, EPO stimulation resulted in similar �-PYreactivity of Jak2 and Jak2Y570F when coexpressed with ELR,with slightly decreased levels of �-PY reactivity in Jak2Y221F.The finding of similar �-Jak2(PY1007,8) reactivity among allthree Jak2 isoforms suggested that the decrease in �-PY reac-tivity of Jak2Y221F may be secondary to the loss of a major siteof phosphorylation in this mutant; this notion was again sup-ported by the finding of similar �-Jak2(PY570) reactivity forJak2 and Jak2Y221F (which was absent in Jak2Y570F). The sim-ilar �-Jak2(PY1007,8) reactivity in Jak2 and Jak2Y221F is con-

FIG. 3. Role of phosphorylation of Jak2 Tyr221 and Tyr570 in ELRsignaling. HEK 293 cells were transfected with ELR and the indicatedJak2 isoform, made quiescent, and incubated in the absence (�) orpresence (�) of EPO (10 U/ml) for 15 min before lysis. Lysates weresubjected to immunoprecipitation with the indicated antibody or di-rectly resolved by SDS-PAGE before transfer to nitrocellulose forimmunoblotting with the indicated antibodies. The migration of de-tected signaling proteins is noted to the right of the panels.



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sistent with the similar ERK and STAT3 signaling mediated bythese isoforms of Jak2.

In the absence of ELR, similar amounts of �-Jak2(CT)-reactive Jak2 and Jak2Y570F were detected. Immunoblottingwith �-PY and �-Jak2(PY1007,8) once again demonstratedincreased phosphorylation of Jak2Y570F compared to Jak2. Im-portantly, the robust tyrosine phosphorylation and activationof Jak2Y570F failed to activate STAT3 and ERK in the absenceof ELR, suggesting that while the presence and phosphoryla-tion of Tyr570 of Jak2 controls the activity of Jak2 indepen-dently of whether the Jak2 molecule is associated with thereceptor, receptor association is still required for signaling bythe activated Jak2.

The increased signaling mediated by Jak2Y570F in the ab-

sence of ligand stimulation suggests a role for Tyr570 in regu-lating the amplitude and duration of cytokine receptor signal-ing. We thus compared the effects of Jak2 and Jak2Y570F onELR-mediated STAT3-dependent transcription in a luciferasereporter assay (Fig. 5, left panel). ELR was transfected withthe STAT3-responsive GAS-luciferase plasmid (4), a controlplasmid expressing Renilla luciferase for normalization, andempty vector, pcDNA3Jak2, or pcDNA3Jak2Y570F. Followingtransfection, cells were switched into serum-free medium ormedium containing EPO. Cells were harvested 12 h later andassayed for luciferase activity. This analysis demonstrated thatEPO stimulated an approximately sevenfold increase in tran-scription from the GAS-luciferase reporter construct in cellsexpressing ELR in the presence of endogenous Jak2; coexpres-sion of Jak2 did not significantly alter EPO-stimulated GAS-luciferase transcription, although it did increase GAS-lucif-erase transcription by approximately twofold in the absence ofcytokine treatment. The finding of increased GAS-luciferaseactivity with overexpression of Jak2 only in the absence ofstimulation is consistent with the idea that small increases inSTAT3 phosphorylation are sufficient to maximally increaseSTAT3-driven reporter activity, and that increases in EPO-stimulated STAT3 phosphorylation with overexpression ofJak2 may thus not result in increased STAT3-mediated tran-scription over that observed in the absence of Jak2 overexpres-sion. In contrast, coexpression of Jak2Y570F with ELR medi-ated greatly increased GAS-luciferase activity in the absence ofligand (comparable to that of ELR plus wild-type Jak2 in thepresence of ligand). EPO stimulation in cells expressingJak2Y570F further increased reporter activity to levels greaterthan those observed with wild-type Jak2 in the presence ofEPO; since STAT3 phosphorylation (Fig. 3 and 4) does notappear different between cells expressing ELR and Jak2 orJak2Y570F, this increase in reporter activity may be secondaryto the increased STAT3 phosphorylation and activity in cellsexpressing Jak2Y570 prior to EPO stimulation. Thus, Jak2Y570F

mediates increased STAT3 activity compared to wild-type Jak2both in the absence and presence of cytokine stimulation. Weperformed a similar analysis comparing the ability of Jak2,Jak2Y221F, and Jak2Y1007F to mediate ELR-stimulated STAT3

FIG. 4. Activation of Jak2Y570F in the absence and presence ofELR. HEK 293 cells were transfected with ELR plus control plasmidor the indicated Jak2 isoform, made quiescent, and incubated in theabsence (�) or presence (�) of EPO (10 U/ml) for an additional 15min before lysis. Lysates were subjected to immunoprecipitation withthe indicated antibody or directly resolved by SDS-PAGE before trans-fer to nitrocellulose for immunoblotting with the indicated antibodies.The migration of detected signaling proteins is noted to the right of thepanels.

FIG. 5. Enhanced STAT3 signaling mediated by Jak2Y570F. HEK 293 cells were transfected with the cDNA for ELR plus control plasmid orthe indicated isoform of Jak2, along with the STAT3-responsive GAS-luciferase and control pRL-TK (constitutively expressing Renilla luciferase)plasmids (4). After transfection, cells were placed in serum free medium (�) or the same medium containing 10 U of EPO/ml (�) for 12 h beforebeing lysed and subjected to a dual luciferase assay. Firefly luciferase activity from the GAS-luciferase construct (normalized to Renilla luciferaseactivity) is shown in the graph. Left and right panels represent independent experiments, each representative of at least two similar experiments.Numbers represent the means of triplicate determinations the standard error of the mean for the single experiment shown.



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reporter activity (Fig. 5, right panel). Once again, overexpres-sion of Jak2 increased the basal levels of STAT3 reporteractivity, while it did not significantly alter the ligand-stimulatedlevels. Similar results were observed with Jak2Y221F: increasedbasal activity with normal stimulated activity, again suggestingthat this Jak2 mutant mediates cytokine-mediated signalingnormally. In contrast, no alteration in basal or stimulated ac-tivity was detected with Jak2Y007,8F compared to vector con-trol, consistent with the inability of this Jak2 mutant to aug-ment the levels of cytokine receptor signaling observed withendogenous Jak2.

In order to understand the role of Tyr570 in the duration aswell as the amplitude of cytokine signaling, we assayed thetyrosine phosphorylation of Jak2 during prolonged ELR acti-vation in cells expressing ELR and Jak2, Jak2Y221F, or

Jak2Y570F (Fig. 6). This analysis demonstrated that phosphor-ylation of Jak2 and Jak2Y2221F decreases toward baseline dur-ing prolonged (24-h) stimulation, with slightly less phosphor-ylation of Jak2Y221F remaining. In contrast, tyrosinephosphorylation of Jak2Y570F failed to decrease after 24 h ofstimulation, suggesting that phosphorylation of Tyr570 of Jak2may participate in the deactivation of Jak2 during prolongedcytokine stimulation.

The suppressor of cytokine signaling-3 (SOCS3) protein hasbeen implicated in the inhibition of Jak2-dependent signalingby LRb and other cytokine receptors (3, 4, 7, 27). SOCS3 is aninhibitory protein that blocks signaling by binding via its phos-photyrosine-binding SH2 domain to specific tyrosine phos-phorylation site targets within signaling complexes (18). Whilethe sensitive inhibition of LRb-STAT3 signaling by SOCS3 ismediated by the binding of SOCS3 to phosphorylated Tyr985

on the intracellular tail of LRb, SOCS3 also appears to binddirectly to phosphorylated Jak2 (4). Mutation of Tyr1007 in theactivation loop of Jak2 blocks SOCS3 binding to Jak2, butsince this Jak2 mutant is inactive, it is not clear whether Tyr1007

is the actual SOCS3 binding site or whether mutation of thissite merely inhibits SOCS3 binding by inactivating the kinase,blocking the phosphorylation of the true SOCS3 binding siteon Jak2. In order to determine whether the inhibitory effects ofTyr570 might be mediated by the binding of SOCS3, we thusexamined the ability of SOCS3 to inhibit Jak2Y570F in complexwith ELR or ELRL985 (in which the direct binding site forSOCS3 on the tail of LRb is mutated) (Fig. 7). In order todetermine whether Tyr570 was dispensable for the SOCS3-mediated inhibition of Jak2 phosphorylation, we analyzed Jak2phosphorylation in cells expressing ELR or ELRL985 plus Jak2

FIG. 6. Prolonged activation of Jak2Y570F. HEK 293 cells weretransfected with ELR and the indicated Jak2 isoform, made quiescent,and incubated in the absence (�) or presence of EPO (10 U/ml) forthe indicated time before lysis. Lysates were immunoprecipitated with�-Jak2(758), and immunoprecipitated proteins were resolved by SDS-PAGE and transferred to nitrocellulose before immunoblotting withthe indicated antibody. The migration of signaling proteins is indicatedto the right of the panels. This figure is representative of multiplesimilar experiments.

FIG. 7. Role of Jak2 Tyr570 in SOCS3-mediated inhibition. HEK 293 cells were transiently transfected with ELR or ELRL985, Jak2 or Jak2Y570F

plus empty vector, and/or the indicated amount of pcDNA3SOCS3 (0.2 or 2 �g; vector plus pcDNA3 totaled 2 �g in each case). Cells wereincubated in the absence or presence of EPO for 5 min before lysis and immunoprecipitation of Jak2. Washed immune complexes or lysates (asindicated) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. The migration of signaling proteins is indicated to theright of the panels. This figure is representative of two independent experiments.



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or Jak2Y570F in the presence or absence of various amounts ofSOCS3 cDNA (Fig. 7B). In cells expressing ELR or ELRL985

plus Jak2, EPO stimulation increased the tyrosine phosphory-lation of Jak2, as seen previously. The addition of SOCS3 tocells expressing ELR plus Jak2 inhibited the EPO-stimulatedincrease in Jak2 phosphorylation, as previously demonstrated(4); in contrast, SOCS3 expression only modestly diminishedthe EPO-stimulated phosphorylation of Jak2 expressed withELRL985, consistent with a role for Tyr985 of LRb in SOCS3-mediated inhibition of signaling. As described previously,Jak2Y570F was phosphorylated in the absence of ligand in cellsexpressing ELR; this was also the case in cells expressingELRL985. The addition of SOCS3 to cells expressing ELR plusJak2Y570F blocked almost all phosphorylation of Jak2Y570F;somewhat greater amounts of SOCS3 were required to simi-larly repress the phosphorylation of Jak2Y570F when expressedwith ELRL985. Thus, as previously reported, Tyr985 of LRb isrequired for the sensitive inhibition of LRb-Jak2 signaling;although SOCS3 also inhibits Jak2 signaling by binding to Jak2independently of LRb Tyr985, Tyr570 of Jak2 is not required forthe binding and inhibition of Jak2 by SOCS3. Indeed, Jak2appears to be more sensitive to SOCS3-mediated inhibition inthe absence of Tyr570.

DISCUSSION

We have identified two previously unreported sites of ty-rosine phosphorylation on Jak2: Tyr221 within the FERM do-main and Tyr570 within the JH2 domain. Both of these residuesbecome phosphorylated upon activation of a chimeric receptorcontaining the intracellular sequences of LRb and by activa-tion of endogenous IL-3 receptor in 32D cells at endogenouslevels of Jak2, suggesting that both sites represent physiologi-cally relevant sites of Jak2 phosphorylation during cytokinesignaling. Both sites also require activation of the Jak2 tyrosinekinase for ligand-stimulated phosphorylation; this and theirrapid phosphorylation during ELR signaling suggests thatTyr221 and Tyr570 represent sites of autophosphorylation onJak2, although we cannot formally exclude the possibility thatthese sites are phosphorylated by another kinase that is depen-dent upon the kinase activity of Jak2 for its own activation.While we failed to detect significant phosphorylation of Tyr221

on Jak2 from unstimulated cells or on the inactive Jak2Y1007,8F,we did detect some phosphorylation of Tyr570 on Jak2 fromunstimulated cells, although its phosphorylation was also in-creased upon Jak2 activation. We similarly detected basalphosphorylation of Tyr570 on the inactive Jak2Y1007,8F, al-though the phosphorylation of Tyr570 did not increase substan-tially upon cytokine stimulation of Jak2Y1007,8F. Consistentwith this finding, our LC-MS/MS analysis consistently detectedphosphorylated Tyr570 on Jak2 and Jak2Y1007,8F from quies-cent cells (data not shown). Combined with the observationthat mutation of Tyr570 increased the basal signaling activity ofJak2, these data suggest that phosphorylation of Tyr570 in theunstimulated state may act to restrain the activity of Jak2.

Jak2 contains a large number of tyrosine phosphorylationsites, including Tyr1007 and Tyr1008 within the activation loop ofthe kinase domain and Tyr966, just NH2-terminal to the cata-lytic loop of the kinase domain (5, 8); the identities of the manyother sites of Jak2 phosphorylation are unknown. Identifica-

tion of phosphorylation sites on Jak2 is likely to be importantfor our understanding of cytokine signaling, since these phos-phorylation events may regulate Jak2 activity or mediate down-stream signaling by recruiting signaling proteins that containphosphotyrosine-binding SH2 domains. Phosphorylation ofTyr1007 and Tyr1008 is required for the activation of Jak2 kinaseactivity in much the same way as activation loop phosphoryla-tion mediates activation of other kinases (6, 8, 15, 26). Phos-phorylated Tyr966 has been reported to mediate the interactionof Jak2 with a 70-kDa protein of unknown function, and thefunction of Tyr966 in Jak2-dependent signaling remains unclear(5).

While Tyr221 lies within the FERM domain of Jak2, Tyr221 isCOOH-terminal to the region of the FERM domain that isthought to be important for interaction of Jak kinases withcytokine receptors (24, 30). Indeed, mutation of Tyr221 did notappreciably affect the ability of activated Jak2 to coimmuno-precipitate with ELR, nor did it affect the amplitude of ELR-dependent signals, such as STAT3 (STAT3 activation duringELR signaling is mediated by the phosphorylation of Tyr1138 ofthe LRb intracellular domain) and ERK, suggesting that thephosphorylation of Tyr221 is unlikely to regulate the interactionof Jak2 with cytokine receptors.

Tyr570 lies at the beginning of the JH2 domain of Jak2; theJH2 domain of Jak kinases regulates tyrosine kinase activity bytonically inhibiting kinase function (10, 19, 22, 28, 29). In orderto test a potential role for Tyr570 in the regulation of Jak2activity, we examined signaling by Jak2Y570F in the absence andpresence of cytokine stimulation. We repeatedly observed thatJak2Y570F exhibited increased general tyrosine phosphoryla-tion and increased basal phosphorylation of Tyr221 andTyr1007,1008 in the absence of cytokine stimulation; this in-creased basal tyrosine phosphorylation of Jak2Y570F correlatedwith increased basal activation of the associated ELR, of ERK,and of STAT3 phosphorylation and transcriptional activity (24,30). Our data show that Jak2Y570F is active in the absence ofreceptor as well as when coexpressed with receptor. While it istheoretically possible that failure to interact properly withELR might underlie the increased basal activity of Jak2Y570F,the increase in receptor-mediated STAT3 and ERK phosphor-ylation by Jak2Y570F and the association of phosphorylatedJak2Y570F with tyrosine-phosphorylated ELR in the absence ofligand suggest that Jak2Y570F associates normally with the in-tracellular domain of LRb and that the constitutive activationof Jak2Y570F occurs independently of any effect on receptorassociation. These data suggest that phosphorylation of Tyr570

acts as a brake upon the activity of Jak2 in the absence ofcytokine stimulation. Jak2Y570F also mediates increasedSTAT3-dependent transcriptional activity during ELR signal-ing. Furthermore, while Jak2 and Jak2Y221F become dephos-phorylated during prolonged EPO stimulation, Jak2Y570F re-mains highly phosphorylated even after 24 h of stimulation,suggesting that the phosphorylation of Tyr570 may also beimportant for the attenuation of Jak2 signaling.

While it is clear that the phosphorylation of Tyr570 on Jak2inhibits Jak2 signaling, the mechanism whereby it does so is notyet clear. The location of Tyr570 within the JH2 domain of Jak2invites the hypothesis that phosphorylation of this site regu-lates the function of the JH2 domain via a conformationalchange in the domain, enhancing JH2-mediated inhibition of



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kinase activity. It is also possible that this site mediates therecruitment of a Jak2 inhibitor, however. Indeed, we haveexamined the possibility that Tyr570 might represent a bindingsite for SOCS3, but we found that SOCS3 inhibited Jak2Y570F

in a manner similar to Jak2, suggesting that another mecha-nism for the activation of Jak2Y570F must apply. Indeed, thephosphorylation of Tyr570 on Jak2Y1007,8F in the context of thepredicted proximity of Tyr570 and the kinase active site in acomputer-modeled structure of Jak2 (13) invites the hypothe-sis that phosphorylated Tyr570 sterically hinders the activationor accessibility of the Jak2 tyrosine kinase. Indeed, this ispotentially consistent with the finding that SOCS3 inhibitsJak2Y570F more potently than wild-type Jak2: were Tyr570 to lienear the active site on Jak2, the phosphorylation of this residuecould interfere with SOCS3-mediated inhibition at the activesite; mutation of Tyr570 would thus increase the susceptibilityof the active site to SOCS3-mediated inhibition. Interestingly,Tyr570 of Jak2 is not conserved in other Jak kinases (e.g., Jak1,Tyk2), suggesting that this mechanism of inhibition is likely tobe specific for Jak2.

Tyr221 and Tyr570 were recently identified as phosphoryla-tion sites on Jak2 during the MS and phosphopeptide mappinganalysis performed by another group (see the accompanyingpaper by L. S. Argetsinger, J.-L. Kouadio, H. Steen, A. Stens-balle, O. N. Jensen, and C. Carter-Su [1]). This group reportedincreased signaling by a Jak2 molecule mutant for Tyr570, as dowe, as well as demonstrating increased autokinase activity bythis mutant, consistent with the notion that phosphorylation ofTyr570 inhibits Jak2 activity. Although we could detect no grossalteration in ELR-activated signaling by a Jak2 molecule mu-tant for Tyr221, the Carter-Su group observed dramaticallydecreased autophosphorylation activity of the Tyr221 mutantexpressed in the absence of cytokine receptors. Our finding ofdecreased total tyrosine phosphorylation of Jak2Y221F withnormal phosphorylation of Tyr570 is consistent with normalactivity but with decreased total phosphorylation due to loss ofa major site of phosphorylation. By the inclusion of ELR in ouranalysis, our methods differ importantly from those of theCarter-Su lab, however: Tyr221 lies in the FERM domain thatcouples cytokine receptors to Jak kinases and which may func-tion in part to control the activity of the Jak tyrosine kinase(30); indeed, some mutations within the FERM domain ofJak3 concomitantly block Jak3 activation and receptor associ-ation. It is thus possible that the apparent discrepancy in ourrespective findings regarding the role of Tyr221 may be second-ary to the presence of ELR in our system and the absence ofcoexpressed cytokine receptor in that of the Carter-Su group;this would suggest that Tyr221 may regulate the activity of Jak2in the absence of cytokine receptor association, or perhaps inassociation with a subset of cytokine receptors that does notinclude LRb. It is also possible that Jak2Y221F is more sensitiveto inhibition during chronic signaling. The resolution of thisissue requires further study.

While determining the exact mechanism by which phosphor-ylation of Tyr570 of Jak2 inhibits Jak2 signaling will requirefurther study, it is clear that the phosphorylation of this siterepresents an important mechanism by which Jak2-dependentsignaling is regulated. Enhancing the phosphorylation ofTyr570 would necessarily decrease the strength of Jak2-depen-dent signals and could underlie impaired signaling by Jak2-

dependent cytokines, such as leptin in the leptin resistance thataccompanies common forms of obesity (9, 11). On the otherhand, impairment of Tyr570 phosphorylation or Tyr570-medi-ated inhibition would be expected to increase cytokine action,as in autoimmunity or tumor promotion (20). Further investi-gation will be required to determine the role of Jak2 inhibitionvia Tyr570 phosphorylation in these and other disease pro-cesses.

ACKNOWLEDGMENTS

This work was supported by NIH DK56731 and a grant from theAmerican Diabetes Association (to M.G.M.), NIH DK 60165 (toE.P.F.), and DK 36836 (Joslin’s Diabetes and Endocrinology ResearchCenter, Proteomics Core Laboratory).

Thanks go to Diane Fingar for helpful discussions and a criticalreading of the manuscript.

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Tyrosine Phosphorylation of Jak2 in the JH2 Domain Inhibits Cytokine Signaling

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