MOLECULAR AND CELLULAR BIOLOGY, May 2004, p. 4557–4570
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 10
Tyrosine 813 Is a Site of JAK2 Autophosphorylation Critical for
Activation of JAK2 by SH2-B?
Jason H. Kurzer,1Lawrence S. Argetsinger,2Yong-Jie Zhou,3Jean-Louis K. Kouadio,2†
John J. O’Shea,3and Christin Carter-Su2*
Graduate Program in Cellular and Molecular Biology1and Department of Molecular and Integrative Physiology,2
University of Michigan Medical School, Ann Arbor, Michigan 48109-0662, and Molecular Immunology and
Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National
Institutes of Health, Bethesda, Maryland 20892-18203
Received 3 October 2003/Returned for modification 2 December 2003/Accepted 9 February 2004
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? 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? to bind JAK3. Taken
together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the
SH2-B?-binding site within JAK2 that is required for SH2-B? to enhance activation of JAK2.
The Janus kinase family of tyrosine kinases (JAK1, JAK2,
JAK3, and Tyk2) plays an essential role in the signaling by all
members of the cytokine receptor superfamily. The JAKs pro-
mote growth, proliferation, and/or differentiation of many cell
types (1, 15). Activation of the JAKs occurs upon ligand bind-
ing to its receptor. Activation is thought to occur as a conse-
quence of two JAK molecules being brought into close enough
proximity to allow for rapid trans-phosphorylation of the acti-
vation loop of each kinase (15). The activated JAK molecules
then phosphorylate multiple targets, including the JAKs them-
selves, the associated receptors, and multiple signaling mole-
cules such as the signal transducers and activators of transcrip-
tion (STATs) (4, 10, 16, 17). Dysregulation of JAKs can lead to
a host of physiological problems, including diseases of the
immune system (6) and cancer (22, 29, 40).
Among the JAKs, JAK2 is activated by more than two-thirds
of the known cytokine receptor ligands, including growth hor-
mone (GH), prolactin, erythropoietin, and leptin, making it
the most studied of the JAK family members (14). Autophos-
phorylation of JAK2 is an important step in regulating signal-
ing as it leads to activation of the kinase. Autophosphorylation
also results in the production of potential docking sites for
downstream signaling molecules containing phosphotyrosine
binding Src homology 2 (SH2) domains, such as the adapter
protein, SH2-B, and the JAK2 inhibitor suppressor of cytokine
signaling 1 (SOCS-1). Identification of the autophosphoryla-
tion sites on JAK2, therefore, is critical for advancing our
understanding of how these kinases signal, and could provide
potential targets for pharmaceutical intervention. To date,
while JAK2 has 49 tyrosines and is highly phosphorylated (3,
36, 39), only two tyrosines have been identified as binding sites
for other proteins. Tyrosine 1007, which is located in the acti-
vation loop of the kinase domain, is critical for full activation
of JAK2 and has been shown to bind SOCS-1 (43) and to serve
as a substrate for protein tyrosine phosphatase-1B (PTP-1B)
(26). Tyrosine 966 has been shown to bind p70, an SH3 domain
containing protein of unknown function (7). To gain additional
insight into the function and/or regulation of JAK2, we sought
both to identify additional tyrosines present in JAK2 that un-
dergo autophosphorylation and to establish a function for
these phosphorylated tyrosines.
Here we show that tyrosine 813 of JAK2 is a site of auto-
phosphorylation. In contrast to most of the previously identi-
fied phosphorylated tyrosines—including 221, 570, 1007, and
1008, which regulate kinase activity—phosphorylation of ty-
rosine 813 seems not to affect the intrinsic activity of JAK2.
Rather, we show that tyrosine 813 is required for JAK2 to bind
the ? splicing variant of SH2-B, and for the ability of SH2-B?
to enhance JAK2 activation as well as increase JAK2-mediated
phosphorylation of STAT5B.
MATERIALS AND METHODS
Reagents. The QuikChange mutagenesis kit was from Stratagene. [?-32P]ATP
(6,000 Ci/mmol) was from ICN. Methylated trypsin was from Promega. Thin-
layer chromatography plates were from EM Science. Bovine serum albumin
(CRG-7) was from Intergen. Dulbecco’s Modified Eagle Medium (DMEM) and
phosphate-free DMEM was from Invitrogen. Recombinant protein A-agarose
* Corresponding author. Mailing address: Department of Molecular
and Integrative Physiology, The University of Michigan Medical
School, Ann Arbor, MI 48109-0622. Phone: (734) 763-2561. Fax: (734)
647-9523. E-mail: firstname.lastname@example.org.
† Present address: Department of Biochemistry and Molecular Bi-
ology, University of Chicago, Chicago, IL 60637.
was from Repligen. Aprotinin, leupeptin, and Triton X-100 were from Roche.
The enhanced chemiluminescence detection system, nitrocellulose paper, and
horseradish peroxidase-conjugated protein A (used at a dilution of 1:7,500) were
from Amersham Pharmacia Biotech. Protein molecular weight standards and
horseradish peroxidase-conjugated anti-mouse immunoglobulin G (used at a
dilution of 1:7,500) and anti-rabbit immunoglobulin G (used at a dilution of
1:7,500) were from Santa Cruz. AlexaFluor680 anti-rabbit and IR800 anti-mouse
antibodies were obtained from Molecular Probes and used at a dilution of
1:20,000. Polyvinylpyrrolidone and phosphoamino acid standards were from
Sigma. X-ray film was from Kodak. Anti-JAK2 antiserum was raised in rabbits
against a synthetic peptide corresponding to amino acids 758 to 776. The anti-
JAK2 used for immunoprecipitation was prepared in conjunction with Pel-Freez
Biologicals (3) and was used at a dilution of 1:250. Antibody to JAK2 used for
immunoblotting was from Upstate Biotechnology, Inc., and used at a dilution of
1:10,000. Antibody recognizing a peptide containing phosphorylated tyrosines
1007 and 1008 of JAK2 [anti-P(Y1007)-JAK2] was kindly provided by Martin
Myers (Harvard, Boston, Mass.). An antibody that recognizes the peptide
CLNSLFTPD[pY]EL, containing phosphorylated tyrosine 813 [anti-P(Y813)-
JAK2], was developed in conjunction with Upstate USA, Inc. Anti-JAK3 anti-
body was raised in rabbits against a synthetic peptide corresponding to amino
acids 1104 to 1124 of human JAK3 (19) and was used at a dilution of 1:3,000 for
immunoblotting. Anti-phospho-JAK3 antibody (anti-P-JAK3) was raised in rab-
bits against the peptide SLISSD[pY]ELLSDP, containing phosphorylated ty-
rosine 785 and used for immunoblotting at a dilution of 1:1,000. Monoclonal
antibody against myc-tag (9E10; anti-myc) and polyclonal anti-STAT5B antibody
raised against amino acids 711 to 727 of murine STAT5B (anti-STAT5B) were
obtained from Santa Cruz Biotechnology, Inc. Anti-myc was used at dilutions of
1:100 for immunoprecipitation and 1:10,000 for immunoblotting. Anti-STAT5B
was used at a dilution of 1:5,000 for immunoblotting. Monoclonal antibody to
phosphorylated tyrosine 699 of STAT5B (anti-P-STAT5) was obtained from
Zymed Laboratories Inc. and used at a 1:7,500 dilution in immunoblotting.
Antiphosphotyrosine antibody 4G10 (anti-PY) was obtained from Upstate USA,
Inc., and was used at 1:7,500 for immunoblotting. Anti-FLAG M2 Affinity gel
(product number A1205) was from Sigma.
Plasmids. The mammalian expression vector prk5 encoding wild-type murine
JAK2 or kinase-inactive murine JAK2(K882E), in which the critical lysine in the
ATP binding domain is mutated to glutamate, were generously provided by J.
Ihle and B. Witthuhn (St. Jude Children’s Research Hospital). The above JAK2
plasmid was used to create JAK2(Y570F) and JAK2(Y813F) via site-directed
mutagenesis using the QuikChange mutagenesis kit from Stratagene. DNAs
encoding FLAG-tagged JAK2(797-1129), containing amino acids 797 to 1129 of
JAK2, and FLAG-tagged JAK2(830-1129), containing amino acids 830 to 1129,
were created by first mutating base pairs in the JAK2 coding sequence from
GCTTTC to GgaTcC (mutated bases lowercase) for FLAG-JAK2(797-1129) and
from GGTGCC to GGatCC for FLAG-JAK2(830-1129) and then subcloning the
JAK2 fragment into a PCMV-tag2B expression vector. JAK2 amino acid num-
bers are numbered according to NCBI accession number NP_032439. Plasmid
encoding rat STAT5B was from L. Yu-Lee (Baylor College of Medicine). Con-
struction of the vector encoding SH2-B? with a myc tag at the N terminus (32)
and the vector encoding myc-SH2-B?(504-670) (33) have been described previ-
ously. SH2-B? amino acid numbers are numbered according to accession num-
Cell culture and transfection. The stock of murine 3T3-F442A fibroblasts was
kindly provided by H. Green (Harvard University). 3T3-F442A cells and 293T
cells were grown in DMEM supplemented with 1 mM L-glutamine, 100 U of
penicillin per ml, 100 ?g of streptomycin per ml, 0.25 ?g of amphotericin per ml,
and 8% calf serum. COS7 cells were grown in the same medium supplemented
with 8% fetal bovine serum rather than 8% calf serum. 3T3-F442A cells were
incubated overnight in serum-free medium containing 1% bovine serum albumin
before adding GH. Both 293T cells and COS7 cells were transiently transfected
using calcium phosphate precipitation (8). At 6 h (293T cells) or 16 h (COS7
cells) after transfection, cells were washed three times and incubated with their
respective medium. 293T cells were used 24 h posttransfection, while COS7 cells
were used 48 h posttransfection.
Immunoprecipitation and immunoblotting. At 48 h after transfection, COS7
cells were washed three times in chilled PBSV (10 mM sodium phosphate, 137
mM NaCl, 1 mM Na3VO4[pH 7.4]) and solubilized in lysis buffer (50 mM Tris
[pH 7.5], 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA, 1 mM Na3VO4, [pH
7.5]), containing 1 mM phenylmethylsulfonyl fluoride, aprotinin (10 ?g/ml), and
leupeptin (10 ?g/ml). Cell lysates were centrifuged at 16,750 ? g for 10 min. The
supernatant was incubated with the indicated antibody on ice for 2 h. The
immune complexes were collected on protein A-agarose (40 ?l) during 1 h of
incubation at 4°C. The beads were washed three times with lysis buffer and boiled
for 5 min in a mixture (80:20) of lysis buffer and sodium dodecyl sulfate-polyac-
rylamide gel electrophoresis (SDS-PAGE) sample buffer (250 mM Tris-HCl [pH
6.8], 10% SDS, 10% ?-mercaptoethanol, 40% glycerol, 0.01% bromophenol
blue). The solubilized proteins were separated by SDS-PAGE (5-to-12% gradi-
ent gels). Proteins in the gel were transferred to a nitrocellulose membrane and
detected by immunoblotting with the indicated antibody using enhanced chemi-
luminescence or by using ODYSSEY Infrared Imaging System (LI-COR Bio-
sciences) software (see Fig. 4B and 6B only). In some experiments, membranes
were incubated in a solution of 100 mM ?-mercaptoethanol, 2% SDS, and 62.5
mM Tris-HCl (pH 6.8) at 50° for 20 min and then reprobed with a separate
In vitro kinase assay. For JAK2, in vitro kinase assays were performed as
described previously (3, 36). Briefly, cells were washed with phosphate-buffered
saline and solubilized in lysis buffer containing 1 mM phenylmethylsulfonyl
fluoride, aprotinin (10 ?g/ml), and leupeptin (10 ?g/ml). Cell lysates were incu-
bated with anti-FLAG affinity gel or anti-JAK2 as appropriate. The anti-JAK2
was precipitated using protein A-agarose. The immune complexes were washed
with lysis buffer and then with kinase buffer (50 mM HEPES, 100 mM NaCl, 5
mM MnCl2, 0.5 mM dithiothreitol, 1 mM Na3VO4[pH 7.6]). The immobilized
JAK2 was incubated in kinase buffer containing 0.5 mCi of [?-32P]ATP, aprotinin
(40 ?g/ml), and leupeptin (40 ?g/ml) at 30°C for 30 min, washed five times with
lysis buffer, and eluted by boiling in a mixture (80:20) of lysis buffer and SDS-
PAGE sample buffer. Proteins were resolved by SDS-PAGE (5-to-12% gradi-
ent), transferred to nitrocellulose, and visualized by autoradiography. For JAK3,
in vitro kinase assays were performed as described previously (45). Briefly, cell
lysates were incubated with anti-JAK3, and the JAK3 immunoprecipitates were
washed once with 100 mM NaCl and 10 mM HEPES, pH 7.5, and resuspended
in JAK3 kinase reaction buffer (20 mM Tris [pH 7.5], 5 mM MgCl2, 5 mM
MnCl2, 1 ?M ATP) containing 10 ?Ci of [?-32P]ATP (Amersham). The reac-
tions were performed at room temperature for 10 min. The reactions were
terminated by addition of lysis buffer containing 100 mM EDTA. The JAK3
immunoprecipitates were also washed once with ice-cold lysis buffer. Beads were
boiled in sample buffer, and the proteins were separated by SDS-PAGE and
transferred to nitrocellulose (Schleicher & Schuell), after which phosphopeptide
mapping was performed.
Phosphopeptide mapping and phosphoamino acid analysis. For JAK2, two-
dimensional (2-D) phosphopeptide mapping and phosphoamino acid analysis
were performed as previously described (5). Briefly,32P-labeled JAK2 was cut
from the nitrocellulose, digested with 5 ?g of sequencing grade methylated
trypsin at 37°C for 4 h, and oxidized with performic acid. Peptides were separated
by thin-layer electrophoresis at pH 1.9 followed by thin-layer chromatography
using phosphochromatography buffer (5). The32P-labeled spots were visualized
using a phosphorimager (Bio-Rad model 505). For phosphoamino acid analysis,
32P-labeled peptides were scraped from the cellulose plate and eluted with pH
1.9 buffer (5). Eluted peptides were mixed with phosphoamino acid standards,
subjected to acid hydrolysis in 6 N HCl at 110°C for 60 min and resolved by
thin-layer electrophoresis at 1,000 V at pH 3.5. Phosphoamino acid standards
were visualized by ninhydrin and32P-labeled spots were visualized with a phos-
For JAK3, reagents for peptide mapping were purchased from J. T. Baker
(Phillipsburg, N.J.) and peptide mapping was performed as described previously
(45). Briefly, samples were electrophoresed on polyacrylamide gels, transferred
to nitrocellulose, and exposed to X-ray film. The band containing JAK3 was
excised and blocked with 1% polyvinylpyrrolidone in 100 mM acidic acid for 1 h
at 37°C, washed three times with digestion buffer (1% NH4CO3, pH 8.4), and
digested with 0.5 ?g of trypsin overnight at 37°C. Peptides were recovered from
the supernatant and dried in a vacuum centrifuge. The dried precipitates were
washed and resuspended in 5 ?l of H2O then subjected to high-voltage electro-
phoresis and thin-layer chromatography (TLC). Tryptic peptides were separated
in the first dimension with pH 4.72 buffer by high-voltage electrophoresis by
using a HTLE-7000 electrophoretic apparatus (C.B.S. Scientific, Del Mar, Calif.)
for 1 h at 1.0 kV. Separation in the second dimension was performed by TLC
using butanol (5%)–pyridine (2.5%)–acetic acid (2.5%)–deionized water (90%)
for 6 h.32P-labeled peptides were visualized by autoradiography.
Tyrosine 813 in JAK2 is phosphorylated in vitro. We have
used mass spectroscopy and phosphopeptide mapping to iden-
tify two tyrosines (Tyr 221 and Tyr 570) that lie in YXXL
motifs as autophosphorylation sites in JAK2 (3a). That finding,
4558 KURZER ET AL.MOL. CELL. BIOL.
and an analysis of identified and hypothesized JAK2 target
sites, suggested that other YXXL motifs might be sites of
autophosphorylation. We therefore examined whether tyrosine
813 (YELL), lying within the pseudokinase domain (JAK ho-
mology domain 2 [JH2]) of JAK2 might be a site of autophos-
phorylation. Wild type JAK2 and JAK2(Y813F) were overex-
pressed in human epithelial kidney 293T cells, highly purified
by immunoprecipitation using anti-JAK2, phosphorylated in
vitro in the presence of [?-32P]ATP, and subjected to 2-D
peptide mapping. When tyrosine 813 in JAK2 was mutated to
phenylalanine, two spots that were present in the map of wild-
type JAK2 (indicated by the arrows in Fig. 1A) were absent in
the 2-D peptide map of JAK2(Y813F) (Fig. 1B), suggesting
that tyrosine 813 in JAK2 is autophosphorylated. To substan-
tiate further that tyrosine 813 in JAK2 is phosphorylated,
FLAG-tagged JAK2(797-1129) and FLAG-tagged JAK2(830-
1129) were subjected to 2-D peptide mapping. The only ty-
rosine present in JAK2(797-1129) and absent in JAK2(830-
1129) is tyrosine 813. Two closely migrating spots are present
in the 2-D peptide map of FLAG-tagged JAK2(797-1129) (Fig.
1C) whose migrations correspond to the migrations of the
peptides containing tyrosine 813 in the map of wild-type JAK2
(Fig. 1A). As would be expected if tyrosine 813 were a site of
autophosphorylation, when the region containing tyrosine 813
is deleted (Fig. 1D), the spots corresponding to the peptides
containing tyrosine 813 disappear [see the peptide map of
FLAG-tagged JAK2(797-1129)]. To rule out the possibility
rosine 813 is due to Ser/Thr phosphorylation by a contaminat-
ing Ser/Thr kinase, phosphoamino acid analysis was performed
to substantiate that the spots associated with tyrosine 813 in
both full-length JAK2 (Fig. 2, lane 1) and JAK2(797-1129)
(Fig. 2, lanes 2 and 3) are primarily phosphorylated on ty-
rosine. Taken together these data provide strong evidence that
JAK2 autophosphorylates tyrosine 813.
Two spots associated with tyrosine 813 are detected in the
2-D peptide maps of JAK2, one above the other. Boyle et al.
(5) indicate that the presence of two spots, one above the
32P incorporated into the spots associated with ty-
FIG. 1. JAK2 is autophosphorylated on tyrosine 813 in vitro. 293T cells expressing the cDNA for JAK2 (A), JAK2 Y813F (B), FLAG-
JAK2(797-1129) (C), or FLAG-JAK2(830-1129) (D) were lysed and immunoprecipitated with either anti-JAK2 (A and B) or anti-FLAG (C and
D). The immobilized JAK2 was incubated in the presence of [?-32P]ATP at 30°C for 30 min. The32P-labeled JAK2 was cut from the nitrocellulose
and subjected to 2-D peptide mapping with the thin-layer electrophoresis step performed at pH 1.9 (5). The arrows in the 2-D peptide maps
indicate the two spots present in JAK2 (A) and FLAG-JAK2(797-1129) (C) that disappear when tyrosine 813 in JAK2 is mutated to phenylalanine
in JAK2(Y813F) (B) and when the region between amino acid 797 and 830 of FLAG-JAK2(797-1129), which contains tyrosine 813, is deleted in
FLAG-JAK2(813-1129) (D). The origin (?) is indicated.
VOL. 24, 2004 P(Y813) OF JAK2 BINDS SH2-B?
other, suggests incomplete oxidation of the methionines in the
peptide during the preparation of the samples. For the spots
associated with tyrosine 813 to migrate in the column of pep-
tides above the origin in the 2-D peptide maps, the peptide
must be uncharged. Trypsin digestion yields a theoretical pep-
tide, DLNSLFTPDpY813ELLTENDMLPNMR, whose theo-
retical charge at pH 1.9 is ?1. Each additional phosphorylation
changes the charge by ?1. Thus, the finding that the two spots
(one above the other) migrate as uncharged peptides suggests
that the peptide containing phosphorylated tyrosine 813 is also
phosphorylated at either the serine or one of the threonines.
When the peptide containing tyrosine 813 was isolated from
the 2-D peptide maps and subjected to phosphoamino acid
analysis, no32P-labeled serine or threonine was detected. The
inability to detect
tion in the in vitro labeled JAK2 is not unexpected because the
JAK2 was highly purified prior to the in vitro kinase assay.
Therefore, the phosphorylation of JAK2 on serine/threonine
must have occurred in vivo, prior to the isolation of JAK2 from
the cells, and indicates that JAK2 is a target for phosphoryla-
tion by cellular serine/threonine kinases.
Tyrosine 813 is autophosphorylated in GH-activated JAK2.
GH binding to GH receptor activates JAK2 (3). Because GH-
dependent phosphorylation of JAK2 can be detected in 3T3-
F442A cells that endogenously express GH receptor and
JAK2, we chose this cell line to assess whether JAK2 auto-
phosphorylation of tyrosine 813 occurs upon activation of
JAK2 by GH. We stimulated 3T3-F442A cells with a 30-ng/ml
concentration (1.4 nM) of GH or vehicle for 15 min. Treated
cells were then solubilized and anti-JAK2 was used to immu-
noprecipitate JAK2. The isolated JAK2 was then subjected to
an in vitro kinase assay in the presence of [?-32P]ATP. We
observed a 4.5-fold increase in the phosphorylation of JAK2 as
32P-labeled serine/threonine phosphoryla-
a result of GH treatment (Fig. 3A, compare lanes 2 and 1). The
2-D map obtained using JAK2 isolated from GH-treated 3T3-
F442A cells (Fig. 3B) is very similar to the 2-D peptide map of
JAK2 immunoprecipitated from 293T cells (Fig. 1A). Spots in
2-D peptide maps of GH-activated JAK2 that are analogous to
those in Fig. 1A that contain tyrosine 813 are indicated by the
arrows in Fig. 3B. Because Fig. 3B is a 2-D peptide map of
JAK2 isolated from GH-treated cells that endogenously ex-
press JAK2 and GH receptor, these results are consistent with
tyrosine 813 in JAK2 being phosphorylated in GH-activated
JAK2 autophosphorylates at tyrosine 813 in response to
GH. To verify further that JAK2 autophosphorylates at ty-
rosine 813, when overexpressed and when stimulated by GH,
an antibody specific to phosphotyrosine 813 [anti-P(Y813)-
JAK2] was developed. Blotting lysates of 293T cells overex-
pressing either JAK2 or JAK2(Y813F) with anti-P(Y813)-
JAK2 demonstrates that anti-P(Y813)-JAK2 is specific for
phosphotyrosine 813 and verifies that overexpressed JAK2 is
indeed phosphorylated at tyrosine 813 (Fig. 4A). To show that
FIG. 2. The peptide containing tyrosine 813 is autophosphorylated
on tyrosine.32P-labeled peptides in 2-D peptide maps corresponding
to the lower of the two spots associated with tyrosine 813 of JAK2
(lane 1), and the upper (lane 2) and lower (lane3) spot associated with
tyrosine 813 in JAK2(797-1129) were scraped from the cellulose plates
and subjected to phosphoamino acid analysis. The origin and the
migration of phosphotyrosine (ptyr), phosphothreonine (pthr), and
phosphoserine (pser) standards are indicated. Spots seen below ptyr
are due to incomplete acid hydrolysis of the peptide.
FIG. 3. JAK2 activated in response to GH autophosphorylates ty-
rosine 813. (A) 3T3-F442A cells were incubated in the absence (lane 1)
or presence of 30 ng of GH/ml (lane 2) for 15 min. The cells were lysed
and JAK2 immunoprecipitated using anti-JAK2. JAK2 was immobi-
lized and incubated in the presence of [?-32P]ATP at 30°C for 30 min.
The JAK2 was resolved by SDS-PAGE, transferred to nitrocellulose
and visualized by autoradiography. (B)32P-labeled JAK2 was cut from
the nitrocellulose and subjected to 2-D peptide mapping with the
thin-layer electrophoresis step performed at pH 1.9 (5). The origin
(?), and spots whose migration are similar to spots identified in Fig.
1A are indicated.
4560KURZER ET AL.MOL. CELL. BIOL.
phosphorylation of JAK2 at tyrosine 813 in 3T3-F442A cells is
a direct result of GH stimulation, 3T3-F442A cells were
treated with vehicle alone for 0 or 180 min or with GH (500
ng/ml) for 5, 15, 30, 60, 90, 120, and 180 min. Immunoblotting
proteins in the harvested cell lysates with anti-P(Y813)-JAK2
demonstrated that tyrosine 813 in JAK2 was phosphorylated
after 5, 15, and 30 min of GH treatment; phosphorylation of
tyrosine 813 returned to basal levels by 60 min, where it re-
mained for the remainder of GH treatment. These data dem-
onstrate that rapid and transient in vivo phosphorylation of
JAK2 at tyrosine 813 occurs as a direct result of GH treatment
(Fig. 4B, top panel). Immunoblotting the lysates with anti-PY
demonstrated that the phosphorylation of tyrosine 813 mimics
the general tyrosine phosphorylation of JAK2 upon GH stim-
ulation (Fig. 4B, middle panel). Immunoblotting with anti-
JAK2 demonstrated that these differences in JAK2 phosphor-
ylation were not a consequence of differences in endogenous
JAK2 expression (Fig. 4B, bottom panel).
Mutation of tyrosine 813 to phenylalanine does not disrupt
JAK2-mediated phosphorylation of STAT5B. Upon establish-
ing tyrosine 813 as a site of GH-stimulated JAK2 autophos-
phorylation, we sought to identify at least one of its roles in
JAK2 signaling. We sought first to determine if tyrosine 813 is
required for the ability of JAK2 to phosphorylate the JAK2
substrate STAT5B. Cytoplasmic STAT5B is a latent transcrip-
tion factor that is recruited to phosphotyrosines on cytokine
receptor/JAK complexes. Phosphorylation of STAT5B on ty-
rosine 699 by JAK2 enables it to dimerize via the SH2 domain
of one STAT molecule and the phosphotyrosine of another.
STAT5B dimers then translocate to the nucleus, bind to reg-
ulatory elements within the DNA, and regulate gene transcrip-
tion (4). STAT5B was expressed alone or coexpressed with
JAK2 or JAK2(Y813F) and cell lysates were prepared. As
illustrated in Fig. 5, immunoblotting the cell lysates with anti-
body specific to phosphotyrosine 699 of STAT5B (anti-P-
STAT5) demonstrated that mutation of tyrosine 813 of JAK2
to phenylalanine did not interfere with the phosphorylation of
STAT5B by JAK2 (top panel). Blotting with anti-STAT5B
(Fig. 5, middle panel) demonstrated that STAT5B was ex-
pressed at similar levels in each condition. Consistent with the
data obtained using anti-P-STAT5, Fig. 5 reveals an additional
slower-migrating band of similar intensity, in the presence of
P-STAT5B. The results suggest that tyrosine 813 is not essen-
tial for JAK2 to phosphorylate STAT5B.
Tyrosine 813 of JAK2 is important for binding of SH2-B? to
JAK2. SH2-B? was previously cloned using a yeast two-hybrid
screen, and shown to be both a binding partner and substrate
of active JAK2 (34). Believed to be an adapter protein,
SH2-B? possesses three proline-rich regions and a pleckstrin
FIG. 4. Autophosphorylation of JAK2 at tyrosine 813 in 3T3-F442A cells in response to GH. (A) 293T cells were transfected with cDNAs
encoding either JAK2 or JAK2 with tyrosine 813 mutated to phenylalanine (Y813F). Cell lysates were immunoblotted with either anti-JAK2,
anti-PY, or anti-P(Y813)JAK2. (B) 3T3-F442A cells were treated either with vehicle for 0 (lane 1) or 180 (lane 9) min or with GH (500 ng/ml)
for 5, 15, 30, 60, 90, 120, or 180 min (lanes 2 through 8, respectively). Cell lysates were immunoblotted with either anti-P(Y813)-JAK2 (top panel),
anti-PY (middle panel), or anti-JAK2 (bottom panel).
VOL. 24, 2004 P(Y813) OF JAK2 BINDS SH2-B?
homology (PH) domain in addition to its SH2 domain. In
accord with its role as an adapter protein, SH2-B? has been
shown to bind the small GTPase, Rac, in a manner dependent
upon its N-terminal proline rich region, the same region re-
quired for SH2-B facilitation of GH-induced cellular motility
(11). SH2-B? also enhances tyrosyl phosphorylation and acti-
vation of JAK2 as well as of downstream JAK2 targets such as
STAT5B (32). Consequently, we were curious to determine if
tyrosine 813 serves as a regulatory site within JAK2 via medi-
ating the stimulatory effects of SH2-B?.
We first sought to determine if tyrosine 813 is necessary for
JAK2 to interact with SH2-B?. SH2-B? has been shown to
bind active JAK2 primarily via its SH2 domain (33, 34). How-
ever, full-length SH2-B? is capable of binding kinase-inactive
JAK2 in a phosphotyrosine-independent manner via a lower-
affinity JAK2-binding site that contains the PH domain of
SH2-B? (33). To determine if the SH2 domain of SH2-B?
binds phosphorylated tyrosine 813 of JAK2, myc-SH2-B?(504-
670), which contains the SH2 domain but lacks the lower-
affinity phosphotyrosine-independent JAK2-binding domain,
was coexpressed in COS7 cells with JAK2, kinase-inactive
JAK2(K882E), JAK2(Y813F), or JAK2(Y570F). JAK2(K882E)
is catalytically inactive due to mutation of the critical lysine in
the ATP binding site to aspartate. Binding of the SH2 domain
of SH2-B? to JAK2 is dependent upon JAK2 autophosphor-
ylation, thus JAK2(K882E) serves as a negative control. We
chose to use tyrosine 570 as a representative of other tyrosines
in JAK2 we have tested because, like tyrosine 813, tyrosine 570
is known to be a site of phosphorylation and resides within the
same sequence motif, YXXL, as tyrosine 813. Myc-SH2-
B?(504-670) was immunoprecipitated with anti-myc and the
precipitated proteins were immunoblotted with anti-JAK2
(Fig. 6A, top panel) to determine if JAK2 or if the JAK2
mutants coimmunoprecipitate with SH2-B?(504-670). As seen
in Fig. 6A, both JAK2 (lane 2) and JAK2(Y570F) (lane 5)
coimmunoprecipitated with SH2-B?(504-670) to a similar ex-
tent. Thus, mutation of a known phosphotyrosine in JAK2,
such as tyrosine 570, does not a priori disrupt the ability of
JAK2 to coimmunoprecipitate with the SH2 domain of SH2-
B?. In contrast, JAK2(Y813F) did not coimmunoprecipitate
with SH2-B?(504-670) (Fig. 6A, lane 4), strongly implicating
tyrosine 813 as being required for the interaction between
JAK2and theSH2 domain
JAK2(Y813F) to coprecipitate with myc-SH2-B?(504-670) is
not believed to be due to a general loss of tyrosine phosphor-
ylation of JAK2, as JAK2(Y813F) phosphorylation under
these conditions is similar to that of wild-type JAK2 (see Fig.
8 and 9). The negative control JAK2(K882E), which is not
phosphorylated on tyrosines, also did not coimmunoprecipi-
tate with myc-SH2-B?(504-670) (Fig. 6A, lane 3), indicating
the phosphotyrosine dependence of JAK2 to coimmunopre-
cipitate with myc-SH2-B?(504-670). Expression of SH2-
B?(504-670) without JAK2 (Fig. 6A, lane 1), or of various
forms of JAK2 without SH2-B?(504-670) (Fig. 6A, lanes 6
through 9) show that coimmunoprecipitation of JAK2 with the
SH2 domain of SH2-B? required expression of both the
SH2-B? mutant and JAK2. Reprobing the membrane with
anti-PY (Fig. 6A, second panel) further demonstrated that
tyrosyl phosphorylated JAK2 and JAK2(Y570F) but not ty-
rosyl phosphorylated JAK2(Y813F) coimmunoprecipitated
with myc-SH2-B?(504-670). Blotting the cell lysates with anti-
JAK2 and anti-myc to detect myc-SH2-B?(504-670) (Fig. 6A,
third and fourth panels, respectively) revealed that the inability
of SH2-B? to coprecipitate JAK2(Y813F) was not due to a
decreased level of expression of JAK2(Y813F) compared to
JAK2, or to a decreased expression level of myc-SH2-B?(504-
670) with JAK2(Y813F) compared to the level expressed with
JAK2. To date, out of thirteen tyrosines in JAK2 tested, ty-
rosine 813 is the only tyrosine we have identified as a potential
binding site for SH2-B? (data not shown).
We further sought to determine if mutation of tyrosine 813
to phenylalanine could disrupt the binding of JAK2 to full-
length SH2-B? to confirm that tyrosine 813 is the primary
binding site within JAK2 for SH2-B?. We expressed myc-
SH2-B? with either JAK2, JAK2(K882E), or JAK2(Y813F) in
COS7 cells; immunoprecipitated myc-SH2-B? from the cell
lysates with anti-myc; and then immunoblotted with anti-JAK2
to detect the presence of any JAK2 that would coprecipitate
with myc-SH2-B? (Fig. 6B). As expected, myc-SH2-B? was
found to precipitate JAK2 to a significantly larger degree than
JAK2(K882E) or JAK2(Y813F) (Fig. 6B, top panel, lanes 1 to
3), supporting the conclusion that tyrosine 813 is the major
binding site in JAK2 for SH2-B?. Indeed, the amount of
JAK2(K882E) and JAK2(Y813F) that coprecipitated with
myc-SH2-B? appeared similar to the levels of each form of
JAK2 that precipitated with anti-myc in the absence of myc-
SH2-B? (Fig. 6B, top panel, lanes 4 to 6), suggesting that most
of the JAK2(K882E) and JAK2(Y813F) seen to coimmuno-
precipitate with myc-SH2-B? was due to nonspecific binding to
either anti-myc or the protein A-agarose. Immunoblotting the
cell lysates with anti-JAK2 and anti-myc (Fig. 6B, third and
fourth panels, respectively) revealed that the inability of
SH2-B? to coprecipitate JAK2(Y813F) was not due to differ-
ences in expression of the JAK2 proteins or myc-SH2-B?.
Tyrosine 785 of JAK3 is important for binding of SH2-B?.
Among the JAK family members, JAK3 is the only other JAK
that possesses a YELL sequence motif. We therefore exam-
ined whether tyrosine 785 in JAK3 that corresponds to tyrosine
of SH2-B?. Failureof
FIG. 5. JAK2(Y813F) is capable of phosphorylating STAT5B. Ly-
sates were prepared of COS7 cells cotransfected with 2.0 ?g of cDNA
encoding STAT5B and either 0.25 ?g of empty vector (lane 1) or
cDNA encoding JAK2 (lane 2) or JAK2(Y813F) (lane 3). The upper
panel demonstrates the degree of phosphorylation of tyrosine 699 of
STAT5B as assessed by immunoblotting with anti-P-STAT5. The mid-
dle panel exhibits the expression level of STAT5B as assessed by
immunoblotting with anti-STAT5B. The lower panel shows the expres-
sion level of JAK2 as assessed by immunoblotting with anti-JAK2.
4562KURZER ET AL.MOL. CELL. BIOL.
813 in JAK2 binds SH2-B?. 2-D phosphopeptide mapping was
used to determine if tyrosine 785 was a site of autophosphor-
ylation of JAK3. To this end, JAK3 or JAK3 (Y785F) were
expressed in COS-7 cells, immunoprecipitated with anti-JAK3,
subjected to in vitro kinase assays, and analyzed via 2-D phos-
phopeptide mapping. The seven most prominent spots of
JAK3 have been labeled a through g (Fig. 7A). Analysis of the
2-D peptide map of JAK3 (Y785F) reveals the disappearance
of spot a, implicating tyrosine 785 as a major site of phosphor-
ylation within JAK3 (Fig. 7A). While, mutation of tyrosine 785
to phenylalanine reduces the anti-PY signal of JAK3 by about
one half, consistent with tyrosine 785 being a major site of
phosphorylation (data not shown) spots b through g remain
present in the 2-D map of JAK3 (Y785F), indicating that
mutation of tyrosine 785 does not alter the global tyrosine
phosphorylation pattern of JAK3. Moreover, mutation of ty-
rosine 785 in JAK3 does not significantly diminish the kinase
activity of JAK3 (data not shown). To verify in vivo phosphor-
ylation at tyrosine 785 in JAK3 within COS7 cells, a phos-
phospecific antibody, anti-P-JAK3, was developed. Cell lysates
from COS7 cells transiently transfected with cDNA for JAK3
or JAK3 (Y785F) were then immunoblotted using anti-P-
JAK3. This antibody recognized JAK3 (Fig. 7B, lane 1), indi-
cating that overexpressed JAK3 is phosphorylated at tyrosine
785 in COS7 cells. As expected, mutation of tyrosine 785 to
phenylalanine dramatically reduced the signal of anti-P-JAK3
(Fig. 7B, lane 2), demonstrating the specificity of the antibody.
To show that tyrosine 785 in JAK3 is phosphorylated in vivo in
response to interleukin-2 (IL-2), NK 3.3 cells were deprived
overnight; stimulated with IL-2 (1,000 U/ml) for 1, 5, 15, or 30
min; and then immunoprecipitated with anti-JAK3 (Fig. 7C).
As shown previously (44) immunoblotting the immunoprecipi-
tated proteins with anti-PY shows that JAK3 is heavily phos-
phorylated in response to treatment with IL-2 (Fig. 7C, top
panel). Blotting with anti-P-JAK3 reveals that JAK3 was phos-
phorylated at tyrosine 785 after 5 min of IL-2 treatment, and
FIG. 6. Tyrosine 813 is required for JAK2 to bind to SH2-B?. (A) COS7 cells were transfected with empty vector (lane 1) or 1.0 ?g of cDNA
encoding either JAK2 (lanes 2 and 6), JAK2(K882E) (lanes 3 and 7), JAK2(Y813F) (lanes 4 and 8), or JAK2(Y570F) (lanes 5 and 9) along with
(lanes 1 to 5) or without (lanes 6 to 9) 2.0 ?g of cDNA encoding myc-SH2-B?(504-670). myc-SH2-B?(504-670) was immunoprecipitated using
anti-myc and immunoblotted using anti-JAK2 (first panel) and anti-PY (second panel). Lysates of the above transfected cells were immunoblotted
with anti-JAK2 (third panel) and anti-myc (fourth panel) to assess levels of expression of JAK2 and myc-SH2-B?(504-670), respectively. (B) COS7
cells were transfected with 1.0 ?g of cDNA encoding either JAK2 (lanes 1 and 4), JAK2(K882E) (lanes 2 and 5), or JAK2(Y813F) (lanes 3 and
6) along with (lanes 1 to 3) or without (lanes 4 to 6) 2.0 ?g cDNA encoding myc-SH2-B?. Myc-SH2-B? was immunoprecipitated using anti-myc
and immunoblotted using anti-JAK2 (first panel) and anti-PY (second panel). Lysates of the above transfected cells were immunoblotted with
anti-JAK2 (third panel) and anti-myc (fourth panel) to assess levels of expression of JAK2 and myc-SH2-B?, respectively.
VOL. 24, 2004P(Y813) OF JAK2 BINDS SH2-B?
FIG. 7. Tyrosine 785 of JAK3 is autophosphorylated and binds the SH2 domain of SH2-B?. (A) cDNAs encoding JAK3 or the mutant Y785F
were transfected into COS-7 cells, and the expressed protein was immunoprecipitated with anti-JAK3, followed by subjection to in vitro kinase
assays. The phosphorylated proteins were subjected to SDS-PAGE, transferred to nitrocellulose, and subjected to autoradiography. The portions
of the membrane containing wild-type and mutant JAK3 proteins were excised and digested in situ with trypsin. Eluted tryptic phosphopeptides
were analyzed by 2-D peptide mapping. The32P-labeled phosphopeptides were visualized by autoradiography, and the most prominent spots were
designated a to g. The orientation of the positive and negative electrodes during electrophoresis is indicated along with the direction of
chromatography; the origin is indicated by the letter O. (B) COS7 cells were transfected with 1.0 ?g of cDNA encoding either JAK3 (lane 1) or
JAK3 with a phenylalanine substitution at tyrosine 785 (lane 2). Lysates of the transfected cells were immunoblotted with anti-P-JAK3 (top panel)
which is specific for phosphorylation of JAK3 at tyrosine 785 and anti-JAK3 (lower panel) to assess protein expression. (C) NK 3.3 cells were
4564 KURZER ET AL.MOL. CELL. BIOL.
remained phosphorylated for the remainder of the assay (Fig.
7C, middle panel). Immunoblotting the immunoprecipitated
proteins with anti-JAK3 verified equal loading of proteins (Fig.
7C, bottom panel).
To determine if SH2-B? binds to tyrosine 785 of JAK3,
SH2-B?(504-670) was expressed with JAK3, JAK3 (Y785F), or
kinase-inactive JAK3 (K855A). Myc-SH2-B?(504-670) was im-
munoprecipitated with anti-myc, and immunoprecipitated pro-
teins were immunoblotted with anti-JAK3 to detect any JAK3
that coimmunoprecipitated with myc-SH2-B?(504-670) (Fig.
7D, top panel). JAK3 clearly coimmunoprecipitated with SH2-
B?(504-670) (Fig. 7D, compare lanes 2 and 5). The non-ty-
rosyl-phosphorylated kinase-inactive JAK3 (K855A) was
found to coprecipitate only poorly with SH2-B?(504-670) (Fig.
7D, lane 4), suggesting that the interaction between JAK3 and
SH2-B? is primarily mediated via a phosphotyrosine/SH2 do-
main interaction. A small amount of JAK3 (Y785F) coprecipi-
tated with myc-SH2-B?(504-670) (Fig. 7D, lane 3). However,
the relative intensity of the observed band for coprecipitated
JAK3 (Y785F) was comparable to the intensity of the band for
JAK3(Y785F) precipitated in the absence of myc-SH2-
B?(504-670) (Fig. 7D, lane 6), suggesting it was nonspecifically
precipitated by either the myc antibody or protein A-agarose
(Fig. 7D, compare lane 3 to lanes 5 to 7). Blotting the cell
lysates with anti-JAK3 and anti-myc (Fig. 7D, middle and
bottom panels, respectively) revealed that the inability of myc-
SH2-B?(504-670) to coprecipitate JAK3 (Y785F) but not
JAK3 was not due to differences in levels of expression of
either JAK3 or myc-SH2-B?(504-670). Thus, these findings
strongly support phosphotyrosine 785 in JAK3 as the primary
binding site for the SH2 domain of SH2-B?.
SH2-B?-mediated enhancement of JAK2 autophosphoryla-
tion requires tyrosine 813 of JAK2. Having obtained evidence
that tyrosine 813 is the primary binding site of SH2-B? in
JAK2, and having supported this conclusion with data ob-
tained with tyrosine 785 of JAK3, we next sought to determine
if mutation of tyrosine 813 would disrupt the stimulatory effect
of SH2-B? on JAK2. To test whether SH2-B? requires ty-
rosine 813 to enhance JAK2 activity, we tested whether muta-
tion of tyrosine 813 to phenylalanine would disrupt the en-
hanced activation of JAK2 reported previously for SH2-B?
(32). COS7 cells were transiently transfected with cDNA en-
coding JAK2, JAK2(Y570F), JAK2(Y813F), or JAK2(K882E)
with or without cDNA encoding myc-SH2-B?. Cell lysates
were blotted with anti-myc to verify equal protein expression of
myc-SH2-B? in the various conditions (Fig. 8, bottom panel).
JAK2 was immunoprecipitated using anti-JAK2, and immuno-
blotted with anti-JAK2(Fig. 8, middle panel) to verify equal
expression and immunoprecipitation of the various JAKs. The
membrane was then stripped and reprobed with anti-PY, to
determine the level of tyrosyl phosphorylation of JAK2 in the
presence and absence of myc-SH2-B? (Fig. 8, top panel). Ty-
rosyl phosphorylation of JAK2 has been shown to correlate
well with JAK2 kinase activity (39). As reported previously
(34), JAK2 was constitutively phosphorylated on tyrosines
when overexpressed in COS7 cells (Fig. 8, lane 1). Kinase-
inactive JAK2(K882E) displayed no tyrosyl phosphorylation
(Fig. 8, lane 3). As reported elsewhere (Argetsinger et al.,
FIG. 8. Tyrosine 813 is required for SH2-B? to enhance phosphorylation of JAK2. COS7 cells were transfected with 1.0 ?g of cDNA encoding
JAK2 (lanes 1 and 2), JAK2(K882E) (lanes 3 and 4), JAK2(Y813F) (lanes 5 and 6), or JAK2(Y570F) (lanes 7 and 8), with (lanes 2, 4, 6, and 8)
or without (lanes 1, 3, 5, and 7) 2.0 ?g of cDNA encoding myc-SH2-B?. In the absence of myc-SH2-B?, cells were transfected with 2.0 ?g of empty
prk5 myc vector. JAK2 was immunoprecipitated (IP) with anti-JAK2 and immunoblotted with anti-PY (upper panel) and anti-JAK2 (middle
panel). For the lower panel, cell lysates of the above transfected cells were immunoblotted with anti-myc to assess levels of expression of
starved overnight and incubated with IL-2 (1,000 U/ml) at indicated time points (lanes 2 to 5). Cell lysates were immunoprecipitated with
anti-JAK3. The immunoprecipitated proteins were analyzed by SDS-PAGE, transferred to membrane and immunoblotted with anti-PY (top
panel), with anti-P-JAK3 (middle panel) or with anti-JAK3 to assess protein loading (bottom panel). (D) Aliquots (1.0 ?g) of empty vector (lane
1) or cDNA encoding either JAK3 (lanes 2 and 5), JAK3 (Y785F) (lanes 3 and 6), or JAK3 (K855A) (lanes 4 and 7) were transfected into COS7
cells with (lanes 1 to 4) or without (lanes 5 to 7) 2.0 ?g of cDNA encodes myc-SH2-B?(504-670). Myc-SH2-B?(504-670) was immunoprecipitated
using anti-myc and immunoblotted with anti-JAK3 (top panel). Cell lysates were immunoblotted with anti-JAK3 (middle panel) and anti-myc
(bottom panel) to assess levels of expression of JAK3 and myc-SH2-B?(504-670), respectively.
VOL. 24, 2004 P(Y813) OF JAK2 BINDS SH2-B?
submitted), JAK2(Y570F) exhibited an elevated level of ty-
rosyl phosphorylation (Fig. 8, compare lane 7 to l). In contrast
to tyrosine 570, mutation of JAK2 at tyrosine 813 did not alter
its constitutive level of tyrosyl phosphorylation (Fig. 8, com-
pare lane 5 to lane 1), indicating that tyrosine 813 is not a site
of intrinsic regulation within JAK2. The unaltered tyrosyl
phosphorylation also suggested that this mutation does not
lead to global structural changes within the protein. As re-
ported previously (32), SH2-B? enhanced the tyrosyl phos-
phorylation of JAK2 (Fig. 8, lanes 1 and 2). Similarly, myc-
SH2-B? enhanced the tyrosyl phosphorylation of JAK2
(Y570F) (Fig. 8, lanes 7 and 8). In contrast, myc-SH2-B? did
not enhance the tyrosyl phosphorylation of JAK2(Y813F) (Fig.
8, lanes 5 and 6), implicating tyrosine 813 as critical for
SH2-B? to enhance JAK2 activity. In addition, myc-SH2-B?
did not enhance the tyrosyl phosphorylation of JAK2(K882E),
verifying the dependence of SH2-B? on functionally active
JAK2 to enhance JAK2 activity. To date, we have tested the
effects of SH2-B? on twelve different JAK2 tyrosine point
mutants, and tyrosine 813 is the only tyrosine we have identi-
fied whose mutation disrupts the ability of SH2-B? to enhance
JAK2 activity (data not shown).
Tyrosine 813 is required for the SH2 domain of SH2-B? to
increase the number of active JAK2 molecules. Because the
SH2 domain has been shown to be both necessary and suffi-
cient for increasing JAK2 activity (32, 33), we examined
whether mutation of tyrosine 813 to phenylalanine would pre-
vent the increased activation of JAK2 by the SH2 domain of
SH2-B?. COS7 cells were transfected with cDNA encoding
JAK2, JAK2(Y813F), or JAK2(K882E) with or without cDNA
encoding an SH2-B? truncation mutant, myc-SH2-B?(504-
670), consisting of the SH2 domain and the C terminus of
SH2-B?. Cell lysates were blotted with anti-myc to verify equal
protein expression of myc-SH2-B?(504-670) for the various
conditions (Fig. 9, bottom panel). JAK2 was immunoprecipi-
tated with anti-JAK2, and immunoblotted with anti-JAK2 to
verify equal protein expression, immunoprecipitation, and
loading between conditions (Fig. 9, third panel). To analyze
JAK2 phosphorylation, membranes were stripped and re-
probed with anti-PY (Fig. 9, first panel). Similar to the effects
observed with SH2-B?, SH2-B?(504-670) was found to en-
hance the overall tyrosyl phosphorylation of JAK2 (Fig. 9,
lanes 1 and 2), as shown previously (33). In contrast, SH2-
B?(504-670) didnot enhance
JAK2(Y813F) (Fig. 9, lanes 3 and 4), suggesting that tyrosine
813 is required for SH2-B?(504-670)-mediated enhancement
of JAK2 activity. The membrane was also stripped and re-
probed with anti-P(Y1007)-JAK2(Fig. 9, second panel). anti-
P(Y1007)-JAK2 recognizes phosphotyrosine(s) 1007 and/or
1008 in the activation loop of JAK2. Because phosphorylation
of tyrosine 1007 is thought to be required for activation of the
kinase (12), an increase in signal observed from anti-P(Y1007)-
JAK2 reflects an increase in the relative number of activated
JAK2 molecules present. Comparison of JAK2 expressed
alone (Fig. 9, lane 1) to JAK2 expressed with myc-SH2-
B?(504-670) (Fig. 9, lane 2) shows that the presence of the
SH2-B?(504-670) increases the number of JAK2 molecules
with phosphorylated Tyr 1007/1008. As expected, because
JAK2(K882E) is inactive, coexpression of JAK2(K882E) with
SH2-B?(504-670) did not enhance overall tyrosyl phosphory-
lation of JAK2(K882E) (Fig. 9, lanes 5 and 6) or phosphory-
lation of tyrosine 1007 and/or 1008. Importantly, the presence
of myc-SH2-B?(504-670) did not increase the number of
JAK2(Fig. 9, lanes 3 and 4). These results support the conclu-
sion that tyrosine 813 is required for the SH2 domain of
SH2-B? to mediate an increase in the number of activated
Tyrosine 813 of JAK2 is required for SH2-B? to enhance the
phosphorylation of STAT5B at tyrosine 699. Enhancement of
JAK2 activity by SH2-B? increases the overall phosphorylation
of JAK2 target proteins such as STAT5B (32). It is therefore
FIG. 9. Tyrosine 813 is required for myc-SH2-B?(504-670) to increase the number of JAK2 molecules phosphorylated at Tyr 1007/1008. COS7
cells were transfected with 1.0 ?g of cDNA for JAK2 (lanes 1 and 2), JAK2(Y813F) (lanes 3 and 4), or JAK2(K882E) (lanes 5 and 6), plus (lanes
2, 4, and 6) or minus (lanes 1, 3, and 5) 2.0 ?g of cDNA encoding myc-SH2-B?(504-670). In the absence of myc-SH2-B?(504-670), cells were
transfected with 2.0 ?g of empty prk5 myc vector. JAK2 was immunoprecipitated with anti-JAK2 and immunoblotted with anti-PY (first panel),
anti-P(Y1007)-JAK2 (second panel), and anti-JAK2 (third panel). The fourth panel shows the expression levels of myc-SH2-B?(504-670) as
assessed by immunoblotting lysates with anti-myc.
4566 KURZER ET AL.MOL. CELL. BIOL.
expected that mutation of tyrosine 813 of JAK2 would not only
disrupt the ability of SH2-B? to enhance JAK2 activity, but
also preclude any enhancement of tyrosyl phosphorylation of
tyrosine 699 on STAT5B. To detect maximally SH2-B?-medi-
ated enhancement of JAK2 phosphorylation of STAT5B,
STAT5B was expressed with or without myc-SH2-B?(504-670)
in the presence of a lower level of JAK2 or JAK2(Y813F) than
used in Fig. 5. Immunoblotting the lysates with anti-myc (Fig.
10, third panel) demonstrates equal protein expression of myc-
SH2-B?(504-670) among the different conditions. Immuno-
blotting cell lysates with anti-STAT5B shows similar levels of
STAT5B were coexpressed with JAK2 and JAK2(Y813F) (Fig.
10, second panel). Immunoprecipitation of the cell lysates with
anti-JAK2, followed by immunoblotting with anti-JAK2 (Fig.
10, fourth panel), demonstrates equal protein expression, im-
munoprecipitation, and loading of JAK2. Reprobing the im-
munoprecipitated JAK2 with anti-PY (Fig. 10, fifth panel)
verifies that myc-SH2-B?(504-670) enhanced the tyrosyl phos-
phorylation of JAK2 but not of JAK2(Y813F). Immunoblot-
ting with antibody to phosphotyrosine 699 in STAT5B illus-
trates that overexpression of STAT5B alone in COS7 cells did
not lead to detectable phosphorylation of STAT5B on tyrosine
699 (Fig. 10, lane 1, top panel), nor did coexpression of
STAT5B with SH2-B?(504-670) (Fig. 10, lane 2, top panel).
Expression of STAT5B with the low levels of JAK2 used in this
experiment also did not lead to detectable STAT5B tyrosyl
phosphorylation (Fig. 10, lane 3, top panel). However, as re-
ported previously (32), coexpression of SH2-B?(504-670) with
STAT5B and JAK2 led to a dramatic increase in phosphory-
lation of tyrosine 699 in STAT5B (Fig. 10, lanes 3 and 4, top
panel). In contrast, coexpression of SH2-B?(504-670) with
JAK2(Y813F) did not enhance the level of phosphorylation of
tyrosine 699 in STAT5B over that detected in the presence of
JAK2(Y813F) alone (Fig. 10, top panel, lanes 5 and 6), further
implicating tyrosine 813 as being important for SH2-B? to
mediate its stimulatory effects on JAK2.
Our laboratory has initiated studies designed to identify
which of the 49 tyrosines within JAK2 are phosphorylated.
Previously, we identified tyrosines 221 and 570, as well as
tyrosine 1007, as phosphotyrosines in active JAK2 that regulate
kinase activity (Argetsinger et al., submitted). Here we show
using 2-D peptide mapping of overexpressed JAK2 from 293T
cells, and phosphoamino acid analysis that tyrosine 813 of
JAK2 is also a site of autophosphorylation. Additionally, we
have observed spots that comigrate similarly to the peptides
containing tyrosine 813 in 2-D maps from JAK2 labeled in vivo
(data not shown). Finally, we made an antibody that specifi-
cally recognizes the phosphorylation of tyrosine 813. Use of
this antibody confirms that tyrosine 813 is phosphorylated in
JAK2 overexpressed in 293T cells and in endogenous JAK2
that is activated in response to GH in 3T3-F442A cells. Phos-
phorylation of tyrosine 813 in response to GH was rapid and
transient, following a time course similar to that of 221, 570,
and 1007 (Argetsinger et al., submitted). Interestingly, tyrosine
813 falls within the amino acid sequence YELL, which con-
forms to the consensus motif, YXXL, shared by both tyrosines
221 and 570 in JAK2. Moreover, JAK2 has been shown to
phosphorylate the JAK2 binding protein, SH2-B?, on tyrosines
439 and 494, both of which are contained within YXXL motifs
(27). Thus, identification of tyrosine 813 as a JAK2 autophos-
phorylation site further implicates YXXL as a common motif
that is recognized and tyrosyl phosphorylated by JAK2.
Mutation of tyrosine 813 to phenylalanine does not substan-
tially alter phosphorylation of other tyrosines in JAK2 (Fig.
1A), the activity of JAK2 (Fig. 8 and 9), or the ability of JAK2
FIG. 10. Tyrosine 813 is required for myc-SH2-B?(504-670) to enhance JAK2-mediated phosphorylation of STAT5B. COS7 cells were
cotransfected with 2.0 ?g of cDNA encoding STAT5B and 2.0 ?g of either empty vector (lane 1) or cDNA encoding myc SH2-B?(504-670) (lanes
2, 4, and 6), without (lanes 1 and 2) or with 0.25 ?g of cDNA encodes either JAK2 (lanes 3 and 4) or JAK2 (Y813F) (lanes 5 and 6). Cell lysates
were immunoblotted with anti-P-STAT5 (first panel), anti-STAT5B (second panel), and anti-myc (third panel). Cell lysates were also immuno-
precipitated with anti-JAK2 and the precipitated proteins were immunoblotted with anti-JAK2 (fourth panel) and with anti-PY (fifth panel). The
nitrocellulose membrane was stripped between blots as outlined in Materials and Methods.
VOL. 24, 2004 P(Y813) OF JAK2 BINDS SH2-B?
to phosphorylate substrates such as STAT5B (Fig. 5). Thus,
mutation of tyrosine 813 does not appear to have any substan-
tial effect on the structural conformation of JAK2. However,
when we investigated the signaling of JAK2 with tyrosine 813
mutated to phenylalanine in the presence of the JAK2 activa-
tor, SH2-B?, major changes were detected. We observed that
mutation of tyrosine 813 in JAK2 to phenylalanine prevents
the coimmunoprecipitation of JAK2 with both full-length
SH2-B? and SH2-B?(504-670). Additionally, we observed that
the activity of JAK2 lacking tyrosine 813 could not be en-
hanced by either SH2-B?(504-670) or full-length SH2-B?. Ad-
ditionally, SH2-B?(504-670) does not enhance JAK2(Y813F)-
mediated phosphorylation of STAT5B on tyrosine 699. In
contrast, mutation of 17 other tyrosines identified as phosphor-
ylation sites within JAK2 or as potential phosphorylation sites,
did not disrupt SH2-B?’s ability to bind to JAK2 (13 tested)
and/or to enhance JAK2 activation (12 tested) (data not
shown). Together, these data strongly suggest that phosphor-
ylated tyrosine 813 is the primary SH2-B?-binding site in
Due to technical difficulties, we have been unable to dem-
onstrate that tyrosine 813 is required for SH2-B? to enhance
the activation of JAK2 in response to GH. Nevertheless, evi-
dence exists to support the conclusion that tyrosine 813 is
important for SH2-B? to enhance GH activation of JAK2.
Specifically, it has been shown previously that SH2-B? can
enhance the GH-stimulated phosphorylation of endogenous
JAK2 in COS7 cells (32) and 293T cells (28). In addition, 2-D
phosphopeptide mapping of overexpressed JAK2 demon-
strates that tyrosines that are autophosphorylated in vitro are
also phosphorylated in vivo (Argetsinger et al., submitted).
Tyrosines 1007,1008, 570, 221, and 813 have all been identified
as sites of JAK2 autophosphorylation by 2-D phosphopeptide
mapping following an in vitro kinase assay. Phosphospecific
antibodies have confirmed that tyrosines 221, 570, 813, and
1007 and/or 1008 are phosphorylated in vivo in overexpressed
JAK2 and in endogenous JAK2 activated by GH (Argetsinger
et al., submitted; this work) (data not shown), suggesting that
activation of JAK2, whether via overexpression or via stimu-
lation by GH, results in similar sites of tyrosyl phosphorylation.
Finally, a variety of findings presented here suggest that ty-
rosine 813 is the primary site in JAK2 for binding SH2-B? and
is required for SH2-B? enhancement of JAK2 activity. We
therefore think it highly likely that GH-mediated phosphory-
lation of tyrosine 813 in JAK2 leads to recruitment of SH2-B?
to GHR-JAK2 complexes, just as phosphorylation of tyrosine
813 recruits SH2-B? to constitutively activated JAK2 com-
Both tyrosine 813 of JAK2 and the corresponding tyrosine
785 of JAK3 are contained within the amino acid sequence
YELL, which conforms to the motif YXXL. It is of interest
that SH2-B? is reported to bind YXXL motifs in other recep-
tors, including the fibroblast growth factor receptor at tyrosine
760 (YLDL) (20) and the erythropoietin receptor at tyrosines
343 (YLVL), 401 (YTIL), and 429 (YLYL) (36a). APS, a
member of the SH2-B? family of proteins, is reported to bind
the erythropoietin receptor at tyrosine 343 (YLVL) (37). Nev-
ertheless, the YXXL motif is not sufficient for protein binding,
as SH2-B? does not bind to other JAK2 tyrosines contained in
YXXL motifs (Fig. 8 and data not shown). In addition,
SH2-B? has been reported to bind other phosphotyrosine con-
taining motifs, including motifs containing tyrosine 724
(YMIM) of the fibroblast growth factor receptor (20), tyrosine
740 (YMDM) in the platelet-derived growth factor receptor
(31), and tyrosines 1158 (YETD), 1162 (YYRK), and 1163
(YRKG) in the catalytic region and tyrosines 960 (YLSA) and
1322 (YTHM) in the juxtamembrane and C-terminal regions
of the insulin receptor, respectively (21, 38). While work re-
mains to determine the exact SH2-B? binding requirements,
tyrosines contained within YXXL and YXXM motifs are
clearly useful starting points when attempting to determine the
binding site for SH2-B? within an SH2-B? binding partner.
As mentioned above, tyrosine 813 of JAK2 is homologous to
tyrosine 785 of JAK3, with both tyrosines found within the
sequence YELL. Because SH2-B? also binds JAK3 (28), we
analyzed whether YELL is a site of phosphorylation in JAK3.
2-D phosphopeptide mapping (Fig. 4A) revealed that tyrosine
785 is a major site of phosphorylation within JAK3, which is
phosphorylated both when overexpressed (Fig. 4B) and when
stimulated by IL-2 (Fig. 4C). As we showed for JAK2 lacking
tyrosine 813, JAK3 lacking the corresponding tyrosine 785
does not coprecipitate with SH2-B?, supporting the conclusion
that SH2-B? binds to phosphorylated tyrosine 785 of JAK3.
These experiments also further refined the interaction of
SH2-B? with JAK3 to amino acids 504-670 of SH2-B?. SH2-
B?(504-670) contains primarily the SH2 domain and the C
terminus of SH2-B?, suggesting that like JAK2, JAK3 interacts
with the SH2 domain of SH2-B?. The finding that myc-SH2-
B?(504-670) binds to kinase-active JAK3 but not to kinase-
inactive JAK3, which is not tyrosyl phosphorylated, further
supports the conclusion that the SH2 domain of SH2-B? binds
one or more phosphotyrosines in JAK3. These results provide
additional evidence that the primary binding sites in JAK2 and
JAK3 for the SH2 domain of SH2-B? is pYELL.
Surprisingly, while SH2-B? binds to both JAK2 and JAK3 at
phosphorylated tyrosines within a YELL motif, SH2-B? en-
hances the activity of only JAK2. Because the SH2-B?-binding
site within JAK3 is analogous to the site within JAK2, it is not
outwardly apparent why SH2-B? has a differential effect on
JAK2 and JAK3. Nevertheless, such discrepancies between
analogous tyrosines within the JAKs are not novel. Indeed,
mutating a conserved tyrosine within the activation loop of the
different JAKs (Y1007 in JAK2, Y980 in JAK3, Y1054 in
Tyk2) eliminates kinase activity in JAK2 but does not abolish
activity in JAK3 and Tyk2, suggesting that catalytic regulation
may be quite different between members of the JAK family
A better understanding for the differential effects of SH2-B?
on JAK2 and JAK3 may come once it is determined how
SH2-B? enhances JAK2 activity. As such, the identification of
tyrosine 813 as the SH2-B? binding site in JAK2 should greatly
help elucidate SH2-B?’s mechanism of action. As further
groundwork for determining how SH2-B? enhances JAK2 ac-
tivity, it is important to define the exact nature of the aug-
mented activity of JAK2 that occurs with SH2-B?. The in-
coexpression of SH2-B? could theoretically be the result of an
increase in the number of phosphotyrosines on JAK2, an in-
crease in the number of phosphorylated JAK2 molecules, or
both. Using antibody to phosphorylated tyrosines 1007 and/or
of JAK2seen with
4568KURZER ET AL.MOL. CELL. BIOL.
1008 in the activation loop of JAK2 we show here that SH2-B?
increases the number of active JAK2 molecules (Fig. 9). No
additional major spots are observed upon 2D phosphopeptide
mapping of JAK2 coexpressed with SH2-B? (Argetsinger et
al., submitted), supporting the conclusion that SH2-B? does
not alter the conformation of JAK2 in such a way that addi-
tional tyrosines are accessible for phosphorylation. Thus, both
of these results suggest that binding of SH2-B? promotes the
activation of JAK2.
Currently, there exist several possibilities for how SH2-B?
might increase JAK2 function. For example, SH2-B? may dis-
rupt binding of JAK2 inhibitors, including the SOCS proteins
and various phosphatases, such as PTP-1B. SOCS-1 has been
shown to bind JAK2 within the kinase activation loop, at ty-
rosine 1007 (43). That tyrosine 813 is required for SH2-B? to
activate JAK2 suggests that SH2-B? does not competitively
inhibit the binding of SOCS-1 to JAK2. Furthermore, prelim-
inary results indicate that SH2-B? does not bind to a phos-
phopeptide containing phosphorylated tyrosine 1007 (data not
shown). In addition, if SH2-B? were to compete with a JAK2
inhibitor for binding at tyrosine 813, it would be expected that
mutation of tyrosine 813 would prevent binding of the inhibi-
tor, and therefore increase the basal activity level of JAK2. As
demonstrated in Fig. 8 and 9, mutation of tyrosine 813 to
phenylalanine does not lead to a significant increase in JAK2
phosphorylation, suggesting that SH2-B? does not directly
compete with a JAK2-inhibitor for binding at tyrosine 813.
Nevertheless, it remains possible that the binding of SH2-B? to
JAK2 allosterically inhibits SOCS-1 binding at the active site,
or affects binding of other SOCS proteins to JAK2. Similarly,
PTP-1B has been shown to bind phosphorylated JAK2 in cells
treated with leptin, gamma interferon, or GH (9, 26) and
appears to dephosphorylate phosphotyrosine 1007 in the acti-
vation loop of JAK2 (26). Thus, it remains plausible that the
enhanced phosphorylation of JAK2 seen in the presence of
SH2-B? results from a decreased affinity of PTP-1B for JAK2.
Another possibility is that binding of SH2-B? may recruit
positive regulators to JAK2. Cross talk between signaling path-
ways, particularly the phosphorylation of tyrosine kinases by
other kinases, has been shown to occur. For instance, GH has
been shown to promote the phosphorylation of the epidermal
growth factor receptor by JAK2, thus enabling the docking of
Grb2 to the epidermal growth factor receptor and subsequent
activation of MAP kinase (42). Similarly, JAK2 has been
shown to be phosphorylated at tyrosine 1007 by the chimeric
oncogene Bcr-Abl in M3.16 cells (41), and Bcr-Abl has been
shown to be in a complex with JAK2 and SH2-B? in 32D cells
expressing Bcr-Abl (40). In this vein, it is possible that SH2-B?
serves as an adapter protein to recruit other tyrosine kinases,
which subsequently phosphorylate and activate JAK2.
JAK dimerization has been proposed to be required for
kinase activation (18, 24). Moreover, an N-terminal multimer-
ization domain has been identified within SH2-B, and has been
implicated in SH2-B-mediated potentiation of TRKA signaling
(30). Thus, it remains possible that multimerization of SH2-B?
may stabilize JAK2 multimers, thereby increasing the overall
kinase activity of the enzyme. However, as shown here and
previously, SH2-B?(504-670), which lacks the N-terminal mul-
timerization domain, is sufficient for the enhancement of JAK2
activity. Thus, while binding of SH2-B? to JAK2 may promote
a conformational change within JAK2 that increases the affin-
ity of JAK2-JAK2 interactions or facilitates JAK2-JAK2
SH2-B? via an N-terminal dimerization domain appears un-
Finally, binding of SH2-B? to JAK2 may cause a conforma-
tional change that maintains JAK2 in an active conformation.
Interestingly, tyrosine 813 resides in JH2 of JAK2. The JH1
domain, or catalytic domain, possesses the kinase activity of
the protein, whereas the JH2 domain, termed the pseudoki-
nase domain, is similar to the kinase domain but contains no
intrinsic activity. It has been shown that deletion of the JH2
domain can lead to hyper-activation of the kinase, suggesting
that the JH2 domain may negatively regulate the JH1 domain
(13, 35). An intriguing possibility, therefore, is that binding of
SH2-B? at tyrosine 813 relaxes the inhibition of JH2 on JH1,
thus enhancing kinase activity. Alternatively, binding of
SH2-B? to JAK2 may be sufficient to stabilize the kinase do-
main in an active conformation. In this regard, it is interesting
that both APS and SH2-B? have been shown to bind to phos-
photyrosines within the active site of the insulin receptor (2, 21,
25). However tyrosine 813 is not in the active site of JAK2 and
the present work provides no evidence that the SH2 domain of
SH2-B? binds to tyrosines in the active site of JAK2.
Summary. We have used 2-D peptide mapping, phos-
phoamino acid analysis, and a phosphospecific antibody to
demonstrate that tyrosine 813 is a site of autophosphorylation
in JAK2. Intriguingly, while mutation of tyrosine 813 in JAK2
does not appear to affect the intrinsic activity of JAK2, it does
disrupt the enhanced activation of JAK2 observed when either
SH2-B? or SH2-B?(504-670) is present. Furthermore, because
SH2-B? does not precipitate JAK2(Y813F) or enhance the
ability of JAK2(Y813F) to phosphorylate STAT5B at tyrosine
699, we conclude that tyrosine 813 is the primary SH2-B?-
binding site within JAK2 and is required to enhance JAK2
activation. This conclusion is further supported by the finding
that mutation of the corresponding tyrosine in JAK3, Y785,
also disrupts the coimmunoprecipitation of JAK3 with SH2-
B?. The binding of SH2-B? to phosphorylated tyrosine 813 in
JAK2 and phosphorylated tyrosine 785 in JAK3, both of which
are found within a YELL sequence, provides additional evi-
dence that the SH2 domain of SH2-B? shows some binding
preference for phosphorylated YXXL motifs.
This work was supported by NIH grants DK34171 and DK54222.
J.H.K. was supported by a Predoctoral Fellowship of the Cellular and
Molecular Approaches to Systems and Integrative Biology Training
Grant T32-GM08322 and the University of Michigan Medical Scientist
Training Program (NIH T32 GM07863). Oligonucleotides were syn-
thesized by the Biomedical Research Core Facility at the University of
Michigan with support from the Michigan Diabetes Research and
Training Center (P60-DK20572), the University of Michigan Multi-
purpose Arthritis Center (P60-AR20557), and the University of Mich-
igan Comprehensive Cancer Center (NIH P30 CA46592). cDNA se-
quencing was supported by the Cellular and Molecular Biology Core of
the Michigan Diabetes Research and Training Center.
We thank Xiaqing Wang for support and assistance with experi-
ments and Barbara Hawkins for assistance with the manuscript.
1. Aaronson, D. S., and C. M. Horvath. 2002. A road map for those who know
JAK-STAT. Science 296:1653–1655.
VOL. 24, 2004P(Y813) OF JAK2 BINDS SH2-B?
2. Ahmed, Z., B. J. Smith, K. Kotani, P. Wilden, and T. S. Pillay. 1999. APS, an Download full-text
adapter protein with a PH and SH2 domain, is a substrate for the insulin
receptor kinase. Biochem. J. 341:665–668.
3. Argetsinger, L. S., G. S. Campbell, X. Yang, B. A. Witthuhn, O. Silven-
noinen, J. N. Ihle, and C. Carter-Su. 1993. Identification of JAK2 as a growth
hormone receptor-associated tyrosine kinase. Cell 74:237–244.
3a.Argetsinger, L. S., J.-L. K. Kouadio, H. Steen, A. Stensballe, O. N. Jensen,
and C. Carter-Su. Autophosphorylation on tyrosines 221 and 570 regulates
its activity, Mol. Cell. Biol., in press.
4. Benekli, M., M. R. Baer, H. Baumann, and M. Wetzler. 2003. Signal trans-
ducer and activator of transcription proteins in leukemias. Blood 101:2940–
5. Boyle, W. J., P. van der Geer, and T. Hunter. 1991. Phosphopeptide mapping
and phosphamino acid analysis by two-dimensional separation on thin-layer
cellulose plates. Methods Enzymol. 201:110–148.
6. Candotti, F., L. Notarangelo, R. Visconti, and J. O’Shea. 2002. Molecular
aspects of primary immunodeficiencies: lessons from cytokine and other
signaling pathways. J. Clin. Investig. 109:1261–1269.
7. Carpino, N., R. Kobayashi, H. Zang, Y. Takahashi, S. T. Jou, J. Feng, H.
Nakajima, and J. N. Ihle. 2002. Identification, cDNA cloning, and targeted
deletion of p70, a novel, ubiquitously expressed SH3 domain-containing
protein. Mol. Cell. Biol. 22:7491–7500.
8. Chen, C., and H. Okayama. 1987. High-efficiency transformation of mam-
malian cells by plasmid DNA. Mol. Cell. Biol. 7:2745–2752.
9. Cheng, A., N. Uetani, P. D. Simoncic, V. P. Chaubey, A. Lee-Loy, C. J.
McGlade, B. P. Kennedy, and M. L. Tremblay. 2002. Attenuation of leptin
action and regulation of obesity by protein tyrosine phosphatase 1B. Dev.
10. Darnell, J. E., Jr. 1997. STATs and gene regulation. Science 277:1630–1635.
11. Diakonova, M., D. R. Gunter, J. Herrington, and C. Carter-Su. 2002.
SH2-B? is a Rac-binding protein that regulates cell motility. J. Biol. Chem.
12. Feng, J., B. A. Witthuhn, T. Matsuda, F. Kohlhuber, I. M. Kerr, and J. N.
Ihle. 1997. Activation of Jak2 catalytic activity requires phosphorylation of
Y1007 in the kinase activation loop. Mol. Cell. Biol. 17:2497–2501.
13. Frank, S. J., W. Yi, Y. Zhao, J. F. Goldsmith, G. Gilliland, J. Jiang, I. Sakai,
and A. S. Kraft. 1995. Regions of the JAK2 tyrosine kinase required for
coupling to the growth hormone receptor. J. Biol. Chem. 270:14776–14785.
14. Herrington, J., L. S. Smit, J. Schwartz, and C. Carter-Su. 2000. The role of
STAT proteins in growth hormone signaling. Oncogene 19:2585–2597.
15. Hou, S. X., Z. Zheng, X. Chen, and N. Perrimon. 2002. The Jak/STAT
pathway in model organisms: emerging roles in cell movement. Dev. Cell
16. Ihle, J. N. 1996. Janus kinases in cytokine signalling. Philos. Trans. R. Soc.
Lond. B Biol. Sci. 351:159–166.
17. Ihle, J. N. 1996. STATs: signal transducers and activators of transcription.
18. Ihle, J. N., and I. M. Kerr. 1995. Jaks and Stats in signaling by the cytokine
receptor superfamily. Trends Genet. 11:69–74.
19. Johnston, J. A., M. Kawamura, R. A. Kirken, Y.-Q. Chen, T. B. Blake, K.
Shibuya, J. R. Ortaldo, D. W. McVicar, and J. J. O’Shea. 1994. Phosphor-
ylation and activation of the Jak-3 Janus kinase in response to interleukin-2.
20. Kong, M., C. S. Wang, and D. J. Donoghue. 2002. Interaction of fibroblast
growth factor receptor 3 and the adapter protein SH2-B. J. Biol. Chem.
21. Kotani, K., P. Wilden, and T. S. Pillay. 1998. SH2-B? is an insulin-receptor
adapter protein and substrate that interacts with the activation loop of the
insulin-receptor kinase. Biochem. J. 335:103–109.
22. Lacronique, V., A. Boureux, V. D. Valle, H. Poirel, C. T. Quang, M.
Mauchauffe, C. Berthou, M. Lessard, R. Berger, J. Ghysdael, and O. A.
Bernard. 1997. A TEL-JAK2 fusion protein with constitutive kinase activity
in human leukemia. Science 278:1309–1312.
23. Leonard, W. J., and J. J. O’Shea. 1998. Jaks and STATs: biological impli-
cations. Annu. Rev. Immunol. 16:293–322.
24. Mizuguchi, R., and M. Hatakeyama. 1998. Conditional activation of Janus
kinase (JAK) confers factor independence upon interleukin-3-dependent
cells. Essential role of Ras in JAK-triggered mitogenesis. J. Biol. Chem.
25. Moodie, S. A., J. Alleman-Sposeto, and T. A. Gustafson. 1999. Identification
of the APS protein as a novel insulin receptor substrate. J. Biol. Chem.
26. Myers, M. P., J. N. Andersen, A. Cheng, M. L. Tremblay, C. M. Horvath,
J. P. Parisien, A. Salmeen, D. Barford, and N. K. Tonks. 2001. TYK2 and
JAK2 are substrates of protein-tyrosine phosphatase 1B. J. Biol. Chem.
27. O’Brien, K. B., L. S. Argetsinger, M. Diakonova, and C. Carter-Su. 2003.
YXXL motifs in SH2-B? are phosphorylated by JAK2, JAK1, and platelet-
derived growth factor receptor and are required for membrane ruffling.
J. Biol. Chem. 278:11970–11978.
28. O’Brien, K. B., J. J. O’Shea, and C. Carter-Su. 2002. SH2-B family members
differentially regulate JAK family tyrosine kinases. J. Biol. Chem. 277:8673–
29. Peeters, P., S. D. Raynaud, J. Cools, I. Wlodarska, J. Grosgeorge, P. Philip,
F. Monpoux, L. Van Rompaey, M. Baens, H. Van den Berghe, and P.
Marynen. 1997. Fusion of TEL, the ETS-variant gene 6 (ETV6), to the
receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and
t(9;15;12) in a myeloid leukemia. Blood 90:2535–2540.
30. Qian, X., and D. D. Ginty. 2001. SH2-B and APS are multimeric adapters
that augment TrkA signaling. Mol. Cell. Biol. 21:1613–1620.
31. Riedel, H., N. Yousaf, Y. Zhao, H. Dai, Y. Deng, and J. Wang. 2000. PSM, a
mediator of PDGF-BB-, IGF-I-, and insulin-stimulated mitogenesis. Onco-
32. Rui, L., and C. Carter-Su. 1999. Identification of SH2-B? as a potent cyto-
plasmic activator of the tyrosine kinase Janus kinase 2. Proc. Natl. Acad. Sci.
33. Rui, L., D. R. Gunter, J. Herrington, and C. Carter-Su. 2000. Differential
binding to and regulation of JAK2 by the SH2 domain and N-terminal region
of SH2-B?. Mol. Cell. Biol. 20:3168–3177.
34. Rui, L., L. S. Mathews, K. Hotta, T. A. Gustafson, and C. Carter-Su. 1997.
Identification of SH2-B? as a substrate of the tyrosine kinase JAK2 involved
in growth hormone signaling. Mol. Cell. Biol. 17:6633–6644.
35. Saharinen, P., K. Takaluoma, and O. Silvennoinen. 2000. Regulation of the
Jak2 tyrosine kinase by its pseudokinase domain. Mol. Cell. Biol. 20:3387–
36. Silvennoinen, O., B. Witthuhn, F. W. Quelle, J. L. Cleveland, T. Yi, and J. N.
Ihle. 1993. Structure of the murine JAK2 protein-tyrosine kinase and its role
in interleukin 3 signal transduction. Proc. Natl. Acad. Sci. USA 90:8429–
36a.Tschirch, E., B. K. Beattie, N. Dowman, C. Carter-Su, and D. L. Barber.
2000. The erythropoietin receptor recruits the adaptor protein SH2-B? to a
region necessary for proliferation and differentiation. Blood 96:568a (Ab-
37. Wakioka, T., A. Sasaki, K. Mitsui, M. Yokouchi, A. Inoue, S. Komiya, and A.
Yoshimura. 1999. APS, an adaptor protein containing pleckstrin homology
(PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in
collaboration with c-Cbl. Leukemia 13:760–767.
38. Wang, J., and H. Riedel. 1998. Insulin-like growth factor-I receptor and
insulin receptor association with a Src homology-2 domain-containing puta-
tive adapter. J. Biol. Chem. 273:3136–3139.
39. Witthuhn, B. A., F. W. Quelle, O. Silvennoinen, T. Yi, B. Tang, O. Miura,
and J. N. Ihle. 1993. JAK2 associates with the erythropoietin receptor and is
tyrosine phosphorylated and activated following stimulation with erythropoi-
etin. Cell 74:227–236.
40. Xie, S., H. Lin, T. Sun, and R. B. Arlinghaus. 2002. Jak2 is involved in c-Myc
induction by Bcr-Abl. Oncogene 21:7137–7146.
41. Xie, S., Y. Wang, J. Liu, T. Sun, M. B. Wilson, T. E. Smithgall, and R. B.
Arlinghaus. 2001. Involvement of Jak2 tyrosine phosphorylation in Bcr-Abl
transformation. Oncogene 20:6188–6195.
42. Yamauchi, T., K. Ueki, K. Tobe, H. Tamemoto, N. Sekine, M. Wada, M.
Honjo, M. Takahashi, T. Takahashi, H. Hirai, T. Tsushima, Y. Akanuma, T.
Fujita, I. Komuro, Y. Yazaki, and T. Kadowaki. 1998. Growth hormone-
induced tyrosine phosphorylation of EGF receptor as an essential element
leading to MAP kinase activation and gene expression. Endocr. J. 45:S27–
43. Yasukawa, H., H. Misawa, H. Sakamoto, M. Masuhara, A. Sasaki, T.
Wakioka, S. Ohtsuka, T. Imaizumi, T. Matsuda, J. N. Ihle, and A. Yo-
shimura. 1999. The JAK-binding protein JAB inhibits Janus tyrosine kinase
activity through binding in the activation loop. EMBO J. 18:1309–1320.
44. Zhou, Y. J., M. Chen, N. A. Cusack, L. H. Kimmel, K. S. Magnuson, J. G.
Boyd, W. Lin, J. L. Roberts, A. Lengi, R. H. Buckley, R. L. Geahlen, F.
Candotti, M. Gadina, P. S. Changelian, and J. J. O’Shea. 2001. Unexpected
effects of FERM domain mutations on catalytic activity of Jak3: structural
implication for Janus kinases. Mol. Cell 8:959–969.
45. Zhou, Y. J., E. P. Hanson, Y. Q. Chen, K. Magnuson, M. Chen, P. G. Swann,
R. L. Wange, P. S. Changelian, and J. J. O’Shea. 1997. Distinct tyrosine
phosphorylation sites in JAK3 kinase domain positively and negatively reg-
ulate its enzymatic activity. Proc. Natl. Acad. Sci. USA 94:13850–13855.
4570 KURZER ET AL.MOL. CELL. BIOL.