From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 49109-0622
Received for publication, October 22, 2002, and in revised form, January 13, 2003
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ABSTRACT |
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SH2-B SH2-B1 is a putative
adapter protein (see Fig. 1 below) implicated in the actions of
multiple cytokines and growth factors. The Four splice variants of SH2-B have been identified (1, 4-6), The domain structure of SH2-B To gain further insight into the mechanisms by which SH2-B Cells and Reagents--
The stocks of COS-7 and 293T cells were
provided by Drs. M. D. Uhler (University of Michigan, Ann Arbor,
MI) and E. R. Fearon (University of Michigan), respectively. The
stock of 3T3-F442A murine fibroblasts was provided by H. Green (Harvard
University, Cambridge, MA). Aprotinin, leupeptin, and Triton X-100 were
from Roche Molecular Biochemicals. Recombinant protein A-agarose was from Repligen. Enhanced chemiluminescence (ECL) detection system was
from Amersham Biosciences. Anti-JAK2 antiserum ( Plasmids--
Construction of the vector encoding rat SH2-B Cell Culture and Transfection--
COS-7, 293T, or 3T3-F442A
murine fibroblast cells were grown in Dulbecco's modified Eagle medium
(DMEM) supplemented with 1 mM L-glutamine, 100 units of penicillin per ml, 100 µg of streptomycin per ml, 0.25 µg
of amphotericin per ml (supplemented DMEM), and 9% fetal calf serum
(COS-7) or 9% calf serum (293T and 3T3-F442A). COS-7 and 293T cells
were transiently transfected using calcium phosphate precipitation
(23). Transfected cells were assayed 24 (293T) or 48 (COS-7) h after
transfection. COS-7 cells overexpressing either PDGFR or JAK2 were
incubated overnight in serum-free medium containing 1% bovine serum
albumin before lysis. For imaging experiments, 3T3-F442A cells were
plated on glass coverslips and transfected with 2.5 µg of cDNA
expression vector using Transfast (Promega) according to the protocol
recommended by the manufacturer. Approximately 36 h after
transfection, cells were incubated overnight in serum-free medium
containing 1% bovine serum albumin, treated with ligands at 37 °C,
and processed for imaging as described below.
Immunoprecipitation and Immunoblotting--
Immunoprecipitations
and immunoblots were performed as described previously (24). Briefly,
24 (293T) or 48 (COS-7) h after transfection, cells were rinsed three
times with 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1 mM Na3VO4.
Cells were then solubilized in lysis buffer (50 mM Tris, pH
7.5, 0.1% Triton X-100, 150 mM NaCl, 2 mM
EGTA, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin) and centrifuged at 14,000 × g for 10 min at
4 °C. The supernatant (cell lysate) was incubated with the indicated
antibody on ice for 2 h. The immune complexes were collected on
protein A-agarose (14 µl, packed volume) for 1 h at 4 °C. The
beads were washed three times with washing buffer (50 mM
Tris, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 2 mM EGTA) and boiled for 5 min in a mixture (80:20) of lysis
buffer and SDS-PAGE sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 10% In Vitro Kinase Assay--
In vitro kinase assays
were performed as described previously (24). SH2-B In Vivo Labeling--
293T cells were transfected as described
above. Twenty-four hours after transfection, cells were washed with
phosphate-free DMEM containing 1% bovine serum albumin. Cells were
treated with 1 mCi of [32P]orthophosphate (ICN) for
4 h followed by pervanadate for 6 or 30 min as indicated.
Pervanadate was prepared by mixing 430 µl of 100 mM
Na3VO4 with 10 µl of 30%
H2O2 and incubating at room temp for 20 min.
The solution was cooled on ice and added to cell medium to yield a
final concentration of 100 µM
Na3VO4, 200 µM H2O2. Cells were lysed. SH2-B Phosphopeptide Mapping and Phosphoamino Acid
Analysis--
Two-dimensional phosphopeptide mapping and phosphoamino
acid analysis were performed as described (25). Briefly, nitrocellulose containing 32P-labeled SH2-B Assessment of Membrane Ruffling--
To measure the effect of
SH2-B JAK2 Phosphorylates Tyrosines 439 and 494 in SH2-B
The region of the nitrocellulose containing 32P-labeled
SH2-B
To determine if peptides 1 and 2 generated from two-dimensional mapping
of SH2-B
Two-dimensional phosphopeptide mapping of SH2-B JAK2 Phosphorylates Tyrosines 439 and 494 in SH2-B
To determine more definitively whether Tyr-494 in SH2-B Tyrosyl Phosphorylation of SH2-B SH2-B JAK1 Phosphorylates Tyr-439 and Tyr-494 in SH2-B
To determine if any additional tyrosines in SH2-B SH2-B
To more closely examine the phosphorylation of SH2-B
To determine whether PDGFR phosphorylates Tyr-439 or Tyr-494 in
SH2-B JAK2 Phosphorylates Tyr-439 and Tyr-494 in SH2-B
Darker exposures of the maps of wild type and mutant forms of SH2-B
Maps of SH2-B SH2-B SH2-B The Function of Tyrosyl Phosphorylation of
SH2-B
Previously, we have shown that overexpression of SH2-B
Interestingly, both Tyr-439 and Tyr-494 reside within the consensus
sequence of YXXL. Thus, candidate interacting proteins should bind phosphorylated tyrosines in the sequence of
YXXL. One such candidate protein is CrkII. CrkII is an
adapter protein implicated in the regulation of the actin cytoskeleton
and known to be tyrosyl-phosphorylated in response to stimulation by GH (32, 33). The preferred binding motif for the SH2 domain of CrkII is
YXXP, however, the Crk SH2 domain has some affinity for pYXXL-containing sequences (34, 35). We overexpressed
hemagglutinin-tagged CrkII with JAK2 in the presence and absence of
SH2-B
Another protein candidate binding partner for tyrosyl-phosphorylated
SH2-B
In summary, using two-dimensional phosphopeptide mapping, we have
determined that Tyr-439 and Tyr-494 in SH2-B binds to the activated form of
JAK2 and various receptor tyrosine kinases. It is a potent stimulator
of JAK2, is required for growth hormone (GH)-induced membrane
ruffling, and increases mitogenesis stimulated by platelet-derived
growth factor (PDGF) and insulin-like growth factor I. Its domain
structure suggests that SH2-B
may act as an adapter protein to
recruit downstream signaling proteins to kinase·SH2-B
complexes. SH2-B
is tyrosyl-phosphorylated in response to GH
and interferon-
, stimulators of JAK2, as well as in response to PDGF
and nerve growth factor. To begin to elucidate the role of tyrosyl
phosphorylation in the function of SH2-B
, we used phosphopeptide
mapping, mutagenesis, and a phosphotyrosine-specific antibody to
identify Tyr-439 and Tyr-494 in SH2-B
as targets of JAK2 both
in vitro and in intact cells. SH2-B
lacking Tyr-439 and
Tyr-494 inhibits GH-induced membrane ruffling but still activates JAK2.
We provide evidence that JAK1, like JAK2, phosphorylates Tyr-439 and
Tyr-494 in SH2-B
and that PDGF receptor phosphorylates SH2-B
on
Tyr-439. Therefore, phosphorylated Tyr-439 and/or Tyr-494 in SH2-B
may provide a binding site for one or more proteins linking cytokine
receptor·JAK2 complexes and/or receptor tyrosine kinases to the actin cytoskeleton.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoform of SH2-B was
originally identified as a JAK2-binding protein that is
tyrosyl-phosphorylated in response to GH and interferon-
, stimulators of the tyrosine kinase JAK2 (1). SH2-B
binds
preferentially via its SH2 domain to the tyrosyl-phosphorylated, active
form of JAK2. It increases dramatically the activity of JAK2 and
enhances the tyrosyl phosphorylation of downstream targets of JAK2 such as STAT5 (2). Thus, SH2-B
would be predicted to act in a positive feedback manner to increase signals by cytokines that activate JAK2.
SH2-B
also binds to and is tyrosyl-phosphorylated by JAK1, suggesting that SH2-B
may also act as an adapter protein in
signaling through cytokines that activate JAK1 (3).
,
,
, and
. They are identical for the first 631 amino acids and
diverge just past the SH2 domain (6). Initial studies suggest that they
have overlapping signaling properties. One or more isoforms have been
shown to be tyrosyl-phosphorylated by activated forms of the receptors
for platelet-derived growth factor (PDGF) (6-8), insulin-like growth
factor-I (6, 8, 9), nerve growth factor (NGF) (10, 11), insulin (5, 9,
12, 13), and fibroblast growth factor (14). SH2-B
has been
implicated in GH- and PDGF-induced changes in the actin cytoskeleton
and/or cell motility (15, 16). SH2-B
and SH2-B
have both been
implicated in NGF-induced neurite outgrowth (10, 11) and SH2-B
has
been shown to increase the NGF-induced tyrosyl phosphorylation of TrkA, the receptor tyrosine kinase for NGF (17). All four isoforms of SH2-B
have been shown to increase DNA synthesis and cellular proliferation
stimulated by PDGF or insulin-like growth factor-I (6, 8), although to
varying degrees. Lastly, SH2-B
has been shown to increase the
phosphorylation and nuclear translocation of STAT5B mediated by a
constitutively active form of fibroblast growth factor receptor
3 (14). These findings suggest that SH2-B mediates or regulates
signaling pathways leading to cell motility, growth, and/or
differentiation induced by multiple growth factors. However, the
mechanisms by which SH2-B
mediates its effects on cell signaling are
largely unknown.
suggests that it may act as an adapter
protein to recruit proteins to cytokine receptor·JAK complexes and
receptor tyrosine kinases. SH2-B
contains an SH2 domain, a
pleckstrin homology domain, three proline rich regions, and nine
tyrosines (see Fig. 1) (1). We have shown that the second proline-rich
region in SH2-B
is required for maximal cell motility induced by GH
and for binding to the small GTPase, Rac (16). Rac is a member of the
Rho family of small GTPases and has been implicated in the formation of
lamellipodia and other structures required for cell motility (18, 19).
Phosphorylated tyrosines in SH2-B
are likely to be binding sites for
downstream molecules containing SH2 or phosphotyrosine binding domains.
elicits
its effects, we sought to identify which tyrosines in SH2-B
are
phosphorylated and the role tyrosyl phosphorylation plays in the
function of SH2-B
. Phosphopeptide mapping determined that Tyr-439
and Tyr-494 in SH2-B
are targets of JAK2 in vitro and
in vivo. We also show that, even though SH2-B
lacking
these tyrosines still activates JAK2, SH2-B
lacking tyrosines 439 and 494 acts as a dominant negative to inhibit GH-induced membrane ruffling demonstrating a critical role for Tyr-439 and Tyr-494 in the
ability of SH2-B
to regulate membrane ruffling. Furthermore, evidence is provided that JAK1 and the receptor tyrosine kinase, PDGFR,
also phosphorylate SH2-B
on Tyr-439. Thus, phosphorylation of
Tyr-439 (pY439) in response to PDGF as well as cytokines that activate
JAK1 and JAK2 may provide a binding site for proteins important in the
regulation of the actin cytoskeleton.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
JAK2) was raised in
rabbits against a synthetic peptide corresponding to amino acids
758-766 of murine JAK2 (20, 21) and was used at a dilution of 1:500
for immunoprecipitation and 1:15,000 for immunoblotting. Antibody to
rat SH2-B
(
SH2-B
) was raised against a glutathione S-transferase fusion protein containing amino acids 527-670
of SH2-B
as described previously (1) and was used at a dilution of
1:100 for immunoprecipitations and 1:15,000 for immunoblotting. Monoclonal anti-phosphotyrosine antibody (
PY, clone 4G10) from Upstate Biotechnology, Inc was used at a dilution of 1:7,500 for immunoblotting. Polyvinylpyrrolidone and phosphoamino acid standards were from Sigma. Methylated trypsin was from Promega. Thin-layer chromatography plates were from EM Science.
with a myc tag at the N terminus has been described previously (2). The cDNA for murine JAK2 was provided by J. Ihle and B. Witthuhn (St. Jude Children's Research Hospital, Memphis, TN) (20). cDNA
encoding murine JAK1 with a myc tag at the C terminus was kindly
provided by R. Schreiber (Washington University, St. Louis, MO).
cDNA encoding platelet-derived growth factor receptor (PDGFR)
subunit was provided by A. Kazlauskas (Harvard University, Cambridge,
MA) (22). Individual tyrosines in SH2-B
were mutated using the QuikChange site-directed mutagenesis kit (Stratagene). The following primers (sense strand) were used to mutate each tyrosine to
phenylalanine: Tyr-48 (5'-CGTTTTCGCCTCTTTCTGGCCTCCCACCC-3'), Tyr-55
(5'-CCCACCCACAATTTGCAGAGCCGGGAGC-3'), Tyr-354
(5'-GGTAGAAGGCCCTTCAGAGTTCATCCTGGAGACAACTG-3'), Tyr-439 (5'-GTCGCAGGGAGCTTTTGGAGGCCTCTCAGACC-3'), Tyr-494
(5'-CCCCTCTCTACCCCGTTCCCTCCCCTGGATAC-3'), Tyr-525
(5'-CCCCTCTCAGGCTTCCCTTGGTTCCACGGC-3'), Tyr-564
(5'-GACGTGGTGAATTTGTCCTCACTTTCAACTTCC-3'), Tyr-624
(5'-CCTTGTCAGCTTTGTGCCCTCCCAGCGG-3'), and Tyr-649
(5'-CGACCGATGCTTCCCCGATGCTTCTTCC-3'). The double mutant,
SH2-B
(Y439F,Y494F), was created by using SH2-B
(Y439F) as a
template and mutating Tyr-494. All nine tyrosines in SH2-B
were
mutated (SH2-B
(9YF)) using the above primers with the QuikChange multisite-directed mutagenesis kit (Stratagene).
SH2-B
(Y439F,Y494F) was subcloned in-frame into pEGFP
(Clontech) using the
BamHI/XbaI sites to create
GFP-SH2-B
(Y439F,Y494F). Mutations were confirmed by sequencing by
the University of Michigan DNA Sequencing Core.
-mercaptoethanol, 40% glycerol, 0.01% bromphenol
blue). The solubilized proteins were separated by SDS-PAGE (5-12%
gradient) followed by immunoblotting with the indicated antibody and
visualization with the ECL detection system.
was
immunoprecipitated with
SH2-B
and immune complexes were collected
using protein A-agarose. Bound proteins were washed twice with
lysis buffer (see above) and once with kinase buffer (50 mM
Hepes, pH 7.6, 5 mM MnCl2, 0.5 mM
dithiothreitol, 100 mM NaCl, 1 mM
Na3VO4). Immunoprecipitates were incubated at 30 °C for 30 min in 50 µl of kinase buffer containing 0.5 mCi of
[
-32P]ATP, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin. Immunoprecipitates were washed five times with 500 µl of
lysis buffer. Proteins were eluted by boiling in a mixture (80:20) of
lysis buffer and SDS-PAGE sample buffer. Proteins were then resolved by
SDS-PAGE (5-12% gradient), transferred to nitrocellulose membrane,
and visualized by autoradiography or phosphorimaging (Bio-Rad model 505).
was
immunoprecipitated, resolved by SDS-PAGE, and transferred to
nitrocellulose as described above.
labeled in vivo
or in vitro (see above) were washed twice with deionized
H2O, soaked in 500 µl of 0.5% polyvinylpyrrolidone in
100 mM acetic acid at 37 °C for 30 min, washed five
times with deionized H2O, and digested with 10 µg of
methylated trypsin for 4 h at 37 °C. Approximately 85-90% of
counts were recovered. Next, digested peptides were lyophilized,
oxidized with performic acid, and re-lyophilized. Peptides were
separated by thin-layer electrophoresis at pH 8.9 (in vitro)
or pH 3.5 (in vivo) followed by a second dimension in
thin-layer chromatography using phosphochromatography buffer (25). For
phosphoamino acid analysis, 32P-labeled peptides were
scraped from the cellulose plates and eluted from the cellulose with pH
1.9 buffer. Eluted peptides or full-length SH2-B
were subjected to
acid hydrolysis in 6 N HCl at 110 °C for 60 min and
resolved by thin layer electrophoresis in buffer at pH 3.5 containing
0.5 mM EDTA. Phosphoamino acid standards were visualized by
ninhydrin, and radioactive spots were visualized by autoradiography or
using a PhosphorImager (Bio-Rad model 505).
on membrane ruffling, cells expressing GFP-SH2-B
or
GFP-SH2-B
(Y439F,Y494F) were deprived of serum overnight and treated
with GH as indicated in the figure legends. Cells were rapidly rinsed
three times with PBS (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) and fixed for 30 min at room temperature in 4%
formaldehyde in PBS. Cells were permeabilized with 0.1% Triton X-100
in PBS for 15 min at room temperature and rinsed three times in PBS.
Filamentous actin was stained by incubating samples with Texas
Red-phalloidin (1:60) for 30 min at room temperature. Coverslips were
then rinsed three times with PBS, mounted on slides, and imaged the
same day. Transfected cells expressing GFP-tagged forms of SH2-B
were located with a fluorescein isothiocyanate filter set using a Nikon
TE200 microscope. The number of ruffles, assessed as a concentration of
F-actin at a plasma membrane protrusion, per transfected cell was
determined. Each transfection was repeated three times with similar
results. Between 30 and 119 untransfected cells or cells positive for
GFP, GFP-SH2-B
, or GFP-SH2-B
(Y439F,Y494F) from the combined three
experiments were scored for the presence of ruffles for each
experimental condition.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in
Vitro--
Two-dimensional peptide mapping was used to identify which
of the one or more tyrosines in SH2-B
are phosphorylated by JAK2. Each of the nine tyrosines in SH2-B
was individually mutated to
phenylalanine within the context of full-length, myc-tagged SH2-B
(Fig. 1). Wild type and mutant forms of
myc-tagged SH2-B
were co-expressed with JAK2 in 293T cells. SH2-B
was immunoprecipitated with
SH2-B
. The immobilized
SH2-B
·JAK2 complex was incubated with [
-32P]ATP
in an in vitro kinase assay. Radiolabeled proteins were separated by SDS-PAGE, transferred to nitrocellulose, and visualized by
autoradiography. As reported previously (2, 3), JAK2 is constitutively
active when overexpressed in 293T cells and co-precipitates with
SH2-B
(Fig. 2A). Wild type
SH2-B
was phosphorylated in vitro (Fig. 2A,
lane 1), presumably by JAK2, because SH2-B
is not
phosphorylated when JAK2 is not overexpressed (data not shown). Each of
the individual tyrosine to phenylalanine mutants of SH2-B
was also
phosphorylated in the kinase assay (Fig. 2A, lanes
2-10), suggesting that SH2-B
is phosphorylated on more than
one tyrosine. SH2-B
with tyrosine 439 mutated to phenylalanine (SH2-B
(Y439F)) migrated faster than wild type or other mutant forms
of SH2-B
(Fig. 2A, lane 5). This faster
migration could be due to the loss of phosphorylation of Tyr-439.
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Fig. 1.
Schematic representation of rat
SH2-B . A, potential sites of
tyrosyl phosphorylation are shown (Y). P,
proline-rich region; PH, pleckstrin homology domain;
SH2, Src homology 2 domain. The arrow indicates
divergence of
,
,
, and
isoforms. B, each
tyrosine in SH2-B
with its three downstream amino acids is
shown.
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Fig. 2.
JAK2 phosphorylates Tyr-439 and Tyr-494 in
SH2-B in vitro. A,
plasmid (0.5 µg) encoding JAK2 was transfected into 293T cells with
plasmid (1 µg) encoding wild type SH2-B
(lane 1), the
indicated mutant form of SH2-B
(lanes 2-10), or with
control plasmid (lane 11). SH2-B
was immunoprecipitated
(IP) with
SH2-B
and incubated with 0.5 mCi of
[
-32P]ATP. Proteins were resolved by SDS-PAGE,
transferred to nitrocellulose, and visualized by autoradiography. The
migration of SH2-B
and JAK2 are noted. B, wild type
SH2-B
or mutant forms of SH2-B
from A were cut from
nitrocellulose and subjected to two-dimensional phosphopeptide mapping
at pH 8.9 (panels i and iii-xi). For the
two-dimensional peptide map in panel ii, SH2-B
(1 µg)
was co-expressed with JAK2 (0.5 µg) in COS-7 cells and processed as
the other samples. C, phosphorylated peptides scraped from
two-dimensional maps of SH2-B
(B, panel i)
were eluted and subjected to acid hydrolysis (lanes 2 and
3). As a comparison, full-length, wild type SH2-B
(panel A, lane 1) was also acid hydrolyzed. Amino
acids were separated by thin layer electrophoresis and visualized by
autoradiography. Migration of phosphoserine (pSer),
phosphothreonine (pThr), and phosphotyrosine
(pTyr) standards is indicated.
was excised and digested with trypsin. Tryptic peptides were oxidized in performic acid and then separated first by thin layer electrophoresis and then in the second dimension by ascending chromatography. 32P-Labeled peptides were visualized by
autoradiography. Wild type SH2-B
contained two highly phosphorylated
peptides (Fig. 2B, panel i, Spots 1 and 2) and several more peptides that are phosphorylated less robustly. Darker exposures of the map of wild type SH2-B
revealed at least five more spots (data not shown). The phosphopeptide map generated from SH2-B
overexpressed in COS-7 cells resembles that
seen with SH2-B
expressed in 293T cells (Fig. 2B,
panel i versus panel ii). Thus,
two-dimensional maps of SH2-B
from both 293T and COS-7 cells reveal
that JAK2 phosphorylates SH2-B
in vitro on at least two peptides.
are phosphorylated on tyrosine(s), serine(s), or
threonine(s), we performed phosphoamino acid analysis. Peptides 1 and 2 were isolated from Fig. 2B (panel i). As a
comparison, we also performed phosphoamino acid analysis on the tryptic
digest of full-length, wild type SH2-B
used for this panel.
Phosphoamino acid analysis revealed that full-length SH2-B
is
phosphorylated primarily on tyrosine residues in vitro (Fig.
2C, lane 1). SH2-B
was minimally
phosphorylated on serines and threonines. The 32P
incorporated into Spots 1 (Fig. 2C, lane
2) and 2 (Fig. 2C, lane 3)
contained exclusively phosphotyrosine. No 32P co-migrating
with phosphoserine or phosphothreonine was detected. We also performed
phosphoamino acid analysis on the five lighter spots that are visible
in darker exposures (48 h) of maps of SH2-B
(data not shown). Each
of these spots contained 32P-labeled phosphoserine and
phosphothreonine as well as phosphotyrosine (data not shown).
in which individual
tyrosines were mutated to phenylalanine (Fig. 2B,
panels iii-xi) yielded maps similar to wild type SH2-B
with two exceptions. In the maps of SH2-B
(Y439F), Spot 1 completely disappears (Fig. 2B, panel vi)
suggesting that in wild type SH2-B
, this spot corresponds to a
peptide containing phosphorylated Tyr-439. Spot 2 disappears when Tyr-494 is mutated suggesting that Tyr-494 is phosphorylated by
JAK2 in vitro (Fig. 2B, panel vii).
Neither the presence nor the migration of any of the lighter spots
reproducibly changed when maps of wild type and all mutant forms of
SH2-B
were compared, suggesting that these lighter spots are not
derived from SH2-B
. Taken together, the data from phosphopeptide
mapping of wild type and mutant forms of SH2-B
as well as
phosphoamino acid analysis reveal that JAK2 phosphorylates SH2-B
on
tyrosines 439 and 494 in vitro.
in
Vivo--
To provide insight into whether Tyr-439 and Tyr-494 are also
phosphorylated in vivo, JAK2 was co-expressed with wild type SH2-B
, SH2-B
(Y439F), SH2-B
(Y494F), or the double mutant
SH2-B
(Y439F,Y494F) in 293T (Fig.
3A) or COS-7 (Fig.
3B) cells. SH2-B
was immunoprecipitated and Western
blotted with
PY. SH2-B
is not tyrosyl-phosphorylated when
expressed alone (see Fig. 9B, lanes 1 and
2, below). As reported previously (3), SH2-B
is
tyrosyl-phosphorylated by JAK2 when they are co-expressed in either
293T or COS-7 cells (Fig. 3, A and B, lane
2). When Tyr-439 is mutated to phenylalanine, the tyrosyl
phosphorylation of SH2-B
decreased compared with wild type in both
cell types tested (Fig. 3, A and B, lane
3 versus lane 2). SH2-B
(Y439F) migrates
faster than wild type SH2-B
, consistent with a lower level of
phosphorylation. SH2-B
(Y494F) has, at best, only a modest reduction
in tyrosyl phosphorylation compared with wild type SH2-B
(Fig. 3,
A and B, lane 4 versus lane 2). When both Tyr-439 and Tyr-494 were mutated to
phenylalanine within the context of full-length SH2-B
(SH2-B
(Y439F,Y494F)) (Fig. 3, A and B,
lane 5), the tyrosyl phosphorylation of SH2-B
was similar
to or less than in cells expressing wild type SH2-B
or cells
expressing SH2-B
lacking Tyr-439 or Tyr-494 individually. These data
indicate that Tyr-439 in SH2-B
is phosphorylated by JAK2 in
vivo. Whether or not Tyr-494 is also phosphorylated is more
difficult to discern. The lack of a reproducible, substantial decrease in the
PY signal when Tyr-494 is mutated may indicate that
4G10
PY does not recognize pY494, that phosphorylation at Tyr-494 is
very labile or undergoes dephosphorylation after the cells are lysed,
that Tyr-494 is not phosphorylated to as great an extent as Tyr-439,
that the relatively small decrease in overall phosphorylation upon
mutation of Tyr-494 cannot be reproducibly detected by Western blotting
with
PY, or that Tyr-494 is not phosphorylated by JAK2 in intact
cells.
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Fig. 3.
Tyr-439 in SH2-B is
phosphorylated by JAK2 in intact cells. Plasmid (0.5 µg)
encoding JAK2 was transfected into 293T (A) or COS-7
(B) cells with control plasmid (1 µg, lane 1)
or plasmid (1 µg) encoding wild type SH2-B
(lane 2) or
the indicated mutant form of SH2-B
(lanes 3-5). Proteins
were immunoprecipitated (IP) with
SH2-B
and resolved
by SDS-PAGE. Proteins were visualized by immunoblotting (IB)
with
PY (upper panel) or
SH2-B
(lower
panel).
is
phosphorylated by JAK2 in vivo, and to confirm that Tyr-439
in SH2-B
is phosphorylated by JAK2 in vivo, we used
[32P]orthophosphate to label SH2-B
in vivo
and subjected 32P-labeled SH2-B
to phosphopeptide
mapping. To maximize the amount of 32P-labeled SH2-B
recovered, cells were treated prior to lysis with the phosphatase
inhibitor pervanadate for 6 min (Fig. 4, A-C) or as long as 30 min (Fig. 4, D and
E). We anticipated that SH2-B
might be phosphorylated on
serines and threonines as well as tyrosines in vivo (1).
Therefore, the first dimension (thin layer electrophoresis) was run at
pH 3.5 rather than pH 8.9 as seen in Fig. 2, because we predicted that
pH 3.5 would provide greater resolution of multiply phosphorylated
peptides from SH2-B
. The map of 32P-labeled SH2-B
expressed in the absence of JAK2 contained two detectable
phosphopeptides after a 12-h exposure (Fig. 4A, Spots a and b). A longer exposure (21 h) of this map reveals
one additional peptide (Fig. 4B, Spot c). When
SH2-B
was co-expressed with JAK2, the map of SH2-B
contained at
least two additional phosphopeptides (Fig. 4, C and
D, Spots 1 and 2). Migration of
Spots 1 and 2 is similar to migration of peptides
containing pY439 and pY494, respectively, observed in maps of SH2-B
phosphorylated in vitro. To confirm that Spots 1 and 2 comigrated with peptides containing pY439 and pY494
labeled in vitro, the tryptic peptides from in
vivo labeled SH2-B
were mixed with the tryptic peptides from
in vitro labeled SH2-B
and run on the same thin layer
chromatography plate. Peptides 1 and 2 from in vitro labeled
SH2-B
comigrated (Fig. 4E) with Spots 1 and
2 in the in vivo map of 32P-labeled
SH2-B
. These data indicate that Tyr-439 and Tyr-494 in SH2-B
are
the primary tyrosines within SH2-B
phosphorylated by JAK2 in
vivo. We next asked if SH2-B
is phosphorylated on any other
tyrosine residues in vivo. Phosphoamino acid analysis revealed that Spots a, b, and c
contained 32P exclusively in one or more phosphorylated
serines (Fig. 5). These data are
consistent with earlier reports using 3T3-F442A cells, which indicated
that SH2-B
is Ser/Thr-phosphorylated in the absence of ligand
stimulation (1). Alternatively, Spots a, b, and
c may represent peptides from one or more other proteins that co-precipitates and co-migrates with SH2-B
. Taken together, Figs. 1-5 demonstrate that overexpressed, constitutively active JAK2
phosphorylates SH2-B
on Tyr-439 and Tyr-494 both in vivo and in vitro.
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Fig. 4.
JAK2 phosphorylates Tyr-439 and Tyr-494 in
SH2-B in vivo. Plasmid (2 µg) encoding SH2-B
was transfected into 293T cells with control
plasmid (2 µg) (A and B) or with plasmid (2 µg) encoding JAK2 (C and D). Twenty-four hours
after transfection, cells were incubated with 1 mCi of
[32P]orthophosphate for 4 h. Cells were treated with
pervanadate for 6 min (A-C) or 30 min (D) and
lysed, and SH2-B
was immunoprecipitated with
SH2-B
. Proteins
were separated by SDS-PAGE, transferred to nitrocellulose, and digested
with trypsin. For panel E, tryptic digests used in
panel D were mixed with tryptic digests from in
vitro labeled SH2-B
co-expressed with JAK2. Samples were
subjected to two-dimensional phosphopeptide mapping at pH 3.5. Peptides
in panel C were separated in a different batch of thin layer
chromatography buffer from peptides in panels A,
B, D, and E resulting in slightly
different migration of the peptides. Peptides were visualized by
autoradiography. A and B are the same map exposed
for 12 versus 21 h as indicated. Dotted
circles in panels A and B indicate the
expected migration of Spots 1 and 2 based upon
their migration in in vitro maps.
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Fig. 5.
SH2-B is
phosphorylated on serines in vivo. Phosphorylated
peptides a, b, and c scraped from the
two-dimensional map of SH2-B
in vivo (Fig. 4D)
were eluted and subjected to acid hydrolysis. Amino acids were
separated by thin layer electrophoresis and visualized by
autoradiography. Migration of phosphoserine (pSer),
phosphothreonine (pThr), and phosphotyrosine
(pTyr) standards is indicated.
Is Not Required for the Ability
of SH2-B
to Activate JAK2--
We next asked if tyrosyl
phosphorylation of SH2-B
by JAK2 is required for the ability of
SH2-B
to increase the activity of JAK2. We have shown previously
that overexpression of SH2-B
increases the kinase activity of JAK2
up to 20-fold (2), establishing SH2-B family members as the only known
cytoplasmic activators of JAK2. Consistent with the ability of SH2-B
to activate JAK2, SH2-B
also increases the tyrosyl phosphorylation
of JAK2 and its downstream target, STAT5B, in response to GH, a potent
stimulator of JAK2. To determine if phosphorylation of SH2-B
by JAK2
is required for the ability of SH2-B
to activate JAK2, JAK2 was expressed in 293T cells alone or co-expressed with wild type or mutant
forms of SH2-B
lacking Tyr-439 and/or Tyr-494, or all nine
tyrosines. Proteins from cell lysates were Western-blotted with
PY.
When JAK2 was expressed alone, JAK2 as well as other cellular proteins
were tyrosyl-phosphorylated (Fig. 6,
lane 1). When SH2-B
was co-expressed with JAK2, the
phosphorylation of JAK2 and other cellular proteins was increased
compared with when JAK2 was expressed alone, indicating that the
activity of JAK2 was increased (Fig. 6, lane 1 versus lane 2). The phosphorylation of JAK2 and
other cellular proteins was similarly increased when JAK2 was
co-expressed with SH2-B
(Y439F), SH2-B
(Y494F),
SH2-B
(Y439F,Y494F), or SH2-B
(9YF) (Fig. 6, lane 1 versus lanes 3-6). The phosphorylation of JAK2
was slightly greater when JAK2 was co-expressed with SH2-B
(Y494F) or
SH2-B
(9YF) (Fig. 6, lanes 4 and 6,
respectively) compared with wild type SH2-B
(Fig. 6, lane
2). This difference was not seen in other experiments and most
likely reflects higher levels of JAK2 in those samples (Fig. 6,
bottom panel). Taken together, these data indicate that
tyrosyl phosphorylation of SH2-B
does not affect the ability of
SH2-B
to increase JAK2 activity.
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Fig. 6.
Tyrosyl phosphorylation of
SH2-B is not required for the ability of
SH2-B
to activate JAK2. Plasmid (0.25 µg) encoding JAK2 was transfected into 293T cells with control
plasmid (1 µg, lane 1), or plasmid (1 µg) encoding wild
type SH2-B
(lane 2), or the indicated mutant form of
SH2-B
(lanes 3-6). Cells were lysed, and proteins were
resolved by SDS-PAGE. Proteins were visualized by immunoblotting
(IB) with
PY (upper panel) or
JAK2
(bottom panel). Molecular weights are indicated as well as
the migration of JAK2 and SH2-B
(top panel).
(Y439F,Y494F) Inhibits GH-induced Membrane
Ruffling--
GH, a potent activator of JAK2, stimulates actin
reorganization and membrane ruffling (15, 16). Membrane ruffles are
found on the leading edge of motile cells and are thought to be
required for cell motility (26). SH2-B
has been shown to be required for maximal GH-induced membrane ruffling and cell motility (15, 16). To
determine if Tyr-439 and Tyr-494 in SH2-B
are required for
GH-stimulated membrane ruffling, 3T3-F442A murine fibroblasts were
transfected with cDNA encoding GFP-SH2-B
,
GFP-SH2-B
(Y439F,Y494F), or GFP as a control. Cells were deprived of
serum overnight and treated with vehicle or 50 ng/ml of GH for 15 min.
Filamentous actin was stained with Texas Red-phalloidin, and the number
of ruffles per cell was counted. In the absence of GH, most of the cells had either no ruffles or one ruffle (Fig.
7, A, E, and
I). Overexpression of GFP alone, GFP-SH2-B
, or
GFP-SH2-B
(Y439F,Y494F) had no effect on the number of ruffles in
these unstimulated cells. Addition of GH stimulated ruffling of cells
(Fig. 7, C, G, and I) as shown
previously (15). The average number of ruffles per cell (~2) was
similar in untransfected cells versus cells overexpressing GFP or GFP-SH2-B
. These data are consistent with earlier results indicating that at 50 ng/ml GH, ruffling is maximal and overexpression of GFP-SH2-B
fails to increase ruffling further (15). In contrast to
these data, overexpression of GFP-SH2-B
(Y439F,Y494F) blocked GH-induced cell ruffling (Fig. 7, E-H and I).
Thus, GFP-SH2-B
(Y439,494) acts as a dominant negative suggesting
that Tyr-439 and/or Tyr-494 within SH2-B
are required for GH-induced
membrane ruffling.
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Fig. 7.
SH2-B (Y439F,Y494F)
inhibits GH-induced membrane ruffling. Plasmid (2.5 µg) encoding
GFP-SH2-B
(A-D) or GFP-SH2-B
(Y439F,Y494F)
(E-H) was transfected in 3T3-F442A cells. Thirty-six hours
after transfection, cells were deprived of serum overnight and
stimulated with vehicle (A and B, and
E and F) or 50 ng/ml GH (C and
D, and G and H) for 15 min. Cells were
fixed, permeabilized, stained for F-actin (phalloidin-Texas Red), and
imaged. Images of Texas Red (Actin) and GFP (GFP)
fluorescence for the same field are shown. Arrows indicate
representative ruffles. Scale bar, 20 µm. I,
cells positive for GFP-SH2-B
were scored for the presence of ruffles
for each experimental condition. Bars represent mean ± S.E. ruffles per cell. *, p < 0.001 compared with
cells expressing GFP with the same treatment. n = 30 (
), 90 (GFP), 84 (SH2-B
), 61 (Y439F,Y494F), 37 (
, +GH), 119 (GFP, +GH), 100 (SH2-B-
, +GH), and 93 (Y439F,Y494F, +GH).
--
The
different members of the Janus family of tyrosine kinases have
differing substrate specificities. We have shown that JAK2 and JAK1 but
not JAK3 phosphorylate SH2-B
(3). Because differing sites of
phosphorylation have the potential to activate different signaling
pathways, we asked whether JAK1 and JAK2 phosphorylate the same or
different tyrosines in SH2-B
. Wild type or mutant forms of SH2-B
were co-expressed with JAK1, or JAK2 as a comparison, in 293T cells.
SH2-B
was immunoprecipitated with
SH2-B
and Western-blotted
with
PY to detect in vivo phosphorylation of SH2-B
. As
reported previously (3), SH2-B
is tyrosyl-phosphorylated in the
presence of JAK1 (Fig. 8, lane
1). When compared with wild type SH2-B
, SH2-B
(Y439F)
migrated faster and exhibited reduced tyrosyl phosphorylation (Fig. 8,
lane 1 versus lane 2), consistent with
decreased phosphorylation of SH2-B
(Y439F). Tyrosyl phosphorylation of SH2-B
(Y494F) was only modestly reduced compared with that of wild
type SH2-B
(Fig. 8, lane 1 versus lane
3). However, SH2-B
(Y439F,Y494F) was clearly phosphorylated less
than either SH2-B
(Y439F) or SH2-B
(Y494F) (Fig. 8, lane
4 versus lanes 2 and 3). These
data suggest that both Tyr-439 and Tyr-494 in SH2-B
are
phosphorylated by JAK1.
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Fig. 8.
JAK1 phosphorylates Tyr-439 and Tyr-494 in
SH2-B . Plasmid encoding JAK1 (2 µg,
lanes 1-5) or JAK2 (0.5 µg, lanes 6-8) was
transfected into 293T cells with plasmid (1 µg) encoding wild type
SH2-B
(lanes 1 and 6), or the indicated mutant
form of SH2-B
(lanes 2-5 and 7-8). Proteins
were immunoprecipitated (IP) with
SH2-B
and resolved
by SDS-PAGE. Proteins were visualized by immunoblotting (IB)
with
PY (upper panel) or
SH2-B
(lower
panel).
are phosphorylated
by JAK1, all nine tyrosines in SH2-B
were mutated to phenylalanine.
SH2-B
(9YF) was co-expressed in 293T cells with JAK1, or for
comparison, JAK2. The small amount of phosphorylation associated with
SH2-B
(Y439F,Y494F) was no longer detected when SH2-B
(9YF) was
co-expressed with JAK1 (Fig. 8, lane 5 versus lane 4) suggesting that JAK1 may phosphorylate one or more
tyrosines in SH2-B
in addition to Tyr-439 and Tyr-494. However, when
SH2-B
(9YF) was co-expressed with JAK2, tyrosyl phosphorylation of
SH2-B
(9YF) was similarly reduced (when the
PY signal was
normalized to amount of expressed SH2-B
) (Fig. 8, lane 8 versus lane 7). The inability to detect
phosphorylated tyrosines other than Tyr-439 and Tyr-494 in the more
definitive two-dimensional phosphopeptide maps of SH2-B
from cells
co-expressing JAK2 (Figs. 2 and 4) suggest that the reduced amount of
PY binding to the region corresponding to SH2-B
when all 9 tyrosines are mutated may reflect low level phosphorylation of
tyrosines other than Tyr-439 and Tyr-494. It is also useful to note
that when SH2-B
(9YF) is co-expressed with JAK2, a band that
co-migrates with SH2-B
is detected by immunoblotting with
PY
(Fig. 8, lane 8). Given that all 9 tyrosines are mutated in
the overexpressed SH2-B
(9YF), it is unlikely that this
tyrosyl-phosphorylated protein is overexpressed SH2-B
(9YF). This
band may be endogenous SH2-B
that is expressed at very low levels in
293T cells. Alternatively, this band may represent a comigrating
protein that is tyrosyl-phosphorylated by JAK2.
Is Phosphorylated on Tyr-439 by PDGFR--
In addition to
JAK1 and JAK2, other tyrosine kinases have been reported to
tyrosyl-phosphorylate SH2-B
, including the receptor tyrosine kinase,
PDGFR (6, 7). We next sought to determine whether PDGFR phosphorylates
Tyr-439 and/or Tyr-494 in SH2-B
. SH2-B
was expressed alone or
with PDGFR (
subunit) in COS-7 cells. Cells were deprived of serum
overnight and stimulated with 25 ng/ml PDGF BB for 15 min. Proteins
from cell lysates were Western-blotted with
PY. In the absence of
overexpressed PDGFR, minimal tyrosyl phosphorylation of proteins was
detected in either the absence of presence of PDGF (Fig.
9A, lanes 1 and
2). After transfection of as little as 0.1 µg of cDNA
encoding PDGFR, a slight increase in the phosphorylation of cellular
proteins was detected even in the absence of PDGF (Fig. 9A,
lanes 3 and 4). In particular, a
ligand-independent band that co-migrates with PDGFR is visible when
Western-blotted with
PY, suggesting that PDGFR, like JAK1 and JAK2,
is autophosphorylated and constitutively active when overexpressed.
These data are consistent with other studies in which PDGFR is
overexpressed (2, 27). Stimulation with PDGF failed to increase further
the phosphorylation of cellular proteins or PDGFR (Fig. 9A,
lane 4). When 2.0 µg of PDGFR cDNA was transfected, the phosphorylation of PDGFR and cellular proteins was significantly increased (Fig. 9A, lanes 5 and 6). In
particular, a band that co-migrates with SH2-B
is detected with
PY even in the absence of PDGF stimulation, suggesting that SH2-B
is phosphorylated by the constitutively active PDGFR (Fig.
9A, lanes 5 versus 6).
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Fig. 9.
PDGFR phosphorylates Tyr-439
SH2-B . A, plasmid (1 µg,
lanes 1-6) encoding wild type SH2-B
was transfected into
COS-7 cells with control plasmid (2 µg, lanes 1 and
2) or plasmid encoding PDGFRB (2 µg, lanes
3-6). Twenty-four hours after transfection, cells were deprived
of serum overnight and stimulated with 25 ng/ml PDGF-BB (lanes
2, 4, and 6) or vehicle (lanes 1,
3, and 5) for 15 min. Proteins were resolved by
SDS-PAGE and visualized by immunoblotting (IB) with
PY.
Molecular weights are indicated as well as the migration of PDGFR and
SH2-B
. B, plasmid (1 µg, lanes 1-4)
encoding wild type SH2-B
was transfected into COS-7 cells with
control plasmid (2 µg, lanes 1 and 2) or
plasmid encoding PDGFRB (2 µg, lanes 3 and 4).
Twenty-four hours after transfection, cells were deprived of serum
overnight and stimulated with 25 ng/ml PDGF-BB (lanes 2 and
4) or vehicle (lanes 1 and 3) for 15 min. Proteins were immunoprecipitated (IP) with
SH2-B
and resolved by SDS-PAGE. Proteins were visualized by immunoblotting
(IB) with
PY (upper panel) or
SH2-B
(lower panel). C, plasmid (lanes 1-5)
encoding PDGFR (2 µg) or JAK2 (0.5 µg, lanes 6-8) was
transfected into COS-7 cells with plasmid (1 µg) encoding wild type
SH2-B
(lanes 1 and 6) or the indicated mutant
form of SH2-B
(lanes 2-5 and 7-8).
Twenty-four hours after transfection, cells were deprived of serum
overnight. Proteins were immunoprecipitated (IP) with
SH2-B
and resolved by SDS-PAGE. Proteins were visualized by
immunoblotting (IB) with
PY (upper panel) or
SH2-B
(lower panel).
by PDGFR,
SH2-B
was immunoprecipitated with
SH2-B
and Western blotted with
PY. In the absence of PDGFR, SH2-B
was not detectably
tyrosyl-phosphorylated whether PDGF was present or not (Fig.
9B, lanes 1 and 2). When SH2-B
was
co-expressed with PDGFR, SH2-B
was tyrosyl-phosphorylated in the
absence of PDGF (Fig. 9B, lane 3). Tyrosyl
phosphorylation of SH2-B
was detected even when as little as 0.1 µg of PDGFR cDNA was transfected into the cells (data not shown).
Stimulation with PDGF modestly increased the tyrosyl phosphorylation of
SH2-B
(Fig. 9B, lanes 3 versus
4).
, PDGFR was co-expressed with wild type or mutant forms of
SH2-B
lacking these tyrosines in COS-7 cells (Fig. 9C,
lanes 1-5). Cells were deprived of serum overnight and
SH2-B
was immunoprecipitated with
SH2-B
. When PDGFR was
co-expressed with SH2-B
, SH2-B
was tyrosyl-phosphorylated (Fig.
9C, lane 1). As seen with overexpression of JAK1
or JAK2, tyrosyl phosphorylation of SH2-B
(Y439F) was significantly
reduced compared with that of wild type SH2-B
(Fig. 9C,
lane 1 versus lane 2). The tyrosyl
phosphorylation of SH2-B
(Y494F) was only marginally reduced compared
with wild type SH2-B
(Fig. 9C, lane 3), and
the tyrosyl phosphorylation of SH2-B
(Y439F,Y494F) was not detectably
different from that of SH2-B
(Y439F) (Fig. 9C, lane
4 versus lane 2). No tyrosyl phosphorylation
of SH2-B
(9YF) was detectable with PDGF (Fig. 9C,
lane 5), or in the corresponding experiment using JAK2 (Fig.
9C, lane 8). Combined, these data indicate that,
although PDGFR may also phosphorylate Tyr-494 or other tyrosine(s) in
SH2-B
, the primary site of phosphorylation in SH2-B
by PDGFR is
Tyr-439.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
We have
demonstrated previously that SH2-B
is tyrosyl-phosphorylated by
constitutively active, overexpressed JAK2, as well as by endogenous,
GH-stimulated JAK2 (1, 3, 24). In the current study, we have identified
Tyr-439 and Tyr-494 in SH2-B
as targets of JAK2 both in
vitro and in vivo. This conclusion is based primarily
on two-dimensional phosphopeptide mapping of SH2-B
labeled in
vitro and two-dimensional phosphopeptide mapping of SH2-B
labeled in vivo. Phosphopeptide maps of SH2-B
overexpressed with JAK2 in 293T cells and phosphorylated by
co-precipitating JAK2 in an in vitro kinase assay show that
SH2-B
is predominantly phosphorylated by JAK2 on two peptides. These
peptides are also phosphorylated by JAK2 when SH2-B
and JAK2 are
co-expressed in COS-7 cells. Phosphoamino acid analysis confirmed that
these two peptides were phosphorylated exclusively on tyrosines.
Mutation of Tyr-439 to phenylalanine eliminated one of these spots in
phosphopeptide maps of SH2-B
and mutation of Tyr-494 eliminated the
other spot. Mutation of any of the remaining tyrosines did not affect
the phosphopeptide map. The simplest explanation for these results is
that wild type SH2-B
is phosphorylated on Tyr-439 and Tyr-494.
labeled in vitro show that each of the maps contained at
least five other spots. If any of these spots were peptides from
SH2-B
containing phosphotyrosine, we would expect the spots to
either disappear or shift when tyrosines in SH2-B
are mutated to
phenylalanine. However, none of these spots were reproducibly altered
by mutations of tyrosines in SH2-B
indicating that these spots do
not contain phosphotyrosine(s) from SH2-B
. Digestion of SH2-B
with protein phosphatase 2A, a serine/threonine phosphatase, reduces
the broadly migrating SH2-B
to a tighter migrating band indicating
that SH2-B
is highly phosphorylated on serines and threonines in
293T cells as well as in 3T3-F442A and PC12 cells (1, 7, 28). Thus, one
possible explanation would be that some or all of the other spots
correspond to peptides containing 32P-labeled phosphoserine
or phosphothreonine. However, phosphoamino acid analysis of these spots
indicated that they contain phosphotyrosine and, to a lesser degree,
phosphoserine and phosphothreonine (data not shown). Thus, these spots
are likely to be peptides from other protein(s) that co-migrate with
SH2-B
.
labeled in vivo indicate that both Tyr-439
and Tyr-494 in SH2-B
are phosphorylated by JAK2, whereas results obtained from examining the phosphorylation of SH2-B
(Y494) with
PY were variable. The tyrosyl phosphorylation of SH2-B
(Y494) was
less than that of wild type SH2-B
, and the phosphorylation of
SH2-B
(Y439F,Y494F) was decreased compared with that of
SH2-B
(Y439F) in 293T cells overexpressing JAK2 but not reproducibly
so in COS-7 cells. This variability in detection of pY494 may reflect
its relatively lower amount of phosphorylation compared with Tyr-439 observed in the maps. JAK2 may have a lower affinity for Tyr-494 than
for Tyr-439, or the phosphorylation of Tyr-494 may be more transient
than that of Tyr-439, perhaps indicating that Tyr-494 is targeted by a
phosphatase. Alternatively, pY494 may be poorly recognized by
PY.
Anti-phosphotyrosine antibodies have been observed to exhibit
variability in their ability to detect phosphotyrosines residing in
different amino acid environments (29). For example, Tyr-343 in the
cytoplasmic domain of the receptor for erythropoietin has been reported
to be phosphorylated but not recognized by
PY (30, 31). Thus, for
whatever reason, the phosphorylation at Tyr-494 in SH2-B
, which is
readily detectable in two-dimensional peptide maps from in
vivo and in vitro labeled SH2-B
, is difficult to
discern on Western blots using 4G10
PY.
Is Phosphorylated on Serine(s) in Vivo--
Phosphoamino
acid analysis of peptides from SH2-B
labeled in vivo
support the hypothesis that SH2-B
is phosphorylated on serines in
addition to being phosphorylated on tyrosines. SH2-B
contains 82 serines. Many of these serines lie within consensus target sequences
for ERKs 1 and 2, protein kinase C, and other Ser/Thr kinases. NGF
stimulates the Ser/Thr phosphorylation of SH2-B
in PC12 cells. An
inhibitor of MEK, the kinase that phosphorylates ERKs 1/2, prevents
most of the NGF-induced Ser/Thr phosphorylation of SH2-B
consistent
with Ser/Thr kinases downstream of MEK phosphorylating SH2-B
.
SH2-B
is phosphorylated on Ser-96 by ERKs 1 and 2 in vitro. However, mutation of Ser-96 to alanine failed to abolish Ser/Thr phosphorylation of SH2-B
in response to NGF. Activation of
PKC by PMA also stimulates Ser/Thr phosphorylation of SH2-B
in PC12
cells. However, down-regulation of PKC by prolonged treatment with PMA
did not affect NGF-promoted phosphorylation of SH2-B
on Ser/Thr,
suggesting that activation of PKC is not required for NGF-induced
Ser/Thr phosphorylation of SH2-B
(28). These data suggest that
SH2-B
is phosphorylated on more than one Ser/Thr, consistent with
our observation here of more than one SH2-B
peptide containing
32P-ser. Further investigation will be required to
determine whether ERKs 1 and 2, PKC, or other Ser/Thr kinases
phosphorylate SH2-B
constitutively or in response to activation of
JAK2 as well as to identify which serine(s) in SH2-B
is(are) phosphorylated.
Is Phosphorylated on Tyr-439 by JAK1 and PDGFR--
We
examined the tyrosyl phosphorylation of SH2-B
by another Janus
family kinase, JAK1, as well as by a receptor tyrosine kinase, PDGFR,
to see whether the different kinases phosphorylated the same or
different tyrosines within SH2-B
as JAK2. One can envision that
different tyrosines in SH2-B
phosphorylated by JAK2 or JAK1
or PDGFR could form binding sites for different proteins and lead to
the activation of signaling pathways specific to PDGF or JAK1 or JAK2.
Western blotting of mutant SH2-B
with
PY suggests that the
primary site in SH2-B
phosphorylated by JAK1 and PDGFR in
vivo is Tyr-439 as it is for JAK2. As previously discussed, phosphorylation at Tyr-494 is not easy to detect with
PY, however, Tyr-494 is also likely to be phosphorylated by JAK1, because
phosphorylation of SH2-B
(Y439F,Y494F) was decreased relative to
SH2-B
(Y439F). Similar results were not obtained with PDGFR,
suggesting that Tyr-494 may not be as good a target of PDGFR. Other
tyrosines may also be phosphorylated by JAK2, JAK1, and PDGFR based on
the further decrease in the ability of
PY to recognize a protein corresponding in migration to SH2-B
when these tyrosine kinases are
co-expressed with SH2-B
lacking all nine of its tyrosines. At least
in the case of JAK2, such phosphorylation is not likely to be
substantial, because it was not observed in the phosphopeptide maps.
--
Presumably, tyrosyl phosphorylation of SH2-B
on
Tyr-439 and Tyr-494 serves some function. We have shown that SH2-B
increases the kinase activity of JAK2 thereby increasing the
autophosphorylation of JAK2 as well as the phosphorylation of targets
of JAK2 such as STAT5B (2). Here we demonstrate that tyrosyl
phosphorylation of SH2-B
is not required to activate JAK2. SH2-B
with individual tyrosines mutated or all nine tyrosines mutated to
phenylalanine increase the phosphorylation of JAK2 to levels similar to
those seen when JAK2 is co-expressed with wild type SH2-B
. These
data are consistent with previous results suggesting that the C
terminus of SH2-B
(amino acids 504-670), which lacks Tyr-439 and
Tyr-494, is necessary and sufficient for SH2-B
to increase the
activity of JAK2 (24).
increases
GH-stimulated membrane ruffling. Overexpression of SH2-B
lacking an
arginine in the SH2 domain critical for binding to phosphotyrosines
(SH2-B
(R555E)) inhibited membrane ruffling. Further investigation
revealed that the second proline-rich region in the N terminus of
SH2-B
(Fig. 1, amino acids 85-106) is required for binding of
SH2-B
to the small GTPase, Rac, and for cell motility. These
data suggest that multiple domains in SH2-B
are required for the
role of SH2-B
in membrane ruffling. Here, we demonstrate that
Tyr-439 and/or Tyr-494 in SH2-B
are also required for
GH-in-duced membrane ruffling. Expression of
SH2-B
(Y439F,Y494F) in 3T3-F442A cells inhibits the
ruffling normally observed in response to GH but not basal ruffling
observed in the absence of GH. Mutating Tyr-439 and Tyr-494 to
phenylalanine had no effect upon the ability of SH2-B
to activate
JAK2, thus the dominant negative effect of SH2-B
(Y439F,Y494F)
suggests that GH-dependent phosphorylation of SH2-B
at
either Tyr-439 and/or Tyr-494 forms binding site(s) for one or more
signaling molecules that play a critical role in the formation of cell ruffles.
, immunoprecipitated SH2-B
, and Western-blotted against
hemagglutinin. Our preliminary data suggest that CrkII does not
associate with SH2-B
. Further studies will be required to identify
other cytoskeletal proteins that may bind pY439 and/or pY494 in
SH2-B
.
is the protein-tyrosine phosphatase, SHP-2. SHP-2 has
not yet been implicated in the regulation of the actin cytoskeleton; however, in response to GH, SHP-2 has been found to associate with GH
receptor and signal-regulatory protein-1
. SHP-2 requires pY449 and
pY473 in SIRP for association. These two tyrosines are found within the
sequence of YXXL (Tyr-449) and the related sequence of
YXXV (Tyr-473) (36). Similarly, SHP-2 requires pY487 and pY595 in GH receptor for maximal association with GH receptor. Both of
these tyrosines are found within the similar sequence of
YXXV (37). To determine if SHP-2 also associates with
SH2-B
, SH2-B
was co-expressed with JAK2, SH2-B
was
immunoprecipitated with
SH2-B
, and immunoprecipitated proteins
were Western-blotted with
SHP-2. This approach did not detect
association of SHP-2 with SH2-B
. Because Tyr-439 and Tyr-494 are
both within the consensus sequence of YXXL, one can envision
that they bind proteins that recognize ITAMs (immunoreceptor
tyrosine-based activation motif). However, the traditional definition
of an ITAM is two YXXL sequences separated by 8-10 amino
acids (38). Tyr-439 and Tyr-494 are separated by too many amino acids
to form a traditional ITAM binding site. The three-dimensional
structure may place Tyr-439 and Tyr-494 within a close enough proximity
to form an ITAM. Further studies will be needed to identify proteins
that associate with pY439 and/or pY494 in SH2-B
.
are phosphorylated by
JAK2 in vivo and in vitro. Further, Tyr-439 and
Tyr-494 in SH2-B
are shown to play a significant role in the
regulation of GH-dependent membrane ruffling. Finally, we
demonstrate that JAK1 and PDGFR also phosphorylate Tyr-439. Therefore,
Tyr-439 in SH2-B
may serve as a binding site for a similar set of
signal proteins following stimulation by PDGF as well as by cytokines that activate JAK1 and/or JAK2.
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ACKNOWLEDGEMENTS |
---|
We thank Xiaqing Wang for support and technical assistance with the experiments, Dr. Linyi Chen for assistance with cloning and support, and B. Hawkins for assistance with the manuscript.
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FOOTNOTES |
---|
* This research was supported in part by National Institutes of Health (NIH) Grant RO1-DK54222 and by the Juvenile Diabetes Research Foundation Center for the Study of Complications of Diabetes. DNA sequencing was carried out with the support of the Cellular and Molecular Biology Core of the Michigan Diabetes Research and Training Center (NIH Grant P60-DK20572) and the University of Michigan Comprehensive Cancer Center (NIH Grant P30-CA46592).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a predoctoral fellowship from the Cancer Biology
Training Program (Nancy Newton Loeb Fund) of the University of Michigan
Comprehensive Cancer Center and a student in the Cellular and Molecular
Biology Graduate Program at the University of Michigan.
§ To whom correspondence should be addressed: Dept. of Physiology, The University of Michigan Medical School, Ann Arbor, MI 48109-0622. Tel.: 734-763-2561; Fax: 734-647-9523; E-mail: cartersu@umich.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210765200
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ABBREVIATIONS |
---|
The abbreviations used are:
SH2, Src homology 2;
JAK, Janus tyrosine kinase;
PH, pleckstrin homology;
GH, growth
hormone;
PDGF, platelet-derived growth factor;
PDGFR, platelet-derived
growth factor receptor;
NGF, nerve growth factor;
PY, anti-phosphotyrosine antibody;
JAK, anti-JAK antibody;
SH2-B
, anti-SH2-B
antibody;
SH, Src homology;
MEK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase;
ERK, extracellular signal-regulated kinase;
PC12 cells, rat adrenal
pheochromocytoma cell line;
STAT5, signal transducers and activators of
transcription 5;
GFP, green fluorescent protein;
DMEM, Dulbecco's
modified Eagle's medium;
PBS, phosphate-buffered saline;
PKC, protein
kinase C;
ITAM, immunoreceptor tyrosine-based activation motif.
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