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Institut National de la Santé et de la Recherche Médicale U 145, Faculté de Médecine, 06107 Nice Cédex 2, France
Address all correspondence and requests for reprints to: Emmanuel Van Obberghen, Institut National de la Santé et de la Recherche Médicale U 145, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cédex 2, France. E-mail: vanobberg{at}unice.fr
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Abstract |
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Taking our data together, we conclude that: 1) insulin and IGF-1 lead to phosphorylation and activation of JAK-1 and JAK-2 in intact cells; 2) phosphorylation of IRS-I by JAK-1 seems to occur on sites different from those phosphorylated by the insulin receptor; 3) JAK-1 interacts directly with phosphorylated insulin and IGF-1 receptors; and 4) the JH7-JH6 and JH1 domains of JAK-1 are responsible for the interaction with insulin and IGF-1 receptors.
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Introduction |
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-subunits and two ß-subunits (1, 2, 3, 4).
These receptors possess tyrosine kinase activity in the cytoplasmic
portion of their ß-subunit. After binding of their respective
ligands, the activated receptors autophosphorylate and phosphorylate
several proteins on tyrosine. Endogenous substrates of both receptors
include insulin receptor substrate (IRS)-1 and IRS-2 (5, 6, 7, 8, 9), and SRC
homology/collagen (SHC) (10, 11). Tyrosine-phosphorylated IRS-1 and
IRS-2 act as docking proteins by binding SH2 domain-containing
proteins, such as growth factor receptor-bound protein 2 (Grb2), the
p85 subunit of phosphatidylinositol 3'-kinase, and SH2-containing
protein tyrosine phosphatase-2. Similarly, phosphorylated SHC binds to
the SH2 domain of the Grb2-Son Of Sevenless complex. This Grb2-Son Of
Sevenless complex recruited by SHC or IRS proteins leads to activation
of Ras-dependent signaling pathways. The Janus kinases (JAKs) are cytoplasmic tyrosine kinases, which were first implicated in cytokine-stimulated signal pathways. Four members have been identified so far: JAK-1, JAK-2, JAK-3, and TYK-2 (12, 13, 14, 15, 16, 17). JAK-1, JAK-2, and TYK-2 are ubiquitous (12, 17, 18, 19), whereas JAK-3 is expressed only in T lymphocytes (15, 16).
In searching for novel pathways activated by insulin and IGF-1
receptors, we previously have found that insulin induces tyrosine
phosphorylation of JAK-1 in cells overexpressing insulin receptors
(20). More recently, phosphorylation of JAK-2 was observed in
insulin-sensitive tissues from hormone-treated rats (21). Other
tyrosine kinase receptors have been shown to activate the JAK/STAT
pathway. Indeed, colony-stimulating factor-1 (CSF-1) induces the
activation of TYK-2, JAK-1, and of STAT1, STAT3, and STAT5 (22, 23, 24, 25).
In fibroblasts, platelet-derived growth factor (PDGF) induces
phosphorylation of JAK-1, JAK-2, TYK-2, and of STAT1, STAT3, STAT5, and
STAT6 (23, 26, 27, 28), whereas JAK-1, STAT1 ( and ß), STAT3 ( and
ß), and STAT5 are activated in hepatocytes isolated from epidermal
growth factor-treated rats (29, 30, 31).
The cytokine receptors, which do not possess intrinsic tyrosine kinase
activity, stimulate the JAK/STAT pathway. Two domains on the
cytoplasmic part of the receptors are responsible for the interaction
with JAKs. The first domain is a proline-rich motif (box a) and the
second one is an acidic motif (box b) (32, 33). The association of JAKs
to cytokine receptors can be constitutive or enhanced by ligand binding
(34, 35). Dimerization of the receptors upon ligand binding induces
autophosphorylation and transphosphorylation of JAKs. Importantly,
activated JAKs phosphorylate the receptors, allowing the recruitment of
SH2 domain-containing signal transducers and activators of
transcription (STATs), which in turn, are phosphorylated by the JAKs.
Phosphorylated STATs form homodimeric or heterodimeric complexes and
translocate to the nucleus, where they activate transcription of
specific genes. Recently, it has been shown that JAKs activated by
cytokines such as interleukin (IL)2, IL4, IL7, IL9, IL15, leukemia
inhibitory factor, interferon and 
, and GH also can lead to
phosphorylation of IRS-1 and IRS-2 (36, 37, 38, 39, 40, 41, 42).
In the present study, we have compared the time courses of JAK-1 and JAK-2 phosphorylation in response to insulin and IGF-1. We have followed the activation of these JAKs by measurement of IRS-1 and IRS-2 phosphorylation in vitro. We also have compared the tryptic maps of IRS-1 phosphorylated by the insulin receptor vs. JAK-1. Finally, we have explored which JAK-1 region(s) is (are) required for interaction with insulin and IGF-1 receptors.
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Materials and Methods |
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Cell culture
NIH 3T3 cells, transfected with an expression plasmid encoding,
either the human IGF-1 receptor (2 x 105
receptors/cell) or the human insulin receptor (106
receptors/cell), were grown in DMEM supplemented with 10% (vol/vol)
FCS, 0.72 mM geneticin, and 10 mM
glutamine.
Human kidney cells, constitutively expressing EBNA-1 protein from Epstein-Barr virus (293-EBNA cells, Invitrogen, San Diego, CA), were grown in DMEM supplemented with 5% (vol/vol) FCS, 0.72 mM geneticin, and 10 mM glutamine.
Partial purification of receptors
IGF-1 and insulin receptors were partially purified by
chromatography on WGA (45). Briefly, confluent cells were serum
deprived overnight in DMEM containing 0.2% (wt/vol) BSA (10 dishes of
15-cm diameter). Cells were washed twice with PBS and solubilized for
90 min at 4 C in 50 mM HEPES, 150 mM NaCl, 1%
(vol/vol) Triton X-100, 10% (vol/vol) glycerol, 1 mM EGTA,
pH 7.6. The supernatant from an ultracentrifugation step (60 min,
100,000 x g, 4 C) was applied to a WGA column, and the
receptors were eluted in fractions of 500 µl with 0.3 M
N-acetyl-D-glucosamine in 50 mM
HEPES, 150 mM NaCl, 0.1% (vol/vol) Triton X-100, 10%
(vol/vol) glycerol, pH 7.6. Protease inhibitors were present throughout
the procedure (20 µM leupeptin, 1.25 mM
bacitracin, 100 U/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). The presence of IGF-1 or insulin
receptors in each fraction was tested by IGF-1 or insulin
induced-autophosphorylation followed by SDS-PAGE. Usually, fractions 2
and 3 were found to contain the receptors and were pooled.
Transfection of 293-EBNA cells.
The full-length rat IRS-1 complementary DNA (cDNA), obtained
from M. F. White (Boston, MA), was subcloned into pCEP-4
expression vector (Invitrogen) between XhoI and
NheI sites. The XbaI site was introduced 330 bp
before the stop codon of the IGF-1 cDNA fragment by PCR. The 
121
IGF-1 cDNA was subcloned into pcDNAneo expression vector (Invitrogen)
between XhoI and XbaI sites.
The human JAK-2 cDNA subcloned into pBluescript-SK was a gift from J. Ihle (Department of Biochemistry, St. Jude Children’s Research Hospital, Memphis, TN). Construction of kinase-dead JAK-2 was performed as described elsewhere (46). Site-directed mutagenesis of W1020G and Q1024A within the carboxyl-terminal protein-tyrosine kinase domain was accomplished by unique-site elimination (CLONTECH) using the following primer: 5'-ggaaagcccaatattcggtacgcacctgcatccttgac-3'. The kinase-dead JAK-2 cDNA was subcloned into pcDNA3 expression vector (Invitrogen) between ApaI and NotI sites.
Exponentially growing cells were trypsinized, seeded at 6 x
106 cells per 15 cm plate, and incubated overnight in 20 ml
growth medium. Ten to 20 µg plasmid DNA (IRS-1 or 121 IGF-1
receptor) was mixed with 1 ml of 0.25 M CaCl2
and 1 ml of (N, N'-bis 2 hydroxyethyl)-2 amino
ethanesulfonic acid and was incubated for 30 min at room temperature.
The calcium phosphate-DNA solution was added dropwise to the cells, and
the mixture was swirled gently and incubated overnight at 35 C under
3% CO2. The cells were then incubated with fresh growth
medium for 8 h before starvation in DMEM containing 0.2% (wt/vol)
BSA for 14 h (47, 48). The cells were cooled to 4 C, washed with
ice-cold PBS (140 mM NaCl, 3 mM KCl, 6
mM Na2HPO4, 1 mM
KH2PO4, pH 7.4), and lysed with 50
mM HEPES, 150 mM NaCl, 1% (vol/vol) Triton
X-100, 100 mM NaF, 0.5 mM phenylmethylsulfonyl
fluoride, 100 U/ml aprotinin (pH 7.6) for 30 min at 4 C. The
solubilizates were clarified by centrifugation at 15,000 x
g for 15 min at 4 C.
The 121 IGF-1 receptor was partially purified on WGA, as described
above. IRS-1 and kinase-dead JAK-2 were immunoprecipitated with
specific antibodies.
Fusion proteins
The human JAK-1 cDNA subcloned into pBluescript-KS was a gift
from J. Ihle. The SmaI site was introduced to each end of
the desired cDNA fragment by PCR to allow the in-frame insertion into
the pGEX-2T vector (Pharmacia Biotech Inc., Uppsala, Sweden). The
portion of mouse IRS-2 cDNA coding for the amino acids 591–786 was
subcloned into vector pGEX-3X (Pharmacia). The two constructs were
expressed into Escherichia coli BL21 bacteria (Stratagene,
La Jolla, CA). Expression of the recombinant protein was induced with
0.1 mM isopropyl-ß-D-thiogalactopyranoside
for 3 h. Bacteria were lysed with 20 mM Tris, 1
M NaCl, 0.2 mM EDTA, 0.2 mM EGTA, 1
mg/ml lysozyme, 0.5 mM phenyl-methylsulfonyl fluoride,
100 U/ml aprotinin, 20 mM leupeptine (pH 7.4) for 30 min in
ice, sonicated three times, and frozen in liquid nitrogen. Lysates
containing GST-IRS-2591–786 were submitted to centrifugation
(30,000 x g at 4 C), and the supernatant was incubated
with glutathione-sepharose (Pharmacia) for 1 h at 4 C. The fusion
proteins were eluted with 50 mM glutathione, and 100
mM HEPES (pH 8). Concerning the GST-JAK fusion proteins,
lysates were centrifuged (30,000 x g at 4 C), and the
pellets were treated with 8 M urea for 30 min at 4 C. After
another centrifugation (30,000 x g at 4 C), the
supernatants were dialyzed against 70 mM NaCl, 1.5
mM KCl, 3 mM
Na2P2O7, 0.5 mM
KH2PO4 (pH 7.4). Then, the lysates were
incubated with glutathione-sepharose (Pharmacia) for 1 h at 4 C.
The fusion proteins were eluted with 50 mM glutathione, 100
mM HEPES (pH 8).
Phosphorylation of JAKs in intact cells
Confluent cells growing in 145-mm culture dishes were starved in
DMEM containing 0.2% (wt/vol) BSA for 15 h before being treated
with insulin (10-7 M) or IGF-1
(10-7 M) for different times. Then the cells
were washed with ice-cold buffer containing 50 mM HEPES,
150 mM NaCl, 10 mM EDTA, 10 mM
Na2P2O7, 100 mM NaF,
and 2 mM vanadate (pH 7.4). The cells were solubilized for
30 min at 4 C in lysis buffer (50 mM HEPES, 150
mM NaCl, 10 mM EDTA, 10 mM
Na2P2O7, 100 mM NaF, 2
mM vanadate, 0.5 mM phenylmethylsulfonyl
fluoride, 100 U/ml aprotinin, 20 mM leupeptin, and 1%
(vol/vol) Triton X-100, pH 7.4). The supernatants from a centrifugation
step (15 min at 15,000 x g at 4 C) were incubated for
4 h at 4 C with antibodies preadsorbed on protein-A sepharose
(anti-JAK1, anti-JAK2, anti-TYK2 antibodies at 1 µg/sample; nonimmune
serum at 3 µg/sample). Pellets were washed with lysis buffer,
resuspended in Laemmli sample buffer, and separated by SDS/PAGE (49).
Proteins were transferred to a polyvinylidene fluoride membrane. The
membrane was blocked with saline buffer (10 mM Tris, 140
mM NaCl, pH 7.4) containing 5% (wt/vol) BSA for 2 h
at 22 C and incubated overnight with mouse monoclonal antibody to
phosphotyrosine (1 µg/ml). The membrane was washed three times with
saline buffer containing Tween-20. Rabbit antimouse antibody (1
µg/ml) was added for 60 min at 22 C, followed by several washes. The
membrane was then incubated with 125I-protein A (500,000
cpm/ml) for 60 min at 22 C. The membrane was washed and
autoradiographed. In some cases, the membrane was stripped for 30 min
at 50 C in 62 mM Tris, 100 mM
2-mercaptoethanol, and 2% (wt/vol) SDS, and reprobed with the
indicated antibodies.
Measurement of JAK activation
Cells overexpressing IGF-1 receptors were depleted for 15 h
before being incubated with 500 µM vanadate for 45 min.
The cells were then stimulated, or not, with IGF-1 (10-7
M) for 10 min. Clarified cell lysates were incubated for
4 h at 4 C with anti-JAKs antibodies (1 µg/sample) preadsorbed
on protein-A sepharose. The pellets were washed twice with 50
mM HEPES, 150 mM NaCl, 0.1% (vol/vol) Triton
X-100, (pH 7.5). Autophosphorylation was measured by addition of
phosphorylation buffer (10 mM HEPES, 50 mM
NaCl, 5 mM MnCl2, 5 mM
MgCl2, pH 7.5) containing varying ATP concentrations.
GST-IRS-2 phosphorylation was measured by addition of GST-IRS-2 (2
µg/sample) and phosphorylation buffer containing 60 µM
ATP for 5 min at room temperature. For IRS-1 immunopurification,
lysates of cells overexpressing IRS-1 were subjected to
immunoprecipitation with antibodies to IRS-1 (1/50 dilution)
preadsorbed on protein A-sepharose. After 3 h at 4 C, the pellets
were washed with 50 mM HEPES, 150 mM NaCl, 1%
(vol/vol) Triton X-100 (pH 7.4). The pellets containing IRS-1 were
mixed with pellets containing the immunopurified JAKs. Phosphorylation
buffer containing 60 µM ATP was added to each sample for
5 min at room temperature.
Reactions were stopped by addition of Laemmli sample buffer (49). The samples were analyzed by SDS-PAGE using a 7.5% resolving gel, followed by Western blot analysis with antibody to phosphotyrosine.
In vitro phosphorylation of kinase-dead JAK-2 by WGA-purified IGF-1
receptors
Antibodies to JAK-2 (1 µg/sample) were incubated with protein
A-sepharose for 1 h at 4 C. The pellets were washed twice with 50
mM HEPES, 150 mM NaCl, pH 7.6. Lysates from
transfected cells were incubated with the antibody to JAK-2 for 2
h. The JAK-2-containing pellets were washed twice with Hepes NaCl
Triton buffer containing 1% (vol/vol) Triton X-100. Antibodies to
IGF-1 receptor (1/50) were incubated with protein A-sepharose for
1 h at 4 C. The pellets were washed twice with 50 mM
HEPES, 150 mM NaCl, pH 7.6. Five microliters of
WGA-purified IGF-1 receptor were incubated with the antibody to IGF-1
receptor for 2 h. The IGF-1 receptor-containing pellets were
washed twice with Hepes NaCl Triton buffer containing 0.1% (vol/vol)
Triton X-100.
The pellets with kinase-dead JAK-2 were mixed with pellets containing
the immunopurified IGF-1 receptor. IGF-1 (10-7
M) was added for 30 min at 22 C. Phosphorylation was then
initiated by adding 60 µM [-32P] ATP
(2.5 Ci/mmol), 50 mM MgCl2, and the reaction
was stopped after 15 min by addition of Laemmli sample buffer (49). The
samples were analyzed by one-dimensional SDS-PAGE using a 7.5%
resolving gel.
Phosphopeptide map analysis of IRS-1
IRS-1 overexpressed in 293 EBNA-cells was isolated by a specific
antibody and phosphorylated in vitro either by insulin
receptors or by JAK-1 or JAK-2, as described above, for 2 h with
[-32P] ATP. After SDS-PAGE, 32P-labeled
IRS-1 was localized by autoradiography. The gel pieces corresponding to
the labeled bands were excised and incubated in 50 mM
NH4HCO3 (pH 8) for 2 h at 37 C.
CaCl2 at a final concentration of 1 mM and
trypsine at a final concentration of 50 µg/ml were added for 12
h at 37 C. For each sample, the eluted phosphopeptides were
lyophilized, washed in H2O, and dissolved in
NH3 (N/1000). Phosphopeptides were separated by
two-dimensional analysis on silice thin-layer plates, as described
(50). The plates were dried and subjected to autoradiography.
Coprecipitation of insulin and IGF-1 receptors with GST-JAKs
Precipitation of nonphosphorylated receptors. Five
microliters of WGA-purified IGF-1 or insulin receptors were incubated
with the fusion protein (60 pmol/sample) preadsorbed on
glutathione-sepharose, or with antibodies to receptor preadsorbed on
protein A-sepharose (1/500). After 4 h at 4 C, the pellets were
washed twice with 50 mM HEPES, 150 mM NaCl,
0.1% (vol/vol) Triton X-100, pH 7.4. The presence of IGF-1 or insulin
receptors was detected by ligand-induced autophosphorylation. Briefly,
pellets were incubated with IGF-1 (10-7 M) for
15 min at 22 C or insulin (10-7 M) for 60 min
at 22 C. Receptor phosphorylation was then initiated by adding 15
µM [-32P] ATP (2.5 Ci/mmol), 8
mM MgCl2, 4 mM MnCl2;
and the reaction was stopped after 30 min by addition of Laemmli
sample buffer.
Precipitation of ligand-occupied receptors. Five microliters of WGA-purified insulin receptors, preincubated with insulin (10-7 M) for 60 min at 22 C, were incubated either with the fusion protein (60 pmol/sample) preadsorbed on glutathione-sepharose, or with antibodies to receptor preadsorbed on protein A-sepharose (1/500). After 4 h at 4 C, the pellets were washed twice. Presence of insulin receptors was detected by ligand-induced autophosphorylation, as described above.
Precipitation of phosphorylated receptors. Five microliters
of WGA-purified wild-type or 121 IGF-1 receptors and insulin
receptors were incubated with IGF-1 (10-7 M)
for 15 min at 22 C or with insulin (10-7 M)
for 60 min at 22 C. Receptor phosphorylation was then initiated by
adding 15 µM ATP, 8 mM MgCl2, 4
mM MnCl2; and the reaction was stopped after 30
min by addition of 50 mM HEPES, 150 mM NaCl, 10
mM EDTA, 10 mM
Na2P2O7, 100 mM NaF, 2
mM vanadate (pH 7.5). Phosphorylated receptors were added
to pellets containing fusion proteins (60 pmol/sample of F1 and F2
fusion proteins; 90 pmol/sample of F1–1, F1–2, F2–1, and F2–2
fusion proteins) or antibodies to receptor (1/500). After 4 h at 4
C, pellets were washed twice.
The samples were analyzed by SDS-PAGE. The gel was dried and autoradiographed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2, both insulin and IGF-1 were more
efficient at stimulating phosphorylation of JAK-1 (5.2 ± 0.6 and
4.5 ± 0.4-fold, respectively), compared with JAK-2 (2.4 ±
0.6 and 1.9 ± 0.2-fold, respectively). In addition, there were
differences in the time course of phosphorylation, depending on the
stimulus. In response to IGF-1, JAK-2 was phosphorylated more rapidly
(within 2 min) than JAK-1 (5 min). In contrast, insulin stimulated
JAK-1 phosphorylation before that of JAK-2 (2 min compared with 10
min). In all cases, phosphorylation of both JAKs was transient and
returned to basal levels within 10 min after IGF-1 stimulation and 20
min after insulin stimulation (data not shown).
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JAKs were immunoprecipitated from cells, either stimulated or not (in the presence of vanadate, to preserve the phosphorylation state). Experiments performed in the presence of the phosphatase inhibitor have shown that insulin- or IGF-1-induced phosphorylation of JAKs can be maintained until 20 min (data not shown).
Figure 3A shows a Western blot of
immunoprecipitated JAK proteins. In these conditions, basal
(i.e. from unstimulated cells) tyrosine kinase activity of
JAK is observed, which is frequently seen in cell-free assays of
tyrosine kinases. When JAK-1 was purified from IGF-1-stimulated cells,
strong autophosphorylation of JAK-1 was induced. This result indicates
that JAK-1 isolated from IGF-1 treated-cells was activated. We obtained
the same result with JAK-2 (data not shown). We then examined whether
these activated JAKs also were able to phosphorylate exogenous
substrates such as IRS-1 and IRS-2. GST-IRS-2 was incubated with JAKs
immunoprecipitated from either IGF-1-stimulated or nonstimulated cells.
Phosphorylation of GST-IRS-2 was determined by Western blotting and
probing with antibodies to phosphotyrosine. As shown in Fig. 3B, GST-IRS-2 phosphorylation by JAK-1 and JAK-2 isolated from untreated
cells was observed. This phosphorylation was clearly increased when
JAK-1 and JAK-2 were purified from cells stimulated by IGF-1.
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, IRS-1
was strongly phosphorylated by the JAKs purified from cells incubated
with insulin, compared with IRS-1 phosphorylated by JAKs purified from
unstimulated cells. We obtained the same result with insulin-activated
JAKs (data not shown).
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-32P] ATP. Comparison of the two
32P-peptide maps showed marked differences (Fig. 5). Indeed, a particular panel of
phosphopeptides was found in IRS-1 phosphorylated by the insulin
receptor, whereas a different panel was seen only in IRS-1
phosphorylated by JAK-1. This indicates that phosphorylation of IRS-1
by JAK-1 or by the insulin receptor could involve distinct tyrosine
residues.
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To do this, kinase-dead JAK-2 from lysates of cells overexpressing the
protein and WGA-purified IGF-1 receptors were immunoprecipitated with
specific antibodies. The pellets containing kinase-dead JAK-2 were
mixed with pellets containing the immunopurified IGF-1 receptor. IGF-1
(10-7 M) was added, or not, for 30 min at 22
C. The phosphorylation reaction was then initiated by adding 60
µM [-32P] ATP (2.5 Ci/mmol), 50
mM MgCl2; and the reaction was stopped after 15
min. Figure 6 shows that tyrosine
phosphorylation of kinase-dead JAK-2 was not induced by IGF-1
receptors. This result suggests that there is no direct phosphorylation
of JAK by IGF-1 receptors.
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). We performed
precipitation of unphosphorylated and ligand-occupied insulin receptors
with GST, GST-F1, and GST-F2 fusion proteins preadsorbed on
glutathione-sepharose, or antibodies to receptor preadsorbed on protein
A-sepharose. After receptor incubation with the GST-JAK fragments,
pellets were washed extensively to remove the nonassociated receptors.
To detect the precipitated receptors, we then performed an in
vitro phosphorylation assay in the presence of
[-32P] ATP. In this case, phosphorylation occurs only
if native or ligand-occupied receptors were previously retained, as is
the case using specific antibodies to receptor (Fig. 8, left and middle
panels).
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In a second series of experiments, insulin receptors were
phosphorylated with [-32P] ATP before being incubated
with JAK-1 fusion proteins. As shown in Fig. 8
(right
panel), phosphorylated receptor ß-subunit was detected both in
GST-F1 and in GST-F2 pellets. These results indicate that only the
phosphorylated form of insulin receptor is recognized by JAK-1.
Moreover, as shown in Fig. 9 (leftand middle panels), IGF-1 receptor ß-subunit was
detected in GST-F1 and GST-F2 pellets only when the receptor was
previously phosphorylated. The IGF-1 receptor, deleted of its 121
C-terminal amino acids, still coprecipitated with GST-F1 and GST-F2
(Fig. 9, right panel), indicating that the C-terminal part
of the IGF-1 receptor is not involved in interaction with JAK-1.
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To identify more precisely the JAK-1 domains responsible for the
interaction, we constructed other fusion proteins with distinct parts
of JAK-1 (described in Fig. 7). Insulin receptors or IGF-1 receptors,
phosphorylated in the presence of [-32P] ATP, were
incubated with GST or the JAK-1 fusion proteins (GST-F1–1, GST-F1–2,
GST-F2–1, and GST-F2–2). As shown in Fig. 10, phosphoreceptors were absent in
GST, GST-F1–2, and GST-F2–1 pellets, but present in GST-F1–1 and
GST-F2–2 pellets. This indicates that two domains of JAK-1 (the
NH2-terminal part containing the JH6-JH7 domains, and the
C-terminal part containing JH1 domain) are responsible for binding to
phosphorylated insulin and IGF-1 receptors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Moreover, we found that insulin treatment causes a more rapid tyrosine phosphorylation of JAK-1, whereas IGF-1 causes a more rapid tyrosine phosphorylation of JAK-2. It is possible that JAK-1 and JAK-2 have different affinities for the insulin receptor and the IGF-1 receptor, and this would affect the time course of JAK recruitment by the receptors.
To determine whether JAKs interact directly with insulin or IGF-1 receptors, we constructed fusion proteins containing different portions of JAK-1. By precipitation assays, we show that JAK-1 does not interact with native and ligand-occupied insulin receptors. In contrast, JAK-1 interacts with the phosphorylated forms of insulin and IGF-1 receptors, indicating that receptor phosphorylation is necessary for the interaction. These data are consistent with reports showing that binding of JAK-1 to the PDGF receptor also requires receptor phosphorylation (26). In constrast, JAKs can interact constitutively with cytokine receptors, such as the PRL receptor, in a phosphorylation-independent way (34, 35). Tyrosine kinase receptors lack proline-rich and acidic motifs found in cytokine receptors and shown to be indispensable for interaction with JAK (32, 33). The domain(s) on the tyrosine kinase receptors responsible for the interaction has (have) yet to be identified. However, we found that the C-terminal tail (121 amino acids) of the IGF-1 receptor ß-subunit is not involved in interaction with JAK-1.
Next, we localized the JAK-1 regions required for interaction with
insulin and IGF-1 receptors. We found that two domains of JAK-1 are
involved in binding to phosphorylated receptors. These are: 1) the
JH6-JH7 domains of the N-terminus; and 2) the JH1 kinase domain in the
C-terminus. Further, we observed that the GST-F2 fragment of JAK-1
precipitates the insulin (or IGF-1) receptor less efficiently than the
GST-F1 fragment, whereas the GST-F2–1 fragment is as efficient as
GST-F1–1. This suggests the occurrence of an inhibitory effect of
F2–2 on F2 binding to the receptors. However, this also could be
caused by slight modification of protein conformation after fusion to
the GST moiety. In JAK-2, the JH6-JH7 domains, which are highly
conserved between the different members of the JAK family, also are
implicated in the interaction with the ß-subunit of the
interferon- receptor (51), the ß-subunit of granulocyte macrophage
colony stimulating growth factor receptor (52), and the GH receptor
(53). Concerning the interaction of JAK-2 with the GH and the
granulocyte macrophage colony stimulating growth factor receptors, the
kinase domain (JH1) is not required. Thus, the role of the JH1 domain
in the interaction of JAK-1 with phosphorylated insulin and IGF-1
receptors could be specific for these proteins.
JAK-1 and JAK-2 are not only phosphorylated in response to insulin and IGF-1 but also are activated. However, the mechanism of the latter process is unknown. One hypothesis could be that JAK activation is triggered by direct phosphorylation by insulin or IGF-1 receptor. However, we did not detect tyrosine phosphorylation of a kinase-dead JAK-2 by the receptor. This result suggests that there is no direct phosphorylation of JAK by IGF-1 receptors.
Another explanation could be that JAKs are stimulated by insulin and IGF-1 receptors in a mechanism similar to that of cytokine receptors, lacking tyrosine kinase activity such as erythropoietin receptors (54). Dimerization of the erythropoietin receptors upon ligand binding leads to transphosphorylation and activation of JAKs. Clustering of the kinases, which are constitutively associated with the erythropoietin receptors, is thought to induce this activation (55).
Our results raise the question of the physiological role of JAK activation by insulin and IGF-1 receptors. Quelle et al. (56) show that STATs are potentially immediate substrates of activated JAKs. A recent study has shown that STAT1, STAT3, and STAT5 from fibroblasts overexpressing insulin receptors are phosphorylated and activated after hormone treatment (57). Moreover, the insulin receptor interacts with and phosphorylates STAT5 in vitro and in insulin-sensitive tissues (57, 58). Thus, these data indicate that STATs can be directly activated by the insulin receptor. However, we cannot rule out the possibility that the JAKs are implicated in activation of STATs in intact cells.
Further, we show that JAKs activated by insulin or IGF-1 receptors can directly phosphorylate IRS-1 and IRS-2. Phosphorylated IRS-1 and IRS-2 generate activation of several signaling pathways. In trying to understand the role of IRS-1 phosphorylation by JAKs, we compared the 32P-peptide maps of IRS-1 phosphorylated in vitro by the insulin receptor vs. JAK-1 or JAK-2. The phosphorylation profile of IRS-1 was found to be different, depending on the tyrosine kinase used. These differences suggest that IRS-1 phosphorylation by JAK-1 or by JAK-2 could occur on tyrosine residues distinct from those phosphorylated by insulin receptors. Phosphorylation of IRS-1 by JAKs could lead to generation of interaction sites for SH2 domain-containing proteins, which are not produced by the insulin receptor. Hence recruitment of JAK-specific downstream signaling molecules could occur. A major challenge is to understand how insulin generates its considerable spectrum of cellular responses, some of which are specific for insulin, and concern mainly metabolic effects, whereas others are seen with several growth factors and relate to growth and differentiation. It remains to be shown whether the JAK-specific sites modified on IRS molecules generate branches in the insulin signaling pathways, which are responsible for a specific type of cellular program, i.e. growth/differentiation vs. metabolism.
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Received May 27, 1997.
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, and leukemia
inhibitory factor utilize insulin receptor substrate-2 in intracellular
signaling. J Biol Chem 271:29415–29421 engages the insulin receptor substrate-1 to
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|
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|
|
|
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 |
|
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|
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|
|
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|
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|
|
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|
|
|
|
|
|
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|
|
|
|
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|
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|
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|
|
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|
|
|
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|
|
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|
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|
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|
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|
|
|
|
|
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|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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