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From the
The identification of JAK2 as a growth hormone (GH)
receptor-associated, GH-activated tyrosine kinase has established
tyrosyl phosphorylation as a signaling mechanism for GH. In the present
study, GH is shown to stimulate tyrosyl phosphorylation of insulin
receptor substrate 1 (IRS-1), the principle substrate of the insulin
receptor. Tyrosyl phosphorylation of IRS-1 is a critical step in
insulin signaling and provides binding sites for proteins with the
appropriate Src homology 2 domains, including the 85-kDa regulatory
subunit of phosphatidylinositol (PI) 3`-kinase. In 3T3-F442A
fibroblasts, GH-dependent tyrosyl phosphorylation of IRS-1 was detected
by 1 min and at GH concentrations as low as 5 ng/ml (0.23 nM).
Tyrosyl phosphorylation of IRS-1 was transient, with maximal
stimulation detected at 30 min and diminished signal detected at 60
min. The ability of GH receptor (GHR) to transduce the signal for IRS-1
tyrosyl phosphorylation is mediated by the intracellular region of GHR
between amino acids 295 and 380 by a mechanism not involving the two
tyrosines in this region. This region of GHR is required for
GH-dependent JAK2 association and activation (VanderKuur, J. A., Wang,
X., Zhang, L., Campbell, G. S., Allevato, G., Billestrup, N., Norstedt,
G., and Carter-Su, C.(1994) J. Biol. Chem. 269,
21709-21717). When other cytokines that activate JAK2 were tested
for the ability to stimulate the tyrosyl phosphorylation of IRS-1,
stimulation was detected with interferon-
A major effect of GH
IRS-1 is a
cytosolic protein that becomes tyrosyl phosphorylated at multiple sites
in response to insulin (4) and insulin-like growth factor-1
(IGF-1)(5) . Tyrosyl phosphorylation of IRS-1 mediates biologic
responses to insulin and IGF-1, including mitogenic effects (6, 7) and
activation of PI 3`-kinase(8, 9) , which is thought to
be required for translocation of GLUT 4 glucose
transporters(10, 11, 12) . Tyrosyl
phosphorylation of IRS-1 provides binding sites for specific proteins
containing Src homology-2 domains, including the 85-kDa regulatory
subunit of PI 3`-kinase(4, 13) ,
GRB-2(14, 15) , the tyrosine phosphatase
SHPTP2(16, 17) , and Nck(18) .
Unlike the
receptors for insulin (19) and IGF-1(20, 21) ,
GHR does not contain an intrinsic tyrosine kinase(22) . However,
ligand binding to GHR activates JAK2 tyrosine kinase(23) . In
addition to GHR, a number of other receptors in the
cytokine/hematopoietin receptor superfamily activate JAK2 in response
to ligand binding, including the receptors for erythropoietin,
prolactin, interleukin (IL) 3, granulocyte-macrophage
colony-stimulating factor, granulocyte colony-stimulating factor,
leukemia inhibitory factor (LIF), IL-6, oncostatin M, ciliary
neurotrophic factor, and interferon-
A specific tyrosine in the receptor for
insulin (42) is required for IRS-1 tyrosyl phosphorylation in
response to insulin. Between amino acids 295 and 380, rat GHR contains
tyrosines at amino acids 333 and 338. One or both of these tyrosines
appears to be phosphorylated in response to GH.
The region between amino acids 295 and 380 has been
shown to be required for GH-dependent activation of
JAK2(35, 43, 44) . Thus the role of this region
of GHR in GH-dependent tyrosyl phosphorylation of IRS-1 may reside in
its ability to transduce the signal that activates JAK2. Consistent
with involvement of JAK2 in IRS-1 tyrosyl phosphorylation, the ability
of each mutant GHR to mediate GH-dependent tyrosyl phosphorylation of
JAK2 correlates with the amount of IRS-1 tyrosyl phosphorylation
detected (Fig. 4). Since GH-dependent tyrosyl phosphorylation of
JAK2 has been determined to reflect JAK2 kinase
activity,(
LIF and IFN-
Although our
results indicate that multiple ligands can utilize IRS-1 as a signaling
molecule, the signaling response need not be identical for every
ligand. IRS-1 contains at least 20 potential tyrosyl phosphorylation
sites, at least eight of which are phosphorylated following insulin
stimulation(13) . Consistent with the ability of Src homology
domain 2 domains in cellular proteins to bind with high affinity to
phosphorylated tyrosyl residues in specific sequence motifs (51), it
has been demonstrated that Src homology domain 2 domains from the
85-kDa regulatory subunit of PI 3`-kinase, GRB2, Nck, and the
protein-tyrosine phosphatase SHPTP2 bind to unique residues within
IRS-1(13, 18, 15) . Differences in specificity
of the kinase(s) activated in response to GH versus insulin or
IGF-1 could result in phosphorylation of unique subsets of IRS-1
tyrosyl residues and thereby alter the downstream signaling pathways
activated. In the case of LIF and IFN-
IRS-1-mediated responses unique to a
specific ligand could also arise if signaling pathways unique to that
ligand acted in concert with pathways originating from IRS-1.
Activation of protein kinase C
The
similarity in the ratio of JAK2 tyrosyl phosphorylation to IRS-1
tyrosyl phosphorylation following treatment of 3T3-F442A fibroblasts
with GH, IFN-
The correlation detected between JAK2 tyrosyl
phosphorylation and IRS-1 tyrosyl phosphorylation is not observed with
all proteins that undergo GH-dependent tyrosyl phosphorylation. In
3T3-F442A fibroblasts, GH is much more effective than IFN-
Ligand-dependent tyrosyl phosphorylation of IRS-1
following stimulation with IGF-1, insulin, or IL-4 requires the
sequence NPXY in the respective receptors(58) . In rat
GHR, the closest match for this sequence is NSPY and includes the
tyrosine at amino acid 437. This tyrosine is not believed to be
phosphorylated,
We thank Liying Zhang and Pinyi Du for technical
assistance and David Kim for help with cell culture.
Volume 270,
Number 24,
Issue of June 16, pp. 14685-14692, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
, and Leukemia Inhibitory Factor Promoted Tyrosyl
Phosphorylation of Insulin Receptor Substrate-1 (*)
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and leukemia inhibitory
factor. The correlation between JAK2 tyrosyl phosphorylation and IRS-1
tyrosyl phosphorylation in response to GH, interferon-, and
leukemia inhibitory factor and in cells expressing different GHR
mutants, provides evidence that IRS-1 may interact with JAK2 or an
auxiliary molecule that binds to JAK2. GH is also shown to stimulate
binding of IRS-1 to the 85-kDa regulatory subunit of PI 3`-kinase. The
ability of GH to stimulate tyrosyl phosphorylation of IRS-1 and its
association with PI 3`-kinase provides a biochemical basis for
responses shared by insulin and GH including the well characterized
insulin-like metabolic effects of GH observed in a variety of cell
types.
()in glycemic control
is to oppose the action of insulin. GH stimulates hepatic glucose
production. It also stimulates lipolysis and decreases glucose
utilization in fat tissue. However, GH is also known to have short term
effects that mimic insulin actions in tissues that have been deprived
of GH(1) . This pattern of rapid, transient insulin-like effects
and delayed onset prolonged anti-insulin effects is also detected in
3T3-F442A adipocytes. For example, in cells that have been deprived of
GH, GH initially stimulates glucose uptake. However, with longer term
GH incubation, glucose uptake becomes depressed(2, 3) .
The existence of the insulin-like effects of GH suggests that GH may
utilize some signaling molecules utilized by insulin.
(IFN-) (24-29).
Thus ligand-dependent tyrosyl phosphorylation of cellular substrates is
expected in response to receptor binding by these cytokines.
Ligand-dependent tyrosyl phosphorylation of proteins in the size range
(160-170 kDa) appropriate for IRS-1 has been observed with GH (30) and a number of other ligands that bind to members of the
cytokine/hematopoietin receptor family, including LIF, IL-6, ciliary
neurotrophic factor, and oncostatin M(31, 32) . This
suggests that these ligands might utilize IRS-1 as a signaling
molecule. In this report, we demonstrate that GH rapidly stimulates
both tyrosyl phosphorylation of IRS-1 and the association of PI
3`-kinase with IRS-1. In addition, we determine that the cytoplasmic
region of GHR proximal to the membrane (amino acids 295-380) is
required for IRS-1 tyrosyl phosphorylation; however, within this
region, tyrosines 333 and 338 are not required. The demonstration that
LIF and IFN-, two cytokines that, like GH, activate
JAK2(29, 26) , also stimulate tyrosyl phosphorylation of
IRS-1 suggests that signaling by IRS-1 may be common to multiple
members of the cytokine/hematopoietin family that activate JAK2.
Materials
Recombinant 22,000-Da hGH and porcine
insulin were a gift of the Eli Lilly Co. Human recombinant IGF-1 was
from Calbiochem. Recombinant protein A-agarose was from Repligen.
Protein assay kit (BCA) was from Pierce. Aprotinin, leupeptin, and
Triton X-100 were purchased from Boehringer Mannheim. Bovine serum
albumin was from Intergen. Prestained molecular weight standards were
from Life Technologies, Inc. Nitrocellulose paper was from Schleicher
and Schuell. Chicken egg ovalbumin was purchased from Sigma. The
enhanced chemiluminescence (ECL) detection system and x-ray film were
from Amersham Corp.
Antisera
Antibody to GH (
GH)
(NIDDK-anti-hGH-IC3, lot C11981) came from the National Institute of
Diabetes and Digestive and Kidney Diseases/National Hormone and
Pituitary Program, National Institutes of Health. Anti-phosphotyrosine
antibody (PY) (4G10) and antiserum to the 85-kDa subunit of PI
3`-kinase (p85
), which was used for immunoblotting,
were purchased from Upstate Biotechnology, Inc. Affinity-purified
antibody prepared against a peptide containing amino acids
146-161 of mouse 85-kDa subunit of PI 3`-kinase (p85) (9) was used for immunoprecipitation. Protein A-purified
polyclonal antibody prepared against recombinant IRS-1 (IRS-1) (4) was used for immunoprecipitations. Monoclonal antibody
raised against recombinant IRS-1 (IRS-1
) (8) was used for immunoblotting. Antibody to JAK2 (JAK2)
was prepared in rabbits against a synthetic peptide corresponding to
amino acids 758-776 of murine JAK2(24) .
Mutagenesis, Transfection, and Cell Culture
CHO
cells expressing full-length rat GHR were developed by co-transfecting
CHO cells with plasmids pLM108 and pIBP-1(33, 34) .
Plasmid pIBP-1 contains the thymidine kinase promoter fused to the
bacterial neomycin phosphotransferase gene conferring G418 resistance.
Plasmid pLM108 contains the simian virus 40 enhancer and the
Zn
-inducible human metallothionein IIa promoter
driving expression of cDNA coding for full-length rat liver GHR. For
CHO cells expressing the various GHR mutants, the GHR cDNA in the
pLM108 plasmid was mutated to replace lysine codons 455, 381, 319, or
295 with termination codons, to delete the codons for amino acids
297-311, or to replace tyrosine codons 333 and 338 with codons
for phenylalanine, as described
previously(34, 35, 36, 37) . The amino
acids in GHR are numbered according to Ref. 38. The binding affinity
for hGH was similar for each cell line(33, 34) .
()CHO cells were grown in Ham's F-12 medium
containing 1.8 g/liter glucose and 10% fetal calf serum. G418 (500
µg/ml) was added to the medium of transfected CHO cells used for
passaging. The stock of 3T3-F442A cells was a kind gift of H. Green
(Harvard University, Boston, MA). 3T3-F442A cells were cultured in
Dulbecco's modified Eagle's medium containing 4.5 g/liter
glucose and 8% calf serum as described previously(39) . All
cells were cultured at 37 °C under 5% CO, 95% air.
Media were supplemented with 1 mML-glutamine, 100
units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml
amphotericin B.
GH Binding Assay
Human GH labeled with 
I to an estimated specific activity of 
90
µCi/µg using chloramine-T was prepared by the University of
Michigan Reproductive Sciences Training Grant Core Facility. GH binding
of cell monolayers at 25 °C for a period of 1 h was determined as
described previously(35) . I-GH binding was
corrected for nonspecific binding as determined by incubating cell
monolayers with I-hGH in the presence of 2 µg/ml
unlabeled hGH. Binding was normalized to protein content using the
Pierce BCA protein assay.
Immunoprecipitation and Western Blotting
Confluent
cells were incubated in serum-free medium overnight (16-20 h) as
described previously(30) . The cells were incubated at 37 °C
in a 95% air, 5% CO atmosphere with hGH, cytokine, or
growth factor at the concentrations and for the times indicated. The
cells were rinsed with three changes of ice-cold PBSV (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 1 mM Na
VO
) and scraped in lysis buffer (50
mM Tris, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 2
mM EGTA, 1 mM NaVO, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10
µg/ml leupeptin) on ice. Cell lysates were centrifuged at 14,000
g for 10 min. The supernatants were incubated on ice
for 3 h with the indicated antibody. Immune complexes were collected on
protein A-agarose for 1 h at 8 °C, washed 3 times with wash buffer
(50 mM Tris, pH 7.5, 0.1% Triton X-100, 150 mM NaCl,
2 mM EGTA, 1 mM NaVO) and
boiled for 5 min in a mixture (80:20) of lysis buffer and 250 mM Tris, pH 6.8, 5% SDS, 10% 
-mercaptoethanol, 40% glycerol.
Immunoprecipitated proteins were separated by SDS-polyacrylamide gel
electrophoresis on 3-10% gradient polyacrylamide gels (30:0.5,
acrylamide/bisacrylamide). For experiments using CHO cells, the amount
of sample added to each lane was normalized to the protein content of
the cell supernatant. Proteins were transferred to nitrocellulose at
100 V at 4 °C for 2 h in 25 mM Tris, 190 mM glycine, 20% methanol, 0.02% SDS. Blots were incubated with the
appropriate antibody and visualized by ECL detection as described
previously(30) . To reprobe the blot with a second antibody, the
blot was rinsed, incubated in 2% SDS, 60 mM Tris, pH 6.7, 100
mM -mercaptoethanol at 50 °C for 30 min, reprobed
without adding additional primary antibody to check that all antibody
had been removed, and then reprobed using the second antibody.
Homology Searches
Pearson and Lipman (40) homology searches of sequences from Genbank (murine GHR
(accession number M33324), murine LIF receptor (D26177), murine gp130
(M83336), murine IFN- receptor (M25764), murine IFN- receptor
-chain (S69336), murine JAK1 (S63728), murine JAK2 (L16956),
murine JAK3 (L32955)] were performed with the Wisconsin Package
FASTA program(82) .
GH Stimulates Tyrosyl Phosphorylation of
IRS-1
Signalling utilizing IRS-1 is dependent upon its tyrosyl
phosphorylation(4) . To determine if IRS-1 is tyrosyl
phosphorylated following stimulation of cells with GH, solubilized
proteins from 3T3-F442A fibroblasts were immunoprecipitated with
IRS-1 and immunoblotted with PY. GH-dependent tyrosyl
phosphorylation of a protein with a M
(160, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 0) appropriate
for IRS-1 is detected by 1 min (the earliest time tested) (Fig. 1, laneF) and at physiologically
relevant concentrations of GH as low as 5 ng/ml (0.23 nM) (the
lowest concentration tested) (Fig. 1, lanesB-D). IRS-1 tyrosyl phosphorylation is transient,
with maximal stimulation in response to 500 ng/ml GH detected at 30
min. The signal is diminished by 60 min (Fig. 1, lanesE-J) with only a minimal further decrease detected
at 120 and 240 min (data not shown). The identity of this protein as
IRS-1 is further suggested by its comigration with a protein recognized
by IRS-1 when the blot was probed with
IRS-1 (Fig. 1, lanesP and Q), with a tyrosyl-phosphorylated protein
precipitated by IRS-1 following stimulation with IGF-1 (Fig. 1, laneK), and with insulin (data not
shown), known effectors of IRS-1 tyrosyl
phosphorylation(5, 4) . IGF-1-stimulated tyrosyl
phosphorylation of IRS-1 is maximal at 3 min (data not shown). The
maximal tyrosyl phosphorylation of IRS-1 in response to GH is 50% of
the level observed following treatment with 23 nM IGF-1 for 3
min (Fig. 1) and equivalent to the level observed following 5 min
of treatment with 10 or 50 nM insulin (data not shown).
Figure 1:
GH promoted tyrosyl phosphorylation of
IRS-1. 3T3-F442A fibroblasts were incubated with the indicated
concentrations of hGH at 37 °C for 15 min (lanesA-D), or with vehicle (lanesE and M), 500 ng/ml (23 nM) hGH (lanesF-J, L, N-Q), or 170 ng/ml (23 nM) IGF-1 (laneK) at 37 °C for the times indicated. Whole
cell lysates were immunoprecipitated with IRS-1 (1:250 dilution) (lanesA-K, P, and Q), GH (1:8000 dilution) (laneL), or
JAK2 (1:250 dilution) (lanesM-O). LaneO was loaded
with one-tenth of the volume of sample loaded in the other lanes.
Immunoprecipitated proteins were immunoblotted with PY (1:7500
dilution) (lanesA-P) or
IRS-1 (1:2000 dilution) (laneQ). For laneP, the blot was probed
first with IRS-1, stripped, and reprobed
with PY. The molecular weight (10
) of
protein standards and the migration of IRS-1, JAK2, and GHR are
indicated. LanesA-D, E-O, and P-Q were from three separate experiments. The band
observed at 111 kDa in laneA is from the molecular
weight standard loaded in the adjacent lane. The faint band detected at
120-kDa in laneK was not observed in three other
experiments.
In
addition to IRS-1, tyrosyl-phosphorylated proteins with M 120,000-130,000 are also detected in
IRS-1 immunoprecipitates from GH-treated cells (Fig. 1, lanesC, D, G-I). These
proteins comigrate with the tyrosyl-phosphorylated JAK2
GHR
complex precipitated using JAK2 (Fig. 1, lanesN and O) and GH (Fig. 1, laneL). Similarly, a protein that comigrates with
tyrosyl-phosphorylated IRS-1 is detected in JAK2
immunoprecipitates (Fig. 1, laneN and see Fig. 5, laneL). These results suggest that
IRS-1 is present in a complex with JAK2 and GHR; however insufficient
protein is co-precipitated to permit identification of these proteins
by immunoblotting with the appropriate antibody.
Figure 5:
Ability of IFN-, LIF, and IL-6 to
tyrosyl phosphorylate IRS-1. 3T3-F442A fibroblasts were incubated with
vehicle (C), 10 ng/ml (0.7 nM) IFN-, 25 ng/ml
(1.3 nM) LIF, 10 ng/ml (0.5 nM) IL-6, or 500 ng/ml
(23 nM) GH at 37 °C for 15 min. Whole cell lysates were
immunoprecipitated with IRS-1 (1:250 dilution) (lanesA-E), nonimmune serum (lanesF (1:250 dilution) and G (1:100 dilution)), or JAK2
(1:200 dilution) (lanesH-L).
Immunoprecipitated proteins were immunoblotted with PY (1:7500
dilution). The molecular weight (10) of
protein standards and the migrations of IRS-1 and JAK2 are indicated.
For the ligands that stimulated JAK2 tyrosyl phosphorylation, 15-min
incubations and the ligand concentration used gave maximal JAK2 tyrosyl
phosphorylation (data not shown).
Ability of GH to Induce Association of PI 3`-Kinase with
IRS-1
The 85-kDa regulatory subunit of PI 3`-kinase binds to
IRS-1 following insulin stimulation(4) . To determine if GH
promotes the association of the 85-kDa subunit of PI 3`-kinase with
IRS-1, IRS-1 was immunoprecipitated from solubilized 3T3-F442A
fibroblasts with IRS-1 and immunoblotted with
p85. A GH-dependent increase in the amount of the
85-kDa subunit of PI 3`-kinase, which coprecipitates with IRS-1 was
detected (Fig. 2, lanesA and B). The
identity of this protein as the 85-kDa subunit of PI 3`-kinase is
suggested by its comigration with a protein immunoprecipitated by
p85 and visualized by p85 in immunoblots (Fig. 2, laneD) and the fact that it has a M appropriate for the 85-kDa subunit of PI
3`-kinase. The 85-kDa subunit of PI 3`-kinase does not appear to
associate with JAK2 as evidenced by the inability to detect p85
following immunoprecipitation with JAK2 (Fig. 2, lanesE and F). A GH-dependent increase in PI
3`-kinase associated with IRS-1 demonstrates that not only does GH
stimulate tyrosyl phosphorylation of IRS-1, but binding site(s) for PI
3`-kinase are created. Thus, following stimulation with GH, IRS-1 is
able to recruit PI 3`-kinase in these cells.
Figure 2:
GH-dependent association of the 85-kDa
regulatory subunit of PI 3`-kinase with IRS-1. 3T3-F442A fibroblasts
were incubated in the absence (-) or presence (+) of 500
ng/ml hGH at 37 °C for 15 min. Whole cell lysates were
immunoprecipitated with IRS-1 (1:250 dilution) (lanesA and B), nonimmune serum (1:125 dilution) (laneC), p85 (1:100 dilution) (laneD), or JAK2 (1:200 dilution) (lanesE and F). LaneD was loaded with
one-fifth of the volume of sample loaded in the other lanes.
Immunoprecipitated proteins were immunoblotted with p85 (1:2000 dilution). The molecular weight
(10) of protein standards and the migration
of the 85-kDa subunit of PI 3`-kinase (p85) are
indicated.
Identification of the Region of GHR Necessary for IRS-1
Tyrosyl Phosphorylation
To begin to understand the molecular
basis for the interaction between IRS-1 and GHR, the ability of various
mutated GHR to transduce the signal for tyrosyl phosphorylation of
IRS-1 was evaluated in CHO cells (mutants summarized in Fig. 3).
CHO cells were solubilized and IRS-1 immunoprecipitated using
IRS-1 and then immunoblotted with PY (Fig. 4A). A GH-dependent increase in tyrosyl
phosphorylation of IRS-1 is detected in cells expressing
GHR
(wild-type), GHR
, and
to a lesser extent GHR
(Fig. 4A, lanesA-F). Binding of I-hGH to cells
expressing GHR was only 20% of the level detected
in cells expressing GHR (Fig. 3).
Therefore, the decrease in GH-dependent tyrosyl phosphorylation of
IRS-1 detected in cells expressing GHR could at
least partially be due to the lower level of GHR expression. No
GH-dependent tyrosyl phosphorylation of IRS-1 is detected in cells
expressing GHR
, GHR
, and
GHR
(Fig. 4A, lanesG-L). In GHR, amino acids
297-311 of GHR, which includes the proline-rich
motif(41) , have been deleted. Thus, the intracellular region of
GHR from the membrane to amino acid 380 and within this region, a
region between 297 and 311 and another region between amino acids 318
and 380, are required for GH-mediated tyrosyl phosphorylation of IRS-1.
The same regions of GHR are required for tyrosyl phosphorylation of
JAK2 as indicated by the lack of GH-dependent JAK2 phosphorylation in
CHO cells expressing GHR, GHR,
and GHR (Fig. 4B).
Figure 3:
Wild-type and mutated GHR expressed in CHO
cells. The extracellular domain, transmembrane domain (cross-hatched box), cytoplasmic domain, homology box 1
(proline rich motif) (solid box), and homology box 2 (stippled box) of the rat liver GHR are noted. The relative
ability of each cell line to bind GH is noted. Intracellular tyrosyl
residues are denoted by Y. Tyrosines mutated to phenylalanine
are denoted by F.
Figure 4:
Region of GHR necessary for IRS-1 tyrosyl
phosphorylation. CHO cells expressing the indicated GHR were incubated
in the absence (-) or presence (+) of 500 ng/ml hGH at 37
°C for 15 min. Whole cell lysates were immunoprecipitated with
IRS-1 (1:250 dilution) (panelA) or JAK2
(1:200 dilution) (panelB). Immunoprecipitated
proteins were immunoblotted with PY (1:7500 dilution). The
molecular weight (10) of protein standards
and the migrations of IRS-1 and JAK2 are indicated. LanesA-L and M-P are from separate
experiments.
The increase
in basal IRS-1 tyrosyl phosphorylation in cells expressing
GHR and GHR compared with
GHR (Fig. 4A, lanesA, C, and E) was consistently detected.
This raises the possibility that a binding site/regulatory region for a
phosphatase may be present between amino acids 455 and 638 of GHR.
Increased basal JAK2 activity does not appear to be involved since JAK2
tyrosyl phophorylation is not detected in these cell lines in the
absence of GH(35) .
To determine if these tyrosines are required for
IRS-1 tyrosyl phosphorylation, the tyrosines at 333 and 338 of GHR were
mutated to phenylalanines in full-length GHR and
in GHR. When expressed in CHO cells,
GHRY(333,338)F and
GHRY(333,338)F mediated GH-dependent tyrosyl
phosphorylation of IRS-1 (Fig. 4A, lanesO and P; and data not shown). Therefore, tyrosines 333 and
338 in GHR are not required for GH-dependent IRS-1 tyrosyl
phosphorylation.
)()this correlation further
suggests that, either directly or indirectly, stimulation of JAK2
kinase activity is required for phosphorylation of IRS-1.
Ability of IFN-
If IRS-1 interacts with JAK2, one would
expect other cytokines that activate JAK2 to stimulate tyrosyl
phosphorylation of IRS-1 in direct relationship to their ability to
activate JAK2. To determine if other cytokines, which signal via JAK2,
stimulate tyrosyl phosphorylation of IRS-1, 3T3-F442A fibroblasts were
treated with IFN-, LIF, and IL-6 to Stimulate IRS-1
Tyrosyl Phosphorylation, LIF, or IL-6, IRS-1 was immunoprecipitated with
IRS-1 and immunoblotted with PY. A ligand-dependent increase
in tyrosyl phosphorylation of IRS-1 was observed in response to
IFN- and LIF (Fig. 5, lanesA-C);
IL-6 was not stimulatory (Fig. 5, lanesAand D). As observed with CHO cells expressing
the various mutated GHR (Fig. 4), the amount of tyrosyl
phosphorylation of IRS-1 detected following treatment with the various
cytokines (Fig. 5, lanesA-E)
correlates with the level of JAK2 tyrosyl phosphorylation observed (Fig. 5, lanesH-L).
Identification of IRS-1 as a Signaling Molecule for GH,
LIF, and IFN-
The work presented in this paper provides
strong evidence that IRS-1 is a signaling molecule for GH in 3T3-F442A
fibroblasts and CHO cells expressing GHR. It supports recent findings
using primary cultures of rat adipocytes published as the present
report was being prepared for publication(45) . Interestingly,
the IRS-1 response to GH reported here for 3T3-F442A fibroblasts is
much larger than that reported for adipocytes. Whether this is due to
inherent differences in IRS-1 signaling at the cellular level or
differences in antibody and technique remains to be determined. The
ability of GH to transiently stimulate tyrosyl phosphorylation of IRS-1
and induce its association with PI 3`-kinase provides a biochemical
basis for the insulin-like effects of GH observed in a variety of cell
types, such as the adipocytes previously used in research with
IRS-1(45) . However, GH-dependent insulin-like effects that have
been characterized in the adipocyte form of 3T3-F442A cells are reduced
in magnitude or undetectable in the fibroblasts form of the 3T3-F442A
cells (2) used in the present study. Therefore, GH-dependent
signaling through IRS-1 is likely to encompass pathways in addition to
those mediating insulin-like metabolic effects.
,
which, like GH, activate JAK2(29, 26) , have also been
shown to induce the tyrosyl phosphorylation of IRS-1. IL-6 (10 ng/ml,
0.5 nM), however, failed to induce either JAK2 or IRS-1
tyrosyl phosphorylation (Fig. 5). The finding that in a sister
cell line of the 3T3-F442A cells used in this study, IL-6 stimulates
JAK2 and MAP kinase activity only at very high concentrations (2
µg/ml) (46) that are approximately 1000 times the reported K
(0.3 and 8 ng/ml) for IL-6 receptors in
human CESS cells (47) suggests that the inability of 10 ng/ml
IL-6 to stimulate JAK2 in the present study results from the absence of
high affinity IL-6 receptors in 3T3-F442A fibroblasts. IRS-1 tyrosyl
phosphorylation in response to LIF, IFN-, and GH suggests that
IRS-1 may be an element of signaling cascades for multiple members of
the cytokine/hematopoietin receptor family. The presence of IRS-1
proteins in signaling pathways for members of the
cytokine/hematopoietin receptor family is consistent with previous
findings in FDC myeloid progenitor cells showing that IL-4, insulin,
and IGF-1 stimulate tyrosyl phosphorylation of 4PS (IL-4-induced
phosphotyrosine substrate), a protein antigenically related to IRS-1
that is thought to function similarly to IRS-1. IL-4 is a member of the
cytokine/hematopoietin receptor family that, upon ligand binding,
activates JAK3 and promotes tyrosyl phosphorylation of
JAK1(48) . The finding that IRS-1 is tyrosyl phosphorylated in
response to IL-4 when the common chain (
)
component of the IL-4 receptor is expressed in mouse L cells that
normally lack this chain (49) suggests that IL-4 is capable of
activating IRS-1. Consistent with this, when IRS-1 and the IL-4
receptor are coexpressed in 32D myeloid progenitor cells,
IL-4-dependent mitogenesis (50) and activation of PI 3`-kinase
and p70
kinase are detected(9) .
versus GH, all of
which activate JAK2 in 3T3-F442A fibroblasts, one would expect
differences in the magnitude of the signaling response due to the
differences in the ability of each ligand to activate JAK2. There might
also be differences in the specific response if the number of tyrosines
phosphorylated differs as well.
(52) or inhibition of
protein phosphatases with okadaic acid (53) has been shown to
suppress tyrosyl phosphorylation of IRS-1 and decrease the binding of
PI 3`-kinase to IRS-1. Therefore ligand-dependent activation of
serine/threonine kinases or inhibition of serine/threonine phosphatases
could suppress binding of specific signaling molecules to IRS-1.
Determination of the tyrosyl residues phosphorylated in IRS-1 and of
the particular proteins that bind to IRS-1 and become activated
subsequent to the binding of each ligand to its receptor will begin to
identify similarities and differences in cellular signaling originating
from IRS-1 in response to insulin, IGF-1, and those ligands like GH
that activate JAK2 kinase. Defining the molecular basis for differences
in signaling by these various ligands and the basis for interactions
between the cytokine network and actions regulated by insulin may
identify critical metabolic steps amenable to therapeutic intervention
in the management of diabetes and other metabolic diseases.
Region of GHR Required for GH-dependent Tyrosyl
Phosphorylation of IRS-1
To begin to define the mechanism by
which IRS-1 undergoes GH-dependent tyrosyl phosphorylation, we
determined which regions of GHR are required for tyrosyl
phosphorylation of IRS-1. The intracellular region of GHR from the
membrane to amino acid 380 and within this region, a region between
amino acids 297 and 311 containing the proline-rich motif and another
region between amino acids 318 and 380, was found to be required for
GH-mediated tyrosyl phosphorylation of both IRS-1 and JAK2. In this
region of GHR (amino acids 295-380), homology with IFN- and
LIF receptors is present solely in two short regions, homology box 1
(proline-rich motif, amino acids 298-305) and box 2 (amino acids
334-350)(54, 41, 55, 82) . These
regions of GHR are thought to be necessary for association with JAK2
and the activation of JAK2 by GH (35, 43, 44). Thus, the role of GHR in
GH-dependent tyrosyl phosphorylation of IRS-1 may reside in its ability
to transduce the signal, which activates JAK2 in response to GH.
, and LIF and of CHO cells expressing various GHR
mutants with GH provides additional evidence that IRS-1 may interact
primarily with JAK2 or an accessory molecule common to these cytokine
receptors that binds to JAK2. That JAK2 may contain a recognition site
for IRS-1 is suggested by the fact that one of the proteins that
coprecipitates with IRS-1 isolated from GH-treated cells comigrates
with JAK2 precipitated using JAK2 and vice versa ( Fig. 1and Fig. 5). The association of IRS-1 with JAK2 would make JAK2 an
obvious candidate for the tyrosine kinase responsible for GH-dependent
tyrosyl phosphorylation of IRS-1. This is also feasible kinetically
since GH-dependent JAK2 tyrosyl phosphorylation(23, 30) precedes or is simultaneous with IRS-1 tyrosyl
phosphorylation in 3T3-F442A fibroblasts (Fig. 1). If IRS-1 does
in fact interact with JAK2, it would be likely that many if not all of
the cytokine/hematopoietin receptors that activate JAK2 would also
signal through IRS-1.
at
stimulating tyrosyl phosphorylation of JAK2 (Fig. 5). However,
even though JAK2 has been shown to be required for IFN--dependent
activation of STAT 1(26) , tyrosyl phosphorylation and
activation of STAT 1 in response to GH is actually substantially less
than for IFN-(56, 57) . The lack of correlation
between JAK2 tyrosyl phosphorylation and STAT 1 tyrosyl phosphorylation
following stimulation with INF- and GH would suggest that in
contrast to IRS-1 signaling, there is some interaction between the STAT
1 signaling pathway and the receptors for the ligands responsible for
STAT 1 activation.
is not conserved in bovine, sheep, pig, and
chicken GHR(59, 60, 61, 62) , and is
deleted in GHR, which supports GH stimulation of
IRS-1 tyrosyl phosphorylation (Fig. 4A). Thus, tyrosine
437 is not thought to be critical in interactions leading to tyrosyl
phosphorylation of IRS-1. Indeed, none of the tyrosines in GHR appear
necessary for IRS-1 tyrosyl phosphorylation since IRS-1 is still
tyrosyl phosphorylated in CHO cells expressing
GHRY(333,338)F or
GHRY(333,338)F with the tyrosines at 333 and 338
(the only tyrosines remaining in the cytoplasmic domain of
GHR and the only ones thought to be
phosphorylated in GHR) mutated to phenylalanine.
Nor does murine JAK1 (63), JAK2(24) , JAK3(48) , LIF
receptor(64) , gp130(65) , IFN-
receptor(66) , or the -subunit of the IFN- receptor (67) contain the NPXY motif(82) , therefore
raising the possibilities that 1) in GH, LIF, and IFN- signaling,
IRS-1 interacts with an as yet unidentified protein; 2) IRS-1
recognizes motifs in addition to the NPXY motif; or 3) the
NPXY motif is actually the binding site for an auxiliary
molecule, which acts as a bridge between IRS-1 and the receptors for
insulin, IGF-1, and IL-4. Consistent with the latter possibility,
following IL-4 stimulation of D10 T lymphocytes, 4PS has recently been
shown to be present in a complex with the IL-4 receptor and the
tyrosine kinase JAK1(68) . Thus, JAK1 could serve as an
auxiliary molecule between the IL-4 receptor and 4PS in the IL-4
system. However, two-hybrid analysis suggests a direct interaction
between insulin receptor and IRS-1 requiring the NPXY
motif(69) . Thus the mechanism of the JAK2-IRS-1 interaction may
be distinct from that of the insulin receptor-IRS-1 interaction.
Role of PI 3`-Kinase in Cellular Signaling
PI
3`-kinase has been implicated in cellular signaling for a number of
ligands that activate receptor and nonreceptor tyrosine kinases (70) including erythropoietin, IL-3, IL-5, and
granulocyte-macrophage colony stimulating
factor(71, 72) , cytokines that induce JAK2 tyrosine
kinase activity. PI 3`-kinase consists of two subunits, a regulatory
85-kDa subunit, and a catalytic 110-kDa subunit. In insulin-stimulated
cells, association of PI 3`-kinase with tyrosyl phosphorylated IRS-1
activates PI 3`-kinase (73). PI 3`-kinase phosphorylates
phosphatidylinositol, phosphatidylinositol 4-phosphate, and
phosphatidylinositol 4,5-bisphosphate at the 3`-position of the
inositol ring(74) . Recently PI 3`-kinase has been shown to be a
dual-specificity kinase. In addition to lipid kinase activity, the
activated kinase possesses serine kinase activity (75) capable
of insulin-dependent serine phosphorylation of the 85-kDa subunit of PI
3`-kinase and IRS-1 (76). Autophosphorylation on serine inhibits the
lipid kinase activity of PI 3`-kinase and may serve an autoregulatory
role. The exact function of PI 3`-kinase in the regulation of cellular
metabolism and growth is unknown. However, several lines of evidence
indicate that PI 3`-kinase may regulate intracellular trafficking of
proteins(77, 78, 79, 80) . GH is known
to effect the translocation of GLUT 4 glucose transporters to the
plasma membrane(81) . Therefore, perhaps the most relevant
evidence linking PI 3`-kinase and vesicular transport is the
demonstration in 3T3-L1 adipocytes, a sister cell line of 3T3-F442A
fibroblasts used in this report, that the PI 3`-kinase inhibitor
LY294002 blocks insulin-induced glucose transport by inhibiting
insulin-stimulated GLUT 4 glucose transporter
translocation(10) . PI 3`-kinase activation has also been shown
to be required for insulin-dependent DNA synthesis and stimulation of
p70 kinase, an enzyme implicated in cell cycle
progression(10) . Whether PI 3`-kinase is involved in
GH-dependent regulation of glucose transport, p70 kinase
activation, stimulation of DNA synthesis, or other cellular functions
awaits further investigation.
Conclusion
These results showing that GH
stimulates both tyrosyl phosphorylation of IRS-1 and association of PI
3`-kinase with IRS-1 provide evidence that IRS-1 serves as a signaling
molecule for GH. Furthermore, demonstration that LIF and IFN-, two
cytokines that, like GH, activate JAK2, stimulate tyrosyl
phosphorylation of IRS-1 suggests that signaling through IRS-1 may be
common to multiple members of the cytokine/hematopoietin receptor
family that activates JAK2. Thus at least some of the signaling
pathways originating at IRS-1 are likely to be shared by these ligands.
The determination that the region of GHR required for GH-mediated
tyrosyl phosphorylation and activation of JAK2 is also required for the
tyrosyl phosphorylation of IRS-1, in conjunction with the correlation
between JAK2 tyrosyl phosphorylation and IRS-1 tyrosyl phosphorylation,
suggests that IRS-1 may primarily interact with JAK2 or an auxiliary
protein and at least for GHR have only minor interactions with the
actual ligand receptor that mediates JAK2 activation and ultimately
IRS-1 tyrosyl phosphorylation.
, interferon-; CHO,
Chinese hamster ovary; hGH, human growth hormone; IRS-1, insulin
receptor substrate-1; 4PS, interleukin-4-induced phosphotyrosine
substrate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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T. Yamauchi, Y. Kaburagi, K. Ueki, Y. Tsuji, G. R. Stark, I. M. Kerr, T. Tsushima, Y. Akanuma, I. Komuro, K. Tobe, et al. Growth Hormone and Prolactin Stimulate Tyrosine Phosphorylation of Insulin Receptor Substrate-1, -2, and -3, Their Association with p85 Phosphatidylinositol 3-Kinase (PI3-kinase), and Concomitantly PI3-kinase Activation via JAK2 Kinase J. Biol. Chem., June 19, 1998; 273(25): 15719 - 15726. [Abstract] [Full Text] [PDF]
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R. G. Richards, M. P. Walker, J. Sebastian, and R. P. DiAugustine Insulin-like Growth Factor-1 (IGF-1) Receptor-Insulin Receptor Substrate Complexes in the Uterus. ALTERED SIGNALING RESPONSE TO ESTRADIOL IN THE IGF-1m/m MOUSE J. Biol. Chem., May 8, 1998; 273(19): 11962 - 11969. [Abstract] [Full Text] [PDF]
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T. Zhu, E. L. K. Goh, and P. E. Lobie Growth Hormone Stimulates the Tyrosine Phosphorylation and Association of p125 Focal Adhesion Kinase (FAK) with JAK2. FAK IS NOT REQUIRED FOR STAT-MEDIATED TRANSCRIPTION J. Biol. Chem., April 24, 1998; 273(17): 10682 - 10689. [Abstract] [Full Text] [PDF]
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T.-W. L. Gong, D. J. Meyer, J. Liao, C. L. Hodge, G. S. Campbell, X. Wang, N. Billestrup, C. Carter-Su, and J. Schwartz Regulation of Glucose Transport and c-fos and egr-1 Expression in Cells with Mutated or Endogenous Growth Hormone Receptors Endocrinology, April 1, 1998; 139(4): 1863 - 1871. [Abstract] [Full Text] [PDF]
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D. W. Love, A. J. Whatmore, P. E. Clayton, and C. M. Silva Growth Hormone Stimulation of the Mitogen-Activated Protein Kinase Pathway Is Cell Type Specific Endocrinology, April 1, 1998; 139(4): 1965 - 1971. [Abstract] [Full Text] [PDF]
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S. Ali and S. Ali Prolactin Receptor Regulates Stat5 Tyrosine Phosphorylation and Nuclear Translocation by Two Separate Pathways J. Biol. Chem., March 27, 1998; 273(13): 7709 - 7716. [Abstract] [Full Text] [PDF]
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M. R. Stofega, H. Wang, A. Ullrich, and C. Carter-Su Growth Hormone Regulation of SIRP and SHP-2 Tyrosyl Phosphorylation and Association J. Biol. Chem., March 20, 1998; 273(12): 7112 - 7117. [Abstract] [Full Text] [PDF]
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D. Guo, J. D. Dunbar, C. H. Yang, L. M. Pfeffer, and D. B. Donner Induction of Jak/STAT Signaling by Activation of the Type 1 TNF Receptor J. Immunol., March 15, 1998; 160(6): 2742 - 2750. [Abstract] [Full Text] [PDF]
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C. Le Stunff and P. Rotwein Growth Hormone Stimulates Interferon Regulatory Factor-1 Gene Expression in the Liver Endocrinology, March 1, 1998; 139(3): 859 - 866. [Abstract] [Full Text] [PDF]
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P. Gual, V. Baron, V. Lequoy, and E. Van Obberghen Interaction of Janus Kinases JAK-1 and JAK-2 with the Insulin Receptor and the Insulin-Like Growth Factor-1 Receptor Endocrinology, March 1, 1998; 139(3): 884 - 893. [Abstract] [Full Text] [PDF]
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T.-Z. Su, M. Wang, L.-J. Syu, A. R. Saltiel, and D. L. Oxender Regulation of System A Amino Acid Transport in 3T3-L1 Adipocytes by Insulin J. Biol. Chem., February 6, 1998; 273(6): 3173 - 3179. [Abstract] [Full Text] [PDF]
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C. Ohlsson, B.-A. Bengtsson, O. G. P. Isaksson, T. T. Andreassen, and M. C. Slootweg Growth Hormone and Bone Endocr. Rev., February 1, 1998; 19(1): 55 - 79. [Abstract] [Full Text]
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S.-O. Kim, J. Jiang, W. Yi, G.-S. Feng, and S. J. Frank Involvement of the Src Homology 2-containing Tyrosine Phosphatase SHP-2 in Growth Hormone Signaling J. Biol. Chem., January 23, 1998; 273(4): 2344 - 2354. [Abstract] [Full Text] [PDF]
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J. S. Freeth, C. M. Silva, A. J. Whatmore, and P. E. Clayton Activation of the Signal Transducers and Activators of Transcription Signaling Pathway by Growth Hormone (GH) in Skin Fibroblasts from Normal and GH Binding Protein-Positive Laron Syndrome Children Endocrinology, January 1, 1998; 139(1): 20 - 28. [Abstract] [Full Text] [PDF]
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C. Bjorbak, S. Uotani, B. da Silva, and J. S. Flier Divergent Signaling Capacities of the Long and Short Isoforms of the Leptin Receptor J. Biol. Chem., December 19, 1997; 272(51): 32686 - 32695. [Abstract] [Full Text] [PDF]
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M. Yamada, H. Ohnishi, S.-i. Sano, A. Nakatani, T. Ikeuchi, and H. Hatanaka Insulin Receptor Substrate (IRS)-1 and IRS-2 Are Tyrosine-phosphorylated and Associated with Phosphatidylinositol 3-Kinase in Response to Brain-derived Neurotrophic Factor in Cultured Cerebral Cortical Neurons J. Biol. Chem., November 28, 1997; 272(48): 30334 - 30339. [Abstract] [Full Text] [PDF]
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F. Verdier, S. Chretien, C. Billat, S. Gisselbrecht, C. Lacombe, and P. Mayeux Erythropoietin Induces the Tyrosine Phosphorylation of Insulin Receptor Substrate-2. AN ALTERNATE PATHWAY FOR ERYTHROPOIETIN-INDUCED PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATION J. Biol. Chem., October 17, 1997; 272(42): 26173 - 26178. [Abstract] [Full Text] [PDF]
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 S. Uddin, E. N. Fish, D. Sher, C. Gardziola, O. R. Colamonici, M. Kellum, P. M. Pitha, M. F. White, and L. C. Platanias The IRS-Pathway Operates Distinctively From the Stat-Pathway in Hematopoietic Cells and Transduces Common and Distinct Signals During Engagement of the Insulin or Interferon-alpha Receptors Blood, October 1, 1997; 90(7): 2574 - 2582. [Abstract] [Full Text] [PDF]
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M. S. Burfoot, N. C. Rogers, D. Watling, J. M. Smith, S. Pons, G. Paonessaw, S. Pellegrini, M. F. White, and I. M. Kerr Janus Kinase-dependent Activation of Insulin Receptor Substrate 1 in Response to Interleukin-4, Oncostatin M, and the Interferons J. Biol. Chem., September 26, 1997; 272(39): 24183 - 24190. [Abstract] [Full Text] [PDF]
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P. Berhanu, C. Anderson, M. Hickman, and T. P. Ciaraldi Insulin Signal Transduction by a Mutant Human Insulin Receptor Lacking the NPEY Sequence. EVIDENCE FOR AN ALTERNATE MITOGENIC SIGNALING PATHWAY THAT IS INDEPENDENT OF Shc PHOSPHORYLATION J. Biol. Chem., September 5, 1997; 272(36): 22884 - 22890. [Abstract] [Full Text] [PDF]
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H. Sakaue, W. Ogawa, M. Takata, S. Kuroda, K. Kotani, M. Matsumoto, M. Sakaue, S. Nishio, H. Ueno, and M. Kasuga Phosphoinositide 3-Kinase Is Required for Insulin-Induced but Not for Growth Hormone- or Hyperosmolarity-Induced Glucose Uptake in 3T3-L1 Adipocytes Mol. Endocrinol., September 1, 1997; 11(10): 1552 - 1562. [Abstract] [Full Text]
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E. L. K. Goh, T. J. Pircher, T. J. J. Wood, G. Norstedt, R. Graichen, and P. E. Lobie Growth Hormone-Induced Reorganization of the Actin Cytoskeleton Is Not Required for STAT5 (Signal Transducer and Activator of Transcription-5)-Mediated Transcription Endocrinology, August 1, 1997; 138(8): 3207 - 3215. [Abstract] [Full Text] [PDF]
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Y. Wang, K. K. Kuropatwinski, D. W. White, T. S. Hawley, R. G. Hawley, L. A. Tartaglia, and H. Baumann Leptin Receptor Action in Hepatic Cells J. Biol. Chem., June 27, 1997; 272(26): 16216 - 16223. [Abstract] [Full Text] [PDF]
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D. Sawka-Verhelle, V. Baron, I. Mothe, C. Filloux, M. F. White, and E. Van Obberghen Tyr624 and Tyr628 in Insulin Receptor Substrate-2 Mediate Its Association with the Insulin Receptor J. Biol. Chem., June 27, 1997; 272(26): 16414 - 16420. [Abstract] [Full Text] [PDF]
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W. P. Schiemann, J. L. Bartoe, and N. M. Nathanson Box 3-independent Signaling Mechanisms Are Involved in Leukemia Inhibitory Factor Receptor alpha - and gp130-mediated Stimulation of Mitogen-activated Protein Kinase. EVIDENCE FOR PARTICIPATION OF MULTIPLE SIGNALING PATHWAYS WHICH CONVERGE AT Ras J. Biol. Chem., June 27, 1997; 272(26): 16631 - 16636. [Abstract] [Full Text] [PDF]
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J. J. Berlanga, O. Gualillo, H. Buteau, M. Applanat, P. A. Kelly, and M. Edery Prolactin Activates Tyrosyl Phosphorylation of Insulin Receptor Substrate 1and Phosphatidylinositol-3-OH Kinase J. Biol. Chem., January 24, 1997; 272(4): 2050 - 2052. [Abstract] [Full Text] [PDF]
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M. J. Welham, H. Bone, M. Levings, L. Learmonth, L.-M. Wang, K. B. Leslie, J. H. Pierce, and J. W. Schrader Insulin Receptor Substrate-2 Is the Major 170-kDa Protein Phosphorylated on Tyrosine in Response to Cytokines in Murine Lymphohemopoietic Cells J. Biol. Chem., January 10, 1997; 272(2): 1377 - 1381. [Abstract] [Full Text] [PDF]
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L. S. Argetsinger, G. Norstedt, N. Billestrup, M. F. White, and C. Carter-Su Growth Hormone, Interferon-gamma , and Leukemia Inhibitory Factor Utilize Insulin Receptor Substrate-2 in Intracellular Signaling J. Biol. Chem., November 15, 1996; 271(46): 29415 - 29421. [Abstract] [Full Text] [PDF]
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A. Kowalski-Chauvel, L. Pradayrol, N. Vaysse, and C. Seva Gastrin Stimulates Tyrosine Phosphorylation of Insulin Receptor Substrate 1and Its Association with Grb2 and the Phosphatidylinositol 3-Kinase J. Biol. Chem., October 18, 1996; 271(42): 26356 - 26361. [Abstract] [Full Text] [PDF]
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L. Yenush, K. J. Makati, J. Smith-Hall, O. Ishibashi, M. G. Myers Jr., and M. F. White The Pleckstrin Homology Domain Is the Principle Link between the Insulin Receptor and IRS-1 J. Biol. Chem., September 27, 1996; 271(39): 24300 - 24306. [Abstract] [Full Text] [PDF]
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M. J.A. Saad, C. R.O. Carvalho, A. C.P. Thirone, and L. A. Velloso Insulin Induces Tyrosine Phosphorylation of JAK2 in Insulin-sensitive Tissues of the Intact Rat J. Biol. Chem., September 6, 1996; 271(36): 22100 - 22104. [Abstract] [Full Text] [PDF]
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S. Li, M. Resnicoff, and R. Baserga Effect of Mutations at Serines 1280-1283 on the Mitogenic and Transforming Activities of the Insulin-like Growth Factor I Receptor J. Biol. Chem., May 24, 1996; 271(21): 12254 - 12260. [Abstract] [Full Text] [PDF]
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X. J. Sun, S. Pons, T. Asano, M. G. Myers Jr., E. Glasheen, and M. F. White The Fyn Tyrosine Kinase Binds Irs-1 and Forms a Distinct Signaling Complex during Insulin Stimulation J. Biol. Chem., May 3, 1996; 271(18): 10583 - 10587. [Abstract] [Full Text] [PDF]
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D. Sawka-Verhelle, S. Tartare-Deckert, M. F. White, and E. Van Obberghen Insulin Receptor Substrate-2 Binds to the Insulin Receptor through Its Phosphotyrosine-binding Domain and through a Newly Identified Domain Comprising Amino Acids 591-786 J. Biol. Chem., March 15, 1996; 271(11): 5980 - 5983. [Abstract] [Full Text] [PDF]
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P. A. Ram, S.-H. Park, H. K. Choi, and D. J. Waxman Growth Hormone Activation of Stat 1, Stat 3, and Stat 5 in Rat Liver J. Biol. Chem., March 8, 1996; 271(10): 5929 - 5940. [Abstract] [Full Text] [PDF]
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M. David, E. Petricoin III, and A. C. Larner Activation of Protein Kinase A Inhibits Interferon Induction of the Jak/Stat Pathway in U266 Cells J. Biol. Chem., March 1, 1996; 271(9): 4585 - 4588. [Abstract] [Full Text] [PDF]
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T. C. Yeh, W. Ogawa, A. G. Danielsen, and R. A. Roth Characterization and Cloning of a 58/53-kDa Substrate of the Insulin Receptor Tyrosine Kinase J. Biol. Chem., February 9, 1996; 271(6): 2921 - 2928. [Abstract] [Full Text] [PDF]
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D. Guo and D. B. Donner Tumor Necrosis Factor Promotes Phosphorylation and Binding of Insulin Receptor Substrate 1 to Phosphatidylinositol 3-Kinase in 3T3-L1 Adipocytes J. Biol. Chem., January 12, 1996; 271(2): 615 - 618. [Abstract] [Full Text] [PDF]
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L. C. Platanias, S. Uddin, A. Yetter, X.-J. Sun, and M. F. White The Type I Interferon Receptor Mediates Tyrosine Phosphorylation of Insulin Receptor Substrate 2 J. Biol. Chem., January 5, 1996; 271(1): 278 - 282. [Abstract] [Full Text] [PDF]
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J. A. Johnston, L.-M. Wang, E. P. Hanson, X.-J. Sun, M. F. White, S. A. Oakes, J. H. Pierce, and J. J. O'Shea Interleukins 2, 4, 7, and 15 Stimulate Tyrosine Phosphorylation of Insulin Receptor Substrates 1 and 2 in T Cells J. Biol. Chem., December 1, 1995; 270(48): 28527 - 28530. [Abstract] [Full Text] [PDF]
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P. E. Lobie, G. Allevato, J. H. Nielsen, G. Norstedt, and N. Billestrup Requirement of Tyrosine Residues 333 and 338 of the Growth Hormone (GH) Receptor for Selected GH-stimulated Function J. Biol. Chem., September 15, 1995; 270(37): 21745 - 21750. [Abstract] [Full Text] [PDF]
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G. Piwien-Pilipuk, D. Van Mater, S. E. Ross, O. A. MacDougald, and J. Schwartz Growth Hormone Regulates Phosphorylation and Function of CCAAT/Enhancer-binding Protein beta by Modulating Akt and Glycogen Synthase Kinase-3 J. Biol. Chem., May 25, 2001; 276(22): 19664 - 19671. [Abstract] [Full Text] [PDF]
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