From the Third Department of Internal Medicine,
Faculty of Medicine, University of Tokyo, Tokyo 113, Japan, the
§ Department of Molecular Biology, Research Institute, The
Cleveland Clinic Foundation, Cleveland, Ohio 44195, the
¶ Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom,
the
Second Department of Internal Medicine, Tokyo Women's
Medical College, Tokyo 162, Japan, and the ** Institute for Diabetes
Care and Research, Asahi Life Foundation, Tokyo 100, Japan
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ABSTRACT |
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Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. We recently showed that GH mainly stimulated tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulated mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, we now show that GH was also able to induce tyrosine phosphorylation of insulin receptor substrate (IRS)-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice was IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in physiological target organ of GH, liver. We also show that PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, we show that tyrosine phosphorylation of IRS-3 was induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, we show that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects.
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INTRODUCTION |
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Growth hormone (GH)1 and prolactin (PRL) initiate their wide variety of biological effects by binding and dimerization of their membrane receptors (1, 2). The GH and PRL receptors belong to the cytokine/hematopoietin receptor superfamily, characterized by homologies in the extracellular domains and lack of intrinsic tyrosine kinase activity (1), however, ligands binding to their receptors activate JAK2 tyrosine kinase (3, 4). JAK2 tyrosine phosphorylates their receptors and JAK2 itself along with STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins translocate into the nucleus and bind to DNA, thereby activating transcription of specific genes (5). In addition, a number of intracellular key proteins have been suggested to be involved in their signaling (6).
One of the signaling molecules known to be activated by GH and PRL (7, 8) is the mitogen-activated protein kinase (MAP kinase), which is believed to play a pivotal role in the regulation of cellular growth and differentiation (9). Association of tyrosine-phosphorylated proteins with Grb2 (growth factor receptor bound protein 2) is known to represent a crucial step in the activation of MAP kinase cascade (10). In the case of GH and PRL, epidermal growth factor receptor (EGFR) and Shc have been shown to be tyrosyl phosphorylated by JAK2 and bind Grb2, leading to MAP kinase activation (11, 12).
Another signaling molecule known to be activated by GH and PRL is phosphatidylinositol 3-kinase (PI3-kinase), which may play a role in initiating insulin-like effects of GH and PRL including glucose transport, glycogenesis, anti-lipolysis, and lipogenesis (13-15), because the selective PI3-kinase inhibitor wortmannin can block at least some of these effects such as anti-lipolysis and lipogenesis (16).
Insulin receptor substrate-1 (IRS-1) is the principal substrate of the insulin receptor kinase whose molecular mass is approximately 170 kDa and has many tyrosine phosphorylation sites (17), which provides binding sites for several distinct Src homology 2 (SH2) proteins (e.g. Grb2, the 85-kDa subunit of PI3-kinase (PI3-kinase p85), Syp, Nck, and Csk) and has been shown to mediate multiple signaling pathways (18, 19). IRS-1 binds the PI3-kinase p85 when tyrosine phosphorylated, thereby activating PI3-kinase (20, 21), leading to exert metabolic effects of insulin such as glucose transport, glycogen synthesis, and anti-lipolysis (22-24).
To better understand the role of IRS-1 in vivo, we and others generated mice with a targeted disruption of the IRS-1 gene and demonstrated that they exhibited mild growth retardation and had partial resistance to the glucose-lowering effect of insulin, which was an unexpectedly mild phenotype, suggesting the presence of IRS-1 independent pathways (25, 26). In liver and also muscle extracts from IRS-1-deficient mice, tyrosine phosphorylation of IRS-2, whose molecular mass is approximately 180 kDa (25, 27, 28), was significantly induced in IRS-1-deficient mice compared with that in wild-type mice (29) and has been suggested to be the mechanism of compensation for IRS-1 deficiency in liver and muscle (30, 31). In another target tissue of insulin, adipocytes, both IRS-1 and IRS-3 (pp60) (32, 33) was suggested to play a major role in insulin-induced PI3-kinase activation, and IRS-3 (pp60) was suggested to be involved in regulating this process in the absence of IRS-1 (34).
Recently, IRS-1/IRS-2 have been shown to be tyrosine-phosphorylated and associated with PI3-kinase p85 in response to GH in primary rat adipocytes (35, 36) and in 3T3-F442A fibroblasts (37, 38). However, the following important issues remained unresolved. First, whether GH was able to induce tyrosine phosphorylation of IRS-1/IRS-2 in physiological target organ, liver, in vivo. Second, the relative contributions of IRS-1 and IRS-2 in the signaling pathways for GH to activate PI3-kinase. Third, the identity of the kinase responsible for the GH-stimulated tyrosine phosphorylation of IRS proteins. Fourth, whether additional cytokine receptor superfamily/IRS proteins signaling pathways could also operate. Using specific antibodies, we show here that GH was able to induce tyrosine phosphorylation of IRS-1/IRS-2, and that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in physiological target organ, liver, using IRS-1-deficient mice. Finally, by using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, we show that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3, their association with p85 PI3-kinase and activation of PI3-kinase. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings is to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase. A specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes, as reported in isolated rat adipocytes (16). Thus our data may provide the biochemical fundamentals to understand the insulin-like effects of GH such as anti-lipolysis.
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EXPERIMENTAL PROCEDURES |
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Materials and Mice-- The polyclonal antibody against 85-kDa subunit of PI3-kinase was from Upstate Biotechnology Inc. The monoclonal antibody against hemagglutinin (HA), 12CA5, was from Boehringer Mannheim. All other materials were obtained from the sources described (12, 31). Mice were fasted overnight and then treated as described (31).
Expression of Wild-type and Kinase-inactive JAK2, Epitope-tagged
IRS-3, GHR, and PRLR--
The CHO (Chinese hamster ovary)-PRLR cells
(approximately 1.0 × 104 receptors/cell) and
expression vector of human PRLR were kindly provided by M. Takahashi,
M. Wada, and M. Honjo. 2A/GHR (39), CHO-GHR, or CHO-PRLR cells were
transfected with or without 3 µg of WT-JAK2 plasmids or dominant
negative JAK2 (
JAK2) (40) that lacks the C terminus kinase domain
under the SR
promoter in 6-cm dishes by the LipofectAMINE method
with modification and the cells were stimulated as described (12, 39,
41). The rat IRS-3 cDNA in the pcDNA3 was modified to encode an
additional 9-amino acid HA epitope (YPYDVPDYA), after the C terminus of
IRS-3 via a recombinant polymerase chain reaction strategy (33). COS cells were transfected with 3 µg of GHR or PRLR, with or without HA-tagged IRS-3 and with or without 2 µg of WT-JAK2 or
JAK2 at 60-80% confluence in 3 ml of Dulbecco's modified Eagle's medium in
6-cm dishes by the calcium phosphate precipitation method and the cells
were stimulated as described (12).
Immunoprecipitation, Immunoblotting, and PI3-kinase Activities-- Immunoprecipitation and immunoblotting were performed as described (31). The amount of tyrosine phosphorylation or associated protein was evaluated by densitometry, as described (31). The significance of differences between the two groups was assessed using Student's unpaired t test.
Lipolysis-- 3T3-L1 adipocytes were incubated in serum-free Dulbecco's modified Eagle's medium for 16 h, and then in modified Krebs-Ringer medium at pH 7.5 containing 24 mM HEPES, 119 mM NaCl, 4.95 mM KCl, 2.54 mM CaCl2, 1.19 mM KH2PO4, 1.19 mM MgSO4, 2 mM glucose for 3 h. Lipolysis, stimulated by norepinephrine (NE) (Sigma), was measured with or without pretreatment with 100 nM wortmannin for 20 min, as proportional to glycerol released to the medium during a 30-min incubation and determined as described (16).
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RESULTS |
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GH Stimulates Tyrosine Phosphorylation of IRS-1/IRS-2 in Liver, in
Vivo--
To determine if IRS-1 and/or IRS-2 are
tyrosine-phosphorylated following stimulation with GH in its
physiological target organ, liver, in vivo, mice were
injected with 5 µg/g body weight of GH and 7.5 min after the
injection, solubilized proteins from livers of wild-type and IRS-1
deficient mice were immunoprecipitated with anti-IRS-1 antibody
(IRS-1) or anti-IRS-2 antibody (
IRS-2), and immunoblotted with
PY. IRS-1 (molecular mass 170 kDa) was tyrosine phosphorylated in
response to GH in wild-type mice (Fig. 1A, lane 3),
however, to a much lesser degree than in response to insulin (Fig.
1A, lane 2). IRS-2 was tyrosine phosphorylated in response
to GH most in IRS-1 deficient mice (Fig. 1B, lane 6),
however, to a much lesser degree than in response to insulin (Fig.
1B, lane 5). GH-dependent tyrosine
phosphorylation of IRS-1/IRS-2 reached the maximum level at 7.5 min
(data not shown).
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GH-stimulated PI3-kinase Activity in Liver-- We measured PI3-kinase activity in livers from wild-type and IRS-1-deficient mice in the immunoprecipitates with the monoclonal antibody against phosphotyrosine (4G10). 7.5 min after GH injection and 2 min after insulin injection, maximal levels in kinase activity were reached (data not shown). In liver of both wild-type and IRS-1-deficient mice, insulin injection resulted in a 12-13-fold increase in PI-3 kinase activity in 4G10 immunoprecipitates, while GH caused an only 2-3-fold increase (Fig. 2).
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Immunological Detection of GH-stimulated Tyrosine-phosphorylated
Proteins Associated with PI3-kinase p85--
The molecular mechanism
by which GH activates PI3-kinase were determined by immunoblotting with
the monoclonal anti-phosphotyrosine antibody (4G10) of anti-PI3-kinase
p85 immunoprecipitates of livers 7.5 min after GH injection and 2 min
after insulin injection. Interestingly, while the 180-kDa protein was
mainly tyrosine phosphorylated and associated with Grb2 in wild-type
mice when injected with GH (12), in p85 immunoprecipitates of
GH-injected wild-type mice, a 170-kDa tyrosine-phosphorylated protein
was mainly present (Fig. 3A, lane
4), indicating that tyrosine phosphorylation of IRS-1, but neither
IRS-2 nor EGFR, may be a major mechanism for GH-induced PI3-kinase
activation in physiological target organ, liver. In wild-type mice, the
amount of pp170 associated with PI3-kinase p85 in response to GH was
much less than that in response to insulin, which was consistent with
that the GH-stimulated tyrosine phosphorylation of IRS-1 was much less
than that stimulated with insulin (Fig. 1A, lanes 2 and 3). To confirm these observations, liver extracts
of GH-treated wild-type mice for 7.5 min were incubated with the
glutathione S-transferase fusion proteins containing the
entire p85 PI3-kinase. Glutathione S-transferase-p85 only associated with pp170 (IRS-1) (data not shown). In IRS-1-deficient mice, the amount of pp180 (IRS-2) associated with PI3-kinase p85 in
response to GH was also much less than that in response to insulin
(Fig. 3A, lanes 5 and 6).
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Complementation with a WT-JAK2 Expression Plasmid into
JAK2-deficient Cell Line Restored GH-dependent Tyrosine
Phosphorylation of IRS-1/IRS-2, Their Association with p85 PI3-kinase,
and Activation of PI3-kinase--
We next examined whether GH-induced
tyrosine phosphorylation of IRS-1/IRS-2, IRS-1 association with p85
PI3-kinase, and PI3-kinase activation are dependent on JAK2 by using
2A/GHR cells, which lack JAK2 (39). GH was unable to induce these
responses in
2A/GHR, whereas complementation with a WT-JAK2
expression plasmid restored them (Fig.
4). These data suggested that JAK2 is
required for the GH-dependent tyrosine phosphorylation of
IRS-1 and -2, their association with p85 PI3-kinase, and activation of
PI3-kinase.
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PRL Was Also Able to Induce Tyrosine Phosphorylation of IRS-2-- Recently, IRS-1 also has been shown to be tyrosine phosphorylated in response to another cytokine superfamily member PRL in 293-PRL receptor cells (42). Thus we next examined whether PRL was able to induce tyrosine phosphorylation of IRS-2 by immunoblotting with the monoclonal anti-phosphotyrosine antibody (4G10) of anti-IRS-2 immunoprecipitates of COS cells transiently transfected with PRLR and CHO-PRLR cells treated with PRL for 5 min. We found that PRL was also able to induce tyrosine phosphorylation of IRS-2 (Fig. 5D, lane 2, and Fig. 6C, lane 6).
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Complementation with a WT-JAK2 Expression Plasmid into
JAK2-deficient Cell Line Restored PRL-dependent Tyrosine
Phosphorylation of IRS-1/IRS-2, Their Association with p85 PI3-kinase,
and Activation of PI3-kinase--
We also examined whether PRL-induced
tyrosine phosphorylation of IRS-1/IRS-2, IRS-1 association with p85
PI3-kinase, and PI3-kinase activation are dependent on JAK2 by using
COS cells, in which the level of expression of JAK2 appears to be
sufficiently low that some level of coexpression of wild-type (WT)-JAK2
and PRLR is required in order to detect activation of JAK2 by PRL (Fig. 5A). Neither expression of PRLR alone nor coexpression of
kinase-inactive JAK2 (JAK2) and PRLR, but coexpression of WT-JAK2
and PRLR restored these responses (Fig. 5B, C, D, and
E, and data not shown). These data suggested that JAK2 is required
for the PRL-dependent tyrosine phosphorylation of IRS-1 and
-2, their association with p85 PI3-kinase and activation of
PI3-kinase.
Dominant Negative JAK2 Almost Completely Abolished GH- and
PRL-dependent Tyrosine Phosphorylation of IRS-1/IRS-2,
Their Association with p85 PI3-kinase, and Activation of
PI3-kinase--
We also used a dominant negative JAK2 mutant, since
the possibility of overestimation of the complemented molecule (JAK2) cannot be excluded in the above experiment. Expression of JAK2, which inhibits autophosphorylation of WT-JAK2 in a dominant negative manner (40), almost completely abolished the GH- and PRL-induced tyrosine phosphorylation of IRS-1/IRS-2, IRS-1 association with p85
PI3-kinase, and activation of PI3-kinase in CHO cells expressing their
cognate receptors (Fig. 6 and data not shown). These data suggested
that the GH- and PRL-induced tyrosine phosphorylation of IRS-1/IRS-2,
IRS-1 association with p85 PI3-kinase, and activation of PI3-kinase are
largely dependent on JAK2.
GH and PRL Were Also Able to Induce Tyrosine Phosphorylation of IRS-3 and Its Association with p85 PI3-kinase via JAK2-- Not only IRS-1 but also pp60 was shown to play a major role in insulin-induced activation of PI3-kinase in adipocytes (34). Recently, Lavan et al. (33) reported the isolation of the cDNA of pp60 which was designated as IRS-3. Thus we next examined whether GH and PRL were able to induce tyrosine phosphorylation of IRS-3 and its association with p85 PI3-kinase via JAK2 in COS cells transiently transfected with HA-tagged IRS-3 treated with GH or PRL for 5 min. We found that both GH and PRL were also able to induce tyrosine phosphorylation of IRS-3 and its association with p85 PI3-kinase via JAK2 (Fig. 7, A and B).
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GH Effect on Anti-lipolysis Is Dependent on PI3-kinase-- To study whether GH stimulation of PI3-kinase is linked to GH-induced insulin-like metabolic effect, GH-stimulated anti-lipolysis was measured with pretreatment with a specific PI3-kinase inhibitor, wortmannin. The anti-lipolytic effect of GH was measured as the ability to counteract lipolysis induced by NE at 100 nM in 3T3 L1 adipocytes. GH (0.5 µg/ml) inhibited lipolysis by 60 ± 10% (mean ± S.E., n = 3, p < 0.05), and wortmannin completely blocked this anti-lipolytic effect, as reported in isolated rat adipocytes (16). These data suggested that PI3-kinase activity was indeed required for anti-lipolysis in response to GH.
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DISCUSSION |
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GH Stimulates Tyrosine Phosphorylation of IRS-1/IRS-2 in Liver, in Vivo-- We recently showed that GH mainly stimulated tyrosine phosphorylation of EGFR and its association with Grb2, and concomitantly stimulated MAP kinase activity in liver, a major target tissue (12). Using specific antibodies, we provided the first evidence that GH was also able to induce tyrosine phosphorylation of IRS-1/IRS-2 in physiological target organ, liver, in vivo, although to a lesser extent as compared with that stimulated with insulin or tyrosine phosphorylation of EGFR stimulated with GH (Fig. 1 and Ref. 12). The degree of GH-stimulated activation of MAP kinase and PI3-kinase appeared to be correlated with the relative amount of GH-stimulated tyrosine-phosphorylated EGFR and IRS-1, respectively (Figs. 1 and 2 and Ref. 12).
Tyrosine Phosphorylation of IRS-1, but Neither IRS-2 nor EGFR, May
Be a Major Mechanism for GH-induced PI3-kinase Activation in
Physiological Target Organ, Liver--
IRS-2 couple more
sensitively to the IL-4 receptor system and less to the insulin
receptor system than IRS-1 (28). In the case of the insulin receptor
system, the degree of compensation for IRS-1 deficiency appears to be
correlated with the relative amount of tyrosine-phosphorylated IRS-2
(in IRS-1 deficient mice) to that of IRS-1 (in wild-type mice) (31).
Recently, using specific antibodies, IRS-1/IRS-2 have been shown to be
associated with PI3-kinase p85 in response to GH in primary rat
adipocytes (35, 36) and in 3T3-F442A fibroblasts (37, 38). There has
been no study, however, which reported the relative contributions of IRS-1 and IRS-2 in the signaling pathways for GH to activate
PI3-kinase. Interestingly, although EGFR was mainly tyrosine
phosphorylated and associated with Grb2 when injected with GH (12), the
major tyrosine-phosphorylated protein in anti-p85 PI3-kinase
immunoprecipitate from liver of wild-type mice was IRS-1, and IRS-2 in
IRS-1-deficient mice (Fig. 3 A) but not EGFR. Thus we
provided the evidence that tyrosine phosphorylation of IRS-1, but
neither IRS-2 nor EGFR, may be a major mechanism for GH-induced
PI3-kinase activation in physiological target organ, liver, in
vivo. The observations that GH had little effect on an increase in
PI3-kinase activity in IRS-2 immunoprecipitates from wild-type mice,
while GH caused a 2-3-fold increase in IRS-1-deficient mice (Fig.
3C) may support the central role of IRS-1 in the control of
PI3-kinase activation by GH.
Additional Cytokine Receptor Superfamily/IRS Proteins Signaling Pathways-- Recently, PRL has been shown to be able to tyrosine phosphorylate IRS-1 in 293-PRLR cells (42). We show in this study for the first time that PRL was also able to induce tyrosine phosphorylation of IRS-2 in COS cells transiently transfected with PRLR (Fig. 5D, lane 2) and in CHO-PRLR cells (Fig. 6C, lane 6). In addition to IRS-1, pp60/IRS-3 also has been shown to play a major role in insulin-induced activation of PI3-kinase in adipocytes (34). In the present study, we also show that both GH and PRL were able to induce tyrosine phosphorylation of IRS-3 and its association with p85 PI3-kinase (Fig. 7). This is the first evidence that IRS-3 can be tyrosine phosphorylated by other tyrosine kinase(s) other than insulin receptor tyrosine kinase.
The GH- and PRL-induced Tyrosine Phosphorylation of IRS-1, -2, and -3, Their Association with p85 PI3-kinase, and Activation of PI3-kinase Are Largely Dependent on JAK2-- There has been no study reporting the identity of the kinase responsible for GH-induced tyrosine phosphorylation of IRS-1/IRS-2, although Argetsinger et al. (37, 38) reported that the regions of GHR necessary for IRS-1/IRS-2 tyrosyl phosphorylation were present between amino acids 295 and 380 containing box 1 and box 2, the same as those required for JAK2 association and tyrosyl phosphorylation. By using the JAK2-deficient cell lines or expressing a dominant negative JAK2 mutant, we provided the first direct evidence that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3, their association with p85 PI3-kinase, and activation of PI3-kinase (Figs. 4-7). Tyrosine phosphorylation of IRS-1 induced by insulin, insulin-like growth factor-I, and interleukin-4 appears to require interaction between the phosphotyrosine-binding domain of IRS-1 and the tyrosine-phosphorylated NPXY motif in the ligand receptor (43-45). GHR and JAK2, however, do not contain NPXY motifs (46, 47). This may explain, at least in part, why GH was able to induce tyrosine phosphorylation of IRS-1/IRS-2, their association with p85 PI3-kinase, and activation of PI3-kinase, however, to a lesser extent as compared with that stimulated with insulin. The domains required for interactions between cytokine superfamily-JAK complexes and IRS proteins are now under investigation.
JAK/IRS Family and JAK/EGFR Pathways for Cytokine Receptor
Superfamily--
An essential role of JAK/STAT pathways is now
established for cytokine receptor superfamily (5). The roles of JAKs in
the activation of additional pathways, however, have been less well established. Burfoot et al. (5) reported that tyrosine
phosphorylation of IRS-1 mediated by the Type I interferons is
dependent on JAK1 and TYK2, and by interleukin 4 and oncostatin M is
largely JAK1-dependent, by complementation with JAK into
mutant cell lines lacking JAK (41). The possibility of overestimation
of the complemented molecule, however, cannot be excluded. Thus we also
used a dominant negative JAK2 mutant. Expression of dominant negative
JAK2 almost completely abolished GH- and PRL-induced tyrosine
phosphorylation of IRS-1 and -2, their association with p85 PI3-kinase
and activation of PI3-kinase (Fig. 6), indicating that these responses
are largely dependent on JAK2. Together with others (12, 41)
observations, our findings using a dominant negative molecule provide
the paradigm of JAK/IRS family and JAK/EGFR pathways for cytokine
receptor superfamily. STAT specificity might be determined not by each JAK but in concert with the cytoplasmic structure of the particular receptor with which it is associated. In the case of IRS family, for
example, tyrosine phosphorylation of IRS-1, but not IRS-2, is dependent
on JAK1 and Tyk2 for the -interferons, while neither IRS-1 nor
IRS-2 was detectably tyrosine phosphorylated by interferon-
, which
was able to activate JAK1 and JAK2 (41). Furthermore, we showed that
GH- and PRL-induced tyrosine phosphorylation of IRS-1, -2, and -3 are
largely dependent on JAK2 (Figs. 4-7). Thus like STAT specificity, IRS
specificity might be determined not by each JAK but by each
receptor.
The Role of PI3-kinase in Mediating Insulin-like Effects of GH-- PI3-kinase has been reported not to be required for glucose uptake stimulated by GH (48). GH-induced anti-lipolysis and lipogenesis, however, have been shown to be PI3-kinase-dependent in isolated rat adipocytes (16). We also showed that a specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes (Fig. 8). Thus PI3-kinase may play an important role in initiating at least some insulin-like effects of GH, such as anti-lipolysis and lipogenesis.
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ACKNOWLEDGEMENTS |
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We thank J. Ihle for JAK2 cDNA, K. Arai
for expression vectors of JAK2 and dominant negative JAK2 (JAK2), M. Takahashi, M. Wada, and M. Honjo for CHO-PRLR and CHO-GHR cells, and S. Kakinuma for technical assistance.
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FOOTNOTES |
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* This work was supported by a grant from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (to T. Y.), from the Ministry of Education, Science, Sports, and Culture and the Ministry of Health and Welfare of Japan (to T. K.) and a grant for diabetes research from the Taisho Pharmaceutical Co., Ltd. (to T. K.).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.
To whom correspondence should be addressed: Third Department of
Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-3815-5411 (ext:
3111); Fax: 81-3-5689-7209; E-mail:
kadowaki-3im{at}h.u-tokyo.ac.jp.
1
The abbreviations used are: GH, growth hormone;
PRL, prolactin; JAK, Janus kinase; STAT, signal transducers and
activators of transcription; MAP, mitogen-activated protein; EGFR,
epidermal growth factor receptor; IRS-1, insulin receptor substrate-1;
PI3-kinase; phosphatidylinositol 3-kinase; SH2, Src homology 2;
CHO, Chinese hamster ovary; HA, hemagglutinin; NE,
norepinephrine; PY, anti-phosphotyrosine.
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REFERENCES |
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