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Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, 13081–970 Campinas SP, Brazil
Address all correspondence and requests for reprints to: Mario J. A. Saad, M.D., Departamento de Clínica Médica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, 13081–970 Campinas SP, Brazil. E-mail: pelthi{at}correionet.com.br
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GH is known to have short term effects that mimic the actions of insulin in tissues that have been deprived of GH, including increased amino acid transport, glucose transport, and lipogenesis (9). The insulin-like effects of GH suggest that GH may use some of the same signaling molecules as those used by insulin. Consistent with this, GH has been shown to stimulate the tyrosyl phosphorylation of insulin receptor substrate-1 (IRS-1) in primary cultures of rat adipocytes (10, 11) and in 3T3-F442A fibroblasts (12), and of a related protein, IRS-2, in 3T3-F442A fibroblasts (13). Tyrosyl phosphorylation of IRS-1 and IRS-2 in response to insulin provides binding sites for specific proteins containing SH2 domains, including the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase (PI3K) (14, 15), tyrosine phosphatase SHP2 (16), and Grb2 (17). Similarly, GH promotes the binding of the 85-kDa regulatory subunit of PI3K to IRS-1 and IRS-2 in cultured cells (11, 12, 13). In 3T3-F442A fibroblasts and CHO cells expressing rat GHR, GH promotes rapid tyrosyl phosphorylation of other insulin signaling molecules, the 46-, 52-, and 66-kDa splice variants of Shc, as well as the subsequent Grb2 association with Shc proteins (18).
Specific tyrosines in GHR are not required for IRS-1, IRS-2 or Shc phosphorylation (12, 13, 18) and the nature of the GH-stimulated interaction with IRS-1 is likely to differ from the interaction with the receptors for insulin, IGF-I, and IL-4. Studies using truncated and mutated GHRs expressed in CHO cells suggest that tyrosyl phosphorylation of IRS-1, IRS-2, and Shc is dependent on JAK2 activation (12, 13, 18).
Although a recent study has shown that GH cannot induce the tyrosine phosphorylation of IRS-1 or Shc in animal tissues in vivo (19), this lack of response may represent an effect of hyperinsulinemia, because the experiments were performed with fed animals, and the basal phosphorylation of these proteins was already high before GH stimulation. It is not known whether GH induces the tyrosine phosphorylation of IRSs and Shc proteins in the tissues of fasted animals and whether these phosphorylated proteins can associate with other proteins containing SH2 domains. This aspect is interesting, because it could indicate physiological conditions under which these pathways may be used and may also facilitate the study of the regulation of these signaling reactions in pathological states characterized by GH resistance or chronic GH treatment (20, 21). In this study we examined whether acute exposure to GH could stimulate the tyrosine kinase activity of JAK2 and also assessed the effects of this hormone on IRS-1, IRS-2, Shc, and JAK2 tyrosyl phosphorylation in the liver, heart, muscle, kidney, and adipose tissue of rats in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PY, 4G10) was obtained from
Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-IRS-1
(IRS-1, C-20), anti-IRS-2 (IRS-2, A-19), anti-Shc (Shc, C-20),
anti-JAK2 (JAK2, HR-758), anti-PI3K (PI3K, p85 Z-8), anti-SHP2
(SHP2, C-18), and anti-Grb2 (Grb2, C-23) antibodies were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA).
Animals
Six-week-old male Wistar rats were provided with standard rodent
chow and water ad libitum. Food was withdrawn 12–14 h
before the experiments.
Methods
Rats were anesthetized with sodium amobarbital (15 mg/kg BW, ip)
and were used 10–15 min later, i.e. as soon as anesthesia
was assured by the loss of pedal and corneal reflexes. The abdominal
cavity was opened, the portal vein was exposed, and 0.5 ml normal
saline (zero time) or GH was injected at a dose of 1.8 mg/kg BW. At 1,
5, 15, 30, and 60 min after GH injection, liver, heart, kidney, muscle,
and adipose tissue were removed, minced coarsely, and homogenized
immediately in extraction buffer [1% Triton-X 100, and 100
mM Tris (pH 7.4) containing 100 mM sodium
pyrophosphate, 100 mM sodium fluoride, 10 mM
EDTA, 10 mM sodium vanadate, 2 mM
phenylmethylsulfonylfluoride, and 0.1 mg aprotinin/ml] at 4 C with a
Polytron PTA 20S generator (model PT 10/35, Brinkmann Instruments, Inc., Westbury, NY) operated at maximum speed for
30 sec. The extracts were centrifuged at 30,000 x g
and 4 C in a Beckman Coulter, Inc. 70.1 Ti rotor (Palo
Alto, CA) for 45 min to remove insoluble material, and the supernatant
of these tissues was used for immunoprecipitation with IRS-1,
IRS-2, Shc, JAK-2, and protein A-Sepharose 6 MB or Protein A/G
Plus (Santa Cruz Biotechnology, Inc.).
Protein analysis by immunoblotting
The precipitated proteins were treated with Laemmli sample
buffer (22) containing 100 mM dithiothreitol and heated in
a boiling water bath for 4 min, after which they were subjected to
SDS-PAGE (6% bis-acrylamide) in a Bio-Rad miniature slab gel apparatus
(Mini-Protean, Bio-Rad Laboratories, Inc., Richmond,
CA).
Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant) in a Bio-Rad miniature transfer apparatus (Mini-Protean) as described by Towbin et al. (23) except for the addition of 0.02% SDS to the transfer buffer to enhance the elution of high molecular mass proteins. Nonspecific protein binding to the nitrocellulose was reduced by preincubating the filter overnight at 4 C in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween-20). The nitrocellulose blot was incubated with antiphosphotyrosine, anti-PI3K, anti-SHP2, or anti-Grb2 antibody; diluted in blocking buffer (0.3% BSA instead of nonfat dry milk) overnight at 4 C; and then washed for 60 min with blocking buffer without milk. The blots were subsequently incubated with 2 µCi [125I]protein A (30 µCi/µg) in 10 ml blocking buffer for 2 h at room temperature and then washed again for 30 min as described above. [125I]Protein A bound to the antiphosphotyrosine and antipeptide antibodies was detected by autoradiography using preflashed Kodak XAR film (Eastman Kodak Co., Rochester, NY) with Cronex Lightning Plus intensifying screens at -80 C for 12–48 h. Band intensities were quantitated by optical densitometry (model GS 300, Hoefer Scientific, San Francisco, CA) of the developed autoradio- graphs.
JAK2 in vitro kinase activity assay
Five minutes after the infusion of saline or a very low dose of
GH (1.8 µg/kg BW) into the portal vein of fasted rats, liver extracts
were obtained and immunoprecipitated with JAK2 as described above.
The resulting immune complexes were collected on Protein A/G Plus.
The protein kinase activity of the immunoprecipitates was measured by incubating the immune complexes in 100 µl buffer containing 50 mM Tris (pH 7.5), 0.2 mM sodium vanadate, 0.1% Triton X-100, 3 mM MnCl2, and 15 µM cold ATP for 30 min at room temperature. The complexes were washed twice with cold buffer, then resuspended in Laemmli sample buffer and analyzed by SDS-PAGE (22). The incorporation of phosphate into the separated proteins was visualized by autoradiography using antiphosphotyrosine immunoblots after transfer to nitrocellulose (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IRS-1. These
immunoprecipitates were analyzed for tyrosyl phosphorylation by
immunoblotting with PY. The presence of phosphorylated IRS-1 was
detectable 1 min after GH infusion and was maximal (12-fold above
basal) 5 min after GH injection. By 15 min post-GH, the phosphorylation
was 50% of the maximum and remained elevated (3-fold above basal) for
up to 60 min after administration of the hormone (Fig. 1A
). This increased tyrosyl
phosphorylation was also observed 2 h after GH administration (20, 21).
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IRS-1 antibody were subsequently incubated with antibodies
against the 85-kDa subunit of PI3K, we observed that the intensity of
the bands increased after GH stimulation. This increase paralleled that
in IRS-1 phosphorylation and was consistent with a stable association
of IRS-1 and PI3K (Fig. 1B
). The association was maximal (8-fold above
basal) 5 min after GH infusion and remained elevated (1.2-fold above
basal) for up to 60 min after GH administration.
SHP2 is an SH2 domain-containing tyrosine phosphatase that associates
with the carboxyl-terminal tyrosine phosphorylation sites of IRS-1
after insulin stimulation (16). To determine whether SHP2 could
interact with IRS-1 in a similar manner after stimulation by GH, the
same blots were incubated with antibody against SHP2. There was a clear
increase in the association between IRS-1 and SHP2 that reached a
maximum (15-fold above basal) 5 min after GH injection (Fig. 1C). This
association gradually declined over the next 60 min.
Tyrosyl phosphorylation of IRS-1 in response to insulin provides
binding sites for Grb2, a protein linked to mitogenic pathways (17).
Similarly, there was a GH-dependent increase in the amount of Grb2 that
coprecipitated with IRS-1. This association was maximal (6-fold above
basal) 5 min after GH administration and declined to 50% of the
maximum after 60 min (Fig. 1D).
To determine the expression of IRS-1 in liver during the time course,
we studied the IRS-1 protein levels before and after GH injection.
Figure 1E shows that there was no change in protein levels after
administration of the hormone.
GH stimulates tyrosyl phosphorylation of IRS-2 and the association
of this substrate with PI3K, SHP2, and Grb2
IRS-2 has substantial structural similarity to IRS-1, including
multiple potential tyrosyl phosphorylation sites that can be tyrosyl
phosphorylated after stimulation of 3T3-F442A fibroblasts with GH (13).
To determine whether IRS-2 was tyrosyl phosphorylated in response to
GH, rats received GH via the portal vein, and liver extracts were
prepared and immunoprecipitated with IRS-2 antibody and
immunoblotted with PY. Increased tyrosyl phosphorylation of a
protein with a Mr appropriate for IRS-2
(180,000–190,000) was detected within 1 min post-GH, with maximum
stimulation (10-fold above basal) detected at 5 min (Fig. 1F). IRS-2
tyrosyl phosphorylation subsequently declined to 20% of the maximum
after 30 min.
Conserved sites between IRS-1 and IRS-2 include those previously seen
to bind PI3K, Grb2, and SHP2. PI3K is known to bind to IRS-2 and is
activated after insulin stimulation (15). GH also promotes the
association of IRS-2 with both PI3K and SHP2 in 3T3-F442A fibroblasts
(13). To examine whether GH could induce the association of PI3K, SHP2,
and Grb2 with IRS-2, solubilized proteins from liver stimulated with GH
were immunoprecipitated with IRS-2 antibody and subsequently
incubated for different times with PI3K, SHP2, or Grb2. The
results showed that GH promoted the association of IRS-2 with PI3K
(Fig. 1G), SHP2 (Fig. 1H), and Grb2 (Fig. 1I) and that this response
paralleled the increase in IRS-2 phosphorylation.
As with IRS-1, there was no change in the levels of IRS-2 protein in
liver after GH stimulation during the time-course experiments (Fig. 1J).
GH stimulates tyrosyl phosphorylation of Shc in rat liver
As the involvement of Shc in tyrosine kinase signaling pathways
appears to require its phosphorylation, and considering that GH has
been demonstrated to promote the tyrosyl phosphorylation of Shc
proteins in 3T3-F442A fibroblasts (18), we examined whether GH could
induce Shc tyrosine phosphorylation in rat liver. Liver extracts were
removed and homogenized after portal vein infusion of GH. The
solubilized proteins were immunoprecipitated with Shc, and the
presence of phosphorylated tyrosines was assessed by Western blotting
with PY (Fig. 2A). We have recently
demonstrated that the 52-kDa Shc isoform has a higher level of tyrosine
phosphorylation than the 46-kDa species when stimulated by insulin
(25), probably as a consequence of the higher amounts of the former
compared with those of other Shc isoforms in rat tissues. We observed
similar results when the tyrosyl phosphorylation of this substrate was
induced by GH. Increased tyrosyl phosphorylation of a protein migrating
at a Mr of approximately 52,000 (appropriate for
Shc) was observed within 1 min and was maximal (8-fold above basal) 15
min after the infusion of GH. Shc tyrosine phosphorylation decreased
within 30 min and was not different from the basal level at the end of
the first hour. There was no detectable tyrosyl phosphorylation in the
46- and 66-kDa Shc isoforms. When the same blot was reprobed with
Shc antibody, p52 was the predominant band, and p46 and p66 were
barely detected.
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Shc and Western blotted with
Grb2. A protein recognized by Grb2 in Western blots and migrating
with the appropriate size for Grb2 (Mr, 23,000)
was precipitated by Shc in a GH-dependent fashion (Fig. 2B). The
association of Shc/Grb2 was maximal (12-fold above basal) 15 min after
GH stimulation and had almost vanished at the end of the first
hour.
The effect of GH on Shc protein levels in liver was examined by
immunoprecipitation and immunoblotting techniques, using anti-Shc
antibody. As shown in Fig. 2C, there was no change in the level of this
protein after acute GH stimulation.
GH stimulates tyrosyl phosphorylation of JAK2 and stimulates JAK2
kinase activity toward IRS-1 in rat liver
To determine whether GH stimulates the tyrosine phosphorylation of
JAK2 in vivo, we performed a time-course experiment after
the administration of GH via the portal vein. As shown in Fig. 3A, solubilized proteins from rat liver
were immunoprecipitated with JAK2 and immunoblotted with PY. By 1
min after exposure to GH there was an increase in the phosphorylation
of a protein with a Mr of 130,000 (appropriate
for JAK2). Liver JAK2 tyrosine phosphorylation was maximal (16-fold
above basal) 5 min after GH injection, although the level of
phosphorylation decreased after 15 min and remained 2-fold above basal
at 60 min. Immunoblotting the same membranes with JAK2 showed that
there was no change in the level of JAK2 protein during the time-course
experiments (Fig. 3B).
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JAK2 and blotted
with IRS-1, a band corresponding to IRS-1 was detected in the basal
state. After stimulation with GH, there was a 2.3-fold increase in the
intensity of this band (Fig. 3C), showing that JAK2 interacts with
IRS-1 and forms stable complexes after exposure to GH.
To test whether JAK2 kinase activity could be stimulated by GH, we
measured enzyme autophosphorylation in vitro by
immunoprecipitating liver extracts (with or without a low dose of GH)
with JAK2 and performed an in vitro kinase assay using
ATP, as described above. JAK2 kinase activity was increased
significantly in liver extracts after a portal infusion of GH, as
demonstrated by an increase in JAK2 autophosphorylation. There was also
an upper band that was phosphorylated after GH infusion in
vivo and addition of ATP in vitro (Fig. 3D).
Immunoblotting with IRS-1 showed that the latter band corresponded
to IRS-1, which was probably associated with JAK2 and was tyrosine
phosphorylated by this kinase (data not shown).
GH stimulates tyrosyl phosphorylation of IRS-1, IRS-2, Shc, and
JAK2 in heart, kidney, muscle, and adipose tissue of rats
To determine whether the same effects of GH could be observed in
other tissues, fragments of heart, kidney, muscle, and adipose tissues
were extracted 5 min after the injection of GH and immunoprecipitated
with IRS-1 (Fig. 4A), IRS-2 (Fig. 4B), Shc (Fig. 4C), and JAK2 (Fig. 4D). The behavior of these
proteins in these tissues was similar to that seen in liver,
i.e. an increase in the tyrosyl phosphorylation of IRS-1,
IRS-2, Shc, and JAK2 after GH infusion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IRS-1 is a major cytoplasmic substrate of the insulin receptor (32), and the results presented here show that GH treatment leads to rapid changes in IRS-1 tyrosine phosphorylation in vivo, in agreement with studies using cultured adipocytes (10, 11) and 3T3-F442A fibroblasts (12). In contrast, in a recent study using liver and muscle from fed animals (19), the tyrosine phosphorylation of IRS-1 did not increase after GH stimulation. This discrepancy may reflect the fact that the high levels of insulin in nonfasted animals maintain IRS-1 sufficiently phosphorylated so that no effect of GH is seen. Despite the high basal phosphorylation of IRS-1 in fed animals, the administration of insulin can still increase the tyrosine phosphorylation of this substrate. This observation agrees with the finding that in cultured cells the phosphorylation of IRS-1 was stimulated much more in response to insulin than to a saturating concentration of GH (10, 12). Greater stimulation of IRS-1 by insulin than by GH may also reflect a difference in the relative affinities of IRS-1 for the insulin receptor kinase and GH-activated kinase. In this regard, the interaction of these two hormones may change this pattern. Chronic administration of GH, which increases basal IRS-1 tyrosine phosphorylation in rat liver, attenuates the stimulation of this substrate by insulin (20, 21). The basal increase in IRS-1 tyrosine phosphorylation observed in the liver of rats exposed to chronic GH administration may be related to GH itself or to the high IGF-I levels and/or high insulin levels that these animals presented. Under physiological conditions, GH seems to stimulate IRS-1 tyrosine phosphorylation when plasma insulin levels are low.
The results of the present study and those of a recent report on IRS-2 tyrosyl phosphorylation in 3T3-F442A fibroblasts (13) in response to GH suggest that both IRS family members have a role in GH signaling. Clearly, there is some overlap in function between IRS-1 and IRS-2, as both bind PI3K, Grb2, and SHP2 in response to GH. Although no downstream signaling molecules unique to either IRS-1 or IRS-2 have yet been identified, the potential for such molecules exists, because IRS-2 contains nine possible phosphorylation sites not shared with IRS-1, and IRS-1 contains five such sites not shared with IRS-2 (15). Distinct roles for IRS-1 and IRS-2 in GH signaling could arise from variations in the tissue expression of IRS-1 and IRS-2 (15).
In insulin-stimulated cells, the association of PI3K with
tyrosyl-phosphorylated IRS-1 (33) and tyrosyl-phosphorylated IRS-2 (15)
activates this enzyme. Thus, the ability of GH to stimulate the
association of IRS-1 and IRS-2 with the 85-kDa regulatory subunit
suggests that GH activates PI3K. A potential role for PI3K in
activating the insulin-like metabolic effects of GH is suggested by the
finding that the PI3K inhibitor wortmannin blocks the ability of GH to
stimulate lipid synthesis (34). In contrast, a recent study has shown
that PI3K activity stimulated by GH has no effect on glucose uptake or
on the trans-location of GLUT4 (35). On the other hand, PI3K
may be involved in the regulation of protein kinase C.
Phosphatidylinositol 3,4-biphosphate and phosphatidylinositol
3,4,5-trisphosphate, products of PI3K, have recently been shown to
activate the Ca+2-independent protein kinase C isoforms
, 
, and 
(36). The observations that GH induces the
trans-location of the isoform of protein kinase C from
the cytoplasm to the membrane in 3T3-F442A fibroblasts (37) and that
the PI3K inhibitor wortmannin inhibits GH-dependent diacylglycerol
formation in rat adipocytes (38) raise the possibility that in some
cell types, PI3K may be involved in the GH-dependent activation of
protein kinase C.
The role of SHP2 in GH signaling has been examined by several approaches, but is still not well defined. SHP2 is probably activated during association with IRS-1 and IRS-2 and may dephosphorylate signaling intermediates located either in the IRS-1 signaling complex or at distant sites, thus down-regulating signaling (39). It seems unlikely that SHP2 bound to IRS-1 or IRS-2 is the primary phosphatase responsible for dephosphorylating JAK2 and/or GHR. The overexpression of a catalytically inactive form of SHP2 blocks, rather than stimulates, the induction by PRL of a ß-casein reporter gene; the latter is a JAK2- and PRL-dependent event (40). Thus, the role of SHP2 in GH signaling seems to include the same functions regulated by this protein when activated by insulin. SHP2 has been implicated in insulin-, IGF-I-, and epidermal growth factor-dependent stimulation of Ras, MAP kinase, DNA synthesis, and c-fos reporter gene expression (41, 42, 43, 44). These responses are blocked by the overexpression of a catalytically inactive form of SHP2, suggesting that SHP2 is a positive regulator of these functions.
Our data indicate that in addition to its important role in coupling to IRS-1 signaling pathway, GH activates pathways involving Shc. This conclusion is in accordance with a report showing that in 3T3-F442A fibroblasts there is an increase in Shc tyrosine phosphorylation after GH treatment (18). Shc is thought to function as an adaptor molecule to recruit Grb2-mSos1 complexes to the activated receptor (45). The nucleotide exchange factor mSos1 then promotes the formation of p21 Ras (GTP), thereby initiating a cascade of phosphorylation events that culminates with the phosphorylation of specific transcription factors in the nucleus (45, 46). The finding in this study that Grb2 coprecipitates with both the IRSs and Shc proteins is consistent with GH activation of MAP kinase. These results suggest that in addition to the mitogenic effects that are induced by IGF (insulin growth factor) after stimulation with GH, GH can play a direct pivotal role in the regulation of cellular growth and differentiation.
Some of the phosphorylations induced by GH and described here in animal tissues are catalyzed by a GHR-associated kinase identified as JAK2 (8, 47, 48), whereas others are catalyzed by downstream kinases (7). Our experiments have shown that in liver, the time course of IRS-1 tyrosyl phosphorylation reflected the JAK2 tyrosyl phosphorylation induced by GH, and this correlation suggests that JAK2 activation may be necessary for IRS-1 tyrosyl phosphorylation. Moreover, coimmunoprecipitation between JAK2 and IRS-1 and the use of an in vitro kinase assay demonstrated that a GH-activated kinase, presumably JAK2, was significantly increased after a portal infusion of GH. Consistent with the involvement of JAK2 in IRS-1 tyrosyl phosphorylation, a previous study demonstrated that the abilities of various mutants of the GHR to mediate GH-dependent tyrosyl phosphorylation of JAK2 correlated with the amount of IRS-1 tyrosyl phosphorylation detected (12).
The increased tyrosyl phosphorylation of IRS-1, IRS-2, JAK2, and Shc in liver, heart, kidney, muscle, and adipose tissue after GH stimulation agrees with the finding that the GHR is a protein expressed in the cell membrane of all of these tissues (4, 5). The ability of GH to activate these signaling pathways in different tissues allows this hormone to exert its diverse metabolic and growth effects.
In summary, our results show that IRSs and Shc proteins serve as signaling molecules for GH in fasted rat tissues. Furthermore, the activation by GH of the tyrosine kinase activity of JAK2 toward IRS-1 as well as the correlation between JAK2 tyrosyl phosphorylation and IRS-1 tyrosyl phosphorylation suggest that IRS-1 may interact primarily with JAK2, which mediates the tyrosyl phosphorylation of this substrate.
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Footnotes |
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ao de Amparo a Pesquisa do
Estado de São Paulo and Conselho Nacional de Pesquisa
(PRONEX). 
Received May 4, 1998.
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Y. Mao, P.-R. Ling, T. P. Fitzgibbons, K. C. McCowen, G. P. Frick, B. R. Bistrian, and R. J. Smith Endotoxin-Induced Inhibition of Growth Hormone Receptor Signaling in Rat Liver in Vivo Endocrinology, December 1, 1999; 140(12): 5505 - 5515. [Abstract] [Full Text]
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 C. M. A. dos Santos, P. van Kerkhof, and G. J. Strous The Signal Transduction of the Growth Hormone Receptor Is Regulated by the Ubiquitin/Proteasome System and Continues After Endocytosis J. Biol. Chem., March 30, 2001; 276(14): 10839 - 10846. [Abstract] [Full Text] [PDF]
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C. M. Alves dos Santos, T. ten Broeke, and G. J. Strous Growth Hormone Receptor Ubiquitination, Endocytosis, and Degradation Are Independent of Signal Transduction via Janus Kinase 2 J. Biol. Chem., August 24, 2001; 276(35): 32635 - 32641. [Abstract] [Full Text] [PDF]
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