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Department of Physiology (J.A.V., C.C.-S.) and Department of Biological Chemistry (E.R.B., K.L.G.), The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622; and the Departments of Physiology and Medicine (S.B.W., J.E.P.), The University of Iowa, Iowa City, Iowa 52242
Address all correspondence and requests for reprints to: Dr. Christin Carter-Su, Department of Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622. E-mail: cartersu{at}umich.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our laboratory and others have demonstrated the ability of GH to activate the mitogen-activated protein (MAP) kinases referred to as extracellular signal-regulated kinases (ERKs) 1 and 2 (2, 3, 4, 5). ERK1 and 2 are serine/threonine kinases that have been shown to phosphorylate a number of substrates, including transcription factors, other protein kinases, cytoplasmic phospholipase A2, and cytoskeletal proteins (reviewed in 6 . They are thought to play an important role in regulating cellular growth and differentiation. Since GH is known to regulate cellular differentiation and body growth, in addition to body metabolism, it seems likely that MAP kinases are important signaling molecules for GH.
One of the pathways leading from growth factor receptor tyrosine kinases to MAP kinases involves SHC, growth factor receptor bound 2 (Grb2), son of Sevenless (SOS), Ras, Raf, and MAP-ERK kinase (MEK) (reviewed in 7 . SHC binds to phosphorylated tyrosines on activated receptor tyrosine kinases. Subsequent tyrosyl phosphorylation of SHC generates a binding site for the SH2 domain of Grb2. Since Grb2 binds via its SH3 domains to the mammalian homolog of the Drosophilia gene product SOS in the absence of ligand, Grb2 binding to SHC is thought to generate a SHC-Grb2-SOS complex. SOS is a guanine nucleotide exchange factor that activates the small GTP binding protein Ras. Ras located at the plasma membrane then associates with and activates the serine/threonine kinase Raf. Raf, in turn, phosphorylates and activates the dual functional serine/threonine/tyrosine kinase MEK, which then phosphorylates and activates ERK1 and ERK2. In addition to the linear pathway described above, some of these proteins are beginning to be implicated in other pathways (8, 9).
In previous work we have shown that GH stimulates the ability of GHR-JAK2 complexes to bind to the SH2 domain of SHC, tyrosyl phosphorylation of SHC, and subsequent association of SHC proteins with Grb2. In this work, we examine the involvement of the remaining molecules in the signaling cascade (SOS, Ras, Raf, and MEK) in GH action. We provide evidence of GH stimulation of SHC-Grb2-SOS complexes, increased GTP binding to Ras, and increased Raf and MEK activity. We also demonstrate a rapid transient dissociation of Grb2 from SOS concomitant with a mobility shift in SOS, suggesting that GH promotes a phosphorylation-dependent dissociation of Grb2 from SOS that may be responsible for the rapid termination of Ras, Raf, and MEK activation by GH. These results indicate that the SHC-Grb2-SOS-Ras-Raf-MEK pathway may very likely serve in the activation of MAP kinase in response to GH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SHC) (pAb), c-Raf-1 (Raf), and Grb2 (Grb2)
(mAb) used for Western blotting were from Transduction Laboratories
(Lexington, KY). Antiphosphotyrosine antibody (PY) (4G10) and
antibody to SOS (SOS) were purchased from Upstate Biotechnology,
Inc. (Lake Placid, NY). Grb2 (C23) used for immunoprecipitation was
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The Ras antibody
(Ras, Y13–259) was from Oncogene Science (Manhasset, NY). The MEK1
antibody (MEK1) is a polyclonal antibody to the human MEK1 protein,
which recognizes both MEK1 and MEK2 (10). The MEK inhibitor PD98059 was
a gift from A. Saltiel (Parke Davis, Ann Arbor, MI).
Immunoprecipitation and Western blotting
Confluent 3T3-F442A fibroblasts cultured as previously described
(11) were incubated overnight in the absence of serum (12). Experiments
in which cells were incubated with PD98059 were treated for 1 h
with dimethylsulfoxide or 100 µM PD98059 before GH
stimulation. Cells were incubated for the indicated times with human GH
at 37 C in 95% air, 5% CO2, rinsed with three changes of
ice-cold PBSV (10 mM sodium phosphate, pH 7.4, 137
mM NaCl, 1 mM Na3VO4)
and scraped on ice in lysis buffer (50 mM Tris, pH 7.5,
0.1% Triton X-100, 137 mM NaCl, 2 mM EGTA, 1
mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin). Cell lysates were centrifuged at 12,000 x
g for 10 min, and the resulting supernatants were incubated
on ice for 2 h with the indicated antibody. Immune complexes were
collected on protein A-agarose during a 1-h incubation at 8 C, washed
three times with wash buffer (50 mM Tris, pH 7.5, 0.1%
Triton X-100, 137 mM NaCl, 2 mM EGTA) and
boiled for 5 min in a mixture (80:20) of lysis buffer and 250
mM Tris, pH 6.8, 5% SDS, 10% ß-mercaptoethanol, 40%
glycerol. In some experiments immobilized proteins were incubated with
alkaline phosphatase (60 U) for 1 h at 37 C before boiling for 5
min in a mixture (80:20) of dephosphorylation buffer (0.05
M Tris-HCl, 0.1 mM EDTA, pH 8.5) and 250
mM Tris, pH 6.8, 5% SDS, 10% ß-mercaptoethanol, 40%
glycerol. The immunoprecipitates were subjected to SDS-PAGE followed by
Western blot analysis with the indicated antibody using the enhanced
chemiluminescence detection system (13). In some experiments, the blots
were rinsed in Tris-borate-NaCl-Tween and Western blotted with a second
antibody. All SDS-PAGE gels contained prestained molecular weight
standards: lysozyme (15,100), ß-lactoglobulin (17,900), carbonic
anhydrase (28,250), ovalbumin (43,600), BSA (70,800), phosphorylase B
(105,000), and myosin (203,000).
Determination of MEK and Raf activation
Activation of MEK and Raf was assessed as described previously
(14). Briefly, recombinant MEK1 and ERK1 cDNAs were subcloned into
pGEX-KG. The proteins were expressed as
glutathione-S-transferase (GST) fusion proteins and purified
by glutathione agarose affinity chromatography. ERK1 was cleaved from
GST using thrombin. Cell lysates were immunoprecipitated with Raf or
MEK1 for 2 h on ice. Protein A was then added and the mixture
rotated at 4 C for 1 h. The beads were washed three times with
wash buffer and once with HEPES buffer (25 mM HEPES, 0.5
mM EDTA, and 0.025% ß-mercaptoethanol, pH 8.0). Raf
activity was assessed by incubating Raf immunoprecipitates with
GST-MEK1 at 30 C for 60 min. ERK1 was added for the final 30 min. MEK
activity was measured by incubating MEK1 immunoprecipitates with
ERK1 for 30 min at 30 C. In both assays, ERK1 activity was measured by
adding myelin basic protein and [32P]ATP, incubating for
30 min at 30 C, and measuring 32P incorporation into myelin
basic protein.
Determination of GTP-bound Ras
3T3-F442A fibroblasts were incubated in serum- and
phosphate-free medium containing 0.2 mCi/ml carrier-free
32P for 16 h. The cells were then left untreated or
stimulated with 500 ng/ml human GH at 37 C for the indicated times.
Cells were solubilized in 50 mM HEPES, 1 mM
sodium phosphate, pH 7.4, 1% Triton X-100, 100 mM NaCl, 20
mM MgCl2, 1 mg/ml BSA, 0.1 mM GTP,
0.1 mM GDP, 1 mM ATP, 0.4 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 10 µg/ml soybean trypsin inhibitor, and 10 mM
benzamide. The extract was incubated with Ras for 60 min, and the
immune complexes were washed five times with the above lysis buffer and
five times with 50 mM HEPES, pH 7.4, 20 mM
MgCl2, 150 mM NaCl, and 0.005% SDS.
Ras-associated guanylnucleotides were eluted at 65 C for 20 min in 20
µl of 2 mM EDTA, pH 8.0, 2 mM dithiothreitol,
0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP. Eluted
GDP and GTP were separated on polyethyleneimine cellulose plates
(Baker, Phillipsburg, NJ) by TLC using 1 M
KH2PO4 (pH 3.4) as the solvent. Labeled
nucleotides were visualized by autoradiography and quantified using a
radioanalytic imager (AMBIS ß detector 1991, AMBIS, Inc., San Diego,
CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Grb2, and Western blotted with SHC (Fig. 1
). The association of all three SHC
isoforms (46, 52, and 66 kDa) with Grb2 was found to increase following
GH treatment. This association was relatively rapid but transient with
detectable increases by 1 or 2 min (lanes B and C) and a maximal effect
at 5–15 min (lanes D–F). However, even in the continuous presence of
GH, all three SHC-Grb2 complexes had dissociated to basal levels or
below within 1 h (lanes H and I).
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Grb2.
Association of SOS with Grb2 was detected by Western blotting with
SOS (Fig. 2). Grb2 was found to be in
a complex with SOS in the absence of ligand in 3T3-F442A cells (lane
A). GH caused a partial dissociation of SOS from Grb2 (lanes D-G). This
dissociation as determined by densitometry was first detectable at 2
min after GH addition and was maximal after 5–15 min. By 60 min SOS
and Grb2 were reassociated. GH-induced disruption of Grb2-SOS complexes
was accompanied by a decrease in the mobility of SOS in the Grb2
immunoprecipitates. This mobility change has been observed in response
to other growth factors such as epidermal growth factor (EGF) and has
been attributed to an increase in SOS phosphorylation (17, 18). We
examined whether the mobility of all the SOS in 3T3-F442A fibroblasts
is altered in response to GH or whether only the SOS associated with
Grb2 is altered. Proteins from solubilized 3T3-F442A cells were
separated by electrophoresis and Western blotted with SOS (Fig. 3A). At 2 min after GH addition, a
portion of SOS migrated with decreased mobility. By 5 min, all SOS
showed a decrease in mobility, with two bands being discernible (lanes
D-F). This mobility shift was transient, and SOS returned to its faster
mobility form by 60 min (lanes H and I). This result suggests that the
entire cellular pool of SOS undergoes phosphorylation, with a portion
of SOS being more highly phosphorylated than the rest. The mobility
shift of SOS in response to GH corresponds in time to the dissociation
of the SOS-Grb2 complex, raising the possibility that a partial
uncoupling of SOS and Grb2 occurs as a consequence of SOS
phosphorylation.
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SOS. Immunoprecipitated proteins were incubated for 1 h
with alkaline phosphatase, separated by electrophoresis, and Western
blotted with SOS (Fig. 3B
, lanes A-D). As predicted from the SOS
blot of whole cell lysates (Fig. 3A), after 10 min stimulation with GH,
a shift in SOS mobility was detected (lane B). Treatment with alkaline
phosphatase caused all the SOS to migrate as the higher mobility form
(lane D), indicating that the lower mobility forms were phosphorylated.
To gain insight into whether MEK or a downstream kinase such as ERKs 1
or 2 may be the SOS kinase, 3T3-F442A cells were incubated with the MEK
inhibitor PD98059 for 1 h before GH (500 ng/ml) stimulation for 5
min. Proteins were solubilized, separated by electrophoresis, and
Western blotted with SOS (Fig. 3B, lanes E-H). Treatment with
PD98059 blocked the shift in SOS mobility (lane H vs. lane
F), consistent with MEK or a downstream kinase being the SOS
kinase.
GH promotes the transient association of SHC proteins with SOS
Having determined that GH promotes both the association of SHC
proteins with Grb2 and the dissociation of Grb2 from SOS, we examined
the effect of GH on SHC-Grb2-SOS complexes. Proteins solubilized from
3T3-F442A cells were immunoprecipitated with SOS, and the
association of SOS with SHC proteins was detected by Western blotting
with SHC (Fig. 4). GH promoted a rapid
increase (within 1 min) in association of all three SHC protein
isoforms with SOS, indicating that GH promotes the formation of
SHC-Grb2-SOS complexes with a time of onset consistent with that of SHC
phosphorylation (15) and SHC-Grb2 association (Fig. 1). However,
GH-dependent SHC association with SOS was more transient (decreasing by
10 min vs. 60 min) and of lesser magnitude than GH-dependent
SHC association with Grb2 (Fig. 1). This suggests that SHC binds to SOS
indirectly via Grb2 and that Grb2 and SHC dissociate as a complex from
SOS.
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Ras. Ras-associated guanylnucleotides were eluted, separated by TLC,
visualized by autoradiography, and quantified using a radioanalytic
imager (Fig. 5). GH caused a rapid
(detectable within 1 min) increase in the amount of GTP bound to Ras.
GTP binding peaked somewhere between 3 and 30 min and was back to basal
values by 30 min.
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Raf and incubated with GST-MEK1 and then ERK1 (Fig. 6). Raf activity was determined by
incubating activated ERK1 with myelin basic protein and
[32P]ATP and measuring 32P incorporation into
myelin basic protein. Raf activity was maximal somewhere between 1 and
10 min after GH addition, rapidly decreased by 10 min, and was back to
near control values by 15 min. To measure MEK activity, 3T3-F442A cells
were stimulated with GH, immunoprecipitated MEK1 and 2 were incubated
with ERK1 (Fig. 7), ERK1 was incubated
with myelin basic protein and [32P]ATP, and
32P incorporation into myelin basic protein was assessed.
In response to GH, there was a rapid increase in MEK activity. Like Raf
activity, MEK activity peaked somewhere between 1 and 10 min, rapidly
decreased by 10 min, and returned to near basal values by 15 min.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of the different proteins in the Ras-MAP kinase pathway in mediating the actions of GH is only beginning to be understood. GH has been shown to activate phospholipase A2 in rat hepatocytes (23) and the S6 kinase, p90rsk, in 3T3-F442A fibroblasts (3), proteins shown to be phosphorylated and/or activated by MAP kinases either in vivo or in vitro. Increased phospholipase A2 activity has been implicated in the Ca2+-dependent GH induction of the P450–2C12 gene (23). p90rsk is known to phosphorylate serum response factor, a transcription factor that binds to the serum response element (SRE) of the c-fos promoter (24). GH stimulates the expression of c-fos (25, 26, 27, 28), and the SRE confers GH-dependent transcriptional regulation of a c-fos reporter construct (25). Another substrate of MAP kinases that binds (constitutively) to the SRE, the ternary complex factor p62TCF/ELK1, is also likely to be involved in GH action. Transcriptional activation of p62TCF/ELK1 requires ERK2 phos-phorylation (29, 30). Consistent with MAP kinases playing a role in the GH induction of c-fos expression, regions of GHR implicated in MAP kinase activation have been implicated in c-fos gene expression (31). Whether proteins in the SHC-Grb2-SOS-Ras-Raf-MEK-ERK pathway regulate primarily ERKs 1 and 2 in response to GH or contribute to responses in other GH signaling pathways is not yet known.
GH promotes the dissociation of Grb2-SOS complexes
Ras activation via the SHC-Grb2-SOS pathway is generally quite
transient, often lasting less than 15 or 30 min (32, 33) even in the
presence of continued tyrosine kinase activity. This implies the
existence of pathways designed to rapidly terminate Ras activation.
However, whereas SOS activation of Ras as a result of Grb2 being
recruited to the membrane is thought to be shared by receptors with
intrinsic or associated tyrosine kinase activity, initial studies
indicate that some diversity may exist in the way that Ras is
deactivated. Deactivation of Ras has been studied mostly for insulin
and EGF, ligands that appear to use different mechanisms of regulating
SOS activation of Ras. Current data support the hypothesis that in
response to insulin, insulin receptors are tyrosyl phosphorylated and
recruit SHC proteins. SHC proteins are tyrosyl phosphorylated by the
insulin receptor and bind Grb2-SOS complexes via the SH2 domain of
Grb2. These SHC-Grb2-SOS complexes do not form a tight complex with the
insulin receptor but appear to activate Ras nevertheless. The active,
GTP-bound form of Ras then returns shortly thereafter to the inactive
GDP-bound state as a consequence of SOS-Grb2 dissociation. SOS-Grb2
dissociation occurs concomitantly with a decrease in the amount of SOS
coprecipitating with SHC and an upward mobility shift of SOS due to
phosphorylation on serines and/or threonines (16, 17, 34), suggesting
that dissociation may be a consequence of SOS phosphorylation. The
rapid dissociation of SOS from Grb2 has also been shown in response to
platelet-derived growth factor and serum and phorbol esters (17).
A second paradigm is presented by EGF. Upon EGF stimulation, Grb2-SOS complex is believed to associate with the EGF receptor either directly through the SH2 domain of Grb2 or indirectly via the SH2 domain of SHC (34, 35). Like insulin, EGF induces SOS phosphorylation. In contrast to insulin, EGF does not induce dissociation of SOS-Grb2 complexes, but rather a dissociation of the Grb2-SOS complex from SHC proteins, the latter remaining in a complex with EGF receptor (18). It is hypothesized that this difference in the effects of insulin and EGF on SHC-Grb2-SOS dissociation may result in part from recruitment of different pools of Grb2-SOS complexes (33), different amino acids in SOS being phosphorylated in response to insulin and EGF and/or the fact that the receptor for EGF, but not for insulin, forms a tight complex with SHC proteins (18).
The data presented in this study indicate that GH most closely
resembles insulin in its regulation of Ras by SOS because GH clearly
promotes the transient dissociation of Grb2 from SOS. In addition, like
insulin receptor and unlike EGF receptor, SHC proteins do not appear to
form a tight complex with either GHR or JAK2 (15). Consistent with
GH-dependent Ras activation being terminated, at least in part, as a
consequence of Grb2 dissociation from SOS,
Ras,4 Raf, and MEK are
inactivated with a time course more closely following SHC-Grb2
dissociation from SOS than that of SHC dephosphorylation or the
dissociation of SHC from Grb2 (Fig. 8).
Curiously, Grb2-SOS dissociation is quite transient, with Grb2-SOS
complexes being reformed by 60 min, about the time that SHC is being
dephosphorylated and SHC-Grb2 complexes are dissociating. The net
result is that SHC-SOS complexes, which are presumably necessary for
Ras activation, start to dissociate when Grb2 and SOS start to
dissociate and remain dissociated even after Grb2 and SOS reassociate.
Together, these events may be sufficient to return Ras activity rapidly
to basal levels, although the possibility that other factors, such as
GTPase activating protein, play a role has not been discounted.
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Conclusion
These studies are the first to show, in a normal cell, activity of
endogenous signaling molecules, GH-promoted formation of Grb2 with all
three SHC isoforms, formation of SHC-SOS complexes, and activation of
Ras, Raf, and MEK. When combined with our previous observations that GH
promotes association of GHR-JAK2 complexes with the SH2 domain of SHC
proteins and tyrosyl phosphorylation of all three SHC isoforms, they
provide strong support for GH activating MAP kinases via the
SHC-Grb2-SOS-Ras-Raf-MEK pathway. They also provide evidence that GH
promotes the premature dissociation of SHC-SOS complexes by
dissociating Grb2 from SOS, thereby terminating the signal sooner than
would be anticipated based upon the rate of SHC dephosphorylation and
SHC-Grb2 dissociation. These studies also raise a number of interesting
issues. One issue is whether tyrosyl-phosphorylated SHC-Grb2 complexes,
once released from SOS, carry out some other function in the cell. A
second question arises from the observation that only a fraction of the
SOS appears to be in a complex with Grb2, and therefore presumably lies
in the SHC-Grb2-SOS-Ras-Raf-MEK-MAPK pathway, yet all of the SOS in the
cell appears to be phosphorylated on serines and/or threonines in
response to ligand stimulation. Does serine/threonine phosphorylation
regulate SOS activity in ways other than promoting its dissociation
from Grb2? Does SOS regulate proteins other than Ras? Does
GH-stimulated phosphorylation of the entire cellular pool of SOS affect
whether other ligands can stimulate Ras? Studies are currently being
conducted to address these issues.
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Footnotes |
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2 Recipient of Postdoctoral Fellowships from the Arthritis
Foundation.
3 Recipient of National Institute of Aging Training Grant
5T32AG00114.
4 The apparent difference in time course for Raf
and MEK vs. Ras inactivation is believed to result from
fewer time points taken for Ras vs. Raf and MEK
assays.
Received March 26, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) of
this paper and Fig. 1
) and Fig. 7
) of Ref. 15). The above data were
obtained by scanning autoradiographs with an Agfa Arcus II scanner
(Agfa/Bayer Co., Wilmington, MA) and analyzing the results using
Molecular Analyst densitometry software from Bio-Rad (Hercules, CA).
The mean ± range for two separate experiments is shown. B, Data
from Fig. 5