(Received for publication, October 3, 1995; and in revised form, November 29, 1995)
From the
In response to stimulation with epidermal growth factor (EGF), the guanine nucleotide exchange factor human SOS1 (hSOS1) promotes the activation of Ras by forming a complex with Grb2 and the human EGF receptor (hEGFR). hSOS1 was phosphorylated in cells stimulated with EGF or phorbol 12-myristate 13-acetate or following co-transfection with activated Ras or Raf. Co-transfection with dominant negative Ras resulted in a decrease of EGF-induced hSOS1 phosphorylation. The mitogen-activated protein kinase (MAPK) phosphorylated hSOS1 in vitro within the carboxyl-terminal proline-rich domain. The same region of hSOS1 was phosphorylated in vivo, in cells stimulated with EGF. Tryptic phosphopeptide mapping showed that MAPK phosphorylated hSOS1 in vitro on sites which were also phosphorylated in vivo. Phosphorylation by MAPK did not affect hSOS1 binding to Grb2 in vitro. However, reconstitution of the hSOS1-Grb2-hEGFR complex showed that phosphorylation by MAPK markedly reduced the ability of hSOS1 to associate with the hEGFR through Grb2. Similarly, phosphorylated hSOS1 was unable to form a complex with Shc through Grb2. Thus phosphorylation of hSOS1, by affecting its interaction with the hEGFR or Shc, down-regulates signal transduction from the hEGFR to the Ras pathway.
The guanine nucleotide exchange factor human SOS1
(hSOS1)()(1) , the homologue of Drosophila Son of sevenless, promotes the activation of Ras following
stimulation with epidermal growth factor (EGF) or with other growth
factors acting through receptor tyrosine kinases(2) . The
molecular mechanism of Ras activation by EGF involves the interaction
of hSOS1 with the activated human EGF receptor (hEGFR) through the
adapter protein Grb2(3) . Grb2 links the hEGFR to hSOS1 by
binding phosphotyrosine 1068 on the cytosolic tail of the hEGFR with
its SH2 domain, and the carboxyl-terminal proline-rich region of hSOS1
with its SH3 domains(4, 5) . It has been suggested,
that upon formation of the hSOS1-Grb2-hEGFR complex, hSOS1 is recruited
to the plasma membrane where it activates Ras by promoting GDP release
and GTP binding(6, 7) .
Stimulation with EGF also triggers the interaction of the hEGFR with the Shc proteins. The mammalian Shc gene encodes three proteins of 46, 52, and 62 kDa, and upon treatment with EGF Shc binds the activated hEGFR and undergoes tyrosine phosphorylation(8) . Phosphorylation of Shc on tyrosine residue 317 creates a binding site for the SH2 domain of Grb2 and promotes the formation of the complex hSOS1-Grb2-Shc which participates in the activation of Ras(9) ,(10) . Active, GTP-bound Ras binds and causes the translocation of the serine threonine kinase Raf to the plasma membrane where Raf is activated by an event as yet unidentified(11, 12) . Active Raf phosphorylates Mek which phosphorylates and activates the mitogen-activated protein kinases (MAPK) Erk1 and Erk2(13) . MAPK phosphorylates a number of cytosolic kinases and nuclear transcription factors which contribute to elicit the cellular responses following growth factor stimulation(14) .
EGF induced
activation of the Ras signaling pathway is often short lived,
RasGTP loading and MAPK activation reach a maximum within
2-5 min and return to a basal level in 1 h(15) . Several
mechanisms which cooperate to the down-regulation of Ras signaling have
been described. Among these mechanisms phosphorylation by MAPK or by
other serine/threonine kinases acting downstream of Ras results in
desensitization of the hEGFR (16) and in down-regulation of Raf
and Mek (17) . In cells treated with EGF or insulin hSOS1 as
well as its murine counterpart, mSOS1(18) , undergoes
serine/threonine phosphorylation (19, 20) . These
observations, and the fact that MAPK phosphorylates Drosophila SOS in vitro on sites which are also phosphorylated in vivo(21) , have suggested that MAPK phosphorylates
hSOS1 in response to growth factors activating receptor tyrosine
kinases. It has been speculated that phosphorylation of hSOS1 by MAPK
constitutes a negative feedback mechanism participating in the
down-regulation of Ras signaling(22) .
Here we show that active MAPK phosphorylates hSOS1 in vitro within the carboxyl-terminal proline-rich domain, on sites which are also phosphorylated in cells stimulated with EGF. Phosphorylation by MAPK does not affect hSOS1 interaction with Grb2 in vitro, nevertheless, phosphorylated hSOS1 demonstrates a markedly decreased ability to form a complex with the hEGFR or Shc through Grb2. Although other serine/threonine kinases cooperate with MAPK in hSOS1 phosphorylation in vivo, our data suggest that hSOS1 phosphorylation participates in the down-regulation of EGF induced activation of Ras.
Figure 1: A, hSOS1 phosphorylation in COS1 cells. COS1 cells were transfected with plasmids encoding Glu-Glu (Met-Glu-Tyr-Met-Pro-Met-Glu)-tagged hSOS1 or cotransfected with Glu-Glu hSOS1 and H-Ras G12V, or Raf-CAAX. Following transfection the cells were grown for 3 days, serum starved, and treated with 10 nM EGF for 10 min or 1 µM PMA for 30 min as indicated. hSOS1 was immunoprecipitated using a rabbit antiserum raised against the full-length hSOS1 protein and analyzed by Western immunoblotting using an anti-Glu-Glu mAb. B, co-transfection of hSOS1 with dominant negative Ras inhibits EGF-induced phosphorylation of hSOS1. COS1 cells were co-transfected with plasmids encoding Glu-Glu hSOS1 and H-Ras S17N, and stimulated with 3 nM EGF for the indicated times. Glu-Glu hSOS1 was immunoprecipitated using a rabbit antiserum raised against the full-length hSOS1 protein and analyzed by Western immunoblotting using an anti-Glu-Glu mAb.
Figure 2:
A, hSOS1 deletion mutants. Dark shaded
areas, CDC25 homology domain; light shaded areas,
proline-rich region. B, GST-Erk1 phosphorylates hSOS1 within
the carboxyl-terminal proline-rich region. Epitope (Glu-Glu)-tagged
hSOS1 deletion mutants were transiently expressed in COS1 cells and,
following overnight serum deprivation, immunoprecipitated using an
anti-Glu-Glu mAb prebound to protein G-Sepharose. Immunoprecipitates
were phosphorylated in vitro using
[-
P]ATP and GST-Erk1 which had been
activated in vitro using a reconstituted Raf-Mek-MAPK
signaling pathway. Immunoprecipitates were resolved by SDS-PAGE and
phosphorylated proteins were detected by autoradiography. C,
hSOS1 is phosphorylated within the proline-rich region in EGF-treated
COS1 cells. COS1 cells, transiently expressing epitope (Glu-Glu)-tagged
hSOS1 deletion mutants, were metabolically labeled with
P
in phosphate-free minimum essential
Eagle's medium (0.5 mCi/ml, 2 ml/6 cm dish). At the end of the
incubation with the radiolabel, cells were stimulated with 10 nM EGF for 5 min. hSOS1 deletion mutants were immunoprecipitated
using an anti-Glu-Glu mAb. Immunoprecipitates were resolved on SDS-PAGE
and phosphorylated proteins were detected by
autoradiography.
Next we studied which region of hSOS1 was phosphorylated in vivo in response to stimulation with EGF. COS1 cells
expressing the hSOS1 deletion mutants were metabolically labeled with P
and stimulated with EGF. Deletion mutants
were immunoprecipitated and resolved on SDS-PAGE which demonstrated
that hSOS1 was phosphorylated in vivo within the region
containing amino acids 1066 and 1333, thus within the same region that
was phosphorylated in vitro by GST-Erk1 (Fig. 2C). A low amount of hSOS1 phosphorylation was
also detected in unstimulated cells (Fig. 2C): this
could be due to the high basal level of MAPK activity which has been
described in COS1 cells(21) . We also studied which region of
hSOS1 was phosphorylated in response to stimulation with PMA. These
experiments showed that, as with EGF-stimulated cells, treatment with
PMA resulted in phosphorylation of hSOS1 only within the proline-rich
domain (not shown).
Figure 3:
Tryptic phosphopeptide mapping of
phosphorylated hSOS1. A, recombinant hSOS1 was phosphorylated in vitro using activated GST-Erk1 and
[-
P]ATP. Phosphorylated hSOS1 was resolved
by SDS-PAGE and transferred onto a poly(vinylidene difluoride)
membrane. The band corresponding to hSOS1 was identified by
autoradiography, cut out, and digested with trypsin. Tryptic peptides,
corresponding to 10,000 cpm, were applied on cellulose plates,
separated by electrophoresis (horizontal) followed by chromatography
(vertical), and visualized by autoradiography. The site of application
is situated near the bottom right corner of the panel. B, COS1
cells transiently expressing Glu-Glu-tagged hSOS1 were metabolically
labeled with
P
and stimulated with 10 nM EGF for 2 min. hSOS1 was immunoprecipitated with an anti-Glu-Glu
mAb, resolved by SDS-PAGE and analyzed as described in A. A+B, tryptic phosphopeptides from A and B (corresponding to 7,000 cpm from each sample) were mixed and
analyzed as described in A. Seven phosphopeptide species
(phosphopeptides 1 to 7) showed identical migration whether they were
generated from hSOS1 phosphorylated in vitro or in
vivo.
Figure 4:
Phosphorylation by MAPK does not affect
hSOS1 binding to GST-Grb2. Bacterially produced GST-Grb2 (10 pmol) was
mixed with P-labeled phosphorylated hSOS1 (6.5 pmol) and
the indicated amounts of unmodified hSOS1 (
) or
cold-phosphorylated hSOS1 (
). Tubes were rotated at 4 °C for
15 min then the GST-Grb2-hSOS1 complexes were captured using
glutathione-agarose beads and resolved on SDS-PAGE. The amount of
P-labeled phosphorylated hSOS1 bound to GST-Grb2 was
quantified using an AMBIS
scanner (100% = 1,000 cpm).
Under the conditions of this assay,
P-labeled
phosphorylated hSOS1 saturated only 50% of GST-Grb2 binding capacity.
The averages (±S.D.) of three independent experiments are
shown.
Figure 5:
A and B, effect of hSOS1 phosphorylation on the assembly of the
hSOS1-GST-Grb2-hEGFR complex in vitro. HER14 fibroblasts were
serum starved and stimulated for 2 min with the indicated
concentrations of EGF, or treated with PMA (1 µM) for 30
min prior to EGF stimulation. Cells were lysed and the hEGFR was
immunoprecipitated using an anti-hEGFR mAb. Immunoprecipitates were
mixed with 3 pmol of recombinant Glu-Glu hSOS1 or in vitro phosphorylated Glu-Glu hSOS1 and with 3 pmol of GST-Grb2.
Formation of the hSOS1-GST-Grb2-hEGFR complex was detected by resolving
the hEGFR immunoprecipitates on SDS-PAGE followed by Western
immunoblotting using an anti-Glu-Glu mAb. C, effect of
phosphorylation of hSOS1 proline-rich region (SOS4) on the
assembly of the Sos
4-GST-Grb2-hEGFR complex in vitro. hSOS1 proline-rich region (amino acid residue 1066 to 1333,
Sos
4) was expressed in Escherichia coli as a GST fusion
protein and epitope (KT3) tagged(40) . Unmodified or in
vitro phosphorylated SOS
4 (3 pmol) was mixed with GST-Grb2 (3
pmol) and hEGFR immunoprecipitates. Association of Sos
4 with
GST-Grb2 and the hEGFR was detected by Western immunoblotting using an
anti-KT3 mAb(40) . D, treatment with PMA prevents EGF
induced co-immunoprecipitation of mSOS1 and the hEGFR. HER14 cells were
serum starved for 16 h and stimulated with the indicated concentrations
of EGF for 2 min or treated with PMA (1 µM) for 30 min
prior to EGF stimulation. Cells were lysed and the hEGFR or mSOS1 was
immunoprecipitated using a polyclonal antibody to the hEGFR or to mSOS1
(both from Upstate Biotechnology Inc., Lake Placid, NY).
Immunoprecipitates were resolved on SDS-PAGE and analyzed by Western
immunoblotting using a mAb to mSOS1 (Transduction Laboratories) or with
2 antibodies to phosphotyrosine (both from Upstate Biotechnology Inc.)
which detected a tyrosine phosphorylated protein of approximately 180
kDa corresponding to the activated hEGFR.
Figure 6: Effect of hSOS1 phosphorylation on the formation of the hSOS1-Grb2-Shc complex. A, COS1 cells were transfected with Shc cDNA and stimulated with the indicated concentrations of EGF. Shc was immunoprecipitated using a rabbit antiserum raised against the full-length Shc protein. The immunoprecipitates, immobilized on Protein A-Sepharose beads, were mixed with GST-Grb2 (3 pmol) and with 100 µl of a soluble fraction (S100) of COS1 cells expressing Glu-Glu hSOS1 which were either untreated or treated with PMA (1 µM) for 30 min, to induce hSOS1 phosphorylation. The formation of the Shc-GST-Grb2-hSOS1 complex was detected by analyzing the immunoprecipitates on SDS-PAGE followed by Western immunoblotting with an anti-Glu-Glu mAb. B, HER14 cells were stimulated with the indicated concentrations of EGF for 2 min or treated with PMA (1 µM) for 30 min prior to EGF stimulation. Cells were lysed and Shc was immunoprecipitated using a rabbit antiserum raised against the full-length Shc protein. Immunoprecipitates were analyzed by Western immunoblotting using a mAb to mSOS1.
To establish if phosphorylation of mSOS1 affected its interaction with Shc in vivo, we attempted to co-immunoprecipitate mSOS1 and Shc from HER14 fibroblasts. Although we were able to co-immunoprecipitate Shc and mSOS1 from cells stimulated with EGF, we failed to co-immunoprecipitate Shc and mSOS1 from cells which were pretreated with PMA and stimulated with EGF (Fig. 6B). Treatment of HER14 cells with PMA caused a decrease of Shc tyrosine phosphorylation which could account for our failure of co-immunoprecipitating mSOS1 and Shc. Nevertheless, a decrease of Shc tyrosine phosphorylation was not sufficient to prevent the formation of the hSOS1-GST-Grb2-Shc complex in vitro, as Shc immunoprecipitated from PMA-treated cells was still able to bind GST-Grb2 and unmodified hSOS1 (not shown).
The formation of complexes of the guanine nucleotide exchange factor hSOS1, the activated hEGFR or Shc, and the adapter Grb2 is critical in coupling EGF stimulation to the activation of Ras. This paper proposes a novel negative feedback mechanism by which the ability of hSOS1 to participate in such signaling complexes is impaired by serine/threonine phosphorylation of its carboxyl-terminal proline-rich domain.
hSOS1 was phosphorylated in vivo as a result of the activation of the Ras signaling pathway by growth factors acting through receptor tyrosine kinases or following co-transfection with activated Ras or Raf. Several lines of evidence suggested that among the serine/threonine kinases acting downstream of Ras, MAPK was a good candidate for phosphorylating hSOS1 in vivo. Blocking EGF-induced MAPK activation by co-transfecting a dominant negative version of Ras resulted in reduction hSOS1 phosphorylation. The carboxyl-terminal proline-rich domain of hSOS1 contains potential MAPK phosphorylation sites and was phosphorylated in vivo, in cells stimulated with EGF. The same proline-rich region of hSOS1 was also phosphorylated in vitro, by activated MAPK. In addition, phosphorylation by MAPK caused a decrease of hSOS1 electrophoretic mobility similar to that of in vivo phosphorylated hSOS1. The study of the two-dimensional tryptic phosphopeptide map confirmed the involvement of MAPK in hSOS1 phosphorylation. Seven phosphopeptides which were detected in the tryptic map of hSOS1 phosphorylated in vitro by GST-Erk1, were also found in the map of hSOS1 phosphorylated in vivo. A further 6 tryptic phosphopeptides were generated only when analyzing hSOS1 phosphorylated in vivo, thereby suggesting that serine/threonine kinases other than MAPK also participated in hSOS1 phosphorylation. Which serine/threonine kinases phosphorylate hSOS1 in addition to MAPK is not yet clear although it is likely that, as MAPK, these enzymes are downstream targets of Ras.
Phosphorylation of hSOS1 occurred within the region containing the binding sites for the SH3 domains of Grb2. Nevertheless, we did not detect any significant difference between the ability of either unmodified or in vitro phosphorylated hSOS1 to bind GST-Grb2. Different from our data, it has recently been suggested that stimulation with insulin of 3T3-L1 adipocites or Chinese hamster ovary cells induced phosphorylation of mSOS1 and disassociation of the mSOS1-Grb2 complex(20) . Stimulation with insulin results in the activation of the Ras pathway and of other insulin-specific signaling systems which operate distinct negative feedback mechanisms(35) . Therefore it is possible that in response to stimulation with insulin, mSOS1 is phosphorylated by kinases different from those activated by EGF, or that mSOS1-Grb2 interacts with yet unidentified proteins, this resulting in disassociation of the mSOS1-Grb2 complex.
Recombinant hSOS1, phosphorylated using GST-Erk1, showed a decreased capacity to form a complex with GST-Grb2 and the activated hEGFR in vitro. Similarly phosphorylated hSOS1 failed to form a complex with GST-Grb2 and tyrosine-phosphorylated Shc in vitro. These data suggest that one effect of MAPK phosphorylation of hSOS1 was to impair its participation in signaling complexes with the hEGFR or Shc, thus uncoupling signal transduction from the hEGFR to Ras. Down-regulation of EGF signaling also occurs through the serine/threonine phosphorylation of the hEGFR which results in inhibition of the tyrosine kinase activity of the receptor and in the decrease of its affinity for EGF(16) . In addition, serine/threonine phosphorylation of the hEGFR cooperated with hSOS1 phosphorylation in blocking the assembly of the hSOS1-GST-Grb2-hEGFR complex in vitro.
The mechanism by which phosphorylation of hSOS1 by MAPK interferes with the participation of hSOS1 in signaling complexes is not clear. It is possible that phosphorylation by MAPK decreases the stability and induces the disassembly of the hSOS1-GST-Grb2-hEGFR complex. It is also possible that, binding of Grb2 to the hyperphosphorylated carboxyl terminus of hSOS1 results in alteration of Grb2 structure and loss of its affinity for binding tyrosine-phosphorylated proteins. The study of the crystal structure of Grb2 suggests that a conformational change affecting the SH3 domains of Grb2 would be unlikely to alter the SH2 domain(36) . Nevertheless, binding of the SH2 domain of Grb2 to tyrosine-phosphorylated Shc positively regulates the interaction of the SH3 domains with the proline-rich region of mSOS1(37) . Thus, conformational changes appear to be transmitted through Grb2 and these changes are involved in regulating Grb2 interaction with target proteins.
MAPK is not the only kinase that phosphorylates hSOS1 in vivo, therefore, to fully understand the biochemical
function of hSOS1 phosphorylation, it will be necessary to identify the
other serine/threonine kinases as well as the sites that are
phosphorylated. It is plausible that phosphorylation of hSOS1 in
vivo, by analogy with the effects of phosphorylation by MAPK in vitro, also impairs the participation of hSOS1 in complexes
with hEGFR or Shc. Indeed, EGF failed to induce co-immunoprecipitation
of mSOS1 and hEGFR or Shc in cells which were pretreated with PMA. In
conclusion our data suggest that hSOS1 phosphorylation, by limiting the
access of hSOS1 to Ras, contributes to regulate the duration or the
amplification of signals originating from activated tyrosine kinases.
In agreement with this view, prolonged RasGTP loading has been
detected in insulin-stimulated cells in which phosphorylation of mSOS1
was inhibited using a specific Mek inhibitor(38) . It has been
proposed that the duration of the activation of the Ras pathway
(sustained versus transient) is critical to enact specific
cellular responses(39) . According to this model, hSOS1
phosphorylation contributes to secure the execution of the appropriate
cellular programs in response to growth factor stimulation.