From the Eukaryotic Genetics Group, Biotechnology
Research Institute, National Research Council, 6100 Royalmount,
Montreal, Quebec H4P 2R2, Canada, the ¶ Department of Experimental
Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada, and the
Biology Department and Department of Anatomy and Cell Biology,
McGill University, Montreal, Quebec H3A 2B2, Canada
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ABSTRACT |
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Grb10 and its close homologues Grb7 and Grb14,
belong to a family of adapter proteins characterized by a proline-rich
region, a central PH domain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their interaction with a variety of activated tyrosine kinase receptors is well documented, but their actual function remains
a mystery. The Grb10 SH2 domain was isolated from a two-hybrid screen
using the MEK1 kinase as a bait. We show that this unusual SH2 domain
interacts, in a phosphotyrosine-independent manner, with both the Raf1
and MEK1 kinases. Mutation of the MEK1 Thr-386 residue, which is
phosphorylated by mitogen-activated protein kinase in
vitro, reduces binding to Grb10 in a two-hybrid assay. Interaction of Grb10 with Raf1 is constitutive, while interaction between Grb10 and MEK1 needs insulin treatment of the cells and follows
mitogen-activated protein kinase activation. Random mutagenesis of the
SH2 domain demonstrated that the Arg-B5 and Asp-EF2 residues are
necessary for binding to the epidermal growth factor and insulin receptors as well as to the two kinases. In addition, we show that a
mutation in Ser-
B7 affects binding only to the receptors, while a
mutation in Thr-
C5 abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domains induces apoptosis in HTC-IR and COS-7 cells. These effects can
be competed by co-expression of the wild type protein, suggesting that
these mutants act by sequestering necessary signaling components.
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INTRODUCTION |
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The three members of the Grb7 family of signaling proteins, Grb7,
Grb10 and Grb14, were first isolated through the ability of their
SH21 domains to recognize
phosphotyrosine-containing sequences on activated tyrosine kinase
receptors. Grb7 has been shown to interact with the EGF, ErbB2/Her2,
platelet-derived growth factor, and FcRI receptors, the Ret
proto-oncogene, the Syp phosphatase, and the SHC adapter protein
(1-5). Grb10 has a much reduced affinity for the EGF receptor, but its
interactions with the Ret proto-oncogene, ELK receptor, insulin-like
growth factor-I receptor, and insulin receptor (IR) have been
demonstrated in vivo (6-11). Grb14, the remaining member of
this family, interacts in vitro with the platelet-derived growth factor receptor (12).
The function of the Grb7/10/14 proteins remains obscure. They are
currently classified as adapter proteins, since they have no obvious
enzymatic function and contain several conserved polypeptide-binding regions in addition to their carboxyl-terminal SH2 domain (see Fig. 1).
A small proline-rich region in the NH2 terminus fits the
consensus sequence for SH3-binding domains. In fact, in
vitro binding by the Grb10 proline-rich domain to the
cAbl SH3 domain has been demonstrated (10). Furthermore, all
three of these proteins contain a central Pleckstrin homology domain.
Similar elements have been shown to mediate protein-protein and/or
protein-lipid interactions (for a review, see Ref. 13). The Grb7/10/14
genes are expressed in a tissue-specific pattern, and there is ample evidence of alternative splicing of the transcripts (1, 6, 7, 10-12,
14). Some of these splicing events result in the expression of proteins
with altered properties;
hGrb102 contains a
deletion in the amino-terminal half of its Pleckstrin homology domain
(6). Finally, the Grb7/10/14 proteins can be phosphorylated in
vivo, and their levels of phosphorylation are regulated by hormone
treatment (2, 4, 7, 12).
It is still unclear whether Grb10 acts as an inhibitor or an activator
of signal transduction. Overexpression of hGrb10 in Chinese hamster
ovary-IR cells reduces insulin-dependent pp60 and insulin
receptor substrate-1 phosphorylation and diminishes PI-3 kinase
activation (6). Others have observed that microinjection of the Grb10
SH2 domain partially inhibits mitogenesis in insulin- or insulin-like
growth factor-I-treated Rat1 fibroblasts but not in cells treated with
EGF or serum (14). These results appear to be in contradiction with the
work of Morrione et al. (15), whose cell lines
overexpressing mGrb10
show growth reduction in the presence of
insulin growth factor-I but not insulin. Unlike the microinjection
experiments, these cells do not show an inhibition in S phase entry but
rather an accumulation in the S and G2 phases. These
contradictions might be explained by the use of different cell lines,
experimental procedures, or Grb10 splicing variants. As for Grb7 and
Grb14, both have been shown to be overexpressed in breast and
prostate cancer tumors and cell lines (2, 12, 16).
The receptors that are recognized by the Grb7/10/14 family share the ability to activate the mitogenic MAP kinase signal transduction pathway (reviewed in Ref. 17). In this report, we present evidence that at least two members this pathway, Raf1 and MEK1, can interact with the SH2 domain of the Grb10 adapter protein. In some cultured cells, binding to Raf1 is constitutive, while the interaction of Grb10 with MEK1 is insulin-dependent. Interestingly, these interactions are phosphotyrosine-independent, and binding of MEK1 to Grb10 appears to be regulated through the retrophosphorylation of MEK1 by the MAP kinases. Finally, we have identified point mutations that affect the specificity of the Grb10 SH2 domain. Overexpression of these mutants was found to induce apoptosis in cultured cells.
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EXPERIMENTAL PROCEDURES |
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Materials-- Most of our chemicals were purchased from Sigma. Restriction and modification enzymes as well as the plasmids and resins used for the expression and purification of GST and MBP fusions proteins were obtained from New England Biolabs or from Amersham Pharmacia Biotech. Sequencing reactions were performed with a DNA sequencing kit from Perkin-Elmer and separated on an Applied Biosystems DNA Sequencer model 370A. PCRs used the Expand High Fidelity PCR System (Boehringer Mannheim). Western blots were performed on Immobilon membranes (Millipore Corp.). The Grb10 (K-20), and Raf1 (C-12) antibodies were from Santa Cruz Biotechnology, the MEK1-NT antibody was from Upstate Biotechnology, and antibodies against the maltose-binding protein and activated ERK came from New England Biolabs. The anti-Flag (M2) monoclonal antibody was from Eastman Kodak Co. Horseradish peroxidase-conjugated secondary antibodies were purchased from Bio-Rad, and the ECL Western blotting detection reagents were from Amersham Pharmacia Biotech. Protein-A and Protein-G Sepharose were from Amersham Pharmacia Biotech. Finally, the HeLa cell cDNA library and all of our cell culture reagents were obtained from Life Technologies, Inc.
Two-hybrid Assays-- Following PCR amplification with the ANO-35 (GGGGGATCCAAATGCCCAAGAAGCCG) and ANO-36 (GCGCTCGAGGCTCTTTTGTTGCTTCCC) primers, the coding sequences of the full-length human MEK1 gene (a gift from S. Pelech) were introduced between the BamHI and XhoI sites of the pEG202 LexA fusion plasmid, resulting in the pAN104 construct. Two-hybrid screening of 2 × 106 primary transformants, from a human fetal brain cDNA library, was then performed exactly as described (18, 19).
EcoRI and XhoI sites were introduced around the coding sequence of the human Raf1 gene (a gift from S. Meloche) by PCR amplification. The fragment was then subcloned in the same sites of the pEG202 vector, yielding pAN130. A similar PCR amplification was also used to insert the regulatory (aa 1-330) or catalytic (aa 331-649) domains of Raf1, as well as the cytoplasmic domain of the insulin (aa 974-1370) and EGF (aa 672-1210) receptors, between the EcoRI and XhoI sites of pEG202. Subcloning of the partial MEK1 fusions in the pEG202 vector were performed as follows; N308, PCR amplification of the partial MEK1 gene with the ANO-35 and CTCGAGCCATGGGAGGTCGGCTGTCCTTCC primers; N293, subcloned 5' SmaI fragment of pAN104 plasmid; N220, removed 3' NcoI/XhoI fragments from pAN104; C304, subcloned the EcoRI/XhoI insert of the pMB6 cDNA (which encodes aa 304-392 of MEK1). QuantitativeAmplification of a Full-length Grb10 Gene--
Two µg of DNA
from a HeLa Cell cDNA library was used as a template in a PCR
reaction with the
GACGAATTCGAACCCATGGCTTTAGCCGGCTGCCCAG and
GACCTCGAGCACAGACCGCTTCTTCACTCCAG primers. The
resulting 2.2-kilobase pair fragment was fully sequenced on both
strands and contains the coding sequences of the hGrb10 gene flanked
by EcoRI and XhoI sites.
Production of Protein Fusions and Resin-binding
Assay--
EcoRI/XhoI fragments containing the
hGrb10 gene or the SH2 domain encoded by the pMB58 cDNA were
subcloned in the pMAL-c2 plasmid. The MBP-hGrb10
and MBP-SH2 fusion
proteins were then purified from Escherichia coli UT5600
cells by affinity chromatography on amylose resin columns as described
by the manufacturer (New England Biolabs). Plasmids containing the
GST-MEK1 and GST-ERK1 constructs were obtained from S. Pelech, while
the SEK1 gene was obtained from Jim Woodgett. Expression and
purification of GST-MEK1, GST-SEK1, and GST-ERK1 were performed as
described (21). GST-Raf1 was constructed by inserting the
EcoRI/XhoI insert of pAN130 in pGEX-5X-1 and
purified as described by Zhang et al. (22).
Grb10 Mutagenesis--
Site-directed mutagenesis of the
Arg-B5 residue was done by overlap extension PCR (23). Random PCR
mutagenesis of the pMB58 insert was performed according to Fromant
et al. (24). The amplified fragment was then cleaved with
EcoRI and XhoI and ligated back into pJG4-5.
Following transformation in E. coli, colonies from >104 transformants were inoculated in 100 ml of 2YT plus
100 µg/ml ampicillin and grown for 4 h at 37 °C. Plasmids
from this mutagenized library were then purified and used to transform
Saccharomyces cerevisiae RFY206. One thousand yeast colonies
were patched on glucose Trp
plates, which were
subsequently replica-plated to generate four copies. One plate was kept
as a master, while the other three were mated to lawns of S. cerevisiae EGY48 cells previously transformed with the pSH18-34
-galactosidase reporter plasmid as well as one of either the pAN129
(LexA-EGF receptor COOH terminus), pAN138 (LexA-IR COOH terminus),
pAN168 (LexA-RAF NH2 terminus), or pAN104 (LexA-MEK1) bait
plasmids. Following selection of the diploids on glucose
His
Trp
Ura
plates, the cells
were replica-plated to galactose/raffinose His
Leu
Trp
Ura
plates and scored
for interaction. Plasmids from potentially interesting mutants were
reisolated from the RFY206 master plates, sequenced, and retransformed
in haploid EGY48 cells along with the pSH18-34 reporter and LexA
fusion plasmids. Binding specificity was confirmed by quantitative
-galactosidase assays on at least four independent
transformants.
Cell Culture and Transfections--
293 HEK, COS-7, and HTC-IR
(25) cell lines were maintained in DMEM, supplemented with 10% fetal
bovine serum (along with 400 µg/ml G418 for the HTC-IR), at 37 °C
in a 5% CO2 atmosphere. The 8-aa Flag immunological tag
was added at the carboxyl-terminal end of the hGrb10 polypeptide by
PCR amplification followed by subcloning in the
EcoRI/XhoI sites of the pcDNA3 expression
vector (Invitrogen), yielding pAN185. Transient transfections with
lipofectamine were performed according to the manufacturer's
instructions (Life Technologies, Inc.). Incubation with the DNA-lipid
complexes was carried on for 5 h followed by the addition of one
volume of DMEM plus 20% fetal bovine serum. Twenty-four to forty-eight
hours after transfection, the cells were starved overnight in DMEM plus 0.1% bovine serum albumin and then induced for 2-30 min with 100 nM insulin. Following hormone treatment, cells were washed
with cold PBS, lysed for 10 min in lysis buffer (50 mM
Hepes, pH 7.5, 150 mM NaCl, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1.5% Triton X-100,
1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl
fluoride, 2 mM sodium vanadate) and spun down at maximum
speed in a cold microcentrifuge for 10 min. Protein concentration was
estimated with the Bio-Rad protein assay.
Production of Recombinant Adenovirus--
The adenovirus (AV)
vector used in this report is a modification of the human strain 5 in
which the E1a, E1b, and E3 regions are deleted (26). The insulin
receptor cDNA was isolated from the CMVneo plasmid (a gift of A. Ullrich). The resulting 4.2-kilobase pair
ClaI-SpeII fragment was blunted with Klenow and
cloned in the EcoRV site of the pAdcmvpoly(A) transfer
vector (a gift of B. Massie). The 1.6-kilobase pair
EcoRI-XhoI fragment encoding the full-length
hGrb10 was also blunted with Klenow and cloned in the
EcoRV site of the transfer vector. The homologous
recombination of the replication-defective human type 5 AV, large scale
production, purification, and titration of the recombinant AV have been
described in detail (27). All recombinant AV were stored at a
concentration of 1-4 × 108 plaque-forming units/ml
in DMEM supplemented with 10% fetal bovine serum. For the infections,
293 cells were transduced at a multiplicity of infection of 1 (1 plaque-forming unit/cell) for 2 h with stocks of either a control
recombinant AV (Ad DE1/DE3) or recombinant AV (Ad-IR/Ad-Grb).
Transduced cells were incubated for 24 h at 37 °C in 5%
CO2 and then starved for 24 h in DMEM containing 0.1% bovine serum albumin. Cells were stimulated for 5 min with 100 nM insulin. The medium was then aspirated, and the cells
were washed twice with PBS and solubilized in lysis buffer.
Immunoprecipitations-- Protein samples were diluted to equal concentration with lysis buffer and then incubated with the antibodies for 1 h at 4 °C. Protein A- or Protein G-Sepharose beads were then added, and the samples were incubated for another 1 h. The beads were then washed three times with PBS, and the bound proteins were released by boiling in SDS-polyacrylamide gel electrophoresis sample buffer.
Detection of Apoptosis by Microscopy and Flow Cytometry-- The RL (GCTTTTTCTCCTCCTTGACAGCCAGAG), TS (GCATTTGTACTCTCACTGTGTCATCACC) and SC (CTCCTCCGTGACTGCCAGAGTAATCC) primers were used to introduce SH2 domain mutations in the pAN185 plasmid using the Chameleon kit from Stratagene. The resulting pAN200, pAN208, and pAN209 plasmids express Grb10-R520L, Grb10-T531S, and Grb10-S522C, respectively. One million HTC-IR cells or 1-2 × 105 COS-7 cells were inoculated in 35-mm wells and co-transfected with 0.5 µg of pAdcMV5GFPQ (a gift from B. Massie) along with 1 µg of either the pcDNA3 vector alone or one of the Grb10 expression plasmids. Twenty-four hours later, the cells were observed in a fluorescence microscope. Cells that were small, round, and showed nuclear fragmentation or condensation following Hoecht staining were counted as apoptotic. HTC-IR cells were also washed in PBS, trypsinized, and analyzed by flow cytometry. For the detection of apoptotic cells by the TUNEL assay, we omitted the GFP plasmid and used the ApoBrdU kit and MPlus software (Phoenix Flow Systems).
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RESULTS |
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Interaction of Grb10 with MEK1 in a Two-hybrid Screen--
The SH2
domain of Grb10 was isolated from the two-hybrid screen of a human
fetal brain cDNA library using the full-length MAP kinase kinase
MEK1. Our screen of 2 × 106 primary transformants
resulted in the isolation of five independent genes. The pMB58
cDNA3 is almost identical
to the 3'-end of the Grb10 gene (6, 10, 14) including the region that
encodes the SH2 domain (Fig.
1A). A sequence comparison
(Fig. 1B) between pMB58 and published Grb10 sequences
suggests that this cDNA represents a novel splice variant with
modifications in the extreme amino-terminal end of the SH2 domain. The
borders between the regions of high and low sequence homology all
contain sequences that are consistent with intron-exon borders. The
variable region encoded by pMB58 still contains all the residues that
have been shown to be important for SH2 domain function, namely a
Trp-Phe-His motif in the first -sheet as well as a conserved
arginine residue 2 amino acids into the first
-helix (Fig.
1C).
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Isolation of a Novel Grb10 Splice Variant--
Unfortunately, we
were unable to isolate longer Grb10 cDNAs from the fetal brain
library. Using primers based on published sequence data (6), we
isolated by PCR the coding sequence of a full-length human Grb10 gene
from a HeLa cell cDNA library. Sequencing of the 2.2-kilobase pair
fragment revealed that this clone is a hybrid between hGrb10 (6) and
hGrb10
(10, 14) (see Fig. 1A). We named this new human
splice variant, simultaneously isolated by Dong et al. (28),
hGrb10
.2,4 It contains the
longer amino-terminal sequences found in hGrb10
as well as the
complete Pleckstrin homology domain seen in the other human and mouse
variants. Inclusion of the pMB58 SH2 domain variant into this
nomenclature will have to await the isolation of a full-length
cDNA. Two forms of Grb10 were thus used in our experiments. The
full-length Grb10 protein encoded by the hGRB10
variant was used in
most experiments, while for simplicity, experiments using the SH2
domain used the sequences of the pMB58 clone.
Grb10 Interacts with Phosphorylated Raf1 and MEK1--
Both the
full-length hGrb10 protein and the SH2 domain initially isolated in
the pMB58 cDNA were purified from bacterial extracts as fusions
with the E. coli MBP. To confirm that the full-length
hGrb10
could also bind to MEK1, we incubated the MBP-SH2 and the
MBP-hGrb10
fusion proteins with glutathione-Sepharose resins loaded
either with GST control protein or with GST fusions of various kinases.
Interestingly, we found that MBP-SH2 was not only retained by the
GST-MEK1 resin but also by GST-Raf1. We also observed binding by the
full-length MBP-hGrb10
protein to the GST-MEK1 (Fig.
2C) and GST-Raf1 resins (Fig.
3), indicating that the modifications in
the pMB58 SH2 domain are not solely responsible for this binding
specificity.
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Localization of the Grb10-binding Site on Raf1 and MEK1-- We returned to the two-hybrid assay both to confirm the observed Grb10-Raf1 interaction and to map the Grb10-binding sites on the Raf1 and MEK1 kinases. Neither the full-length Raf1 nor its carboxyl-terminal catalytic domain (aa 331-648) interacted with the SH2 domain in the two-hybrid assay. The SH2-binding domain in Raf1 was located in its NH2-terminal regulatory domain (aa 1-330), since a LexA fusion of this region was able to confer growth on media lacking leucine to cells containing the pMB58 cDNA (Table I). A deletion of the 88 amino acids in the carboxyl terminus of MEK1 abolished interaction with the SH2 domain. We established that this absence of interaction is not the result of a two-hybrid artifact, since two of the COOH-terminal mutants (1-308 and 1-293) were still capable of interacting with other MEK1-binding clones that were isolated in our initial screen (results not shown). Due to the high background, most of the results from the amino-terminal deletions were inconclusive except for the 304-393 construct in which growth with the Grb10 SH2 domain was clearly faster than with the vector alone. The carboxyl-terminal domain of MEK1 (aa 304-393) contains 9 Ser/Thr residues including Thr-386, which is phosphorylated by MAP kinases in vitro (41, 42). Another target for MAP kinase phosphorylation, Thr-292, lies very close to the end of the 1-308 mutant. After mutating either residue to alanine, a quantitative two-hybrid assay (Table II) demonstrated that Thr-292 is not involved in the interaction, while the T386A mutation reduced the affinity of MEK1 for Grb10 by almost half. These results thus locate the Grb10-binding site in the extreme COOH terminus of MEK1, a region that, incidentally, is not conserved in the sequence of the SEK1 kinase.
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In Vivo Interaction among Grb10, Raf1, and MEK1--
To determine
whether the interaction of Grb10 with Raf1 and MEK1 also occurs
in vivo, we performed immunoprecipitations in Triton-soluble
protein extracts taken from a variety of sources. Fig.
4A shows the results obtained
from 293 HEK cells in which both hGrb10 and the insulin receptor
were expressed using adenovirus vectors. In these cells, Grb10 was
co-immunoprecipitated by an anti-Raf1 antibody in both starved and
insulin-treated cells (lanes 3 and 4). We also
used the anti-Flag monoclonal antibody to efficiently co-immunoprecipitate p74raf1 from extracts of 293 HEK cells
transiently expressing a hGrb10
-Flag protein (Fig. 4B).
As in the virus-infected 293 HEK cells, this interaction occurred
irrespective of the presence of mitogenic agents. However, the
Grb10-Raf1 interaction appears to be cell type-specific, since we were
unable to co-immunoprecipitate these proteins from transfected NIH-3T3
or HTC-IR cells (not shown).
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Mutagenesis of the Grb10 SH2 Domain--
The structure of several
SH2 domains has revealed that an invariant arginine (Arg-520 in the
case of hGrb10) interacts with the phosphate group of
phosphotyrosine residues. We introduced an Arg-520 to leucine mutation
in the pMB58 SH2 domain and observed that this mutant failed to
interact, in a two-hybrid assay, with LexA fusions of the
carboxyl-terminal domain of the insulin and EGF receptors, the
regulatory domain of Raf1, or the full-length MEK1 kinase.
Immunoblotting of total yeast cell extracts confirmed that expression
of this mutant is not significantly different from that of the wild
type SH2 domain (result not shown). This confirmed that a conserved
residue, which is involved in the interaction of SH2 domains with the
phosphate group of phosphotyrosine residues, is also necessary for the
recognition of Raf1 and MEK1 by Grb10 although neither appears to
be tyrosine-phosphorylated.
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Induction of Apoptosis by Grb10 Mutants--
The Arg-B5
Leu, Ser-
B7
Cys, and Thr-
C5
Ser mutations were introduced
in the full-length hGrb10
gene. These constructs (named Grb10-RL,
Grb10-SC, and Grb10-TS) were co-transfected in HTC-IR cells along with
a reduced amount of a second plasmid containing the gene for the green
fluorescent protein (GFP). Consistent with the results of Morrione
et al. (15), overexpression of wild type Grb10 had little
effect on cell morphology and survival, but initial observations by
fluorescence microscopy, followed by more stringent analysis by flow
cytometry, showed a strong reduction in the number of GFP-positive
adherent cells cotransfected with either of the three SH2 domain
mutants (Fig. 6A). Most of the
GFP-positive cells were dead and could be found floating in the media,
while the remaining adherent cells were smaller, rounder, and more
refractive than normal (results not shown, but see Fig. 8). A TUNEL
assay (49), which detects DNA strand breaks induced by programmed cell
death, also showed an increase in the number of apoptotic cells
following transfection with the Grb10-RL, Grb10-TS, and Grb10-SC
plasmids (Fig. 6B). Because the small size and low transfection efficiencies of the HTC-IR cells made detailed analysis difficult, we repeated the transfections in COS-7. As seen in Fig.
7A, transfection of these
normally large cells with SH2 domain mutants of Grb10 increased the
proportion of small round cells from 17-23 to 42-53%. Co-staining of
these cells with the DNA-binding dye Hoecht 33258 revealed the nuclear
fragmentation that is one of the structural hallmarks of apoptosis
(Fig. 8). Cells transfected with the
pcDNA3 vector were similar to those expressing Grb10, while those
transfected with Grb10-RL or Grb10-SC looked like cells transfected
with Grb10-TS (results not shown). Finally, the effects of the Grb10-RL
mutant can be reversed by co-expression of wild type Grb10 (Fig.
7B).
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DISCUSSION |
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We used the yeast two-hybrid assay, resin-binding assays, and
co-immunoprecipitations to demonstrate that Grb10 interacts with at
least two members of the mitogenic MAP kinase cascade: Raf1 and MEK1.
The extreme 5'-end of the cDNA initially isolated in the two-hybrid
screen differs slightly from other Grb10 clones, possibly the result of
splicing variation. To confirm that the interactions with the kinases
are not limited to this specific SH2 domain variant, we used PCR to
isolate a full-length human Grb10 cDNA, hGrb10, and observed,
through resin-binding assays and immunoprecipitations, that it too can
interact with Raf1 and MEK1. Phosphotyrosine blots, phosphatase
treatment, and mutagenesis of the kinases have demonstrated that the
recognition of Raf1 and MEK1 by the Grb10 SH2 domain is mediated by
phosphothreonine or phosphoserine residues. Examples of
phosphotyrosine-independent binding by SH2 domains have been described
before. These include the recognition of Bcr by the SH2 domains of
cAbl, phospholipase C
, Src, and GTPase-activating protein (35, 36)
and the binding of vAlb to Shc (40). It has also been reported that the
SH2 domains of Fyn and Src interact with a phosphoserine residue on Raf
(39), while a 62-kDa ubiquitin-binding protein is a
phosphotyrosine-independent ligand of the p56lck SH2 domain
(37). There is also evidence for the interaction of the SH2 domain of
the Syp phosphatase with its own catalytic domain in the absence of
phosphotyrosine (38).
In a two-hybrid assay, only the isolated amino-terminal domain of Raf1 binds to the Grb10 SH2 domain. The absence of interaction with the full-length kinase is either an indication that the binding site is masked in the context of the whole protein or simply an artifact (our laboratory and others have frequently observed in two-hybrid assays that, while individual domains will interact with a given protein, the full-length protein will not (49)). In vivo interaction between Raf1 and Grb10 was observed in resting 293 HEK cells, which suggests that binding is mediated via a constitutively phosphorylated residue. For example, in unstimulated Balb/3T3 cells, Ser-43 and Ser-259, both of which are located in the regulatory domain of Raf1, are phosphorylated by means of a mechanism other than autophosphorylation (34). Binding between Grb10 and Raf1 is cell-type specific, since we failed to detect this interaction in NIH-3T3 and HTC-IR cells. The Grb10-binding site on MEK1 appears to be located in its carboxyl-terminal tail. Threonine 386, a residue that is phosphorylated in vivo by MAK kinases (41, 42, 50, 51), was shown to play a partial role in Grb10 recognition. The residual binding of the SH2 domain to the MEK1-T386A mutant indicates that phosphorylation at this residue might not be as critical as it is for phosphotyrosine-containing targets. The difficulty in synthesizing threonine-phosphorylated peptides has greatly hampered further research into this area.
Based on the published structures of SH2 domains bound to
phosphotyrosine targets, we know that Arg-B5 is positioned at the heart of the phosphate-binding pocket and makes hydrogen bonds with the
phosphate residue. This interaction is also critical in the binding of
the SH2 domain to Raf1 and MEK1, thus reinforcing our hypothesis for
the recognition of a phosphoamino acid by Grb10. The equivalent of
another SH2 domain residue necessary for interaction with the tyrosine
kinase receptors and the two kinases, Asp-EF2, is positioned in Syp
next to the polypeptide-binding groove and can theoretically make
interactions with residues downstream of the phosphorylated amino acid.
Structural analysis (43, 44, 46) and random mutagenesis of the
phosphatidylinositol 3-kinase SH2 domain (52) have previously
demonstrated the importance of the EF loop in binding specificity. The
positions of the
B5 and EF2 mutants therefore suggest that the
tyrosine-phosphorylated receptors and the Raf1 and MEK1 kinases
interact with the Grb10 SH2 domain in a very similar fashion, involving
interactions with a phosphorylated amino acid as well as with residues
located further downstream. These results also suggest that the Grb10
SH2 domain cannot interact with more than one of these targets
simultaneously, although this hypothesis has not been tested
experimentally. Other residues in the Grb10 SH2 domain were shown to be
involved in interaction with only some of its targets. One of these is
Ser-
B7, which is conserved between Grb10 and Syp and, in the latter
case, makes hydrogen bonds with the phosphotyrosine. Mutagenesis of this residue into a cysteine has much greater effects on interactions with the receptors. If binding of the Raf1 and MEK1 kinases to Grb10 is
coordinated by a much shorter phosphoserine or phosphothreonine residue, the phosphate group might not be in a position to make significant interactions with Ser-
B7. Another mutant, Thr-
C5
Ser, abrogates binding only to MEK1. Interestingly, almost all SH2
domains contain a serine at position
C5, including those found in
Grb7 and Grb14. Other elements of the Grb10 SH2 domain have not been
studied in detail but might also play a role in its binding
specificity. For example, two residues that are critical for
phosphotyrosine interactions in most SH2 domains, His-
D4 and
Lys-
D6, are not conserved in Grb10. It was suggested, based on the
Syp three-dimensional structure, that the
D-strand prevents shorter
(3.5-Å) phosphothreonines and phosphoserines from coordinating with
the Arg-
B5 without an intervening water molecule, thus permitting interactions with only the 7.0-Å phosphotyrosine (46). In addition, the residue at position
D6 was recently shown to be a major
determinant in the recognition of the tyrosine-phosphorylated ErbB2
receptor by Grb7 (53).
Fath et al. (54) have shown that expression of an isoform of the Grb2 adapter protein that is missing part of its SH2 domain induces apoptosis in Swiss 3T3 cells. We observed a similar phenomenon when the expression of Grb10 point mutants, whose SH2 domains are unable to interact with either MEK1 or with two tyrosine kinase receptors, increased the number of apoptotic cells following lipofectamine-mediated transfection. Reversal of the cell death phenotype by concomitant expression of the wild type Grb10 suggests that these mutants are acting by sequestering necessary signaling components. It has already been demonstrated that some tyrosine kinase receptors and the RAF-MEK-ERK pathway inhibit apoptosis (56, 57) and that the equilibrium between the ERK and JNK pathways determines the choice between proliferation and programmed cell death (57, 58).
In conclusion, our work places Grb10 in the expanding family of kinase-anchoring (or scaffolding) proteins (for reviews, see Refs. 59 and 60). These include the budding yeast Ste5p (49, 61, 62), the Ksr kinase (63, 64), the phosphoserine-binding 14-3-3 protein (65, 66), AKAP79 (67), its close homologue Gravin (68), and the SH2/SH3 adapter proteins Nck (69, 70) and Grb2 (71). The specific role of Grb10 in the transmission of proliferative signals including the possibility that it, like many of these proteins, is involved in the subcellular localization of multiprotein complexes is still being investigated.
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ACKNOWLEDGEMENTS |
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We thank Roger Brent, Bernard Massie, Sylvain Meloche, Steve Pelech, Axel Ullrich, and Jim Woodgett for gifts of plasmids used in this study. We are also grateful to Csilla Csank, Louise Larose, Ekkerhard Leberer, and Malcolm Whiteway for comments and to Lucie Bourget for expertise in flow cytometry.
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FOOTNOTES |
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* This is National Research Council of Canada publication number 41397.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.
§ Recipient of a postdoctoral fellowship from the National Science and Engineering Research Council of Canada. To whom correspondence should be addressed: Tel.: 514-496-6145; Fax: 514-496-6213; E-mail: andre.nantel{at}bri.nrc.ca.
1 The abbreviations used are: SH2 and SH3, Src homology 2 and 3, respectively; AV, adenovirus(es); EGF, epidermal growth factor; GFP, green fluorescent protein; GST, glutathione S-transferase; IR, insulin receptor; MBP, maltose-binding protein; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; aa, amino acids; DMEM, Dulbecco's modified Eagle's medium, TUNEL, TdT-mediated dUTP nick end-labeling.
2 We use the nomenclature for Grb10 splice variants that was agreed upon by several researchers and is maintained on the World Wide Web at www.bri.nrc.ca/thomasweb/grb7.htm.
3 GenBankTM accession number AF000018.
4 GenBankTM accession number AF000017.
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REFERENCES |
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