A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.

Abstract

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.

Full text

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TECHNICAL REPORT
A proteomics strategy to
elucidate functional protein-
protein interactions applied
to EGF signaling
Blagoy Blagoev, Irina Kratchmarova, Shao-En Ong,
Mogens Nielsen, Leonard J.Foster, and Matthias Mann*
Published online 10 February 2003; doi:10.1038/nbt790
Mass spectrometry–based proteomics1can reveal protein-protein
interactions on a large scale2,3, but it has been difficult to separate
background binding from functionally important interactions and
still preserve weak binders. To investigate the epidermal growth
factor receptor (EGFR) pathway4–6, we employ stable isotopic
amino acids in cell culture (SILAC)7to differentially label proteins
in EGF-stimulated versus unstimulated cells. Combined cell
lysates were affinity-purified over the SH2 domain of the adapter
protein Grb2 (GST-SH2 fusion protein) that specifically binds
phosphorylated EGFR and Src homologous and collagen (Shc)
protein. We identified 228 proteins, of which 28 were selectively
enriched upon stimulation. EGFR and Shc, which interact directly
with the bait, had large differential ratios. Many signaling mole-
cules specifically formed complexes with the activated EGFR-
Shc, as did plectin, epiplakin, cytokeratin networks, histone H3,
the glycosylphosphatidylinositol (GPI)-anchored molecule CD59,
and two novel proteins. SILAC combined with modification-based
affinity purification is a useful approach to detect specific and
functional protein-protein interactions.
The ebb and flow of cellular life depends largely on signaling path-
ways that are regulated by specific protein-protein interactions.
These interactions often involve assembly of large signaling com-
plexes containing many different protein kinases, protein phos-
phatases, their substrates, and scaffold proteins. A useful method to
study protein-protein interactions is affinity purification using a bait
molecule, but this is normally confined to verification of suspected
interactions by means of western blotting or selective sequencing of
the most prominent interaction partners. Here we extend these
methods to the study of signaling-dependent interactions.
To study the interactions of endogenous, activated EGFR, we
devised a strategy based on incorporating isotopically-labelled argi-
nine ([13C6]Arg) into the proteins of a cell population and a post-
translational modification-dependent affinity purification (Fig. 1).
This led to the identification of 228 proteins (see Supplementary
Table 1 online),of which 28 were specifically enriched by Grb2-SH2,
with ratios >1.3 (Table 1 and Fig.2).Reproducibility was assessed by
(i) determining the variability of the ratios for different peptides of
the same protein,(ii) determining the variability of the peptide ratio
from individual mass spectra over the elution peak, and (iii) doing
independent runs for some of the fractions,each of which resulted in
an analytical error smaller than the cutoff of 1.3-fold change.Among
the 28 proteins,EGFR and Shc are known to bind directly to the SH2
domain of Grb2 in their phosphorylated state. This direct binding
was reflected in the ratios for these two proteins,in that their magni-
tude clearly differentiated them from the remaining proteins in the
list (Table 1). Because the binding of Grb2 to Shc occurs only after
the latter has already been recruited to and phosphorylated by the
activated EGFR, this strategy specifically enriches only proteins
involved in EGF signaling. Even if the SH2 domain of Grb2 bound
other tyrosine-phosphorylated proteins, these would have to
increase as a result of EGF stimulation to register in this experiment.
To explore further the activation state of EGFR isolated in this man-
ner, we measured receptor autophosphorylation in the unlabeled,
unstimulated versus the labeled,EGF-stimulated states (Fig.2).Two
phosphorylation sites, Tyr1086 and Tyr1173—known binding sites
of Grb2 and Shc, respectively—were quantified by SILAC. Indeed,
autophosphorylated peptides were only observed in the labeled
(activated) state, proving that only activated receptor is enriched in
this scheme.
Endogenous Grb2 and Sos-1 were associated with the activated
complex, as expected (Table 1). In the well-established model of
EGFR signaling5, Grb2 is recruited to the receptor, bringing Sos-1
close to Ras at the plasma membrane. Sos-1 then activates Ras by
GTP exchange,which in turn leads to the activation of the canonical
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Center for Experimental BioInformatics (CEBI),Department of Biochemistry
and Molecular Biology,University of Southern Denmark,Campusvej 55,DK-
5230 Odense M,Denmark.*Corresponding author (mann@bmb.sdu.dk).
Figure 1. Strategy to study activated EGFR complex.One cell population
is grown in normal arginine-containing medium (blue dish), whereas
another population is grown in [13C]arginine ([13C]Arg)–containing medium
(red dish), encoding all proteins with this 6 Da heavier amino acid (red
color).The encoded cells are stimulated with EGF, lysed, and mixed 1:1
with the lysate of unlabeled, untreated cells.The combined cell lysates
are affinity-purified with GST fused to the SH2 domain of Grb2, which
specifically interacts with the activated, phosphorylated form of the
receptor and its associated binders.Proteins eluted from the beads are
digested with trypsin and the resulting peptides analyzed by mass
spectrometry.Arginine-containing peptides occur in doublets, separated
by 6 Da.Proteins that interact with the bait in a stimulation-dependent
manner are mostly in their encoded form and therefore have a more
intense peak for the labeled arginine-containing peptide (red peaks).
These proteins can be distinguished from a large excess of background
binding proteins, which manifest themselves by their 1:1 ratio between
stimulated and unstimulated states.
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Raf-MAPK cascade. Another consequence of Ras activation is the
stimulation of other small GTPases (Rac, Rho, Cdc42) that direct
cytoskeletal rearrangements. In this context we identified Vav-2, a
Rho-regulating protein that binds to phosphorylated EGFR8, and
Vav-3,which is also reported to associate with the activated receptor9.
The IQ motif–containing GTPase-activating protein-1 is a known
binding partner for both Rac1 and Cdc42. This binding is stimulated
by EGF and has importance for cellular polarization and cytoskeletal
remodeling10. To date, Rac GTPase–activating protein-1, another
modulator of small GTPases, has mainly been described in
hematopoietic and germ cell development11,but the fruit fly homolog,
DRacGAP (34% identity),is a negative regulator of EGFR signaling12.
Signaling effects are produced as a result of a balance of activat-
ing and deactivating steps, and shortly after stimulation, EGFR is
internalized and deactivated through a complex process involving
multiple mechanisms5.Among the proteins specifically recruited to
activated EGFR, we found all four subunits of the adapter complex
AP-2, an essential component of receptor-mediated endocytosis by
means of clathrin-coated vesicles13. We have also identified mem-
bers of the ubiquitination pathway, including ubiquitin itself and
Cbl, a signaling molecule that has an E3 ligase function and directs
proteasome-mediated destruction of EGFR5. Ubiquitin and ubiq-
uitin-like molecule C were identified in the same molecular-weight
fractions that contained EGFR,and at high ratios,suggesting that a
proportion of the receptor is ubiquitinated within 10 minutes of
stimulation. Finally, the receptor tyrosine phosphatase SHP-2 also
showed an increased ratio after EGF stimulation14,15.
An additional class of proteins identified in this study are
involved in growth factor–induced cytoskeletal rearrangement.
Association of EGFR and Shc with actin microfilaments is already
known16,but we also find the cytoskeletal keratins 7,8,17,and 18 in
specific association with EGFR complexes—all with similar ratios.
Note that specifically interacting keratins can easily be distin-
guished from contaminating hair or skin keratins,because the latter
do not result in any [13C6]Arg-labeled peptides.In addition,we find
the intermediate filaments binding proteins plectin and epiplakin,
both from the same protein family17.Confocal immunofluorescence
microscopy showed co-localization between Shc and the cytoker-
atin network (Fig.3,top row).Together these findings strongly sug-
gest association of EGFR complexes with intermediate filaments.
Identification of all of the proteins just mentioned shows that our
strategy was effective in pinpointing known,functional members of
the EGFR complex in a large background of >200 proteins. Two
novel proteins were also identified in this extensively studied path-
way (Table 1).The sequence of one has homology to Rho-interacting
protein-3 (RIP-3), suggesting that it may be involved in EGFR-
mediated signaling through small GTPases. The other protein has
homology to Caenorhabditis elegans smu-1 and rat brain–enriched
Figure 2. Quantification of protein ratios from peptide doublets.The panels show a section of the mass spectrum of peptides from different identified
proteins.The lower-mass peak cluster is due to the peptide with normal arginine, whereas the higher-mass peak cluster derives from the same peptide
from the labeled and stimulated cell population.The top left and top middle panels show peptide quantification doublets from specifically enriched
proteins due to direct binding (EGFR and Shc), as seen by the increased peak intensities of the labeled peptide (solid dot) over the unlabeled peptide
(unfilled dot) in the mass spectrum.The top right panel shows a doublet from cytokeratin-17, which is enriched to a lesser degree, indicating secondary
binding to the bait by means of activated EGFR complex.The bottom left panel demonstrates the typical 1:1 ratio observed in peptide doublets from
proteins that are not specifically enriched.Protein abundance ratios are the average of the ratios observed for several mass spectra, similar to the panels
shown here, and the average of several peptides identifying the same protein.The bottom right panel shows identification of the peptide containing the
EGFR autophosphorylation site Tyr1173 (Y1173).Only the [13C6]Arg-labeled form of the phosphopeptide is detected (see intensity of peak marked by
solid dot versus unfilled dot), indicating that activated receptor was purified exclusively.
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WD-repeat protein18,19. It contains several putative WD40 repeats
thought to be involved in mediating protein-protein interactions, a
LisH domain, and an associated CTLH domain (C-terminal to
LisH), suggested to be involved in cytoskeletal dynamics. No func-
tions have been described for the homologs except C. elegans smu-1,
mutations of which affect alternative splicing events18.
EGFR has been found in the nucleus, and there is increasing, if
controversial, evidence of direct nuclear function20,21. In this respect
it is interesting that we found association of activated complex with
snRNP D2 and histone H3. Confocal immunofluorescence
microscopy of histone H3 and EGFR showed a punctate nuclear
localization of histone H3 and partial nuclear localization of EGFR.
In the nucleus EGFR showed extensive co-localization with histone
H3 (Fig. 3, middle row). Further study will be necessary to address
the potential relevance of these findings with respect to the role of
histone H3 in regulating gene expression and reports of EGFR-
mediated histone H3 phosphorylation22.
Surprisingly,we also identified CD59,an extracellular GPI-anchored
molecule, as a component of the activated complex (Table 1).
Functional characterization of CD59,a typical marker for lipid rafts,
has centered on its inhibitory role in the complement system and in
T-cell receptor activation23. Although some studies have reported
increased cellular tyrosine kinase activity upon CD59 stimulation24,25,
connection with EGFR signaling has not been suggested.
Fluorescence resonance energy transfer (FRET) experiments between
antibody-labeled CD59 and EGFR indicated that the two proteins
are,if not directly associated,at least very closely linked in the plasma
membrane (Fig.3,bottom row).In addition,we observed that a sub-
stantial fraction of plasma membrane CD59 disappeared upon EGF
stimulation (data not shown).Further functional studies will be nec-
essary to address the precise role of CD59 in EGF signaling.
Several advantages of the strategy described here are apparent.
Functional protein-protein interactions are revealed through isotope
ratios, avoiding a trade-off between false-positive binding and the
ability to detect weak interactions. In the SH2 affinity purification
reported here,we were able to determine a small number of function-
al binders among hundreds of nonspecific binders.The strategy is rel-
atively straightforward, involving only standard co-precipitation
techniques, and can thus be useful in a wide variety of situations.
Endogenous complexes are captured in an activation-dependent
manner, reducing the chance of artifacts due to overexpression.
Table 1. Proteins recruited to activated Grb2-EGFR complex
Gi no.a
Protein nameRatios.d.b
No. of
peptides
Proteins directly involved in signaling
4885199 Epidermal growth factor
receptor (EGFR)
284403 Shc
4504111Grb2
476780 Sos-1
4506787IQ motif containing GAP1
7019433Rac GAP1
11429601Vav-2
3928847Vav-3
15.8 6.3 23
7.9
1.46
2.01
1.46
1.45
1.75
2.54
2.2
0.12
0.03
0.18
0.19
0.21
0.13
22
1
1
5
4
2
1
Proteins involved in signal attenuation
19913416 AP-2, α1 subunit
4557469AP-2, β1 subunit
11038643 AP-2, σ1 subunit
18554450 AP-2, µ1 subunit
8928017Cbl-Bc
7445542Polyubiquitin
17481486Similar to ubiquitin C
464494SHP-2c
2.73
2.11
2.77
1.85
1.73
1.64
3.21
2
0.4
0.27
0.1
0.16
0.04
0.07
0.38
0.15
4
3
1
1
3
2
2
5
Proteins involved in cytoskeletal functions
4501889Actin
14783596Keratin-7
2506774Keratin-8
4557701Keratin-17
12653819Keratin-18
4505877Plectin-1
13876386 Epiplakin-1
1.48
1.47
1.54
1.43
1.36
1.48
1.43
0.19
0.18
0.19
0.21
0.13
0.24
0.11
5
16
20
20
11
21
9
Other proteins with no previously reported
involvement in EGFR signaling
515118 CD59
4504281Histone H3, member A
4759158snRNP D2
14770022 Novel (similar to RIP-3)
8922679Novel (with WD repeats)
1.48
1.52
1.35
1.4
1.34
0.27
0.09
0.05
0.09
0.1
4
2
1
1
3
aNCBI GenBank accession number.
bStandard deviation determined from quantification of several peptides or from
the variance of the ratios obtained from individual mass spectra in the case of
proteins quantified with a single peptide.
cProteins that also have direct signaling function.
Figure 3. Subcellular co-localization of selected proteins by confocal
microscopy and FRET.HeLa cells stained with antibodies against Shc and
keratin (top row) or EGFR and histone H3 (middle row) and their respective
overlays.Anti-keratin-18 antibody was conjugated to FITC;Cy5-conjugated
anti-rabbit secondary antibodies were used to visualize Shc and histone
H3 labeling, whereas Cy3-conjugated anti-goat antibodies were used for
EGFR.Confocal slices are between 0.5 and 0.6 µm;for EGFR–histone H3,
the image was taken through the center of the nucleus, 3–4 µm from the
bottom of the cell, whereas for keratin-Shc, the image was taken near the
bottom of the cell.Bottom row:Close association of CD59 and EGFR at
the cell surface revealed by FRET.Unpermeabilized HeLa cells were
stained as described with antibodies against EGFR (left) and CD59
(middle), and then stained by Cy3-conjugated anti-sheep (left) and Cy5-
conjugated anti-mouse secondary antibodies (middle).FRET between Cy3
and Cy5 (right) was detected by exciting the sample with 543 nm light and
measuring emission intensity in the 645- to 720-nm range (see
Experimental Protocol).FRET image is color-coded, with lower intensities
colored blue and higher intensities colored red.Confocal slices are
between 0.5 and 0.6 µm.Scale bar, 10 µm.
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Dynamics of complexes could also be studied by carrying out the
experiment at different time points.Combined with the automation
demonstrated in recent large-scale proteomics experiments, the
strategy described here can add crucial data for deciphering not only
EGFR,but also many other dynamic signaling networks.
Experimental protocol
Cell culture and affinity purification. For labeling experiments, 2 × 108
HeLa cells (ATCC; American Type Culture Collection, Manassas, VA) were
grown in medium containing either normal or [13C6]arginine (Cambridge
Isotope Labs,Andover,MA) as described7.After 8 h of serum starvation,the
labeled cells were stimulated with 150 ng/ml EGF (Upstate Biotechnology,
Lake Placid,NY) for 10 min,whereas the unlabeled cells were left untreated.
Cells from both conditions were lysed in lysis buffer containing 1%
(vol/vol) Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM sodium
orthovanadate, and protease inhibitors (Complete tablets; Roche,
Mannheim, Germany). Lysates were mixed in a 1:1 ratio according to their
protein concentrations and incubated at 4 °C with Grb2 fusion protein
bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech,
Uppsala,Sweden).After 4 h of incubation,the beads were washed extensively
with lysis buffer,boiled in LDS sample buffer (Invitrogen,Carlsbad,CA),and
resolved on a 4–12% (wt/vol) Novex gel (Invitrogen). The entire lane was
excised in ten bands and digested enzymatically with trypsin for liquid chro-
matography–tandem mass spectrometry (LC-MS/MS) analyses26.
The plasmid pGEX-2T (Amersham Pharmacia Biotech), containing a
300 bp fragment encoding the SH2 domain of human Grb2 (amino acids
59–158), was a gift from Akhilesh Pandey (Johns Hopkins University,
Baltimore, MD). The GST fusion protein was expressed in Escherichia coli
and subsequently purified on glutathione Sepharose beads (purity >95%)
following the procedure described by the manufacturer (Amersham
Pharmacia Biotech).
Mass-spectrometric analysis. Nanoscale LC-MS and LC-MS/MS was done
with a quadrupole time-of-flight instrument (QSTAR-Pulsar; ABI-SCIEX,
Toronto, Canada) essentially as described27. A linear gradient elution from
95% buffer A (H2O–acetic acid, 100:0.5 vol/vol) to 50% buffer B (H2O–
acetonitrile–acetic acid, 20:80:0.5 vol/vol) in 120 min chromatographically
separated the peptides.Protein identification was done with the Mascot soft-
ware package (Matrix Science, London, UK) using the NCBI nonredundant
protein database. Arginine-containing peptides (approximately half of all
tryptic peptides) appeared in a labeled and unlabeled form separated by 6 Da
and co-elute exactly, aiding quantification. High-resolution extracted ion
chromatograms (±0.03 Da) for the labeled and unlabeled form were
obtained,reducing background contribution.Peptide abundance ratios from
the peak intensities of the extracted ion chromatograms were averaged to give
protein abundance ratios. Peptide ratios were also verified by quantification
directly on the basis of the mass spectra.
Microscopy. The following antibodies were used: goat anti-EGFR (Santa
Cruz Biotechnologies, Santa Cruz, CA), sheep anti-EGFR (Upstate
Biotechnology), rabbit anti–histone H3 (Cell Signaling Technologies,
Beverly,MA),fluorescein isothiocyanate (FITC)–conjugated mouse anti-ker-
atin-18 (Sigma, Copenhagen, Denmark), CD59 (Abcam, Cambridge, UK),
Shc (BD Transduction Laboratories, Lexington, KY), Alexa-488-conjugated
anti-rabbit (Molecular Probes, Eugene, OR), and cyanin-3 (Cy3)- and Cy5-
conjugated anti-goat, -rabbit, -mouse, and -sheep antibodies (Jackson
Immuno Labs,West Grove,PA).
Serum-starved HeLa cells were fixed in 4% (vol/vol) formaldehyde for
10 min on ice, permeabilized where indicated with 0.1% (vol/vol) Triton
X-100 for 20 min,and blocked for 30 min with 3% (vol/vol) BSA in PBS or,in
the case of samples stained for histone H3, with 3% (wt/vol) skim milk
powder–3% horse serum in PBS. Primary and secondary antibody incuba-
tions were 1 h each, and the coverslips were washed 4× with PBS after each
incubation.Coverslips were mounted on glass slides and imaged using a Zeiss
Axiovert 200M laser scanning confocal microscope 510 equipped with a
META polychromatic multichannel detector.FRET between Cy3 (EGFR) and
Cy5 (CD59) was measured as described for sensitized acceptor fluorescence28:
Cy3 was excited using the 543 nm laser line of a He-Ne laser, and Cy5 emis-
sion was detected with the META system configured to measure wavelengths
between 645 and 720 nm.Under conditions identical to those used for FRET
analysis (detector gain, pinhole, laser transmission percentage), no signal in
the FRET channel was seen for cells labeled only for EGFR (with Cy3) or only
for CD59 (with Cy5).
Note: Supplementary information is available on the Nature Biotechnology
website.
Acknowledgments
We thank other members of our laboratory for help and fruitful discussions and
comments on the manuscript.Akhilesh Pandey provided the GST-SH2 (Grb2)
domain construct.Work in the Center for Experimental BioInformatics (CEBI)
is supported by a generous grant by the Danish National Research foundation.
Competing interests statement
The authors declare that they have no competing financial interests.
Received 12 November 2002; accepted 16 January 2003
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