ARTICLE pubs.acs.org/jpr
Quantitative Phospho-Proteomic Profiling of Hepatocyte Growth Factor (HGF)-MET Signaling in Colorectal Cancer Shawna L. Organ,†,‡ Jiefei Tong,§ Paul Taylor,§ Jonathan R. St-Germain,§,|| Roya Navab,† Michael F. Moran,*,§,|| and Ming-Sound Tsao*,†,‡,^ †
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Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Canada ‡ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada § Program in Molecular Structure and Function, Hospital For Sick Children, Toronto, Canada Department of Molecular Genetics, and Banting and Best Department of Medical Research, University of Toronto, Toronto, Canada ^ Department of Medical Biophysics, University of Toronto, Toronto, Canada
bS Supporting Information ABSTRACT: Colorectal cancer (CRC) is the second leading cause of death from cancer. The MET receptor tyrosine kinase and/or its ligand HGF are frequently amplified or overexpressed in CRC. It is known that tyrosine phosphorylated proteins are involved in progression and metastasis of colorectal cancer; however, little is known about the MET phospho-proteome in CRC. High resolution mass spectrometry was used to characterize immunoaffinitypurified, phosphotyrosine (pY)-containing tryptic peptides of the MET-expressing CRC cell model, DLD1. A total of 266 unambiguously identified pY sites spanning 168 proteins were identified. Quantification of mass spectrometry ion currents identified 161 pY sites, including many not previously linked to MET signaling, that were modulated in abundance by HGF stimulation. Overlay of these data with proteinprotein interaction data sets suggested that many of the identified HGF-modulated phospho-proteins may be directly or indirectly associated with MET. Analysis of pY sequence motifs indicated a prevalence of Src family kinase consensus sequences, and reciprocal signaling between Src and MET was confirmed by using selective small molecule inhibitors of these kinases. Therefore, using quantitative phospho-proteomics profiling, kinase modulation by ligand and inhibitors, and data integration, an outline of the MET signaling network was generated for the CRC model. KEYWORDS: MET/HGF, tyrosine phosphorylation, colorectal cancer, mass spectrometry, immunoaffinity enrichment, label-free quantitative analysis
’ INTRODUCTION Colorectal cancer (CRC) is the second leading cause of death from cancer.1 The overall 5-year survival rate is 4060%, but prognosis is highly dependent on tumor grade and stage. Activation of receptor tyrosine kinases (RTKs), such as the hepatocyte growth factor (HGF) receptor MET, is common in CRC. Our laboratory was the first to report that MET mRNA was overexpressed in primary CRC compared to normal mucosa, and that ∼70% of tumors showed at least a 4-fold increase in expression.2 Subsequent reports have largely confirmed that MET mRNA/protein is upregulated in greater than two-thirds of CRC.3,4 DiRenzo et al.5 also reported that CRC liver metastases commonly show MET mRNA and protein overexpression, which has been significantly correlated with higher stage in CRC patients.6,7 Thus, available data indicate that MET overexpression is important in CRC progression. Reversible phosphorylation is a pivotal mechanism that regulates the activity and function of signal transduction proteins. r 2011 American Chemical Society
Dysregulated tyrosine kinase activity is prevalent in many types of cancers,8 and can drive malignancy and tumor survival signals.9 The analysis of protein-phosphotyrosine (pY), which is relatively rare when compared with phosphorylated serine and threonine,10 has been facilitated by sensitive methods in mass spectrometry (MS) and pY-peptide affinity purification.11 Such approaches have been applied to outline cancer-associated, RTK-driven signaling networks such as the epidermal growth factor receptor (EGFR) in glioblastoma12 and fibroblast growth factor receptor-3 (FGFR3) in multiple myeloma.13 Although many studies have dissected RTK, and EGFR in particular, signaling networks in various cancers,10,1419 MET signaling in colorectal cancer has not been fully delineated. The MET RTK is a disulfide-linked heterodimer composed of an extracellular 50 kDa R chain and a transmembrane 145 kDa Received: March 15, 2011 Published: May 24, 2011 3200
dx.doi.org/10.1021/pr200238t | J. Proteome Res. 2011, 10, 3200–3211
Journal of Proteome Research β chain.20 HGF binds to the extracellular domain of the β chain,21,22 which causes trans autophosphorylation of tyrosines 1230, 1234, and 1235 in the MET kinase domain.23 Unique to the MET receptor is a multifunctional consensus sequence in the C-terminal tail.24 Phosphorylation of tyrosines 1349 and 1356 in this sequence create binding sites for the SH2 domains of downstream signaling mediators.24,25 Overexpression of the MET protein by either gene amplification or deregulated protein expression or degradation can cause or augment ligand-independent MET activation, resulting in uncontrolled downstream signaling.5,2628 Overexpression of HGF by stroma29 or tumor30 can also lead to deregulated MET signaling and further tumor progression. In this study, HGF/MET signaling in CRC was investigated and measured by label-free MS analysis of protein-pY in the DLD1 human CRC model,31 in which overexpression of MET enhances tumorigenicity32 and metastatic potential.30 A comprehensive set of HGF-regulated tyrosine phosphorylations was discovered that provides insight into mechanisms involved in CRC oncogenesis and tumor progression.
’ MATERIALS AND METHODS Cell Culture and Cell Lysis
The colorectal cancer DLD1 cell line was cultured routinely in DMEM supplemented with 10% FBS at 37 C under 5% CO2. One 108 cells per group were serum starved with serum-free DMEM for 24 h, then either lysed immediately or treated with 20 ng/mL HGF for 10 or 60 min. For MS analysis, cells were lysed in 8 M urea buffer then processed according to Rush et al.11 to enrich for pY-containing peptides. For MET and Src inhibitor experiments, DLD1 cells were plated at 50% confluence in 6 well dishes and serum starved as described above. Cells were then treated with either 10 μM PP2 (Calbiochem, La Jolla, CA) or 200 nM PHA-665752 (Pfizer, New York, NY) for 1 h then stimulated with HGF as described above, then lysed for Western blotting. LCMS/MS Analysis
Triplicate experiments were run sequentially on an LTQOrbitrap hybrid analyzer (LTQ-Orbitrap, ThermoFisher, San Jose, CA) outfitted with a nanoelectrospray source and EASYnLC split-free nano-LC system (Proxeon Biosystems, Odense, Denmark). The peptides were trapped on a homemade 150 μm ID trapping column containing 5 μm Magic C18 resin (Michrom Bioresources, Auburn, CA). The peptides were eluted using a 75 μm ID analytical column containing an emitter tip. The gradient applied was 040% acetonitrile in 0.1% formic acid at 300 nL/min. The instrument method consisted of one MS full scan (4001400 m/z) in the Orbitrap mass analyzer, and automatic gain control target of 500 000 ions with a maximum ion injection of 500 ms, one microscan, and a resolution of 60 000. Three data-dependent MS/MS scans were performed in the linear ion trap using the three most intense ions at 35% normalized collision energy. The MS and MS/MS scans were obtained in parallel fashion. In MS/MS mode, automatic gain control targets were 10 000 ions with a maximum ion injection time of 100 ms. A minimum ion intensity of 1000 was required to trigger an MS/MS spectrum. The dynamic exclusion was applied using a maximum exclusion list of 500 with one repeat count with a repeat duration of 30 s and exclusion duration of 45 s. Database Searching
Tandem mass spectra were extracted by Bio-Works version 3.3. Charge state deconvolution and deisotoping were performed. All
ARTICLE
MS/MS samples were analyzed using Sequest (ThermoFinnigan, San Jose, CA; version SRF v. 5) and X! Tandem (www.thegpm. org; version 2007.01.01.2). Both search engines were set up to search the IPI human database (version 3.41, 72,155 entries) assuming trypsin digestion, allowing two missed cleavages, and using a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 7.0 ppm. Iodoacetamide derivative of cysteine was specified as a fixed modification. Oxidation of methionine and phosphorylation of tyrosine, serine and threonine were specified in Sequest and X! Tandem as variable modifications. Modification by sulfation was not distinguishable from phosphorylation with the mass accuracy of these experiments. However, these isobaric modifications at tyrosines were assumed to be phosphorylation since the analyzed peptides were purified by using antiphosphotyrosine enrichment. The assignment of post-translational modifications was made by the search engines (described above) and verified by manual inspection. Ascore values, which indicate the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra,33 were determined by using the software program ScaffoldPTM (version ScaffoldPTM1.0.12, Proteome Software Inc., Portland, OR, www.proteomesoftware.com). Scores of g19 indicate a 99% probability of correct phosphorylation assignment. In 27 instances, the localization of phosphorylation within a phospho-peptide remained ambiguous following manual inspection and by Ascore, and these phospho-sites are listed in bold (Supporting Information (SI) Table 1). Criteria for Protein Identification
Scaffold34 (version Scaffold.3.00.03, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm.35 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. To identify novel phosphosites, all phosphosites were compared with the Phosphosite (www.phosphosite.org) and PhosphoElm (http://phospho.elm.eu.org/) databases. All data relating to MS/MS analysis and or phosphorylations described in this study will be made freely available through databases including: Human Proteinpedia (humanproteinpedia.org), TRANCHE (tranche.proteomecommons.org), PRIDE (ebi.ac.uk/ pride), Global Proteome Machine (thegpm.org), and PhosphoSite (phosphosite.org). Label-free Quantification
Mascot search results were used for peptide identification. Peptides were quantified by using IDEAL-Q software,36 which uses elution time alignment strategies to quantify extracted MS ion current (XIC) values between multiple experiments. Peptides that increased >2-fold or decreased
