Mass Spectrometry Mapping of Epidermal Growth Factor Receptor Phosphorylation Related to Oncogenic Mutations and Tyrosine Kinase Inhibitor Sensitivity

Zhang, Guolin; Fang, Bin; Liu, Richard Z.; Lin, Hui‐Yi; Kinose, Fumi; Bai, Yun; Oguz, Umut; Remily-Wood, Elizabeth; Li, Jiannong; Altiok, Soner; Eschrich, Steven; Koomen, John M.; Haura, Eric B.

doi:10.1021/pr1006203

Cited by 56 publications

(52 citation statements)

References 76 publications

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“…Left-hand and right-hand bars represent all and unique peptides, respectively. EGFR tyrosine residues, along with Tyr1092 and Tyr1197 (found in cluster 4 in the present study), were inhibited by erlotinib in a concentration-dependent manner (33). Other highly down-regulated phosphosites were Tyr771 of phospholipase C-gamma-1 (PLCG1) and Tyr427 of SHC-transforming protein 1 (SHC1).…”

Section: Fig 4 Identified and Quantitated Tyr-phosphorylated Peptidessupporting

confidence: 52%

“…Tyr-phosphorylated Sites Modulated by Erlotinib-Erlotinib is a reversible inhibitor of the EGFR kinase that competes with ATP for its binding site. Therefore, the phosphorylation of EGFR, its substrates, and downstream molecules is expected to be down-regulated (32)(33)(34). For the present experiment, an erlotinib concentration of 5 M was selected because it corresponds approximately to the measured plasma level in humans (35).…”

Section: Resultsmentioning

confidence: 99%

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Development of a 5-plex SILAC Method Tuned for the Quantitation of Tyrosine Phosphorylation Dynamics

Tzouros

Golling

Ávila

et al. 2013

Molecular & Cellular Proteomics

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The propagation of phosphorylation downstream of receptor tyrosine kinases is a key dynamic cellular event involved in signal transduction, which is often deregulated in disease states such as cancer. Probing phosphorylation dynamics is therefore crucial for understanding receptor tyrosine kinases' function and finding ways to inhibit their effects. MS methods combined with metabolic labeling such as stable isotope labeling with amino acids in cell culture (SILAC) have already proven successful in deciphering temporal phosphotyrosine perturbations. However, they are limited in terms of multiplexing, and they also are time consuming, because several experiments need to be performed separately. Here, we introduce an innovative approach based on 5-plex SILAC that allows monitoring of phosphotyrosine signaling perturbations induced by a drug treatment in one single experiment. Using this new labeling strategy specifically tailored for phosphotyrosines, it was possible to generate the time profiles for 318 unique phosphopeptides belonging to 215 proteins from an erlotinib-treated breast cancer cell line model. Hierarchical clustering of the time profiles followed by pathway enrichment analysis highlighted epidermal growth factor receptor (EGFR or ErbB1) and ErbB2 signaling as the major pathways affected by erlotinib, thereby validating the method. Moreover, based on the similarity of its time profile to those of other proteins in the ErbB pathways, the phosphorylation at Tyr453 of protein FAM59A, a recently described adaptor of EGFR, was confirmed as tightly involved in the signaling cascade. The present investigation also demonstrates the remote effect of EGFR inhibition on ErbB3 phosphorylation sites such as Tyr1289 and Tyr1328, as well as a potential feedback effect on Tyr877 of ErbB2. Overall, the 5-plex SILAC is a straightforward approach that extends sample multiplexing and builds up the arsenal of methods for tyrosine phosphorylation dynamics. Molecular & Cellular Proteomics 12: 10.1074/mcp.O113.027342, 3339-3349, 2013.The transmission of information in a cell is largely driven by protein phosphorylation (1, 2). It is used to convey a signal within the cell and modulate different biological outcomes such as transcription regulation or cytoskeleton rearrangement. The receptor tyrosine kinases represent key players at the plasma membrane interface because they operate as initiators of phosphorylation cascades (3, 4). Among the 58 receptor tyrosine kinases encoded by the human genome, the ErbB family members-composed of ErbB1 (also called epidermal growth factor receptor (EGFR)), 1 ErbB2, ErbB3, and ErbB4 -play a pivotal role because they control important processes such as cell differentiation, proliferation, and apoptosis (5). Deregulation of ErbB signaling networks is known to be connected to various diseases such as cancer (6, 7). Thus, the ErbB family members represent an important target class for understanding and curing disease-indeed, both small-molecule tyrosine kinase inhibitors (TKIs) and monoclonal a...

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Section: Fig 4 Identified and Quantitated Tyr-phosphorylated Peptidessupporting

confidence: 52%

Section: Resultsmentioning

confidence: 99%

Development of a 5-plex SILAC Method Tuned for the Quantitation of Tyrosine Phosphorylation Dynamics

Tzouros

Golling

Ávila

et al. 2013

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…Multiplexed protein quantitation by multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) are powerful tools for systems characterization (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39). These multiplexed assays can interrogate coordinated expression of proteins in functional protein networks, such ␤-catenin signaling (35), nuclear factor-B signaling (36), protein expression changes because of EGFR signaling (37,38), and phosphotyrosine quantitation in EGFR signaling (39).…”

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confidence: 99%

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer

Hutton

Wang

Zimmerman

et al. 2016

Molecular & Cellular Proteomics

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Metabolic reprogramming, in which altered utilization of glucose and glutamine supports rapid growth, is a hallmark of most cancers. Mutations in the oncogenes KRAS and BRAF drive metabolic reprogramming through enhanced glucose uptake, but the broader impact of these mutations on pathways of carbon metabolism is unknown. Global shotgun proteomic analysis of isogenic DLD-1 and RKO colon cancer cell lines expressing mutant and wild type KRAS or BRAF, respectively, failed to identify significant differences (at least 2-fold) in metabolic protein abundance. However, a multiplexed parallel reaction monitoring (PRM) strategy targeting 73 metabolic proteins identified significant protein abundance increases of 1.25-twofold in glycolysis, the nonoxidative pentose phosphate pathway, glutamine metabolism, and the phosphoserine biosynthetic pathway in cells with KRAS G13D mutations or BRAF V600E mutations. These alterations corresponded to mutant KRAS and BRAF-dependent increases in glucose uptake and lactate production. Metabolic reprogramming and glucose conversion to lactate in RKO cells were proportional to levels of BRAF V600E protein.In DLD-1 cells, these effects were independent of the ratio of KRAS G13D to KRAS wild type protein. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutant status. Metabolic reprogramming is driven largely by modest (<2-fold) alterations in protein expression, which are not readily detected by the global profiling methods most commonly employed in proteomic studies. The results indicate the superiority of more precise, multiplexed, pathway-targeted analyses to study functional proteome systems. Data are available through MassIVE Accession MSV000079486 at

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“…The advantage of analyzing purified protein complexes is the ability to identify specific interacting proteins and posttranslational modifications that may otherwise go undetected in large-scale global analyses. This can be achieved by performing an immunoprecipitation (IP) with a bait protein and analyzing the prey proteins using microcapillary-tandem mass spectrometry (LC-MS/MS) (6)(7)(8)(9)(10)(11)(12)(13). These experiments have included various types of affinity tags and have been overlaid with RNAi genetic screens (14,15).…”

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confidence: 99%

Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS)

Breitkopf

Yuan

Pihán

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Hypothesis directed proteomics offers higher throughput over global analyses. We show that immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) in H929 multiple myeloma (MM) cancer cells led to the discovery of a rare and unexpected BCR-ABL fusion, informing a therapeutic intervention using imatinib (Gleevec). BCR-ABL is the driving mutation in chronic myeloid leukemia (CML) and is uncommon to other cancers. Three different IP-MS experiments central to cell signaling pathways were sufficient to discover a BCR-ABL fusion in H929 cells: phosphotyrosine (pY) peptide IP, p85 regulatory subunit of phosphoinositide-3-kinase (PI3K) IP, and the GRB2 adaptor IP. The pY peptides inform tyrosine kinase activity, p85 IP informs the activating adaptors and receptor tyrosine kinases (RTKs) involved in AKT activation and GRB2 IP identifies RTKs and adaptors leading to ERK activation. Integration of the bait-prey data from the three separate experiments identified the BCR-ABL protein complex, which was confirmed by biochemistry, cytogenetic methods, and DNA sequencing revealed the e14a2 fusion transcript. The tyrosine phosphatase SHP2 and the GAB2 adaptor protein, important for MAPK signaling, were common to all three IP-MS experiments. The comparative treatment of tyrosine kinase inhibitor (TKI) drugs revealed only imatinib, the standard of care in CML, was inhibitory to BCR-ABL leading to down-regulation of pERK and pS6K and inhibiting cell proliferation. These data suggest a model for directed proteomics from patient tumor samples for selecting the appropriate TKI drug(s) based on IP and LC-MS/MS. The data also suggest that MM patients, in addition to CML patients, may benefit from BCR-ABL diagnostic screening.targeted therapies | personalized medicine | protein-protein interactions P roteomics studies over the last decade have provided significant advancements in our understanding of the functional landscape of proteins, their posttranslational modifications and the protein-protein interactions (PPI) (1-3). This is especially important for diseases such as cancer because proteins and their transient posttranslational modifications (PTMs) reflect the changes cells undergo during pathogenic development. Modern high-resolution mass spectrometers are well suited for these analyses and have led the efforts in this field of proteomics, allowing researchers to analyze dynamic changes in cell lines or tissue and to distinguish between normal and unhealthy states (4, 5). PPI networks and phosphoproteomics are important for understanding the function and regulation of pathways leading to cell growth and proliferation in diseases such as cancer. The advantage of analyzing purified protein complexes is the ability to identify specific interacting proteins and posttranslational modifications that may otherwise go undetected in large-scale global analyses. This can be achieved by performing an immunoprecipitation (IP) with a bait protein and analyzing the prey proteins using microcapillary-tandem mass spectrometry (LC-MS/MS) (6-1...

show abstract

Mass Spectrometry Mapping of Epidermal Growth Factor Receptor Phosphorylation Related to Oncogenic Mutations and Tyrosine Kinase Inhibitor Sensitivity

Cited by 56 publications

References 76 publications

Development of a 5-plex SILAC Method Tuned for the Quantitation of Tyrosine Phosphorylation Dynamics

Development of a 5-plex SILAC Method Tuned for the Quantitation of Tyrosine Phosphorylation Dynamics

Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer

Detection of a rare BCR–ABL tyrosine kinase fusion protein in H929 multiple myeloma cells using immunoprecipitation (IP)-tandem mass spectrometry (MS/MS)

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