SH2 domains recognize specific phosphopeptide sequences

Songyang, Zhou; Shoelson, Steven E.; Chaudhuri, Manas; Gish, Gerald; Pawson, Tony; Haser, Wayne G.; King, Fred L.; Roberts, Tom; Ratnofsky, Sheldon; Lechleider, Robert J.; Neel, Benjamin G.; Birge, Raymond B.; Fajardo, J. Eduardo; Chou, Margaret M.; Hanafusa, Hidesaburô; Schaffhausen, Brian; Cantley, Lewis C.

doi:10.1016/0092-8674(93)90404-e

Cited by 2,438 publications

(389 citation statements)

References 38 publications

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“…The p85 subunit contains two SH2 domains, which bind phosphotyrosine residues in the cytoplasmic domains of receptor tyrosine kinases. The consensus p85 binding site, YXXM (Zhou et al, 1993), is repeated seven times in the C-terminal region of ErbB3, suggesting an important role for PI3-K in ErbB3 signaling. While p85 itself can be tyrosine phosphorylated, this phosphorylation is not considered as critical for activation as membrane translocation (Klippel et al, 1996).…”

Section: Pi3-k Is Assembled Into An Activation Complexmentioning

confidence: 99%

“…Another salient feature of ErbB3 is its C-terminal tail, which is substantially longer and more divergent in sequence than the analogous regions of other family members (Kraus et al, 1989;Plowman et al, 1990). This cytoplasmic region bears many potential docking sites for src homology-2 (SH2) domain-containing proteins, which bind to phosphotyrosine residues in a speci®c sequence context (Zhou et al, 1993). Putative binding sites exist for the p85 subunit of phosphatidylinositol 3-kinase (PI3-K), the adaptor proteins SHC and GRB2, the src family tyrosine kinases, and the SHP-2/Syp tyrosine phosphatase.…”

Section: Introductionmentioning

confidence: 99%

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ErbB kinases and NDF signaling in human prostate cancer cells

et al. 1997

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Section: Pi3-k Is Assembled Into An Activation Complexmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

ErbB kinases and NDF signaling in human prostate cancer cells

et al. 1997

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“…Secondly, spectral TIC was used to calculate the relative quantitative ratios. Figure 1 shows the experimental workflow.From the label-free pTyr screen, we show nine proteins including eight known src homology 2 domain (SH2) domain-containing proteins [21] and pyruvate kinase M2 (PKM2), a glycolytic protein that is critical for proliferating cancer cells [22]. To demonstrate an example of a specific pTyr binding protein acquired by the spectral TIC method, the 85kDa regulatory beta subunit of phosphoinositide 3-kinase (p85) resulted in a spectral counting ratio of 15.0:1 (15 spectra from pTyr column and 1 spectrum from the Tyr column) while the spectral TIC ratio was 16:1 from the average TIC values 1.60E from the pTyr column and 1.0E 4 from the Tyr column.…”

mentioning

confidence: 99%

“…The binding of SH2 domains to pTyr is influenced by the amino acid sequence Nterminal and C-terminal of the pTyr residue [21]. Also, MS/ MS TIC values from shotgun DDA experiments can be subjective based on several factors during the analysis, including previous peptide ions that were attacked for MS/MS in data dependent cycles, the chromatographic elution point when a peptide ion is selected for MS/MS and differences in ionization efficiencies.…”

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confidence: 99%

A label‐free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

et al. 2008

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In order to assess the biological function of proteins and their modifications for understanding signaling mechanisms within cells as well as specific biomarkers to disease, it is important that quantitative information be obtained under different experimental conditions. Stable isotope labeling is a powerful method for accurately determining changes in the levels of proteins and PTMs; however, isotope labeling experiments suffer from limited dynamic range resulting in signal change ratios of less than ,20:1 using most commercial mass spectrometers. Label-free approaches to relative quantification in proteomics such as spectral counting have gained popularity since no additional chemistries are needed. Here, we show a label-free method for relative quantification based on the TIC from peptide MS/MS spectra collected from data-dependent runs can be used effectively as a quantitative measure and expands the dynamic range over isotope labeling experiments allowing for abundance differences up to ,60:1 in a screen for proteins that bind to phosphotyrosine residues. Keywords:Differential expression / Quantitative analysis / Shotgun proteomics / Signal transduction profiling 994 Proteomics 2008, 8, 994-999 Methods for acquiring quantitative proteomics data are continually developing with very accurate stable isotope labeling (SIL) and label-free approaches. SIL provides chemically equivalent but isotopically different internal standards for each peptide/protein for direct comparison of mass spectral signal intensities that represent relative abundance. Common SIL strategies include protein level labeling strategies such as stable isotope labeling of amino acids in cell culture (SILAC) [1], a global method whereby all translated proteins have isotope labels metabolically incorporated at selected amino acid residues, and isotope-coded affinity tags (ICAT) [2], a technique that labels cysteine residues at the protein level. Peptide level labeling strategies include multiplexed isobaric tags for relative and absolute quantification (iTRAQ) [3], global internal standard technology (GIST) [4], a global post-digestion labeling method that labels primary amine groups (peptide N-terminus and lysine residues) and an extension of GIST called in-gel stable isotope labeling (ISIL) [5,6], a method that labels primary amine groups of proteins (protein N-terminus and lysine residues) directly from gel separated samples. Each isotope labeling strategy has its advantages and disadvantages depending upon the experimental questions but most suffer from an experimental Proteomics 2008, 8, 994-999 Cell Biology 995 dynamic range limitation of ,20:1. Protein level differences greater than this limit usually suffer from very large errors in ratio determination [7]. Label-free approaches to quantitative proteomics have gained prominence in recent years since no additional chemistry or sample preparation steps are required. These include biostatistical profiling [8,9] in clinical proteomics whereby peptide ions that show intensity chang...

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“…Because of past results in a number of systems, it is clear that Grb2 is an activator of the Ras pathway. Grb2 binds to the consensus sequence YVNV when the tyrosine residue is phosphorylated (Zhou et al, 1993). This sequence is found in the ®rst exon coding region of BCR.…”

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confidence: 99%

Bcr phosphorylated on tyrosine 177 binds Grb2

Dai

et al. 1997

Oncogene

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We and others have shown that the Bcr-Abl oncoprotein binds activators of the Ras pathway such as Grb2 and Shc. Grb2 binding is mediated through a phosphorylated tyrosine residue (Y177) located within a consensus Grb2 binding site encoded by the ®rst exon of the BCR gene. Our results indicate that P160 BCR is tyrosine phosphorylated at the same site by Bcr-Abl in kinase assays (Puil et al., 1994). We performed experiments to determine whether Bcr, which was tyrosine phosphorylated within cells by activated c-Abl, could also bind Grb2, and whether phosphotyrosine 177 was the major binding site. Complexes between Bcr and Abl were detected in a hemopoietic cell line lacking Bcr-Abl and in COS1 cells coexpressing both Bcr and Abl proteins. P160 BCR was tyrosine phosphorylated in COS1 cells coexpressing Abl and Bcr proteins. Similarly, various deletion mutants of Bcr including BCRN553, BCRN413 and BCRN221 were tyrosine phosphorylated by activated c-Abl whereas BCRN159 was not. Wild-type Bcr and Bcr Y177F were examined under these conditions for their ability to co-precipitate with Grb2. The results showed that while wild-type tyrosine phosphorylated Bcr e ciently bound Grb2, tyrosine phosphorylated Bcr Y177F had greatly reduced Grb2-binding ability. Studies with GST-SH2 (Grb2) revealed that tyrosine phosphorylated Bcr was able to bind to GST SH2 (Grb2) but tyrosine phosphorylated Bcr Y177F was de®cient in binding. These results indicate that the Bcr protein when phosphorylated at tyrosine 177 binds Grb2, thereby implicating Bcr as a potantial activator of the Ras pathway.

show abstract

SH2 domains recognize specific phosphopeptide sequences

Cited by 2,438 publications

References 38 publications

ErbB kinases and NDF signaling in human prostate cancer cells

ErbB kinases and NDF signaling in human prostate cancer cells

A label‐free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

Bcr phosphorylated on tyrosine 177 binds Grb2

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