PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse

Hornbeck, Peter; Kornhauser, Jon M.; Tkachev, Sasha; Zhang, Bin; Skrzypek, Elźbieta; Murray, Beth; Latham, Vaughan M.; Sullivan, Michael L.

doi:10.1093/nar/gkr1122

Cited by 1,446 publications

(1,670 citation statements)

References 45 publications

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“…The function of these sites is unclear and these residues are not conserved in the TACC family (20,21). However, Y867 is a conserved tyrosine residue in the TACC family and our data identify it as a novel phosphorylation site for the FGFR3(K650E)-TACC3 fusion protein.…”

Section: Lc-ms/ms Analysis Identifies Elevated and Novel Phosphorylatmentioning

confidence: 71%

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Nelson

Meyer

Siari

et al. 2016

Molecular Cancer Research

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Fibroblast growth factor receptors (FGFR) are critical for cell proliferation and differentiation. Mutation and/or translocation of FGFRs lead to aberrant signaling that often results in developmental syndromes or cancer growth. As sequencing of human tumors becomes more frequent, so does the detection of FGFR translocations and fusion proteins. The research conducted in this article examines a frequently identified fusion protein between FGFR3 and transforming acidic coiled-coil containing protein 3 (TACC3), frequently identified in glioblastoma, lung cancer, bladder cancer, oral cancer, head and neck squamous cell carcinoma, gallbladder cancer, and cervical cancer. Using titanium dioxide-based phosphopeptide enrichment (TiO 2 )-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), it was demonstrated that the fused coiled-coil TACC3 domain results in constitutive phosphorylation of key activating FGFR3 tyrosine residues. The presence of the TACC coiled-coil domain leads to increased and altered levels of FGFR3 activation, fusion protein phosphorylation, MAPK pathway activation, nuclear localization, cellular transformation, and IL3-independent proliferation. Introduction of K508R FGFR3 kinase-dead mutation abrogates these effects, except for nuclear localization which is due solely to the TACC3 domain.Implications: These results demonstrate that FGFR3 kinase activity is essential for the oncogenic effects of the FGFR3-TACC3 fusion protein and could serve as a therapeutic target, but that phosphorylated tyrosine residues within the TACC3-derived portion are not critical for activity.

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Section: Lc-ms/ms Analysis Identifies Elevated and Novel Phosphorylatmentioning

confidence: 71%

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Nelson

Meyer

Siari

et al. 2016

Molecular Cancer Research

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“…org) (29), which included 155,027 sites of phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, O-N-acetylgalactosamine, O-N-acetylglucosamine, etc. As described in their web site, these data were manually collected from the published experiments in PubMed or from the unpublished data generated at the Cell Signaling Technology (http://www.cellsignal.com).…”

Section: Methodsmentioning

confidence: 99%

“…Here, we extended the co-evolution measure to the modification level, which reflects the level of co-occurrence of PTM at a pair of residues across species. This analysis was restricted to human, house mouse, and brown rat, whose PTM data were downloaded from the database PhosphoSitePlus ® (29). The total PTM sites are 101,844 for house mice and 16,567 for brown rats.…”

Section: Methodsmentioning

confidence: 99%

Systematic Characterization and Prediction of Post-Translational Modification Cross-Talk *

Huang

Xu²,

Zhou

et al. 2015

Molecular & Cellular Proteomics

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Post-translational modification (PTM) 1 plays an important role in regulating the functions of proteins. PTMs of multiple residues on one protein may work together to determine a functional outcome, which is known as PTM crosstalk. Identification of PTM cross-talks is an emerging theme in proteomics and has elicited great interest, but their properties remain to be systematically characterized. To this end, we collected 193 PTM cross-talk pairs in 77 human proteins from the literature and then tested location preference and co-evolution at the residue and modification levels. We found that cross-talk events preferentially occurred among nearby PTM sites, especially in disordered protein regions, and cross-talk pairs tended to co-evolve. Given the properties of PTM cross-talk pairs, a naïve Bayes classifier integrating different features was built to predict cross-talks for pairwise combination of PTM sites. By using a 10-fold cross-validation, the integrated prediction model showed an area under the receiver operating characteristic (ROC) curve of 0.833, superior to using any individual feature alone. The prediction performance was also demonstrated to be robust to the biases in the collected PTM cross-talk pairs. The integrated approach has the potential for large-scale prioritization of PTM cross-talk candidates for functional validation and was implemented as a web server available at

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“…Determine the predicted isoelectric point for the target protein using a suitable proteomics database. For CrkL, the basal isoelectric point is 6.26, although high phosphorylation shift to pI 3.57 is possible (www.phosphosite.org 43 ) 27. Use default assay settings with a simple assay plate design (Fig.…”

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confidence: 99%

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

et al. 2014

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This protocol describes a highly reproducible antibody-based method providing protein level and phosphorylation status information from nanogram quantities of protein cell lysate. Nanocapillary isoelectric focusing (cIEF) combines with UV activated linking chemistry to detect changes in phosphorylation status. We describe how to detect changes in response to tyrosine kinase inhibitors (TKIs) in the phosphorylation status of the adapter protein CrkL a major substrate of the oncogenic tyrosine kinase BCR/ABL in chronic myeloid leukaemia (CML), using highly enriched CML stem cells and mature cell populations in vitro. This protocol provides a 2.5pg/nL limit of protein detection (<0.2% of a stem cell sample containing <10 4 cells). Additional assays are described for pTyr207-CrkL and PTPRC/CD45, developed using this protocol and applied to CML patient samples. This method is high throughput and can act as a screen for in vitro cancer stem cell response to drugs and novel agents. SummaryThis protocol uses a highly sensitive and reproducible antibody-based capillary isoelectric focusing approach to establish protein phosphorylation status from nanogram quantities of protein cell lysate from cell line or primary stem cell material. Is-protocol-to

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PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse

Cited by 1,446 publications

References 45 publications

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Oncogenic Gene Fusion FGFR3-TACC3 Is Regulated by Tyrosine Phosphorylation

Systematic Characterization and Prediction of Post-Translational Modification Cross-Talk *

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

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