A semisynthetic epitope for kinase substrates

Allen, Jasmina J.; Li, Manqing; Brinkworth, Craig S.; Paulson, Jennifer; Wang, Dan; Hübner, Anette; Chou, Wen Hai; Davis, Roger J.; Burlingame, Alma L.; Messing, Robert O.; Katayama, Carol D.; Hedrick, Stephen M.; Shokat, Kevan M.

doi:10.1038/nmeth1048

Cited by 284 publications

(309 citation statements)

References 30 publications

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“…We have recently reported a method for identification of askinase targets by using a monoclonal antibody that recognizes a chemically derivatized thiophosphorylation adduct (14,43). Although the antibody-based approach does not immediately identify the phosphorylation site targeted by an as-kinase, it is complementary to the method reported here because it leads to purification of the entire substrate protein.…”

Section: Many Phosphorylation Sites Identified Lack An Optimal Cdk Comentioning

confidence: 99%

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Blethrow

Glavy²,

Morgan³

et al. 2008

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

291

290

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We describe a method for rapid identification of protein kinase substrates. Cdk1 was engineered to accept an ATP analog that allows it to uniquely label its substrates with a bio-orthogonal phosphate analog tag. A highly specific, covalent capture-andrelease methodology was developed for rapid purification of tagged peptides derived from labeled substrate proteins. Application of this approach to the discovery of Cdk1-cyclin B substrates yielded identification of >70 substrates and phosphorylation sites. Many of these sites are known to be phosphorylated in vivo, but most of the proteins have not been characterized as Cdk1-cyclin B substrates. This approach has the potential to expand our understanding of kinase-substrate connections in signaling networks.P rotein kinases regulate a vast array of biological processes through phosphorylation of protein substrates. A comprehensive map of all phosphorylation sites and kinase-substrate pairs would greatly facilitate the study of signaling networks. This goal faces two fundamental challenges. First, all protein kinases use ATP as a cofactor to phosphorylate their targets, and thus the direct substrates of a single kinase cannot be easily traced in protein mixtures containing multiple kinases. Second, phosphorylation often occurs at low stoichiometry and on low-abundance proteins. This makes substrate and phosphorylation site identification very challenging. Powerful methods have been developed to address these dual problems (1, 2). Kinase-substrate pairs can be tested in multiplexed phosphorylation assays by using immobilized arrays of purified proteins. These high-throughput chip-based assays have the added benefit of presenting low-abundance proteins at easily detectable levels and have provided a first-generation map of protein phosphorylation in Saccharomyces cerevisiae (3). However, these assays do not currently allow identification of phosphorylation sites and have not been adapted to organisms with more complex proteomes. Prediction of high-likelihood kinase substrates can sometimes be achieved by using knowledge of the substrate sequence motif preferences of individual kinases, but only when these preferences are strong (4, 5). Finally, thousands of in vivo phosphorylation sites from metazoan organisms have been identified in proteomic screens (6-9), but for most of these sites the responsible upstream kinases remain unknown.We have demonstrated a chemical and genetic solution to the common use of ATP by all kinases. Our approach relies on engineering a kinase to accept unnatural ATP analogs by modification of the ATP-binding pocket (10, 11). The analogs are very poor substrates for wild-type kinases; thus, an analog-sensitive kinase (or as-kinase) can be used to specifically radiolabel its substrates in cell extracts while preserving important aspects of biological context, such as the integrity of protein complexes. Coupling this approach with the use of libraries of genetically encoded affinity-tagged proteins has facilitated identification of low-abundanc...

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Section: Many Phosphorylation Sites Identified Lack An Optimal Cdk Comentioning

confidence: 99%

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Blethrow

Glavy²,

Morgan³

et al. 2008

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

291

290

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“…An antibody was raised against the thiophosphorylhistidine derivative 2 but the antibody so raised could not distinguish pHis from other phosphoamino acids. 94,95 Pirrung et al 96 reported the preparation of malonate 3 and fluoromalonate 4 derivatives of His as τ-pHis analogues. However, no biochemical studies using these analogues have been reported.…”

Section: Phosphohistidine Analogues and Antibodiesmentioning

confidence: 99%

Advances in development of new tools for the study of phosphohistidine

Makwana

Muimo

Jackson

2018

Laboratory Investigation

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Protein phosphorylation is an important post-translational modification that is an integral part of cellular function. The O-phosphorylated amino-acid residues, such as phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr), have dominated the literature while the acid labile N-linked phosphorylated amino acids, such as phosphohistidine (pHis), have largely been historically overlooked because of the acidic conditions routinely used in amino-acid detection and analysis. This review highlights some misinterpretations that have arisen in the existing literature, pinpoints outstanding questions and potential future directions to clarify the role of pHis in mammalian signalling systems. Particular emphasis is placed on pHis isomerization and the hybrid functionality for both pHis and pTyr of the proposed τ-pHis analogue bearing the triazole residue.

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“…Here, we characterize thiophosphopeptide and thiophosphoprotein enrichment using iodoacetyl enrichment using both pH 4 and pH 8. Finally, we performed the enrichment using anti-thiophosphate ester antibodies, which were designed to purify the substrates of the analog-specific (AS) kinase (33). AS kinases can utilize N 6 -alkylated ATP-␥-S to produce thiophosphorylated substrates.…”

Section: Enrichment Of Thiophosphorylated Peptides Via Differentmentioning

confidence: 99%

“…The Shokat lab also developed an anti-thiophosphate ester antibody for specific kinase substrate purification (33). For this method to be effective, thiophosphopeptides are first alkylated using p-nitrobenzylmesylate (PNBM), producing a phosphoryl-conjugate that is then recognized by the antibody.…”

Section: Enrichment Of Thiophosphorylated Peptides Via Differentmentioning

confidence: 99%

Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level

Han

Yuan

Molden

et al. 2016

Molecular & Cellular Proteomics

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Protein phosphorylation, one of the most common and important modifications of acute and reversible regulation of protein function, plays a dominant role in almost all cellular processes. These signaling events regulate cellular responses, including proliferation, differentiation, metabolism, survival, and apoptosis. Several studies have been successfully used to identify phosphorylated proteins and dynamic changes in phosphorylation status after stimulation. Nevertheless, it is still rather difficult to elucidate precise complex phosphorylation signaling pathways. In particular, how signal transduction pathways directly communicate from the outer cell surface through cytoplasmic space and then directly into chromatin networks to change the transcriptional and epigenetic landscape remains poorly understood. Here, we describe the optimization and comparison of methods based on thiophosphorylation affinity enrichment, which can be utilized to monitor phosphorylation signaling into chromatin by isolation of phosphoprotein containing nucleosomes, a method we term phosphorylation-specific chromatin affinity purification (PS-ChAP). We utilized this PS-ChAP 1 approach in combination with quantitative proteomics to identify changes in the phosphorylation status of chromatin-bound proteins on nucleosomes following perturbation of transcriptional processes. We also demonstrate that this method can be employed to map phosphoprotein signaling into chromatin containing nucleosomes through identifying the genes those phosphorylated proteins are found on via thiophosphate PS-ChAP-qPCR. Thus, our results showed that PS-ChAP offers a new strategy for studying cellular signaling and chromatin biology, allowing us to directly and comprehensively investigate phosphorylation signaling into chromatin to investigate if these pathways are involved in altering gene expression. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD002436. Molecular & Cellular Proteomics

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A semisynthetic epitope for kinase substrates

Cited by 284 publications

References 30 publications

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates

Advances in development of new tools for the study of phosphohistidine

Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level

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