A comparative study of ATP analogs for phosphorylation-dependent kinase–substrate crosslinking

Garre, Satish; Senevirathne, Chamara; Pflum, Mary Kay H.

doi:10.1016/j.bmc.2014.01.034

Cited by 18 publications

(15 citation statements)

References 32 publications

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“…Again, DNA-PK was observed in the crosslinked complex, but no other controls (Figure 5A, lane 6 versus lane 3). Due to the presence of higher order crosslinked complexes with ATP-BP, which were absent with ATP-ArN 3 (Figure 4A, lane 6 compared to lane 5), as was previously reported, 9 ATP-ArN 3 was preferred for kinase-catalyzed crosslinking. These results confirm that kinase-catalyzed crosslinking with either ATP-ArN 3 or ATP-BP analogs can be used to validate an active p53 kinase.…”

Section: Resultssupporting

confidence: 82%

Identification of Kinases and Interactors of p53 Using Kinase-Catalyzed Cross-Linking and Immunoprecipitation

et al. 2018

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Kinase enzymes phosphorylate protein substrates in a highly ordered manner to control cell signaling. Unregulated kinase activity is associated with a variety of disease states, most notably cancer, making the characterization of kinase activity in cellulo critical to understand disease formation. However, the paucity of available tools has prevented a full mapping of the substrates and interacting proteins of kinases involved in cellular function. Recently we developed kinase-catalyzed crosslinking to covalently connect substrate and kinase in a phosphorylation-dependent manner. Here, we report a new method combining kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP) to identify kinase-substrate pairs and kinase-associated proteins. K-CLIP was applied to the substrate p53, which is robustly phosphorylated. Both known and unknown kinases of p53 were isolated from cell lysates using K-CLIP. In follow-up validation studies, MRCKbeta was identified as a new p53 kinase. Beyond kinases, a variety of p53 and kinase-associated proteins were also identified using K-CLIP, which provided a snapshot of cellular interactions. The K-CLIP method represents an immediately useful chemical tool to identify kinase-substrate pairs and multi-proteins complexes in cells, which will embolden cell signaling research and enhance our understanding of kinase activity in normal and disease states.

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Section: Resultssupporting

confidence: 82%

Identification of Kinases and Interactors of p53 Using Kinase-Catalyzed Cross-Linking and Immunoprecipitation

et al. 2018

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“…To address this issue, various groups have designed approaches to screen for kinase substrates using purified active kinases to phosphorylate cell lysates in vitro , followed by MS analysis to identify phosphoproteins [68–74]. A major pitfall of these approaches is that they only identify potential kinase substrates in vitro .…”

Section: Ms-based Investigations Of Kinase Substratesmentioning

confidence: 99%

Protein networks and activation of lymphocytes

Helou

Salomon

2015

Current Opinion in Immunology

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The signal transduction pathways initiated by lymphocyte activation play a critical role in regulating host immunity. High-resolution mass spectrometry has accelerated the investigation of these complex and dynamic pathways by enabling the qualitative and quantitative investigation of thousands of proteins and phosphoproteins simultaneously. In addition, the unbiased and wide-scale identification of protein-protein interaction networks and protein kinase substrates in lymphocyte signaling pathways can be achieved by mass spectrometry-based approaches. Critically, the integration of these discovery-driven strategies with single-cell analysis using mass cytometry can facilitate the understanding of complex signaling phenotypes in distinct immunophenotypes.

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“…ABPP probes covalently target the active site of enzymes for their detection or purification. Substrate ID describes a set of tools for the capture of the downstream substrates of a given enzyme . While ABPP and substrate ID are powerful tools for assessing the functional status and output of enzymes, we sought a way to not only identify active kinases but also to capture the kinases as they bind to a specific substrate, a method we term substrate‐based protein profiling (SBPP).…”

Section: Introductionmentioning

confidence: 99%

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

et al. 2019

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The proteomic mapping of enzyme–substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide‐based substrate analog capable of reprogramming the wild‐type phosphoryl‐transfer reaction to produce a kinase‐acrylamide‐based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP‐based methacrylate crosslinker (ATP‐MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP‐MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP‐based methacrylate (ADP‐MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP‐MA over ATP‐MA is due to reduced inhibition of the second step of the kinase–substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP‐MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site‐specific manner. The kinase–substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.

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A comparative study of ATP analogs for phosphorylation-dependent kinase–substrate crosslinking

Cited by 18 publications

References 32 publications

Identification of Kinases and Interactors of p53 Using Kinase-Catalyzed Cross-Linking and Immunoprecipitation

Identification of Kinases and Interactors of p53 Using Kinase-Catalyzed Cross-Linking and Immunoprecipitation

Protein networks and activation of lymphocytes

Chemically reprogramming the phospho‐transfer reaction to crosslink protein kinases to their substrates

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