K-CLASP: A Tool to Identify Phosphosite Specific Kinases and Interacting Proteins

Dedigama-Arachchige, Pavithra; Pflum, Mary Kay H.

doi:10.1021/acschembio.6b00289

Cited by 19 publications

(21 citation statements)

References 27 publications

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“…Methods that make use of modified forms of substrates and/or ATP in an effort to crosslink substrates to their cognate kinases in cell extracts are promising. However, where tested, the specificity of kinase crosslinking has been low, these approaches require additional steps such as mass spectrometry to identify the crosslinked kinase [23][24][25] , and they have not yet been used to identify unknown kinases. Tracking a specific kinase activity through biochemical enrichment steps can also be carried out, but this often requires large amounts of cell extract, multiple steps, and protein sequencing methods to identify candidate kinases [26][27][28] .…”

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confidence: 99%

Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites

et al. 2020

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There are thousands of known cellular phosphorylation sites, but the paucity of ways to identify kinases for particular phosphorylation events remains a major roadblock for understanding kinase signaling. To address this, we here develop a generally applicable method that exploits the large number of kinase inhibitors that have been profiled on near-kinomewide panels of protein kinases. The inhibition profile for each kinase provides a fingerprint that allows identification of unknown kinases acting on target phosphosites in cell extracts. We validate the method on diverse known kinase-phosphosite pairs, including histone kinases, EGFR autophosphorylation, and Integrin β1 phosphorylation by Src-family kinases. We also use our approach to identify the previously unknown kinases responsible for phosphorylation of INCENP at a site within a commonly phosphorylated motif in mitosis (a non-canonical target of Cyclin B-Cdk1), and of BCL9L at S915 (PKA). We show that the method has clear advantages over in silico and genetic screening.

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confidence: 99%

Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites

et al. 2020

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“…For example, kinases collaborate with ATP-biotin ( Figure 1A) to attach a phosphoryl biotin tag to substrates, facilitating protein visualization and purification. Importantly for K-BIPS, the extent of protein biotinylation by ATP-biotin in lysates was reduced significantly in the presence of phosphatase inhibitors, [21][22][23][24] indicating that active phosphatases are required for full kinase-catalyzed biotinylation of cellular proteins. Based on this phosphatase dependence, K-BIPS compares the relative biotinylation of proteins in untreated and phosphatase-inactivated cell lysates after the ATP-biotin reaction.…”

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confidence: 99%

l‐Lactate Dehydrogenase Identified as a Protein Tyrosine Phosphatase 1B Substrate by Using K‐BIPS

Acharige

Pflum

2020

ChemBioChem

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Kinases and phosphatases are major players in a variety of cellular events, including cell signaling. Aberrant activity or mutations in kinases and phosphatases can lead to diseases such as cancer, diabetes, and Alzheimer's. Compared to kinases, phosphatases are understudied; this is partly a result of the limited methods for identifying substrates. As a solution, we developed a proteomics-based method called kinase-catalyzed biotinylation to identify phosphatase substrates (K-BIPS) that previously identified substrates of Ser/Thr phosphatases using small molecule inhibitors. Here, for the first time, K-BIPS was applied to identify substrates of a tyrosine phosphatase, protein tyrosine phosphatase 1B (PTP1B), under siRNA knockdown conditions. Eight possible substrates of PTP1B were discovered in HEK293 cells, including the known substrate pyruvate kinase. In addition, l-lactate dehydrogenase (LDHA) was validated as a novel PTP1B substrate. With the ability to use knockdown conditions with Ser/Thr or Tyr phosphatases, K-BIPS represents a general discovery tool to explore phosphatases biology by identifying unanticipated substrates.

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“…In prior work, we used a biotinylated peptide substrates as a handle to isolate kinases specific to a single phosphosite. 10 Here we combined crosslinking with immunoprecipitation in a method called K-CLIP ( K inase-catalyzed C ross L inking and I mmuno P recipitation) to isolate and characterize select full-length substrate-conjugated complexes from a complex cellular mixture. Immunoprecipitation (IP) with an antibody selective to a desired substrate or kinase of interest will isolate only substrate- or kinase-bound complexes (Figure 1E).…”

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confidence: 99%

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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K-CLASP: A Tool to Identify Phosphosite Specific Kinases and Interacting Proteins

Cited by 19 publications

References 27 publications

Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites

Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites

l‐Lactate Dehydrogenase Identified as a Protein Tyrosine Phosphatase 1B Substrate by Using K‐BIPS

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

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