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Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics.

Okerberg ES, Hainley A, Brown H, Aban A, Alemayehu S, Shih A, Wu J, Patricelli MP, Kozarich JW, Nomanbhoy T, Rosenblum JS - PLoS ONE (2016)

Bottom Line: Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1.To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics.The tumorigenic potential of this mutation remains to be determined.

View Article: PubMed Central - PubMed

Affiliation: ActivX Biosciences, Inc., La Jolla, CA, United States of America.

ABSTRACT
We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined.

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Related in: MedlinePlus

Analysis of a CSNK1A1 active site peptide from wild-type and mutant CSNK1A1 expressed in HEK293 cells.A) Amino acid sequence of the mutant peptide. B) LC-MS/MS spectra for wild-type and mutant protein. The mutant sample is off-set by 50 units for clarity and the region of interest is highlighted. C) Region of interest in the LC-MS/MS spectra for colon tumor and matched control samples. Each spectrum is normalized to the highest intensity ion. D) Extracted ion chromatograms from HEK293 lysates transfected with either wild-type or mutant CSNK1A1. The wild-type sample is normalized to the peak eluting at 63.0 minutes and the mutant sample is normalized to the peak eluting at 59.8 minutes.
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pone.0152934.g003: Analysis of a CSNK1A1 active site peptide from wild-type and mutant CSNK1A1 expressed in HEK293 cells.A) Amino acid sequence of the mutant peptide. B) LC-MS/MS spectra for wild-type and mutant protein. The mutant sample is off-set by 50 units for clarity and the region of interest is highlighted. C) Region of interest in the LC-MS/MS spectra for colon tumor and matched control samples. Each spectrum is normalized to the highest intensity ion. D) Extracted ion chromatograms from HEK293 lysates transfected with either wild-type or mutant CSNK1A1. The wild-type sample is normalized to the peak eluting at 63.0 minutes and the mutant sample is normalized to the peak eluting at 59.8 minutes.

Mentions: Wild-type CSNK1A1 and CSNK1A1 D136N were transiently expressed in HEK293 cells and analyzed as the colon samples were. The observed MS2 spectrum and elution time for the engineered CSNK1A1 mutant were identical to that observed in the colon tumor sample (Fig 3). This verified that, in the tumor sample, the catalytic amino acid for CSNK1A1 had been converted to an asparagine from an aspartic acid.


Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics.

Okerberg ES, Hainley A, Brown H, Aban A, Alemayehu S, Shih A, Wu J, Patricelli MP, Kozarich JW, Nomanbhoy T, Rosenblum JS - PLoS ONE (2016)

Analysis of a CSNK1A1 active site peptide from wild-type and mutant CSNK1A1 expressed in HEK293 cells.A) Amino acid sequence of the mutant peptide. B) LC-MS/MS spectra for wild-type and mutant protein. The mutant sample is off-set by 50 units for clarity and the region of interest is highlighted. C) Region of interest in the LC-MS/MS spectra for colon tumor and matched control samples. Each spectrum is normalized to the highest intensity ion. D) Extracted ion chromatograms from HEK293 lysates transfected with either wild-type or mutant CSNK1A1. The wild-type sample is normalized to the peak eluting at 63.0 minutes and the mutant sample is normalized to the peak eluting at 59.8 minutes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4816389&req=5

pone.0152934.g003: Analysis of a CSNK1A1 active site peptide from wild-type and mutant CSNK1A1 expressed in HEK293 cells.A) Amino acid sequence of the mutant peptide. B) LC-MS/MS spectra for wild-type and mutant protein. The mutant sample is off-set by 50 units for clarity and the region of interest is highlighted. C) Region of interest in the LC-MS/MS spectra for colon tumor and matched control samples. Each spectrum is normalized to the highest intensity ion. D) Extracted ion chromatograms from HEK293 lysates transfected with either wild-type or mutant CSNK1A1. The wild-type sample is normalized to the peak eluting at 63.0 minutes and the mutant sample is normalized to the peak eluting at 59.8 minutes.
Mentions: Wild-type CSNK1A1 and CSNK1A1 D136N were transiently expressed in HEK293 cells and analyzed as the colon samples were. The observed MS2 spectrum and elution time for the engineered CSNK1A1 mutant were identical to that observed in the colon tumor sample (Fig 3). This verified that, in the tumor sample, the catalytic amino acid for CSNK1A1 had been converted to an asparagine from an aspartic acid.

Bottom Line: Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1.To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics.The tumorigenic potential of this mutation remains to be determined.

View Article: PubMed Central - PubMed

Affiliation: ActivX Biosciences, Inc., La Jolla, CA, United States of America.

ABSTRACT
We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined.

Show MeSH
Related in: MedlinePlus