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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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Analysis of a CSNK1A1 active-site peptide in both colon tumor and matched control samples.A) Amino acid sequence of the wild type peptide. B) LC-MS/MS spectra for the CSNK1A1 peptide. The colon tumor sample is off-set in the Y-axis by 50 units for clarity and the region of interest (ROI) 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 for CSNK1A1 from both colon tumor and matched control samples. Signals are normalized to the peak eluting at 59.8 minutes.
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pone.0152934.g002: Analysis of a CSNK1A1 active-site peptide in both colon tumor and matched control samples.A) Amino acid sequence of the wild type peptide. B) LC-MS/MS spectra for the CSNK1A1 peptide. The colon tumor sample is off-set in the Y-axis by 50 units for clarity and the region of interest (ROI) 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 for CSNK1A1 from both colon tumor and matched control samples. Signals are normalized to the peak eluting at 59.8 minutes.

Mentions: An unusual circumstance was observed for CSNK1A1in the tumor sample derived from one patient, sample 815zp (Fig 2). Under our experimental conditions, the CSNK1A1 peptide elutes at 63.0 minutes, but signal apparently corresponding to CSNK1A1 was observed at both 59.8 minutes and 63.0 minutes in the tumor samples of this patient. The observed elution time shift of ~3 minutes for the first extracted ion peak is well outside our expected standard deviation for elution times of ± 0.98 minutes. Moreover, in the 59.8 minute extracted ion peak, a fragment ion in the MS2 spectrum corresponding to the peptide sequence “DIK*” (where K* is the desthiobiotinylated lysine) of CSNK1A1 was shifted from the expected m/z of 553.4 to 552.4 atomic mass units (amu). This fragment ion contains both the conserved catalytic aspartic acid (D) and an adjacent, probe-labeled lysine (K*). This “DIK” (or the mass equivalent “DLK”) sequence is largely conserved among protein kinases, and the 553.4 amu fragment can be diagnostic for the presence of a probe-labeled kinase peptide. All fragment ions that did not contain the catalytic aspartic acid were unchanged, which suggested that a specific alteration was present in fragments containing the aspartic acid from “DIK*”. The observed mass shift was reproduced on replicate analyses, confined to CSNK1A1, and was not present in any normal colon samples, demonstrating that the one amu shift was not a simple artifact of sample procurement, processing, etc. Moreover, the observed one amu mass shift was consistent with a tumor-specific missense mutation (D→N), and a similar elution time shift had previously been observed in our labs upon mutation of a kinase active site aspartic acid to asparagine (in a different kinase). As such, we sought verification of a missense mutation through probe-based analysis of a recombinant CSNK1A1 D→N mutant.


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 in both colon tumor and matched control samples.A) Amino acid sequence of the wild type peptide. B) LC-MS/MS spectra for the CSNK1A1 peptide. The colon tumor sample is off-set in the Y-axis by 50 units for clarity and the region of interest (ROI) 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 for CSNK1A1 from both colon tumor and matched control samples. Signals are 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.g002: Analysis of a CSNK1A1 active-site peptide in both colon tumor and matched control samples.A) Amino acid sequence of the wild type peptide. B) LC-MS/MS spectra for the CSNK1A1 peptide. The colon tumor sample is off-set in the Y-axis by 50 units for clarity and the region of interest (ROI) 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 for CSNK1A1 from both colon tumor and matched control samples. Signals are normalized to the peak eluting at 59.8 minutes.
Mentions: An unusual circumstance was observed for CSNK1A1in the tumor sample derived from one patient, sample 815zp (Fig 2). Under our experimental conditions, the CSNK1A1 peptide elutes at 63.0 minutes, but signal apparently corresponding to CSNK1A1 was observed at both 59.8 minutes and 63.0 minutes in the tumor samples of this patient. The observed elution time shift of ~3 minutes for the first extracted ion peak is well outside our expected standard deviation for elution times of ± 0.98 minutes. Moreover, in the 59.8 minute extracted ion peak, a fragment ion in the MS2 spectrum corresponding to the peptide sequence “DIK*” (where K* is the desthiobiotinylated lysine) of CSNK1A1 was shifted from the expected m/z of 553.4 to 552.4 atomic mass units (amu). This fragment ion contains both the conserved catalytic aspartic acid (D) and an adjacent, probe-labeled lysine (K*). This “DIK” (or the mass equivalent “DLK”) sequence is largely conserved among protein kinases, and the 553.4 amu fragment can be diagnostic for the presence of a probe-labeled kinase peptide. All fragment ions that did not contain the catalytic aspartic acid were unchanged, which suggested that a specific alteration was present in fragments containing the aspartic acid from “DIK*”. The observed mass shift was reproduced on replicate analyses, confined to CSNK1A1, and was not present in any normal colon samples, demonstrating that the one amu shift was not a simple artifact of sample procurement, processing, etc. Moreover, the observed one amu mass shift was consistent with a tumor-specific missense mutation (D→N), and a similar elution time shift had previously been observed in our labs upon mutation of a kinase active site aspartic acid to asparagine (in a different kinase). As such, we sought verification of a missense mutation through probe-based analysis of a recombinant CSNK1A1 D→N mutant.

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