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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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Genomic DNA sequencing of CSNK1A1.A) PCR sequencing results for control using intronic primer. B) PCR sequencing results for patient 815zp using an intronic primer. In both cases, nucleotide of interest is highlighted with an asterisk.
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pone.0152934.g005: Genomic DNA sequencing of CSNK1A1.A) PCR sequencing results for control using intronic primer. B) PCR sequencing results for patient 815zp using an intronic primer. In both cases, nucleotide of interest is highlighted with an asterisk.

Mentions: To sequence the heterogeneous genomic DNA, two primers were designed that anneal to intronic sequences on either side of the exon that contains the CSNK1A1 active-site peptide. PCR product was generated from a tumor that did not give signal for the mutant in the mass spectrometry experiment as well as patient 815zp. Direct sequencing of PCR product from the control tumor yielded the expected GAC codon for the active site aspartic acid. In contrast, sequencing the PCR product from patient 815zp yielded a small, but distinct peak of A in the first position of this codon concomitant with a smaller G peak relative to the control tumor (Fig 5, compare the height of the highlighted guanosine to the adjacent adenosines in panels A and B). Replacing G with A at this position would result in an AAC/asparagine codon. Though highly suggestive of a mutation, sequencing of the PCR product using this approach yielded chromatograms that were too noisy to prove the existence of the mutation.


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)

Genomic DNA sequencing of CSNK1A1.A) PCR sequencing results for control using intronic primer. B) PCR sequencing results for patient 815zp using an intronic primer. In both cases, nucleotide of interest is highlighted with an asterisk.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152934.g005: Genomic DNA sequencing of CSNK1A1.A) PCR sequencing results for control using intronic primer. B) PCR sequencing results for patient 815zp using an intronic primer. In both cases, nucleotide of interest is highlighted with an asterisk.
Mentions: To sequence the heterogeneous genomic DNA, two primers were designed that anneal to intronic sequences on either side of the exon that contains the CSNK1A1 active-site peptide. PCR product was generated from a tumor that did not give signal for the mutant in the mass spectrometry experiment as well as patient 815zp. Direct sequencing of PCR product from the control tumor yielded the expected GAC codon for the active site aspartic acid. In contrast, sequencing the PCR product from patient 815zp yielded a small, but distinct peak of A in the first position of this codon concomitant with a smaller G peak relative to the control tumor (Fig 5, compare the height of the highlighted guanosine to the adjacent adenosines in panels A and B). Replacing G with A at this position would result in an AAC/asparagine codon. Though highly suggestive of a mutation, sequencing of the PCR product using this approach yielded chromatograms that were too noisy to prove the existence of the mutation.

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