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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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Sequence analysis of cloned PCR products from the 815zp tumor sample.A) A representative clone showing the mutated nucleotide (*). B) Alignment of all 41 sequences. Differences between pseudogene and CSNK1A1 are indicated by a plus symbol.
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pone.0152934.g006: Sequence analysis of cloned PCR products from the 815zp tumor sample.A) A representative clone showing the mutated nucleotide (*). B) Alignment of all 41 sequences. Differences between pseudogene and CSNK1A1 are indicated by a plus symbol.

Mentions: In order to prove the existence of a mutation at this position, primers annealing to the ends of the active-site containing exon were used to amplify and clone the exon. Several dozen clones were individually sequenced, resulting in the unambiguous identification of an AAC codon in CSNK1A1 in the 815zp tumor sample (Fig 6A). The active-site exon of CSNK1A1 is almost exactly duplicated on chromosome 15 in a CSNK1A1P1 pseudogene. As a result, this approach resulted in the sequencing of a number of pseudogene sequences (Fig 6B). Interestingly, the pseudogene is also modified at the codon for amino acid 136, in this case the GCC codes for a catalytically incompetent alanine (Fig 6B, plus symbol adjacent to the asterisk). Of the 41 clones sequenced, 19 were derived from the pseudogene, almost exactly the 1:1 ratio to CSNK1A1 expected based on their equal chromosomal representation. Of the 22 CSNK1A1 clones sequenced, 10 were from the mutant. We have not validated the ability of this assay to quantify the relative abundance of the different sequences, but the mutant sequence likely made up a substantial fraction of the tumor from patient 815zp.


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)

Sequence analysis of cloned PCR products from the 815zp tumor sample.A) A representative clone showing the mutated nucleotide (*). B) Alignment of all 41 sequences. Differences between pseudogene and CSNK1A1 are indicated by a plus symbol.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0152934.g006: Sequence analysis of cloned PCR products from the 815zp tumor sample.A) A representative clone showing the mutated nucleotide (*). B) Alignment of all 41 sequences. Differences between pseudogene and CSNK1A1 are indicated by a plus symbol.
Mentions: In order to prove the existence of a mutation at this position, primers annealing to the ends of the active-site containing exon were used to amplify and clone the exon. Several dozen clones were individually sequenced, resulting in the unambiguous identification of an AAC codon in CSNK1A1 in the 815zp tumor sample (Fig 6A). The active-site exon of CSNK1A1 is almost exactly duplicated on chromosome 15 in a CSNK1A1P1 pseudogene. As a result, this approach resulted in the sequencing of a number of pseudogene sequences (Fig 6B). Interestingly, the pseudogene is also modified at the codon for amino acid 136, in this case the GCC codes for a catalytically incompetent alanine (Fig 6B, plus symbol adjacent to the asterisk). Of the 41 clones sequenced, 19 were derived from the pseudogene, almost exactly the 1:1 ratio to CSNK1A1 expected based on their equal chromosomal representation. Of the 22 CSNK1A1 clones sequenced, 10 were from the mutant. We have not validated the ability of this assay to quantify the relative abundance of the different sequences, but the mutant sequence likely made up a substantial fraction of the tumor from patient 815zp.

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