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Identification of potential yeast casein kinase 1 substrates via a comparative phosphoproteomic analysis
Author(s) -
Brame Cynthia Jan,
Robinson Lucy C.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1004
Subject(s) - yeast , casein kinase 2 , biochemistry , tandem affinity purification , saccharomyces cerevisiae , biology , phosphoproteomics , kinase , proteome , cytokinesis , protein kinase a , computational biology , chemistry , affinity chromatography , enzyme , mitogen activated protein kinase kinase , protein phosphorylation , cell , cell division
Yck1/2 are essential and redundant casein kinase 1 proteins required for endocytic trafficking and cytokinesis in budding yeast. Although much has been learned about Yck function, it has proven difficult to discern substrates through traditional approaches. We therefore applied a comparative phosphoproteomics approach to identify substrates of these kinases. Proteins from wild‐type and conditional Yck mutant ( yck ts ) yeast were proteolyzed and the peptides differentially labeled during conversion to methyl esters. Labeled peptides from the two yeast strains were combined and subjected to immobilized metal affinity chromatography and analysis via nanoflow HPLC/microelectrospray ionization tandem mass spectrometry. We determined the sequence and relative abundance of more than 100 phosphopeptides in duplicate analyses. In yeast grown at the restrictive temperature, five peptides from three proteins were determined both to be significantly more abundant in the wild‐type than the yck ts protein samples and to have a sequence consistent with the consensus sequence for casein kinase 1 recognition. The potential Yck substrates are being investigated by genetic analyses, and preliminary data support their identification. We conclude that this approach, when expanded to identify more phosphopeptides, will provide an unbiased means to identify a large set of substrates. Work supported by NSF grant 0517204.