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KMID : 0578320080250010043
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Molecules and Cells
2008 Volume.25 No. 1 p.43 ~ p.49
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Proteomics Analysis of Immunoprecipitated Proteins Associated with the Oncogenic Kinase Cot
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Binhui Wu
Rupert C. Wilmouth
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Abstract
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Cancer Osaka thyroid, also known as Tpl-2 (Cot) is a member of the MAP3K kinase family and plays a key role in the regulation of the immune response to pro-inflammatory stimuli such as lipopolysaccharide (LPS) and tumour necrosis factor-? (TNF-?). A series of Cot constructs with an N-terminal 6xHis tag were transiently expressed in HEK293 cells: Cot130-399 (kinase domain), Cot1-388 (N-terminal and kinase do-mains), Cot1 413, Cot1 438 (containing a putative PEST sequence), Cot1-457 (containing both PEST and degron sequences) and Cot1-467 (full-length protein). These Cot proteins were pulled down using an anti-6xHis antibody and separated by 2D electrophoresis. The gels were silver-stained and 21 proteins were detected that did not appear, or had substantially reduced intensity, in the control sample. Three of these were identified by MS and MS/MS analysis as Hsp90, Hsp70 and Grp78. Hsp90 appeared to bind to the kinase domain of Cot and this interaction was further investigated using co-immuno-precipitation with both overexpressed Cot in HEK293 cells and endogenous Cot in Hela cells.
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KEYWORD
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Chaperone, Cot, Hsp90, MAP3K
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