Proteomics analysis of immunoprecipitated proteins associated with the oncogenic kinase cot.
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-alpha (TNF-alpha). A series of Cot constructs with an N-terminal 6xHis tag were transiently expressed in HEK293 cells: Cot(130-399) (kinase domain), Cot(1-388) (N-terminal and kinase domains), Cot(1-413), Cot(1-438) (containing a putative PEST sequence), Cot(1-457) (containing both PEST and degron sequences) and Cot(1-467) (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.
Wu
Binhui
B
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore.
Wilmouth
R C
RC
D000818:Animals; D002460:Cell Line; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*
D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D020543:Proteome; D011518:Proto-Oncogene Proteins; D020930:MAP Kinase Kinase Kinases; C069937:MAP3K8 protein, human