Analysis of ubiquitin degradation and phosphorylation of proteins.
Both ubiquitination and phosphorylation are crucial mediators involved in controlling the functions of numerous proteins belonging to the Toll-like receptor (TLR) signaling pathways. Altering the aforementioned post-translational events can be detrimental to the host survival. Therefore, the importance of these modifications cannot be overestimated. This chapter describes techniques used to examine if a protein is ubiquitinated and/or phosphorylated. In addition, a method is provided to identify the modified amino acids. We have previously shown using these techniques that the protein MyD88 adapter-like (Mal) is phosphorylated and ubiquitinated following activation of the TLR2 and TLR4 signaling pathways. Both post-translational modifications are essential for the activation and degradation of Mal, and thus are crucial steps, in regulating these TLR signaling cascades and consequently the innate immune response.
Division of Pediatric Infectious Diseases, University of California Los Angeles, Los Angeles, CA 90048, USA. grayp@cshs.org
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Gray
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007422:Intestines / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D011506:Proteins; D000469:Alkaline Phosphatase