title: Immunoaffinity enrichment coupled to quantitative mass spectrometry reveals ubiquitin-mediated signaling events.
abstract: Ubiquitination is one of the most prevalent posttranslational modifications in eukaryotic cells, with functional importance in protein degradation, subcellular localization and signal transduction pathways. Immunoaffinity enrichment coupled with quantitative mass spectrometry enables the in-depth characterization of protein ubiquitination events at the site-specific level. We have applied this strategy to investigate cellular response triggered by two distinct type agents: small molecule inhibitors of the tumor-associated kinases MEK and PI3K or the pro-inflammatory cytokine IL-17. Temporal profiling of protein ubiquitination events across a series of time points covering the biological response permits interrogation of signaling through thousands of quantified proteins, of which only a subset display significant and physiologically meaningful regulation. Distinctive clusters of residues within proteins can display distinct temporal patterns attributable to diverse molecular functions, although the majority of differential ubiquitination appears as a coordinated response across the modifiable residues present within an individual substrate. In cells treated with a combination of MEK and PI3K inhibitors, we found differential ubiquitination of MEK within the first hour after treatment and a series of mitochondria proteins at later time points. In the IL-17 signaling pathway, ubiquitination events on several signaling proteins including HOIL-1 and Tollip were observed. The functional relevance of these putative IL-17 mediators was subsequently validated by knockdown of HOIL-1, HOIP and TOLIP, each of which decreased IL-17-stimulated cytokine production. Together, these data validate proteomic profiling of protein ubiquitination as a viable approach for identifying dynamic signaling components in response to intracellular and extracellular perturbations.
authors: Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors: Kebing
authors: K
authors: Yu
authors: Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors: Lilian
authors: L
authors: Phu
authors: Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors: Eugene
authors: E
authors: Varfolomeev
authors: Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors: Daisy
authors: D
authors: Bustos
authors: Department of Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: domagoj@gene.com.
authors: Domagoj
authors: D
authors: Vucic
authors: Department of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: donaldk@gene.com.
authors: Donald S
authors: DS
authors: Kirkpatrick
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001384:Azetidines / Q000494:pharmacology; D045744:Cell Line, Tumor / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007191:Indazoles / Q000494:pharmacology; D020381:Interleukin-17 / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010880:Piperidines / Q000494:pharmacology; D011506:Proteins / Q000276:immunology* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014157:Transcription Factors; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D001384:Azetidines; D004791:Enzyme Inhibitors; D007191:Indazoles; D020381:Interleukin-17; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases; D024101:Mitochondrial Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010880:Piperidines; D011506:Proteins; D013449:Sulfonamides; D014157:Transcription Factors; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; C532162:2-(1H-indazol-4-yl)-6-(4-methanesulfonylpiperazin-1-ylmethyl)-4-morpholin-4-ylthieno(3,2-d)pyrimidine; C410602:TOLLIP protein, human; C504507:RBCK1 protein, human; C558338:RNF31 protein, human; C574276:cobimetinib
keywords: LC-MS/MS; immunoaffinity enrichment; mitochondria; proteomics; ubiquitination