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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.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Yu K,Phu L,Varfolomeev E,Bustos D,Vucic D,Kirkpatrick DS

doi

10.1016/j.jmb.2015.03.018

subject

Has Abstract

pub_date

2015-06-05 00:00:00

pages

2121-34

issue

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(15)00214-4

journal_volume

427

pub_type

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Immunoaffinity enrichment coupled to quantitative mass spectrometry reveals ubiquitin-mediated signaling events.