Article

Quantitative Proteomics Analysis of Macrophage Rafts Reveals Compartmentalized Activation of the Proteasome and of Proteasome-mediated ERK Activation in Response to Lipopolysaccharide

Laboratories of Respiratory Biology, NIEHS, National Institutes of Health, United States Department of Health and Human Services, Research Triangle Park, North Carolina 27709, USA.
Molecular &amp Cellular Proteomics (Impact Factor: 7.25). 10/2008; 8(1):201-13. DOI: 10.1074/mcp.M800286-MCP200
Source: PubMed

ABSTRACT Lipopolysaccharide (LPS), a glycolipid component of the outer membrane of Gram-negative bacteria, is a potent initiator of the innate immune response of the macrophage. LPS triggers downstream signaling by selectively recruiting and activating proteins in cholesterol-rich membrane microdomains called lipid rafts. We applied proteomics analysis to macrophage detergent-resistant membranes (DRMs) during an LPS exposure time course in an effort to identify and validate novel events occurring in macrophage rafts. Following metabolic incorporation in cell culture of heavy isotopes of amino acids arginine and lysine ([(13)C(6)]Arg and [(13)C(6)]Lys) or their light counterparts, a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative, liquid chromatography-tandem mass spectrometry proteomics approach was used to profile LPS-induced changes in the lipid raft proteome of RAW 264.7 macrophages. Unsupervised network analysis of the proteomics data set revealed a marked representation of the ubiquitin-proteasome system as well as changes in proteasome subunit composition following LPS challenge. Functional analysis of DRMs confirmed that LPS causes selective activation of the proteasome in macrophage rafts and proteasome inactivation outside of rafts. Given previous reports of an essential role for proteasomal degradation of IkappaB kinase-phosphorylated p105 in LPS activation of ERK mitogen-activated protein kinase, we tested for a role of rafts in compartmentalization of these events. Immunoblotting of DRMs revealed proteasome-dependent activation of MEK and ERK specifically occurring in lipid rafts as well as proteasomal activity upon raft-localized p105 that was enhanced by LPS. Cholesterol extraction from the intact macrophage with methyl-beta-cyclodextrin was sufficient to activate ERK, recapitulating the LPS-IkappaB kinase-p105-MEK-ERK cascade, whereas both it and the alternate raft-disrupting agent nystatin blocked subsequent LPS activation of the ERK cascade. Taken together, our findings indicate a critical, selective role for raft compartmentalization and regulation of proteasome activity in activation of the MEK-ERK pathway.

Download full-text

Full-text

Available from: Kenneth Tomer, Jun 23, 2015
0 Followers
 · 
59 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The trichothecene mycotoxin deoxynivalenol (DON) targets the innate immune system and is of public health significance because of its frequent presence in human and animal food. DON-induced proinflammatory gene expression and apoptosis in the lymphoid tissue have been associated with a ribotoxic stress response (RSR) that involves rapid phosphorylation of mitogen-activated protein kinases (MAPKs). To better understand the relationship between protein phosphorylation and DON's immunotoxic effects, stable isotope dimethyl labeling-based proteomics in conjunction with titanium dioxide chromatography was employed to quantitatively profile the immediate (≤30 min) phosphoproteome changes in the spleens of mice orally exposed to 5 mg/kg body weight DON. A total of 90 phosphoproteins indicative of novel phosphorylation events were significantly modulated by DON. In addition to critical branches and scaffolds of MAPK signaling being affected, DON exposure also altered phosphorylation of proteins that mediate PI3K/AKT pathways. Gene ontology analysis revealed that DON exposure affected biological processes such as cytoskeleton organization, regulation of apoptosis, and lymphocyte activation and development, which likely contribute to immune dysregulation associated with DON-induced RSR. Consistent with these findings, DON impacted phosphorylation of proteins within diverse immune cell populations, including monocytes, macrophages, T cells, B cells, dendritic cells and mast cells. Fuzzy c-means clustering analysis further indicated that DON evoked several distinctive temporal profiles of regulated phosphopeptides. Overall, the findings from this investigation can serve as a template for future focused exploration and modeling of cellular responses associated with the immunotoxicity evoked by DON and other ribotoxins.
    Toxicological Sciences 06/2013; DOI:10.1093/toxsci/kft145 · 4.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cell membranes are composed of many different lipids and protein receptors, which are important for regulating intracellular functions and cell signaling. To orchestrate these activities, the cell membrane is compartmentalized into microdomains that are stably or transiently formed. These compartments are called "lipid rafts". In gamete cells that lack gene transcription, distribution of lipids and proteins on these lipid rafts is focused during changes in their structure and functions such as starting flagella movement and membrane fusion. In this paper, we describe the role of lipid rafts in gamete maturation, fertilization, and early embryogenesis.
    01/2011; 2011(2090-3030):264706. DOI:10.1155/2011/264706
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pre-B cell colony enhancing factor (PBEF) is a highly conserved, pleiotropic protein reported to be an alternate ligand for the insulin receptor (IR). We evaluated the effects of PBEF on the localization of the IR β chain to lipid rafts in A549 epithelial cells. Methods: We isolated lipid rafts from A549 cells and detected the IR by immunoprecipitation from raft fractions or whole cell lysates. Cells were treated with rPBEF, its enzymatic product nicotinamide adenine dinucleotide (NAD), or the Nampt inhibitor, daporinad, to study the effect of PBEF on IRβ movement. We used co-immunoprecipitation studies in cells transfected with PBEF and IRβ constructs to detect interactions between PBEF, the IRβ, and caveolin-1 (Cav-1). Results: PBEF was present in both lipid raft and non-raft fractions, while the IR was only found in lipid raft fractions of resting A549 cells. The IR, PBEF and Cav-1 co-immunoprecipitated. rPBEF treatment resulted in the movement of IRβ and tyrosine phosphorylated Cav-1 from lipid rafts to non-rafts, an effect that could be blocked by daporinad, suggesting that this effect was facilitated by the Nampt activity of PBEF. The addition of PBEF to insulin-treated cells resulted in reduced Akt phosphorylation of both Ser473 and Thr308. Conclusions: PBEF can inhibit insulin signaling through the IR by Nampt-dependent promotion of IR translocation into the non-raft domains of A549 epithelial cells. PBEF-induced alterations in the spatial geometry of the IR provide a mechanistic explanation for insulin resistance in inflammatory states associated with upregulation of PBEF. Copyright © 2014, American Journal of Physiology - Endocrinology and Metabolism.
    AJP Endocrinology and Metabolism 12/2014; 308(4):ajpendo.00006.2014. DOI:10.1152/ajpendo.00006.2014 · 4.09 Impact Factor