ABSTRACT
The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E(2), that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-alpha, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor kappaB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates.
Subject(s)
A Kinase Anchor Proteins/immunology , Cyclic AMP-Dependent Protein Kinases/immunology , Cyclic AMP/immunology , Lipopolysaccharides/pharmacology , Macrophages/immunology , NF-kappa B p50 Subunit/immunology , Tumor Necrosis Factor-alpha/immunology , A Kinase Anchor Proteins/genetics , A Kinase Anchor Proteins/metabolism , Animals , Cell Line , Cyclic AMP/genetics , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Dinoprostone/genetics , Dinoprostone/immunology , Dinoprostone/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Macrophages/metabolism , Mice , NF-kappa B p50 Subunit/genetics , NF-kappa B p50 Subunit/metabolism , Phosphorylation/drug effects , Phosphorylation/immunology , RNA Interference , Second Messenger Systems/drug effects , Second Messenger Systems/immunology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.
