TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFα and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

The enteroinvasive bacterium Shigella flexneri uses multiple secreted effector proteins to downregulate interleukin-8 (IL-8) expression in infected epithelial cells. Yet, massive IL-8 secretion is observed in Shigellosis. Here we report a host mechanism of cell-cell communication that circumvents the effector proteins and strongly amplifies IL-8 expression during bacterial infection. By monitoring proinflammatory signals at the single-cell level, we found that the activation of the transcription factor NF-κB and the MAP kinases JNK, ERK, and p38 rapidly propagated from infected to uninfected adjacent cells, leading to IL-8 production by uninfected bystander cells. Bystander IL-8 production was also observed during Listeria monocytogenes and Salmonella typhimurium infection. This response could be triggered by recognition of peptidoglycan and is mediated by gap junctions. Thus, we have identified a mechanism of cell-cell communication that amplifies innate immunity against bacterial infection by rapidly spreading proinflammatory signals via gap junctions to yet uninfected cells.

BackgroundDuring pathogen infection, innate immunity is initiated via the recognition of microbial products by pattern recognition receptors and the subsequent activation of transcription factors that upregulate proinflammatory genes. By controlling the expression of cytokines, chemokines, anti-bacterial peptides and adhesion molecules, the transcription factor nuclear factor-kappa B (NF-κB) has a central function in this process. In a typical model of NF-κB activation, the recognition of pathogen associated molecules triggers the canonical NF-κB pathway that depends on the phosphorylation of Inhibitor of NF-κB (IκB) by the catalytic subunit IκB kinase β (IKKβ), its degradation and the nuclear translocation of NF-κB dimers.MethodologyHere, we performed an RNA interference (RNAi) screen on Shigella flexneri-induced NF-κB activation to identify new factors involved in the regulation of NF-κB following infection of epithelial cells by invasive bacteria. By targeting a subset of the human signaling proteome, we found that the catalytic subunit IKKα is also required for complete NF-κB activation during infection. Depletion of IKKα by RNAi strongly reduces the nuclear translocation of NF-κB p65 during S. flexneri infection as well as the expression of the proinflammatory chemokine interleukin-8. Similar to IKKβ, IKKα contributes to the phosphorylation of IκBα on serines 32 and 36, and to its degradation. Experiments performed with the synthetic Nod1 ligand L-Ala-D-γ-Glu-meso-diaminopimelic acid confirmed that IKKα is involved in NF-κB activation triggered downstream of Nod1-mediated peptidoglycan recognition.ConclusionsTaken together, these results demonstrate the unexpected role of IKKα in the canonical NF-κB pathway triggered by peptidoglycan recognition during bacterial infection. In addition, they suggest that IKKα may be an important drug target for the development of treatments that aim at limiting inflammation in bacterial infection.

BackgroundNuclear factor-κB (NF-κB) is a transcription factor that regulates the transcription of genes involved in a variety of biological processes, including innate and adaptive immunity, stress responses and cell proliferation. Constitutive or excessive NF-κB activity has been associated with inflammatory disorders and higher risk of cancer. In contrast to the mechanisms controlling inducible activation, the regulation of basal NF-κB activation is not well understood. Here we test whether clathrin heavy chain (CHC) contributes to the regulation of basal NF-κB activity in epithelial cells.MethodologyUsing RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression. Immunofluorescence staining showed constitutive nuclear localization of the NF-κB subunit p65 in absence of stimulation after CHC knockdown. Elevated basal p65 nuclear localization is caused by constitutive phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) through an IκB kinase α (IKKα)-dependent mechanism. The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown. Disruption of clathrin-mediated endocytosis by chemical inhibition or depletion of the μ2-subunit of the endocytosis adaptor protein AP-2, and knockdown of clathrin light chain a (CHLa), failed to induce constitutive NF-κB activation and IL-8 expression, showing that CHC acts on NF-κB independently of endocytosis and CLCa.ConclusionsWe conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells. Furthermore, our data suggest a potential link between a defect in CHC expression and chronic inflammation disorder and cancer.