ABSTRACT
Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor ßPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration.
Subject(s)
Cell Movement , Dendritic Cells/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Neoplasms/metabolism , Semaphorins/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Animals , Genotype , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mass Spectrometry , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/pathology , Nerve Tissue Proteins/metabolism , Phenotype , RNA Interference , Receptors, Cell Surface/metabolism , Rho Guanine Nucleotide Exchange Factors/metabolism , Semaphorins/deficiency , Semaphorins/genetics , Time Factors , Transfection , Tumor Suppressor Proteins/genetics , cdc42 GTP-Binding Protein/metabolism , rac GTP-Binding Proteins/metabolismABSTRACT
The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKß-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which â¼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKß substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKß on serine 298. We provide evidence that IKKß-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo.
Subject(s)
Cell Adhesion Molecules/drug effects , Gene Expression Regulation, Neoplastic , I-kappa B Kinase/drug effects , Phosphorylation/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Blotting, Western , Cell Adhesion Molecules/metabolism , Chromatin Immunoprecipitation , Chromatography, Liquid , HEK293 Cells , Humans , I-kappa B Kinase/metabolism , I-kappa B Proteins , Immunoprecipitation , MCF-7 Cells , Mass Spectrometry , Membrane Proteins , NF-KappaB Inhibitor alpha , NF-kappa B , Phosphoproteins , RNA-Binding Proteins , Serine , Tumor Stem Cell Assay , Up-RegulationABSTRACT
Plexins are widely expressed transmembrane proteins that mediate the cellular effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still poorly understood. Here we show that signalling via B-family plexins leading to the activation of the small GTPase RhoA requires activation of the IκB kinase (IKK)-complex. In contrast, plexin-B-dependent regulation of R-Ras activity is not affected by IKK activity. This regulation of plexin signalling depends on the kinase activity of the IKK-complex, but is independent of NF-κB activation. We confirm that the IKK-complex is active in tumour cells and osteoblasts, and we demonstrate that plexin-B-dependent tumour cell invasiveness and regulation of osteoblast differentiation require an active IKK-complex. This study identifies a novel, NF-κB-independent function of the IKK-complex and shows that IKK directs plexin-B signalling to the activation of RhoA.
Subject(s)
Cell Adhesion Molecules/metabolism , I-kappa B Kinase/metabolism , Nerve Tissue Proteins/metabolism , rhoA GTP-Binding Protein/metabolism , Cell Differentiation/genetics , Cell Line , Cell Line, Tumor , HEK293 Cells , Humans , MCF-7 Cells , NF-kappa B/metabolism , Signal Transduction/geneticsABSTRACT
OBJECTIVE: To evaluate standardized lung recruitment strategy during both high frequency oscillation (HFO) and volume-targeted conventional ventilation (CV+V) in spontaneously breathing piglets with surfactant washout on pathophysiologic and inflammatory responses. DESIGN: Prospective animal study. SETTING: Research laboratory. SUBJECTS: Twenty-four newborn piglets. INTERVENTIONS: We compared pressure support and synchronized intermittent mandatory ventilation, both with targeted tidal volumes, (PSV+V, SIMV+V) to HFO. Animals underwent saline lavage to produce lung injury, received artificial surfactant and were randomized to one of the three treatment groups (each n=8). After injury and surfactant replacement, lung volumes were recruited in all groups using a standard protocol. Ventilation continued for 6 h. MEASUREMENTS AND MAIN RESULTS: Arterial and central venous pressures, heart rates, blood pressure and arterial blood gases were continuously monitored. At baseline, post lung injury and 6 h we collected serum and bronchoalveolar lavage samples for proinflammatory cytokines: IL 6, IL 8 and TNF-alpha, and performed static pressure-volume (P/V) curves. Lungs were fixed for morphometrics and histopathologic analysis. No physiologic differences were found. Analysis of P/V curves showed higher opening pressures after lung injury in the HFO group compared to the SIMV+V group ( p<0.05); no differences persisted after treatment. We saw no differences in change in proinflammatory cytokine levels. Histopathology and morphometrics were similar. Mean airway pressure (P(aw)) was highest in the HFO group compared to SIMV+V ( p<0.002). CONCLUSIONS: Using a standardized lung recruitment strategy in spontaneously breathing animals, CV+V produced equivalent pathophysiologic outcomes without an increase in proinflammatory cytokines when compared to HFO.