Deregulated expression of TANK in glioblastomas triggers pro-tumorigenic ERK1/2 and AKT signaling pathways

Signal transmission by the noncanonical IkappaB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IKKɛ, requires interaction with adapter proteins such as TRAF associated NF-κB activator (TANK). Although increased expression or dysregulation of both kinases has been described for a variety of human cancers, this study shows that deregulated expression of the TANK protein is frequently occurring in glioblastomas (GBMs). The functional relevance of TANK was analyzed in a panel of GBM-derived cell lines and revealed that knockdown of TANK arrests cells in the S-phase and prohibits tumor cell migration. Deregulated TANK expression affects several signaling pathways controlling cell proliferation and the inflammatory response. Interference with stoichiometrically assembled signaling complexes by overexpression or silencing of TANK prevented constitutive interferon-regulatory factor 3 (IRF3) phosphorylation. Knockdown of TANK frequently prevents constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). TANK-mediated ERK1/2 activation is independent from the canonical MAP kinase or ERK kinase (MEK) 1/2-mediated pathway and utilizes an alternative pathway that uses a TBK1/IKKɛ/Akt signaling axis, thus identifying a novel pathway suitable to block constitutive ERK1/2 activity.Peer reviewe

Ca2+-Transport through Plasma Membrane as a Test of Auxin Sensitivity

Auxin is one of the crucial regulators of plant growth and development. The discovered auxin cytosolic receptor (TIR1) is not involved in the perception of the hormone signal at the plasma membrane. Instead, another receptor, related to the ABP1, auxin binding protein1, is supposed to be responsible for the perception at the plasma membrane. One of the fast and sensitive auxin-induced reactions is an increase of Ca2+ cytosolic concentration, which is suggested to be dependent on the activation of Ca2+ influx through the plasma membrane. This investigation was carried out with a plasmalemma enriched vesicle fraction, obtained from etiolated maize coleoptiles. The magnitude of Ca2+ efflux through the membrane vesicles was estimated according to the shift of potential dependent fluorescent dye diS-C3-(5). The obtained results showed that during coleoptiles ageing (3rd, 4th and 5th days of seedling etiolated growth) the magnitude of Ca2+ efflux from inside-out vesicles was decreased. Addition of ABP1 led to a recovery of Ca2+ efflux to the level of the youngest and most sensitive cells. Moreover, the efflux was more sensitive, responding from 10−8 to 10−6 M 1-NAA, in vesicles containing ABP1, whereas native vesicles showed the highest efflux at 10−6 M 1-NAA. We suggest that auxin increases plasma membrane permeability to Ca2+ and that ABP1 is involved in modulation of this reaction

Improving Identification of In-organello Protein-Protein Interactions Using an Affinity-enrichable, Isotopically Coded, and Mass Spectrometry-cleavable Chemical Crosslinker

An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein- protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain.Proteomic