12 research outputs found
Probing kinase activation and substrate specificity with an engineered monomeric IKK2
Catalytic subunits of the IB kinase (IKK), IKK1/IKKalpha, and IKK2/IKKbeta function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKK. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein-protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IBalpha substrate. Thus, the NF-B-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers. 2014 American Chemical Societ
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Form Factors and Two-Photon Exchange in High-Energy Elastic Electron-Proton Scattering.
We present new precision measurements of the elastic electron-proton scattering cross section for momentum transfer (Q^{2}) up to 15.75ââ(GeV/c)^{2}. Combined with existing data, these provide an improved extraction of the proton magnetic form factor at high Q^{2} and double the range over which a longitudinal or transverse separation of the cross section can be performed. The difference between our results and polarization data agrees with that observed at lower Q^{2} and attributed to hard two-photon exchange (TPE) effects, extending to 8ââ(GeV/c)^{2} the range of Q^{2} for which a discrepancy is established at >95% confidence. We use the discrepancy to quantify the size of TPE contributions needed to explain the cross section at high Q^{2}
AIM 2 inflammasomes regulate neuronal morphology and influence anxiety and memory in mice
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First Measurement of the EMC effect in B10 and B11
The nuclear dependence of the inclusive inelastic electron scattering cross section (the EMC effect) has been measured for the first time in B10 and B11. Previous measurements of the EMC effect in Aâ€12 nuclei showed an unexpected nuclear dependence; B10 and B11 were measured to explore the EMC effect in this region in more detail. Results are presented for Be9, B10, B11, and C12 at an incident beam energy of 10.6 GeV. The EMC effect in the boron isotopes was found to be similar to that for Be9 and C12, yielding almost no nuclear dependence in the EMC effect in the range A=4-12. This represents important new data supporting the hypothesis that the EMC effect depends primarily on the local nuclear environment due to the cluster structure of these nuclei