SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Authors: Kasirer-Friede, Ana; Tjahjono, Winson; Eto, Koji; Shattil, Sanford J.
Abstract
Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin alpha IIb beta 3 as a fibrinogen receptor in hemostasis is well defined, but the roles of alpha IIb beta 3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin a cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-kappa B activation. SHARPIN interacted with aIIb in pull-down and co-immunoprecipitation assays. SHARPIN was partially localized, as was alpha IIb beta 3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also co-immunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-kappa B pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-kappa B. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to alpha IIb beta 3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.
Exclusivity and Compensation in NF kappa B Dimer Distributions and I kappa B Inhibition
BIOCHEMISTRY
Authors: Ramsey, Kristen M.; Chen, Wei; Marion, James D.; Bergqvist, Simon; Komives, Elizabeth A.
Abstract
The NF kappa B transcription factor family members RelA, p50, and cRel form homo- and heterodimers that are inhibited by I kappa B alpha, I kappa B beta, and I kappa B epsilon. These NF kappa B family members have diverse biological functions, and their expression profiles differ, leading to different concentrations in different tissue types. Here we present definitive biophysical measurements of the NF kappa B dimer affinities and inhibitor affinities to better understand dimer exchange and how the presence of inhibitors may alter the equilibrium concentrations of NF kappa B dimers in the cellular context. Fluorescence anisotropy binding experiments were performed at low concentrations to mimic intracellular concentrations. We report binding affinities much stronger than those that had been previously reported by non-equilibrium gel shift and analytical ultracentrifugation assays. The results reveal a wide range of NF kappa B dimer affinities and a strong preference of each I kappa B for a small subset of NF kappa B dimers. Once the preferred I kappa B is bound, dimer exchange no longer occurs over a period of days. A mathematical model of the cellular distribution of these canonical NF kappa B transcription factors based on the revised binding affinities recapitulates intracellular observations and provides simple, precise explanations for observed cellular phenomena.