1. SHARPIN at the nexus of integrin, immune, and inflammatory signaling in human platelets.
- Author
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Kasirer-Friede A, Tjahjono W, Eto K, and Shattil SJ
- Subjects
- Cell Lineage, Gene Knockdown Techniques, Homeostasis, Humans, I-kappa B Kinase metabolism, Induced Pluripotent Stem Cells metabolism, Inflammation pathology, Megakaryocytes metabolism, Models, Biological, NF-kappa B metabolism, Platelet Membrane Glycoprotein IIb metabolism, Protein Binding, Ubiquitin metabolism, Ubiquitin-Protein Ligases metabolism, Ubiquitination, Blood Platelets metabolism, Signal Transduction, Ubiquitins metabolism
- Abstract
Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbβ3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbβ3 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 α 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-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbβ3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated 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-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbβ3 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., Competing Interests: The authors declare no conflict of interest.
- Published
- 2019
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