Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium

0455873 - ÚMG 2016 RIV US eng J - Journal Article
Sulimenko, Vadym - Hájková, Zuzana - Černohorská, Markéta - Sulimenko, Tetyana - Sládková, Vladimíra - Dráberová, Lubica - Vinopal, Stanislav - Dráberová, Eduarda - Dráber, Pavel
Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium.
Journal of Immunology. Roč. 194, č. 9 (2015), s. 4099-4111. ISSN 0022-1767. E-ISSN 1550-6606
R&D Projects: GA ČR GAP302/12/1673; GA ČR GPP302/11/P709; GA ČR(CZ) GA14-09807S; GA ČR GA15-22194S; GA MŠMT(CZ) LD13015; GA MŠMT LH12050; GA MZd NT14467
Institutional support: RVO:68378050
Keywords : Bone Marrow-Derived Mast Cells * Microtubule Nucleation * GIT1/beta PIX Proteins * Calcium
Subject RIV: EB - Genetics ; Molecular Biology
Impact factor: 4.985, year: 2015

Ag-mediated activation of mast cells initiates signaling events leading to Ca2+ response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by gamma-tubulin and gamma-tubulin complex proteins. In this study, we report that, in bone marrow-derived mast cells (BMMCs), gamma-tubulin interacts with p21-activated kinase interacting exchange factor beta (beta PIX) and G protein-coupled receptor kinase-interacting protein (GIT) 1. Microtubule regrowth experiments showed that the depletion of beta PIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that beta PIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca2+ affects gamma-tubulin properties and stimulates the association of GIT1 and gamma-tubulin complex proteins with gamma-tubulin. Microtubule nucleation also was affected by Ca2+ level. Moreover, in activated BMMCs, gamma-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and beta PIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/beta PIX proteins, and Ca2+ in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.
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Permanent Link: http://hdl.handle.net/11104/0256475