title: Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium.
abstract: Ag-mediated activation of mast cells initiates signaling events leading to Ca(2+) 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 γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow-derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor β (βPIX) and G protein-coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of βPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that βPIX 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 Ca(2+) affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca(2+) level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and βPIX 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/βPIX proteins, and Ca(2+) in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic;
authors.forename: Vadym
authors.initials: V
authors.lastname: Sulimenko
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic; Department of Cell Biology, Faculty of Science, Charles University Prague, CZ-128 43 Prague 2, Czech Republic; and.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Hájková
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic; Department of Cell Biology, Faculty of Science, Charles University Prague, CZ-128 43 Prague 2, Czech Republic; and.
authors.forename: Markéta
authors.initials: M
authors.lastname: Černohorská
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic;
authors.forename: Tetyana
authors.initials: T
authors.lastname: Sulimenko
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic;
authors.forename: Vladimíra
authors.initials: V
authors.lastname: Sládková
authors.affiliation: Department of Signal Transduction, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic.
authors.forename: Lubica
authors.initials: L
authors.lastname: Dráberová
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic;
authors.forename: Stanislav
authors.initials: S
authors.lastname: Vinopal
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic;
authors.forename: Eduarda
authors.initials: E
authors.lastname: Dráberová
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic; paveldra@img.cas.cz.
authors.forename: Pavel
authors.initials: P
authors.lastname: Dráber
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002118:Calcium / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D008407:Mast Cells / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008870:Microtubules / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors / Q000378:metabolism*
chemical_list: C576607:Arhgef7 protein, mouse; D018797:Cell Cycle Proteins; D020690:GTPase-Activating Proteins; C120766:Git1 protein, mouse; D064067:Rho Guanine Nucleotide Exchange Factors; D002118:Calcium