Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells.

• Klíčová slova
• Centrosome, GIT1/βPIX signaling proteins, Microtubule nucleation, PAK1 kinase, γ-tubulin,
• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• centrozom metabolismus   MeSH
• faktory zaměňující Rho guanin nukleotidy genetika   metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• imunoblotting MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• nádorové buněčné linie MeSH
• p21 aktivované kinasy genetika   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• signální transdukce MeSH
• transformované buněčné linie MeSH
• tubulin metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• faktory zaměňující Rho guanin nukleotidy MeSH
• GIT1 protein, human MeSH Prohlížeč
• p21 aktivované kinasy MeSH
• proteiny buněčného cyklu MeSH
• tubulin MeSH

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.

• MeSH
• buňky kostní dřeně cytologie   MeSH
• faktory zaměňující Rho guanin nukleotidy metabolismus   MeSH
• mastocyty cytologie   MeSH
• mikrotubuly metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteiny aktivující GTPasu metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Arhgef7 protein, mouse MeSH Prohlížeč
• faktory zaměňující Rho guanin nukleotidy MeSH
• Git1 protein, mouse MeSH Prohlížeč
• proteiny aktivující GTPasu MeSH
• proteiny buněčného cyklu MeSH
• vápník MeSH