title: GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.
abstract: 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.
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: 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: 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.
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.
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 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. Electronic address: paveldra@img.cas.cz.
authors.forename: Pavel
authors.initials: P
authors.lastname: Dráber
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D018385:Centrosome / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D064067:Rho Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014404:Tubulin / Q000378:metabolism; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; C485647:GIT1 protein, human; D064067:Rho Guanine Nucleotide Exchange Factors; D014404:Tubulin; D054462:p21-Activated Kinases
keywords: Centrosome; GIT1/βPIX signaling proteins; Microtubule nucleation; PAK1 kinase; γ-tubulin