c-Abl kinase-mediated phosphorylation of γ-tubulin promotes γ-tubulin ring complexes assembly and microtubule nucleation

Abstract

Cytoskeletal microtubules (MTs) are nucleated from γ-tubulin ring complexes (γTuRCs) located at MT organizing centers (MTOCs), such as the centrosome. However, the exact regulatory mechanism of γTuRC assembly is not fully understood. Here, we showed that the nonreceptor tyrosine kinase c-Abl was associated with and phosphorylated γ-tubulin, the essential component of the γTuRC, mainly on the Y443 residue by in vivo (immunofluorescence and immunoprecipitation) or in vitro (surface plasmon resonance) detection. We further demonstrated that phosphorylation deficiency significantly impaired γTuRC assembly, centrosome construction, and MT nucleation. c-Abl/Arg deletion and γ-tubulin Y443F mutation resulted in an abnormal morphology and compromised spindle function during mitosis, eventually causing uneven chromosome segregation. Our findings reveal that γTuRC assembly and nucleation function are regulated by Abl kinase-mediated γ-tubulin phosphorylation, revealing a fundamental mechanism that contributes to the maintenance of MT function. Cytoskeletal microtubules (MTs) are nucleated from γ-tubulin ring complexes (γTuRCs) located at MT organizing centers (MTOCs), such as the centrosome. However, the exact regulatory mechanism of γTuRC assembly is not fully understood. Here, we showed that the nonreceptor tyrosine kinase c-Abl was associated with and phosphorylated γ-tubulin, the essential component of the γTuRC, mainly on the Y443 residue by in vivo (immunofluorescence and immunoprecipitation) or in vitro (surface plasmon resonance) detection. We further demonstrated that phosphorylation deficiency significantly impaired γTuRC assembly, centrosome construction, and MT nucleation. c-Abl/Arg deletion and γ-tubulin Y443F mutation resulted in an abnormal morphology and compromised spindle function during mitosis, eventually causing uneven chromosome segregation. Our findings reveal that γTuRC assembly and nucleation function are regulated by Abl kinase-mediated γ-tubulin phosphorylation, revealing a fundamental mechanism that contributes to the maintenance of MT function. Microtubules (MTs) are one of the most important cytoskeleton elements and play crucial roles in cell shape maintenance, organelle movement, and cell division. The minus ends of MTs are nucleated from and anchored to the MT organizing center (MTOC), a specialized structure responsible for MT assembly from the centrosome or Golgi apparatus during interphase and from the spindle during mitosis (1Kellogg D.R. Moritz M. Alberts B.M. The centrosome and cellular organization.Annu. Rev. Biochem. 1994; 63: 639-674Crossref PubMed Scopus (327) Google Scholar, 2Chabin-Brion K. Marceiller J. Perez F. Settegrana C. Drechou A. Durand G. Pous C. The Golgi complex is a microtubule-organizing organelle.Mol. Biol. Cell. 2001; 12: 2047-2060Crossref PubMed Scopus (230) Google Scholar). In mammalian cells, the centrosome is the major organelle that functions as an MTOC and consists of a pair of centrioles with an orthogonal orientation to each other, which are embedded in a tightly clustered electron-dense pericentriolar material (PCM) (1Kellogg D.R. Moritz M. Alberts B.M. The centrosome and cellular organization.Annu. Rev. Biochem. 1994; 63: 639-674Crossref PubMed Scopus (327) Google Scholar). The PCM is an organized lattice-like structure containing more than 100 different proteins (3Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling.Nature. 2003; 426: 570-574Crossref PubMed Scopus (1036) Google Scholar), including several scaffold proteins, such as pericentrin (PCNT), CDK5RAP2, Cep152, Cep192, and CPAP, effector proteins, such as γ-tubulin, and other kinases or phosphatases (4Woodruff J.B. Wueseke O. Hyman A.A. Pericentriolar material structure and dynamics.Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2014; 369: 20130459Crossref PubMed Scopus (145) Google Scholar). Scaffold proteins are localized at the core matrix of the PCM, while effector proteins serve as sites of centrosomal MT nucleation (4Woodruff J.B. Wueseke O. Hyman A.A. Pericentriolar material structure and dynamics.Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2014; 369: 20130459Crossref PubMed Scopus (145) Google Scholar). The γ-tubulin ring complex (γTuRC), which functions as an MT nucleator in the PCM, is an ∼2.2 MDa complex composed of γ-tubulin and several γ-tubulin complex proteins (GCPs, GCP2∼6) belonging to a conserved protein family (5Zheng Y. Wong M.L. Alberts B. Mitchison T. Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex.Nature. 1995; 378: 578-583Crossref PubMed Scopus (715) Google Scholar). The γ-tubulin small complex (γTuSC) is the elemental unit of the γTuRC, consisting of two molecules of γ-tubulin and one molecule each of GCP2 and GCP3 (6Oegema K. Wiese C. Martin O.C. Milligan R.A. Iwamatsu A. Mitchison T.J. Zheng Y. Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.J. Cell Biol. 1999; 144: 721-733Crossref PubMed Scopus (249) Google Scholar). The γTuRC is composed of two highly asymmetric but similarly sized halves: a core of four γTuSCs and an arrangement of different γTuSC-like subunits (GCP4/5 and GCP4/6) capped by a terminal γTuSC that together form a large and diverse binding surface for regulatory factors (7Kollman J.M. Merdes A. Mourey L. Agard D.A. Microtubule nucleation by gamma-tubulin complexes.Nat. Rev. Mol. Cell Biol. 2011; 12: 709-721Crossref PubMed Scopus (333) Google Scholar, 8Wieczorek M. Urnavicius L. Ti S.C. Molloy K.R. Chait B.T. Kapoor T.M. Asymmetric molecular architecture of the human gamma-tubulin ring complex.Cell. 2020; 180: 165-175.e16Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). MT nucleation at MTOCs requires a regulatory mechanism to recruit γTuRCs. The regulators might be PCM components, such as PCNT, CDK5RAP2, and NEDD1, which always function as γTuRC-associated proteins (9Teixido-Travesa N. Roig J. Luders J. 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The microtubule protein of the tyrosine kinase Abl in 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). reveal in MT by Abl family how MT nucleation at MTOC is regulated by the kinases is not fully understood. In we showed that c-Abl associated with and phosphorylated γ-tubulin on tyrosine phosphorylation, c-Abl regulated the nucleation of centrosomal and Golgi defects of on γ-tubulin by c-Abl impaired the assembly of both MTs and PCM in interphase and the spindle structure in mitosis by γTuRC In to the mechanism of Abl family kinases in the of the MT we that with in the centrosome in c-Abl was to be in not in from cell which that c-Abl might be associated with a c-Abl was in the from in which in both abl1 and abl2 by and c-Abl was to with γ-tubulin, the most important protein for MT nucleation and a component of the γTuRC, in Moreover, c-Abl and Arg demonstrated to with γ-tubulin and was to with In in was with an by and to with or demonstrated that directly with γ-tubulin but not The interaction was further demonstrated by surface plasmon and γ-tubulin was to bind similarly with c-Abl in the or of c-Abl in a Moreover, an in showed that c-Abl was associated with γ-tubulin in the cell during and the was in in the spindle and during and further the interaction between c-Abl and γ-tubulin, from and with or of the with showed that with c-Abl but not also demonstrated that directly with c-Abl but not findings that c-Abl associated with γ-tubulin in the The that γ-tubulin c-Abl that γ-tubulin was a of c-Abl. the phosphorylation of γ-tubulin, was with or of the with demonstrated that was phosphorylated by c-Abl Moreover, cell with or and the of the with showed that a of γ-tubulin in the cell was tyrosine phosphorylated The phosphorylation of γ-tubulin was in and The γ-tubulin phosphorylation was significantly in was to reveal the phosphorylation sites in γ-tubulin in the of and tyrosine sites or the was with and the that the mutation in compromised the phosphorylation of γ-tubulin the of phosphorylation of γ-tubulin was further demonstrated by the that phosphorylation was by or c-Abl in the of the c-Abl or also showed compromised phosphorylation c-Abl in the in the of c-Abl or or by c-Abl demonstrated that c-Abl the phosphorylation of γ-tubulin, on Y443 its which is important for γTuRC assembly, and γ-tubulin phosphorylation significantly the GCP2 was in γ-tubulin in the of c-Abl/Arg with or when tyrosine Y443 was to which that phosphorylation of γ-tubulin is in γTuRC The binding between γ-tubulin and was on the structure of the γTuRC with M. Urnavicius L. Ti S.C. Molloy K.R. Chait B.T. Kapoor T.M. Asymmetric molecular architecture of the human gamma-tubulin ring complex.Cell. 2020; 180: 165-175.e16Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). at γ-tubulin Y443 a in the binding with the at most on the γTuRC ring and and resulted in an increased interaction between γ-tubulin and However, in the binding binding in when Y443 was to that the mutation not the interaction and it is to the Y443F to γ-tubulin In of the molecular γ-tubulin was in in than in γTuRCs in the of cell γ-tubulin or at with and significantly more was to be γTuRCs than as by the of and γ-tubulin and Moreover, or subunits by and γTuRC was in and in with and and in and as by the of γ-tubulin and and the of of the that Y443 phosphorylation of γ-tubulin by c-Abl/Arg kinases the of the The PCM of the centrosome a for cellular MT nucleation. deficient in c-Abl/Arg showed a significantly PCM by than also PCM with the at a in which γ-tubulin was by Moreover, more CDK5RAP2, an essential component of PCM, its to 100 was to bind γTuRC at MTOCs T. L. L. Zheng J.Y. R. of its localization to and the Golgi Biol. 2010; Full Text Full Text PDF PubMed Scopus Google and was to be associated with γTuRC in with or with PCNT, an component of PCM, was in a of and than in that PCM proteins the PCM at in the of γ-tubulin the and centriole structure by The of containing increased from in to in and from in to in that c-Abl and Arg PCM through γ-tubulin phosphorylation and regulate centrosome The centrosome in to the of PCM proteins as from interphase to mitosis J.M. D.R. Biol. 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In for the the nonreceptor tyrosine kinase c-Abl/Arg was demonstrated to bind γ-tubulin and to γ-tubulin on and Y443 residue of in γ-tubulin in E. was demonstrated to bind c-Abl by or c-Abl showed on such interaction a kinase activity interaction between c-Abl and other c-Abl γ-tubulin not bind the c-Abl domain and which to the interaction one of the of the is the ability to interaction c-Abl was in the in the of c-Abl or kinase other tyrosine the between c-Abl and γ-tubulin was significantly by tyrosine binding when c-Abl was in the c-Abl than γ-tubulin but the binding was significantly than that of γ-tubulin in the of c-Abl sites other than Y443 also be phosphorylated by c-Abl that c-Abl with γ-tubulin and that is significantly by phosphorylation of phosphorylation in that γ-tubulin might also be phosphorylated by other tyrosine kinases PCM protein to the centriole during mitosis through their the centrosomal MTs on the centrosome by complexes R. C.E. for microtubules in centrosome in 1999; PubMed Scopus Google Scholar). PCM on the MT assembly of the centrosome. The nucleation of MTs on the centrosome is on the of γTuRC to the and γTuRC is a PCM component that is tightly associated with scaffold proteins such as In and phosphorylation of γ-tubulin γTuRC assembly and PCM and centrosome and MT in interphase and spindle in mitosis The PCM an function in centriole assembly and to the centriole and a centrosome J. P. A. of centriole by the pericentriolar Cell Biol. PubMed Scopus Google Scholar). the MT of the centriole to be impaired in and and which that c-Abl might the assembly of the that nucleation of MTs of the centriole requires the γTuRC complex localized in the PCM of the centrosome C.J. with a centrosomal protein which is associated with the gamma-tubulin Cell Biol. PubMed Scopus Google Scholar), we that the PCM by c-Abl/Arg deletion centriole assembly MTs play a in cellular cell and in MT assembly can to a of defects in mitosis and other on the MT is highly in the and is important for The development of γ-tubulin embryos at the in mice mitosis is to A. A. M. S. of two gamma-tubulin in Biol. PubMed Scopus Google Scholar). in chromosome during mitosis to which contributes to and is to be a of human of PubMed Scopus Google Scholar). MTs an important for F. C. and 2003; PubMed Scopus Google Scholar). In the MT can to and such as and S. J. S. P. S. T.J. C. A. M. M. A. M. C. and J. Scopus Google Scholar). In γ-tubulin be related to by the or the binding of γ-tubulin and the assembly of the γTuRC K. N. L. G. Y. C. E. S. M. D. D. E. F. C. in and of development and PubMed Scopus Google Scholar). also to in in K.S. P. P. of J. PubMed Scopus Google Scholar). and show of γ-tubulin and cytoskeletal of the Golgi apparatus and of cells, which that abnormal γ-tubulin the of and related M. K. N. of gamma-tubulin protein and of the to of and PubMed Scopus Google Scholar). c-Abl also in the of the cell and the development and function of the in and mice from defects in neurulation and before 11 days post The cytoskeleton in but MT is (20Koleske A.J. Gifford A.M. Scott M.L. Nee M. Bronson R.T. Miczek K.A. Baltimore D. Essential roles for the Abl and Arg tyrosine kinases in neurulation.Neuron. 1998; 21: 1259-1272Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). c-Abl is also in such as and S.D. P. c-Abl in Mol. 2011; PubMed Scopus Google Scholar, tyrosine kinase in 2011; PubMed Scopus Google Scholar, S. E. C. M. G. and c-abl the in J. Cell Biol. 2012; PubMed Scopus Google Scholar, M.L. C.E. of and of in Mol. 22: PubMed Scopus Google Scholar). The findings in that is regulated by Abl kinases a mechanism to MT dynamics also for the development of c-Abl such as and in the of and and in of and human and human and and fibroblast in with and The at with In the with the of or γ-tubulin was from an by or by the γ-tubulin gene with the sequence the a The was from The to the γ-tubulin gene in was from and the sequence was was by two mutation in the sequence to using the and with also using a using to the and or by of the and The with to the abl1 and abl2 by the and cloned the which the using and the with selected by and with using cell and from proteins to with or of the was as the by and and as to the or was as the and directly to the The protein using an at the with a The on in for at by using in for MT was before the by a of and for at the with for and with or for at by with or or for at The with at the the by a using a and at and for the on an with a 100 with a the of in as a which was further to a using as of the PCM selected for or on on for or to MT was by the in at for the the in for at for and to with an with with and with and with and with a at with and and at in with and The by in a at for and further by at the for of the was in a in and or and to and in complex with γTuRC was by from the proteins by the for with in The was to each was by and to In cell for at with of or protein with The with and to by of the was as a in In binding and The with or proteins for at The binding of the proteins to was using an The in was to the between γ-tubulin and c-Abl in on with and with in the with γ-tubulin and c-Abl and with and to the for The by the with a was on at a of using a and composed of 3 and of through at from to was using from of with and by which the protein and to using a and the the using the for phosphorylation The binding in γ-tubulin with by the using a of the structure of the The different in using from protein structure with The for the structure of human γTuRC from the The of is in that are as The of using using to each and in each was using as in the and and are by and not contains supporting The that is of with the of We J. Koleske and for and and was by by the of and by the of L. and C. C. G. J. Y. C. C. and G. L. and L. G. Y. J. R. and N. C. T. D. Y. and Y. J. J. Y. C. and L. G. was by of Wang was by

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