title: Phosphorylation of targeting protein for Xenopus kinesin-like protein 2 (TPX2) at threonine 72 in spindle assembly.
abstract: The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr(72)) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr(72), we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr(72) does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr(72) regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.
authors: From the Departments of Clinical Neurosciences, Cell Biology & Anatomy, and Biochemistry & Molecular Biology and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada T2N4N1.
authors: Su Yeon
authors: SY
authors: Shim
authors: the Cell Division and Cancer Group, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid 28029, Spain.
authors: Ignacio
authors: I
authors: Perez de Castro
authors: From the Departments of Clinical Neurosciences, Cell Biology & Anatomy, and Biochemistry & Molecular Biology and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada T2N4N1.
authors: Gernot
authors: G
authors: Neumayer
authors: From the Departments of Clinical Neurosciences, Cell Biology & Anatomy, and Biochemistry & Molecular Biology and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada T2N4N1.
authors: Jian
authors: J
authors: Wang
authors: the Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.
authors: Sang Ki
authors: SK
authors: Park
authors: the Molecular Genetics Research Laboratory, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan, and.
authors: Kamon
authors: K
authors: Sanada
authors: From the Departments of Clinical Neurosciences, Cell Biology & Anatomy, and Biochemistry & Molecular Biology and Hotchkiss Brain Institute, University of Calgary, Calgary, Canada T2N4N1, mdnguyen@ucalgary.ca.
authors: Minh Dang
authors: MD
authors: Nguyen
mesh_terms: D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D008941:Spindle Apparatus; D013912:Threonine / Q000737:chemistry / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D017931:DNA Primers; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; C486551:TPX2 protein, Xenopus; D029867:Xenopus Proteins; D013912:Threonine
keywords: Antibody; Cell Cycle; Microtubule-associated Protein (MAP); Mitotic Spindle; Protein Phosphorylation