Nguyen, Minh DangShim, Su Yeon2014-04-302015-10-282014-04-302014http://hdl.handle.net/11023/1459The Targeting Protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein required for mitotic spindle assembly. In interphase cells, TPX2 regulates the amplification of the -H2AX signal in the nucleus during the DNA damage response. TPX2 functions are likely to be regulated by the putative phosphorylation sites identified by several mass spectrometry-based screenings. However, most of these 41 sites have not been validated and their roles have not been characterized. In my thesis, I characterized the phosphorylation of TPX2 at Threonine 72 (Thr72) in vivo. First, I confirmed the specificity of our homemade phospho-Thr72 antibodies. I then found out that phosphorylation at Thr72 is cell cycle-dependent, peaking at the M phase of the cell cycle. I also confirmed the existence of Thr72 phosphorylation in mitotic HeLa cells by mass spectrometry. Using a pharmacological approach, I discovered that cyclin-dependent kinases can mediate phosphorylation at this site in mitotic HeLa cells. Finally, using TPX2 siRNAs and phospho-mutants, I examined the role of Thr72 in mitotic spindle assembly and DNA damage response. Understanding the significance of phosphorylation of Thr72 may provide new insights into the roles of TPX2 in healthy and diseases conditions, such as cancers.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.Biology--MolecularBiochemistryTPX2, HeLa cells, Cancer, mitotic spindle assembly, DNA damage responsemaster thesis10.11575/PRISM/25295