Moorhead, GregNasa, Isha2016-11-022016-11-0220162016Nasa, I. (2016). Regulation of Protein Phosphatase One During Cell Cycle (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/27589http://hdl.handle.net/11023/3446Protein phosphatase 1 (PP1) is a highly conserved enzyme that controls the majority of serine/threonine (Ser/Thr) dephosphorylation reactions in eukaryotes. PP1 gains substrate specificity through binding to a large number (> 200) of regulatory proteins, which control PP1 localization, activity, and substrate interaction. PP1 recognizes the majority of these regulatory proteins via well-characterized RVxF binding motif generating hundreds of distinct PP1 holoenzymes. The main objective of this research was to uncover the regulatory mechanisms that govern the interaction of PP1 with its regulatory proteins during the cell cycle. The progression of cell cycle is largely governed by reversible protein phosphorylation. I showed that a subset of the RVxF binding motifs, in which x is a phosphorylatable amino acid (RV[S/T]F), are phosphorylated specifically during mitosis and that this phosphorylation event abrogates the interaction of PP1 with the regulatory protein. This phosphorylation is primarily governed by mitotic protein kinase Aurora B and is crucial to maintain phosphorylation of PP1 substrates during mitosis. In addition, I showed that PP1 itself dephosphorylates RVp[S/T]F motifs during mitotic exit, which allows the phosphatase to re-associate with the regulatory proteins and dephosphorylate other mitotic substrates. To gain further insight into the regulation of PP1 function in cell cycle, I characterized the novel cell cycle dependent interactome of PP1. Using quantitative mass spectrometry, I identified 113 novel RVxF containing potential PP1 binding partners including 17 mitosis-specific partners. Furthermore, using immunoblotting, I validated 9 of the PP1 interactions both in asynchronous and mitotic populations with proteins involved in cell cycle regulation (Aurora B, Aurora A, TPX2, CDCA2 (RM), TACC3, GCN2, DBC1, BRCA1 and RIF1). In addition, I demonstrated a novel interaction of PP1 with centrosomal protein, CEP192 via its ‘KHVTF’ motif. The work presented here expands our understanding of the regulation of PP1 in the cell cycle, and also suggests a novel regulatory mechanism by which the coordinated activities of Aurora B kinase and PP1 drive mitotic progression, which is crucial to maintain the genomic stability.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.BiochemistryProtein phosphatasesCell cyclePP1doctoral thesis10.11575/PRISM/27589