University of Windsor Scholarship at UWindsor Electronic Theses and Dissertations Theses, Dissertations, and Major Papers 2011 Regulation of MTMR2 by Phosphorylation: Subcellular Characterization and Functional Role in Vesicular Trafficking Norah Franklin University of Windsor Follow this and additional works at: https://scholar.uwindsor.ca/etd Recommended Citation Franklin, Norah, "Regulation of MTMR2 by Phosphorylation: Subcellular Characterization and Functional Role in Vesicular Trafficking" (2011). Electronic Theses and Dissertations. 71. https://scholar.uwindsor.ca/etd/71 This online database contains the full-text of PhD dissertations and Masters’ theses of University of Windsor students from 1954 forward. These documents are made available for personal study and research purposes only, in accordance with the Canadian Copyright Act and the Creative Commons license—CC BY-NC-ND (Attribution, Non-Commercial, No Derivative Works). 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Regulation of MTMR2 by Phosphorylation: Subcellular Characterization and Functional Role in Vesicular Trafficking By Norah Franklin A Thesis Submitted to the Faculty of Graduate Studies through the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Master of Science at the University of Windsor Windsor, Ontario, Canada 2011 © 2011 Norah Franklin i Regulation of MTMR2 by Phosphorylation: Subcellular Characterization and Functional Role in Vesicular Trafficking by Norah Franklin APPROVED BY: ____________________________________________________ Dr. Andrew Hubbersty Department of Biological Sciences ____________________________________________________ Dr. Sirinart Ananvoranich Department of Chemistry and Biochemistry ________________________________________ Dr. Panayiotis Vacratsis, Advisor Department of Chemistry and Biochemistry ________________________________________ J.R. Green, Chair of Defense Department of Chemistry and Biochemistry September 8, 2011 ii DECLARATION OF CO-AUTHORSHIP / PREVIOUS PUBLICATION I. Co-Authorship Declaration I hereby declare that this thesis incorporates material that is the result of joint research, as follows: This thesis incorporates the outcome of joint research undertaken in collaboration with Dr. Gregory S. Taylor, Department of Biochemistry and Molecular Biology, University of Nebraska. The collaboration is covered in Chapter 2 of the thesis. In all cases, the key ideas, primary contributions, experimental designs, data analysis and interpretation, were performed by the authors and the contribution of co-authors was through the provision of experimental assistance, analysis and data interpretation.; Dr. Gregory S. Taylor and Dr. Panayiotis Vacratsis performed the [32P] orthophosphate labelling and the matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis for the identification of the phosphorylation site Ser58 on FLAG-MTMR2. Dr. Gregory S. Taylor provided all of the original wild-type pCNF- FLAG-MTMR2 and pET-MTMR2-His6 constructs. Dr. Panayiotis Vacratsis analyzed the MS samples for Ser58 stoichiometry. My contribution to this manuscript was preparation of the MS samples from immunoprecipitated mammalian cells that were utilized in the studies for the identification of Ser58 stoichiometry by MS, protein purification for in vitro phosphatase assays, co-immunoprecipitation experiments, localization experiments and characterization of the phenotype, as well as cell signaling analysis. I am aware of the University of Windsor Senate Policy on Authorship and I certify that I have properly acknowledged the contribution of other researchers to my iii thesis, and have obtained written permission from each of the co-author(s) to include the above material(s) in my thesis. I certify that, with the above qualification, this thesis, and the research to which it refers, is the product of my own work. II. Declaration of Previous Publication This thesis includes 1 original paper that has been previously published/submitted for publication in peer reviewed journals, as follows: Thesis Chapter Publication/full citation Publication status Chapter 2 Norah E. Franklin, Gregory S. Published Taylor, and Panayiotis O. Vacratsis (2011). Endosomal Targeting of the Phosphoinositide-3-Phosphatase MTMR2 Is Regulated by an N- terminal Phosphorylation Site. JBC 286, 15841–15853 I certify that I have obtained a written permission from the copyright owner(s) to include the above published material(s) in my thesis. I certify that the above material describes work completed during my registration as graduate student at the University of Windsor. I declare that, to the best of my knowledge, my thesis does not infringe upon anyone‟s copyright nor violate any proprietary rights and that any ideas, techniques, quotations, or any other material from the work of other people included in my thesis, published or otherwise, are fully acknowledged in accordance with the standard referencing practices. Furthermore, to the extent that I have included copyrighted material that surpasses the bounds of fair dealing within the meaning of the Canada iv Copyright Act, I certify that I have obtained a written permission from the copyright owner(s) to include such material(s) in my thesis. I declare that this is a true copy of my thesis, including any final revisions, as approved by my thesis committee and the Graduate Studies office, and that this thesis has not been submitted for a higher degree to any other University or Institution. v ABSTRACT MTMR2 is an active member of the MTM family of inositol lipid phosphatases and the physiological substrates for MTMR2 play key roles in endosomal trafficking. Additionally, loss of function mutations in MTMR2, have been associated with the peripheral neuromuscular disorder CMT4B1. In these studies we used mass spectrometry (MS) based methods to identify a novel high stoichiometry phosphorylation site at Ser58 on MTMR2. Further analysis has illustrated that a phosphorylation deficient MTMR2 (S58A) displayed constitutive localization to early endocytic structures. Moreover, we determined that the subcellular targeting of S58A was accompanied by depletion of PI(3)P and an increase in the phosphorylation of ERK1/2. Recently we have used in vitro kinase assays to determine that ERK1/2 phosphorylates Ser58. Furthermore, we have recently confirmed a C- terminal phosphorylation site at Ser631 and generated combinatorial mutants which have shown to display characteristic localization to a distinct subset of endocytic compartments as well as sustained ERK1/2 activation. vi DEDICATION À ma famille Je t’aime gros come le ciel toujours et à jamais Et Je ne pourrais jamais l'avoir fait sans vous Je dis vraiment merci tous du fond de mon coeur pour votre amour et soutien Vous êtes ma roche de Gibraltar vii ACKNOWLEDGEMENTS Some say that there are people that come into your lives and you will never be the same. I first and foremost have to extent full gratitude to my supervisor Dr. Vacratsis. He has provided me with the framework and knowledge for me to become successful not only in research but in life as well. The support throughout the years cannot be expressed in words how much it has been appreciated. I truly thank you for all that you have done for me both as a teacher and as a friend. I would like to express my appreciation towards my committee members Dr. Ananvoranich and Dr. Hubbersty for offering suggestions and words of encouragement throughout our meetings in addition to reading my thesis. Also, thanks are extended to all of the professors in the biochemistry department; Dr. Ananvoranich, Dr. Boffa, Dr. Lee, Dr. Mutus, and Dr. Pandey. Special thanks go to Dr. Mutus for letting me spend hours and days using his microscope, and Dr. Ananvoranich for providing me the use of countless times occupying the imager. I have considered it a privilege that I have been endowed use of any equipment I‟ve ever required for my research throughout the years and without this support my project would have not have become to its full potential. Several people have been instrumental in allowing this project to be near completion and of course the first thanks go out to my fellow lab members; Colleen I thank you for always providing me with encouragement in addition to the unforgettable laughs we have shared and the mischief we‟ve seemed to get ourselves into time after time, Chris for having an open ear and mind for anything I‟ve ever needed and being prepared for the „not-so-common‟ questions I always seemed to ask (and of course for all the help with last minute formatting), Besa for sharing joint excitement and frustrations viii will all that comes with research, and Anna for her guidance and wisdom. Thanks go out to Catherine and Justin for there assistance in getting this project underway. I would also like to extent thanks to Dr. Gregory S. Taylor for his joint collaborations with the MTM projects. Last but not least our undergraduate students that we have had the pleasure of working with over the past few years: Esther, Jordan,