The Role of Leucine Carboxyl Methyltransferase 1 in Cell Growth and Mouse Development Público

Abstract

Protein Phosphatase 2A (PP2A), a multifunctional Serine/Threonine phosphatase, has
been implicated in the regulation of cell growth and proliferation, primarily at the G2/M
transition, and in the development of human cancers. PP2A is methylated on its catalytic subunit C-
terminal leucine alpha-carboxy group by Leucine Carboxyl Methyltransferase-1 (LCMT-
1) and is demethylated by Protein Methylesterase-1 (PME-1). Reversible methylation is
the most specific cellular mechanism for regulating PP2A, and thus may have promise
as a mechanism-based therapeutic target. In this study we have analyzed the
importance of LCMT-1 for PP2A methylation and function in cell cycle regulation and
mouse development. We have found that downregulation of LCMT-1 in cultured cells,
results in cell cycle defects specifically during mitosis, including mitotic arrest and
apoptosis. Moreover, we have found that gene-trap knockout of lcmt-1 in
mice results in embryonic lethality due to severe defects in the developing liver and
brain. Loss or downregulation of LCMT-1 results in up to a 60% reduction in PP2A
methylation in the developing embryo as well as cultured cells, demonstrating that
LCMT-1 is the sole mammalian PP2A methyltransferase. We have observed a dramatic
reduction of PP2A heterotrimers containing the B family regulatory B-type subunit,
the subunit that is the most affected by methylation. Lastly, we have observed a
dramatic increase in the phosphorylation of microtubule-associated protein tau, a
hallmark of Alzheimer's disease, in the developing embryo brain at known PP2A-
regulated residues. Taken together, our results indicate that LCMT-1 is a key regulator
of growth and development and is important for cell proliferation and differentiation.

The Role of Leucine Carboxyl Methyltransferase 1 in Cell Growth and Mouse
Development


By
Jocelyn Anne Lee
M.S. Biology, Virginia Commonwealth University, 2004
B.A. Biology, University of Virginia, 2000
Advisor: David C. Pallas, Ph.D.





A dissertation submitted to the Faculty of the
James T. Laney School of Graduate Studies of Emory University
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in
Graduate Division of Biological and Biomedical Science
Biochemistry, Cell, and Developmental Biology
2011

Table of Contents

Table of Contents










Page
Introduction .................................................................................................................... 1
LCMT-1 and PP2A Methylation ...................................................................................... 1
Interactions of LCMT-1 with Ligands and Other Proteins ............................................. 4
Regulation of LCMT-1 Activity ....................................................................................... 5
Major Sites of LCMT-1 Expression ................................................................................. 7
Phenotypes of loss of LCMT-1 ........................................................................................8
PP2A and the Cell Cycle ..................................................................................................8
PP2A and Mitotic Entry ................................................................................................ 10
PP2A and the Metaphase-Anaphase Transition ............................................................ 11
PP2A and Mitotic Exit .................................................................................................. 12
LCMT-1, PP2A, and Apoptosis ..................................................................................... 12
Liver Development and Hematopoiesis ....................................................................... 16
PP2A, Tau, and Alzheimer's Disease ........................................................................... 20
Introduction Literature Cited ....................................................................................... 23


Chapter 1: Leucine Carboxyl Methyltransferase 1 is Necessary for Normal Progression
Through Mitosis in Mammalian Cells ......................................................................... 32
Introduction .................................................................................................................. 32
Methods and Materials ................................................................................................. 35
Results .......................................................................................................................... 40
Discussion ..................................................................................................................... 47
Figure Legends .............................................................................................................. 54
Figures .......................................................................................................................... 63
Chapter 1 Literature Cited ............................................................................................ 71

Chapter 2: Loss of Leucine Carboxyl Methyltransferase 1 Results in Embryonic Lethality
and Fetal Liver Apoptosis ........................................................................................... 74
Introduction .................................................................................................................. 74
Methods and Materials ................................................................................................. 77
Results ........................................................................................................................... 81
Discussion .................................................................................................................... 89
Figure Legends .............................................................................................................. 95
Figures ........................................................................................................................ 102
Chapter 2 Literature Cited .......................................................................................... 110


Chapter 3: Leucine Carboxyl Methyltransferase 1 Regulates Tau Phosphorylation in the
Developing Mouse Embryo ........................................................................................ 113
Introduction ................................................................................................................. 113
Methods and Materials ............................................................................................... 116
Results ......................................................................................................................... 118
Discussion ................................................................................................................... 120
Figure Legends ............................................................................................................ 122
Figures ........................................................................................................................ 124
Chapter 3 Literature Cited .......................................................................................... 125

Discussion of Study ..................................................................................................... 129
Literature Cited ........................................................................................................... 137

About this Dissertation

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• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Palavra-chave	LCMT-1
Committee Chair / Thesis Advisor	Pallas, David C, Emory University
Committee Members	Moberg, Kenneth H, Emory University Vertino, Paula M, Emory University Cheng, Xiaodong, Emory University Moreno, Carlos S, Emory University

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