rheb dynamics on lysosomal membranes determines mtorc1 activity after loss of p53 or activation ...

252 Pages·2016·16.14 MB·English

Virginia Commonwealth University
VCU Scholars Compass
Theses and Dissertations Graduate School
2015
RHEB DYNAMICS ON LYSOSOMAL
MEMBRANES DETERMINES MTORC1
ACTIVITY AFTER LOSS OF P53 OR
ACTIVATION OF AMPK
Catherine M. Bell
Virginia Commonwealth University, [email protected]
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Copyright 2015
Catherine Macdonald Bell
ALL RIGHTS RESERVED
RHEB DYNAMICS ON LYSOSOMAL MEMBRANES DETERMINES
MTORC1 ACTIVITY AFTER LOSS OF P53 OR ACTIVATION OF AMPK

A dissertation submitted in partial fulfillment of the requirements for the degree
of Doctor of Philosophy at Virginia Commonwealth University

by
CATHERINE MACDONALD BELL

Virginia Commonwealth University
Richmond, Virginia
November 2015
ACKNOWLEDGEMENTS

“Motho ke motho ka batho babang (a person is a person because of other people)” - Jeremy Cronin
It is with the deepest gratitude that I thank all those who have supported and guided me throughout my graduate
education and dissertation research. To my graduate mentor, Professor Richard G. Moran, I cannot thank you
enough for your guidance throughout this process. Your voice was with me in the interviews for graduate school
admission, during my move from South Africa, and remained a constant source of wisdom and validation
throughout my degree. Thank you for challenging me, for forcing me outside my comfort zone, for teaching me
how to think and write critically about my data and others, and above all, for teaching me to be bold and have true
grit. This has been an incredible, life-changing experience.
To my committee, thank you for your judicious and reassuring counsel. Dr Shirley Taylor, I will always remember
your creative and ingenious approach to research, and your undeniable kindness and stoicism. Dr Darlene Brunzell,
you are an incredible inspiration for my career and for my life. Dr Kurt Hauser, thank you for our wonderful
discussions and your insight into being a successful academic. Dr Daniel Conrad, your Immunobiology lectures are
my reason for staying the course through my first year of graduate school. I am also indebted to the kindness and
backing of Dr Steven Sawyer, Dr Hamid Akabarali, and Dr William Dewey. It has been an honor to be part of your
department. To Dr Henderson and Frances White, thank you for your invaluable training on the confocal
microscope.
Thank you to my colleagues in the lab. Importantly, Dr Stuti Agawral for our time spent slaying the same science
dragons. Dr Chen Yang, thank you for being a dear and wise friend. And to my lab family over the years - Dr Lisa
Shock, Dr Timothy Lochmann, Dr Alexandra Racanelli, Dr Cortney Lawrence, Dr Scott Lawrence, Chuck Lyons, Elliot
Burton, Audrey Thacker, Jason Robinson – thank you for making me laugh, feeding me wine and doughnuts,
teaching me countless techniques, and for occasionally doing my lab duties without complaint! Finally, thank you
to Dr John Hackett and his wife Rajani, for your friendship and support of both Gareth and I.
To my wonderful Richmond family, I love you so much. Thank you for your friendship and kindness. I will be
heartbroken to leave you: Dan, Kim T., Satti, Heidi, Joyce, Manuel, Matt, Katya, Jacy, Devin, Sean, Tamara, Tomas,
Maria, John, Kim S., and my life coach, Jo-Lynne. To Mr and Mrs Dillehay, thank you for your generosity of spirit
and for providing us with a beautiful home (and wedding venue). You will always be so special to us. To my South
African besties, here is to continued adventures all over the world, with wine and sushi in hand! Thank you for
everything: Sheena, Gill, Bridgette, Marta, Alexia, Nicolette, Natasha. To the Bongers family, Helen, Eldie, Sam, and
Anton, thank you for helping me get to back on my feet and to the USA. To my crazy, beautiful family, thank you
for molding me into the person I am today. I love you mum and dad, I am so blessed to come from a home so filled
with love and laughter and character-building drama. Also, Alex, Josie, Meryn, Jeremy, Bruce, Billy, Sarah,
Llewellyn, Ewan, Steph, Harrison, Charlotte, Harry, Olivia, Chloe, Molly, and Max. To my parents-in-law, Silver
Snake and grazing Sheep thank you accepting me into your family. I love you so much.
Finally, Gareth, my husband, best friend, partner-in-crime, and international man of mystery, you are my twin soul.
Thank you for the love, support, and kindness. This has been the start of a grand adventure! Ek herhaal jou, sonder
begin of einde.

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List of figures
Page
1.1 Overview of mTORC1 signaling 4
1.2 Translational control by mTORC1 11
1.3 mTORC1 control by Sestrins, Gators, and Rags 18
1.4 AMPK is a heterotrimeric enzyme complex containing a catalytic α-subunit (α1 and α2),
and regulatory β- (β1 and β2) and γ-subunits (γ1, γ2, and γ3) 21
1.5 Overview of AMPK signaling 24
2.1 The endomembrane system of the mammalian cell 34
2.2 GAPS, GEFs, and GDIs regulate small GTPase proteins 37
2.3 Ras core structure and C-terminal lipidation motifs of posttranslational completely
processed N-Ras, H-Ras, K-Ras4A, and K-Ras4B 43
2.4 Crosstalk between p53, AMPK, and mTORC1 53
2.5 Representative chromatogram of a standard aqueous mixture of GDP and GTP 59
2.6 Detection and quantitation limits of the HPLC protocol for quantitation of Rheb
guanine nucleotides 60
2.7 Setting the thresholds for colocalization analysis after confocal microscopy 65
2.8 Single color antibody controls for immunofluorescence 66
2.9 Estimation of the efficiency of endogenous Rheb Immunoprecipitation 71
2.10 Analysis of whole cell nucleotide, and Rheb-GDP/GTP levels by HPLC 73
2.11 Scintillation counting of HPLC fractions collected every minute after sample injection 76
2.12 Scintillation counting for quantitation of tritium in Rheb IP 77
2.13 GDP and GTP can be resolved by TLC 79
2.14 FLAG-Rheb overexpressing HCT116s have a higher degree of 32P-phosphate labeling
in pulldowns than untransfected cells 80
2.15 Standard curves for GTP and GDP for quantitation by coupled enzyme assays 81
2.16 TSC2 null and p53 null cells have significantly more Rheb-GTP 83
2.17 TSC2 GAP assay shows no difference in GTP converted to GDP in p53+/+ and p53-/- cells 84
2.18 Total membrane fractionation showed less TSC2 and more Rheb in membrane fractions 87
2.19 Heavy and light membrane fractionation show the loss of TSC2 from heavy
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membranes after insulin stimulation 89
2.20 TSC2 is lost and Rheb increases in the heavy membrane fraction of p53 wild type
and null cells 90
2.21 TSC2 null MEFs have a marked increase in heavy membrane Rheb levels 92
2.22 Loss of p53 decreases the distribution of TSC2 to lysosomal membranes 94
2.23 Loss of p53 decreases the distribution of TSC2 to and increases the level of
Rheb in lysosomal membranes 95
2.24 Silencing of p53 augments mTORC1 signaling 97
2.25 siRNA knockdown of p53 in H460 and A549 cells recapitulates the changes
in lysosomal TSC2 and Rheb seen in HCT116 cells 98
2.26 Forced expression of TSC2 and/or Sestrin2 decreases the elevated mTORC1
activity in p53 null cells down to levels seen in p53 wt cells 99
2.27 Transfection efficiencies for HA-TSC2 and FLAG-SESN2 101
2.28 Transfection of HA-TSC2 into HCT116 cells null for p53 restores lysosomal
TSC2 and diminishes Rheb at lysosomal membranes 102
2.29 Transfection of Flag-Sestrin2 into HCT116 cells null for p53 does not change
TSC2 localization with lysosome 103
2.30 Transfection of Flag-Sestrin2 into HCT116 cells null for p53 decreases the
partitioning of mTOR to the lysosome 105
2.31 Proposed mechanism of p53 involvement in the control of mTORC1
lysosomal dynamics 107
3.1 Structures of the three different classes of AMPK activating compounds 115
3.2 Overview of folate metabolism 119
3.3 Chemical structures of oxidized and reduced folates 120
3.4 Summary of AMPK subcellular localization 128
3.5 PTX-activated AMPK does not activate TSC2 132
3.6 PTX does not activate TSC2 because it causes a deficiency in p53 transactivation 134
3.7 The PTX+TdR induced loss of TSC2 is reflected in lysosomal fractions 136
3.8 PTX+TdR decreases the distribution of TSC2 to lysosomal membranes 137
3.9 AMPK activation by AICAR increases the distribution of TSC2 to lysosomal
membranes, and the increases total TSC2 139
3.10 Both AICAR and PTX+TdR cause a reduction of Rheb in heavy membrane fractions 141
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3.11 Quantitation of Rheb in heavy membrane fractions after AMPK activation
shows a significant decrease 142
3.12 Both AICAR and PTX+TdR cause a reduction in the occupancy of Rheb at lysosomes 144
3.13 AICAR and PTX+TdR cause separate effects on lysosomal mTOR 146
3.14 Phosphorylation of Raptor is necessary and sufficient for inhibition of mTORC1
by PTX-activated AMPK 147
3.15 AMPK activation by PTX results in a slight increase in lysosomal Raptor 149
3.16 AMPK activation by AICAR results in no significant change to lysosomal Raptor 150
3.17 AMPK activation by AICAR or PTX+TdR does not increase AMPK in heavy membranes 152
3.18 AMPK activation decreases lysosomal levels of AMPK 153
3.19 AMPK translocates to the nucleus after activation by ZMP 154
3.20 Proposed model for orientation of Rheb-GDP and Rheb-GTP in lipid-bilayer
nanodiscs based on NMR measurements 159
3.21 Model for the subcellular dynamics of AMPK and mTORC1 components after
PTX (+TdR) or AICAR treatment 164
4.1 Chemical structures of the four classical antifolates compared to folic acid 166
4.2 Cellular targets of antifolate drugs 169
4.3 AMPK is activated by all four antifolates, MTX, RTX, LTX, and PTX over the no
drug control 178
4.4 AMPK is activated to varying degrees over 48 h by the four antifolates 179
4.5 AMPK activation over 48 h 180
4.6 AMPK activation by the direct activator 182
4.7 mTORC1 signaling is blunted by all four antifolates 184
4.8 p53 is stabilized and accumulates with all four antifolates, but p53 driven
transcription is blocked by LTX and PTX 186

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List of tables
5.1 Summary of results in Chapter 2, effects of p53 loss on mTORC1 lysosomal dynamics 195
5.2 Summary of results in Chapter 3, effects of AMPK activation by AICAR and Pemetrexed
on mTORC1 lysosomal dynamics, and AMPK localization 195
5.3 Summary of results in Chapter 4, effects of classical antifolate drug treatment on AMPK,
mTORC1, and p53 in carcinoma cells 196

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Abbreviations:

2-DG 2-Deoxyglucose
4EBP1 4E (eIF4E)-binding protein 1
5-FU 5-Fluorouracil
ACC Acetyl CoA carboxylase
AICAR 5’-Aminoimidazole-4-carboxamide riboside
AICART Aminoimidazolecarboxamide ribonucleotide formyltranferase
ALL Acute lymphoblastic leukemia
AMPK 5’-AMP-activated protein kinase
AMPKK AMPK kinase
ATM Ataxia telangiectasia mutated
BCRP Breast cancer-resistance protein
BNIP3s Bcl-2 homology 3 domain-containing proteins
C2 5-(5-hydroxyl-isoxazol-3-yl)-furan-2-phospahte
CAAX C = cysteine, A = aliphatic amino acid, X = any amino acid
CAMKKβ Ca2+/calmodulin-dependent kinase kinase β
CBM Carbohydrate-binding module
DDATHF 6R-5,10-dideazatatrahydrofolate
DEP Dishevelled, egl-10, pleckstrin
Deptor DEP domain containing mTOR interacting protein
dFBS Dialyzed fetal bovine serum
DHF Dihydrofolate
DHFR Dihydrofolate reductase
DR5 Death receptor 5
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dTMP Deoxythymidine monophosphate
DTT Dithiothreitol
dUMP Deoxyuridine monophosphate
EGF Epithelial growth factor
EGFR Epithelial growth factor receptor
eIF Eukaryotic translation initiation factor
eIF4E/F/G Eukaryotic translation initiation factor 4E/F/G
ER Endoplasmic reticulum
ERK1/2 Extracellular-signal-regulated kinase 1/2
FKBP12 12 kDa FK506-binding protein
FPGS Folypoly-γ-glutamate synthetase
FR Folate receptor
FRAP Fluorescence Recovery after Photobleaching
FRET-FLIM Fluorescence Resonance Energy Transfer and Fluorescence
Lifetime Imaging
GAP GTPase activating protein
GART Glycinamide ribonucleotide transferase
GDI Guanine nucleotide dissociation inhibitors
GEF Guanine nucleotide exchange factor
GSK3β Glycogen synthase kinase 3β
HEAT Huntington, EF3, PP2A and TOR
HMG-CoA 3-Hydroxy-3-methyl-glutaryl-CoA reductase
HVR Hypervariable domain
Icmt Isopenylcysteine carboxyl methyltransferase
IF immunofluorescence
IGF1 Insulin-like growth factor 1
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