Nucleotide regulation of AMP-activated protein kinase

294 Pages·2013·5.66 MB·English

by  Lizzi Underwood

Nucleotide regulation
of AMP-activated
protein kinase

Elizabeth Ann Underwood

A thesis submitted for the degree of Doctor of Philosophy
October 2012

Division of Molecular Structure,
MRC National Institute for Medical Research, London

Division of Biosciences,
UCL, London

1
I, Elizabeth Ann Underwood, confirm that the work presented in this thesis is my
own. Where information has been derived from other sources, I confirm that this
has been indicated in the thesis.

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Abstract
AMP-activated protein kinase (AMPK) acts as the cell’s master energy regulator, sensing and
maintaining the concentration of ATP in a narrow range irrespective of energy demand.
This kinase has received significant attention as a drug-target for type-2 diabetes, obesity
and cancer. Although historically the AMP:ATP ratio has been considered the signal for
AMPK activation, we have recently demonstrated that ADP is likely to be an important
physiological regulator of AMPK in both mammals and yeast.

The binding of adenine nucleotides and staurosporine to the full-length 111
heterotrimer, both phosphorylated and unphosphorylated, is described. Binding was
monitored through displacement of fluorescently labelled nucleotides (coumarin-AXP),
either via direct coumarin excitation or Forster Resonance Energy Transfer (FRET) in which
tryptophan residues were excited. Mg.ATP was found to bind more weakly than ADP, a
feature which is likely key to AMPK regulation.

A -Nicotinamide adenine dinucleotide (NADH) coupled
spectrophotometric assay was used to monitor AMPK kinetics and
its regulation by nucleotides. NADH binds at Site-1, within the
-subunit (pictured right), and competes with allosteric activation
by AMP, but not the protective effect of AMP/ADP against T172
dephosphorylation. Therefore it seems that AMP binding at Site-1
mediates allostery whilst AMP/ADP binding at Site-3 affords
protection against dephosphorylation. In order to explore this idea
further, AXP binding constants were used to model binding site occupancies over the
concentration ranges used in vitro. The modelling demonstrates that, in vitro, Site-1 is
occupied by AMP and Site-3 by AMP/ADP in a manner consistent with their assigned
regulatory functions. This modelling study was also extended to consider in vivo binding site
occupancy.

It was important to verify that coumarin-ADP bound in a homologous fashion to ADP,
specifically in the same exchangeable binding pockets. X-ray crystallography was used to
determine the structure of a truncated form of AMPK in complex with coumarin-ADP.
This structure is compared to an ADP-bound form.

SNF1 is the Saccharomyces cerevisiae AMPK ortholog. The binding of nucleotides to SNF1,
and its regulation by ADP, was also characterised. As with the mammalian enzyme AXP
bound at two exchangeable sites, and interacted with Mg.ATP more weakly than ADP.
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Acknowledgments

First, I must thank Steve Martin without whom this thesis, and the work it contains, would
literally not have been possible. His support, guidance and friendship over the past three
years has been invaluable. I could not have wished for a better mentor.

I must also thank Steve Gamblin for giving me the opportunity to work in his lab, for his help
and direction, and for giving me so much freedom during my time at the NIMR.

I extend this thanks to the rest of the Gamblin empire lab, past and present. Especially Bing
Xiao, Matt Sanders and Richard Heath with whom I have worked, in parallel, on AMPK. They
have offered me a lot of practical advice, listened to my chattering, but perhaps most
importantly, have become good friends.

To the members of Molecular Structure and Physical Biochemistry, the cohort of students
that I started with (Dan, Darren, Leonard, Sorrel, Christina), those which have come and
gone (Alice, Jo, Mateo, Laura, James, Aylin, Gem, Kat, Lucy), the people who periodically ask
“so, how goes the science?” (Sarah, Sarah, Ross, Cath, Avneet, Julz, Kelly, Kate, Caroline, Jen)
and anyone else that I temporarily forget; thank-you for being such good friends to me.

To my parents (Mike and Di) and brothers (Charles and Matt) I hope that you know how
much your favourite daughter/sister loves you all, and how grateful I am for your support
over the past 25 years (or at least the bits my younger brothers were around for).

Finally, thanks to those who were brave enough to proof-read my thesis (Smartin, Gamblin,
Dave Carling and Matt), and to those who are just about to venture in. I owe you all a pint.

Cheers,
Lizzi (aka Bob)
October 2012

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Publications

Work described in this thesis has been presented in the following publications:

Xiao, B.*, Sanders, M. J.*, Underwood, E.*, Heath, R.*, Mayer, F. V.*, Carmena, D., Jing, C.,
Walker, P. A., Eccleston, J. F., Haire, L. F., Saiu, P., Howell, S. A., Aasland, R., Martin, S. R.,
Carling, D., and Gamblin, S. J. (2011) Structure of mammalian AMPK and its regulation by
ADP, Nature 472, 230-233.
(1)

Mayer, F. V.*, Heath, R.*, Underwood, E.*, Sanders, M. J.*, Carmena, D., McCartney, R. R.,
Leiper, F. C., Xiao, B., Jing, C., Walker, P. A., Haire, L. F., Ogrodowicz, R., Martin, S. R.,
Schmidt, M. C., Gamblin, S. J., and Carling, D. (2011) ADP regulates SNF1, the
Saccharomyces cerevisiae homolog of AMP-activated protein kinase, Cell Metabolism
14, 707-714.
(2)

* These authors contributed equally to the work.
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Abbreviations

ACC Acetyl-CoA Carboxylase
AD Alzheimer's Disease
AICAR Aminoimidazole-4-carboxamide ribotide
AID Autoinhibitory domain
ADP Adenosine Diphosphate
AID Autoinhibitory Domain
Aq Aqueous
AgRP Agouti-Related Peptide
AMP Adenosine Monophosphate
AMPK Adenosine Monophosphate-activated Protein Kinase
ATP Adenosine Triphosphate
AU Absorbance Unit
CaMKK Calmodulin-dependent protein Kinase Kinase
CPT1 Carnitine palmitoyltransferase 1
CD Circular Dichroism
CCD Charge-Coupled Device
C-ADP (7-diethylaminocoumarin-3-carbonylamino)-3’-deoxyadenosine 5’diphosphate
C-ATP (7-diethylaminocoumarin-3-carbonylamino)-3’-deoxyadenosine 5’triphosphate
CBS Cystathionine--synthase
CIDEA Cell-death Inducing DFFA-like Effector A
CLC2 Chloride Channel 2
CNTF Ciliary Neurotrophic Factor
COX2 Cyclooxygenase-2
CPT1 Carnitine Palmitoyltransferase 1
Cr Creatine
CREB cAMP Response Element-Binding protein
CRTC-1 cAMP-Regulated Transcriptional Co-activator-1
DLS Dynamic Light Scattering
DNA Deoxyribonucleic acid
DNP 2,4-dinitrophenol
DR Dietary Restriction
DUB Deubiquitinating enzymes
EDTA Ethylenediaminetetraacetic acid
EF2 Elongation Factor 2
EM Electron Microscopy
ER Endoplasmic Reticulum
FABP Fatty Acid Binding Protein
FAS Fatty Acid Synthase
FAT Fatty Acid Translocase
FRET Förster Resonance Energy Transfer
GAP Guanosine triphosphate Activating Protein
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GBD Glycogen Binding Domain
GLUT Glucose Transporter
GP Glycogen Phosphorylase
GPAT Glycerol-3-phosphate acyl-transferase
GS Glycogen Synthase
GSK-3 Glycogen Synthase Kinase 3
GST Glutathione S-transferase
HDAC5 Histone Deacetylase 5
HNF-4 Hepatic Nuclear Factor 4
HPLC High Performance Liquid Chromatography
HSL Hormone-Sensitive Lipase
HMGR 3-hydroxy-3-methyl-glutaryl-CoA reductase
IC Inhibitory Concentration
IGF-1 Insulin-like Growth Factor-1
IMAC Immobilised Metal ion Affinity Chromatography
IMPDH IMP-dehydrogenase
IRS-1 Insulin Receptor Substrate-1
IPTG Isopropyl β-D-1-thiogalactopyranoside
LB Luria-Bertoni
LDH Lactate Dehydrogenase
LKB1 Liver kinase B1
MCD Malonyl-CoA Decarboxylase
MALLS Multiangle Laser Light Scattering
MEF Myocyte Enhancer Factor
MES 2-(N-morpholino)ethanesulfonic acid
MO25 Mouse protein 25
MPD 2-methyl-2,4-pentanediol
MRW Mean Residue Weight
mTOR Mammalian Target of Rapamycin
MW Molecular Weight
NADH Nicotinamide Adenine Dinucleotide
NADPH Nicotinamide Adenine Dinucleotide Phosphate
nd not determined
NE Non-Exchangeable
NF Nuclear Factor kappa-light-chain-enhancer of activated B cells
NMR Nuclear Magnetic Resonance
NPY Neuropeptide Y
NRF Nuclear Respiratory Factors
OD Optical Density
PCr Phosphocreatine
PDB Protein Data Bank
PEG Polyethylene Glycol
PEP Phosphoenol Pyruvate
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PFK2 Phosphofructose kinase-2
PGC-1 Proliferator-activated receptor gamma coactivator-1 
PK Pyruvate Kinase
PKA Protein Kinase A
POMC Proopiomelanocortin
PP2C Protein Phosphatase 2C
RBS Ribosome Binding Site
ROS Reactive Oxygen Species
SAK1 SNF1 Activating Kinase-1
SD Standard Deviation
SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
SEC Size-Exclusion Chromatography
SHREBP1c Sterol Regulatory Element Binding Protein 1c
SNF1 Sucrose-NonFermenting 1
SNP Single Nucleotide Polymorphism
STRE Stress Response Element
TCEP Tris(2-carboxyethyl)phosphine
TLS Translation, Libration and Screw
TSC1/TSC2 Tuberous Sclerosis 1/2 Complex
ULK1 Unc-51-Like Kinase
UV Ultraviolet
WPWS Wolff-Parkinson-White Syndrome
ZMP AICAR 5’-monophosphate

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Table of Contents

Declaration 2
Abstract 3
Acknowledgments 4
Publications 5
Abbreviations 6
Table of Contents 9
List of Figures 13
List of Tables 15

1 Introduction 16
1.1 AMPK
1.2 AMPK activity 20
1.2.1 Carbohydrate Metabolism
1.2.2 Lipid Metabolism 23
1.2.3 Protein Synthesis 25
1.2.4 Cell Growth and Apoptosis 27
1.3 AMPK in Health and Disease 29
1.3.1 Obesity
1.3.2 Type-2 Diabetes 31
1.3.3 Cancer 32
1.3.4 Cardiac Function and Wolff-Parkinson-White Syndrome (WPWS) 34
1.3.5 Alzheimer’s Disease (AD) 37
1.3.6 Ageing 38
1.3.7 Concluding Remark 40
1.4 The  Heterotrimer 41
1.4.1 Overall Architecture
1.4.2 The  subunit 43
1.4.3 The  subunit 46
1.4.4 The  subunit 47
1.5 Regulation of AMPK 54
1.5.1 Post-translational Modifications
1.5.1.1 T172 phosphorylation by upstream kinase
1.5.1.2 Dephosphorylation by phosphatases 56
1.5.1.3 Other phosphorylation sites 57
1.5.1.4 Myristoylation 58
1.5.1.5 Ubiquitination and Acetylation 59
1.5.2 Direct Regulators 60
1.5.2.1 Nucleotide Regulation
1.5.2.2 Glycogen Regulation 61
1.5.2.3 Pharmacological agents 64
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1.5.3 Indirect Regulators 66
1.5.3.1 Natural Compounds
1.5.3.2 Metformin and thiazolidinediones
1.5.3.3 Exercise 67
1.5.3.4 Endocrine Regulation of AMPK 69
1.5.4 Concluding Remark 71
1.6 Aims

2 Methods 72
2.1 Molecular Biology
2.1.1 DNA constructs
2.1.2 Bacterial Strains 73
2.1.3 Agarose gel electrophoresis 74
2.1.4 DNA sequencing
2.1.5 Transformation 75
2.2 Protein Biochemistry
2.2.1 Protein Expression
2.2.2 Cell Lysis 76
2.2.3 Protein Purification
2.2.3.1 Nickel affinity purification
2.2.3.2 Ion exchange chromatography 77
2.2.3.3 Size exclusion chromatography
2.2.4 In vitro phosphorylation 78
2.2.5 His-tag cleavage
2.2.6 Sodium Dodecyl sulphate-Polyacrylamide Gel Electrophoresis
2.2.7 Determination of protein concentration 79
2.2.8 Dynamic Light Scattering

3 Nucleotide binding to AMPK 80
3.1 Abstract
3.2 Introduction 81
3.2.1 Previous reports of nucleotide binding to AMPK
3.2.2 Fluorescence 83
3.2.3 Fluorescent nucleotide derivatives 85
3.2.4 Förster Resonance Energy Transfer (FRET) 88
3.3 Methods 89
3.3.1 Binding Experiments
3.3.1.1 1:1 Interactions
3.3.1.2 2:1 Interactions 90
3.3.1.3 Competition/Displacement 92
3.3.1.4 General Methods 93
3.3.1.5 Staurosporine binding titrations
3.3.1.6 NADH binding titrations 94
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