University of Dundee DOCTOR OF PHILOSOPHY Canonical and non-canonical regulation of AMP

169 Pages·2017·3.29 MB·English

University of Dundee
DOCTOR OF PHILOSOPHY
Canonical and non-canonical regulation of AMP-activated protein kinase
Auciello, Francesca Romana
Award date:
2015
Awarding institution:
University of Dundee
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Download date: 17. Feb. 2017
Canonical and non-canonical
regulation of AMP-activated
protein kinase

Francesca Romana Auciello

A thesis presented for the degree of Doctor of Philosophy
University of Dundee
September 2015
Table of Contents
List of figures ................................................................................................................................. 6
List of tables .................................................................................................................................. 9
Abbreviations .............................................................................................................................. 10
Acknowledgements ..................................................................................................................... 13
Declaration .................................................................................................................................. 15
Summary ..................................................................................................................................... 16
Publications ................................................................................................................................. 19
Chapter 1: Introduction .............................................................................................................. 20
1.1 Protein phosphorylation ................................................................................................... 20
1.2 AMP-activated protein kinase (AMPK): discovery and historical background ................. 21
1.3 AMPK structure: the α, β and the γ subunits ................................................................... 22
1.3.1 The α subunit ............................................................................................................. 23
1.3.2 The β subunit ............................................................................................................. 24
1.3.3 The γ subunit .............................................................................................................. 25
1.4 AMPK activation by upstream kinases .............................................................................. 27
1.4.1 Phosphorylation by LKB1 ........................................................................................... 27
1.4.2 Phosphorylation by CaMKKβ ..................................................................................... 28
1.5 AMPK regulation by adenosine nucleotides: promotion of phosphorylation .................. 29
1.6 AMPK regulation by adenosine nucleotides: allosteric stimulation and protection from
dephosphorylation .................................................................................................................. 30
1.7 AMPK regulation by ADP ................................................................................................... 31
1.8 AMPK activators ................................................................................................................ 33
1.8.1 Exercise activates AMPK ............................................................................................ 33
1.8.2 Activation of AMPK by hormones .............................................................................. 34
1.8.3 Activation of AMPK by drugs and xenobiotics ........................................................... 36
1.9 Downstream events regulated by AMPK .......................................................................... 45
1.9.1 Glucose metabolism ................................................................................................... 46
1.9.2 Lipid metabolism ........................................................................................................ 50
1.9.3 Protein metabolism .................................................................................................... 51
1.9.4 Regulation of cell cycle............................................................................................... 52
1.9.5 Mitochondrial biogenesis and autophagy ................................................................. 53
1.10 AMPK and human diseases ............................................................................................. 54
1.10.1 Type 2 diabetes ........................................................................................................ 55
1.10.2 Cancer ...................................................................................................................... 56
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1.10.3 Inflammatory disease............................................................................................... 58
1.11 Experimental aims........................................................................................................... 58
Chapter 2: Materials and Methods ............................................................................................. 60
2.1 Materials ........................................................................................................................... 60
2.1.1 Chemicals ................................................................................................................... 60
2.1.2 Molecular biology reagents ....................................................................................... 61
2.1.3 Plasmids ..................................................................................................................... 61
2.1.4 Primers ....................................................................................................................... 62
2.1.5 Protein biochemistry reagents ................................................................................... 63
2.1.6 Proteins ...................................................................................................................... 64
2.1.7 Peptides ..................................................................................................................... 64
2.1.8 Antibodies .................................................................................................................. 64
2.1.9 Buffers ........................................................................................................................ 66
2.1.10 Media ....................................................................................................................... 67
2.2 Methods ............................................................................................................................ 68
2.2.1 Site-directed mutagenesis ......................................................................................... 68
2.2.2 Transformation of E.coli ............................................................................................. 68
2.2.3 Purification of plasmid DNA from E.coli ..................................................................... 69
2.2.4 DNA quantification ..................................................................................................... 70
2.2.5 DNA sequencing ......................................................................................................... 70
2.2.6 Expression of recombinant proteins in E.coli ............................................................. 70
2.2.7 Purification of GST-fusion proteins ............................................................................ 71
2.2.8 Purification of His-fusion proteins ............................................................................. 71
2.2.9 Phosphorylation of α1β1γ1 complex by CaMKKβ ..................................................... 72
2.2.10 General mammalian tissue culture .......................................................................... 73
2.2.11 Freezing and thawing cell lines ................................................................................ 73
2.2.12 HEK-293 cell line ...................................................................................................... 73
2.2.13 HeLa cell line ............................................................................................................ 74
2.2.14 G361 human melanoma cell line ............................................................................. 74
2.2.15 Generation of HEK-293 cells expressing wild-type, K29A, K31A and K29A/K31A
AMPK-α2 ............................................................................................................................. 74
2.2.16 Lysis of mammalian cells .......................................................................................... 75
2.2.17 Estimation of protein concentration........................................................................ 75
2.2.18 SDS-PAGE ................................................................................................................. 76
2.2.19 Comassie Blue staining of gels ................................................................................. 76
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2.2.20 Immunoblotting (Western blotting) ........................................................................ 77
2.2.21 Non covalent-coupling of antibodies to protein G-sepharose beads ...................... 77
2.2.22 Immunoprecipitation and assay of AMPK from cell lysates .................................... 78
2.2.23 Allosteric stimulation of AMPK immunoprecipitated from cell lysates ................... 79
2.2.24 AMPK assays using bacterially expressed AMPK heterotrimers .............................. 79
2.2.25 Protection against dephosphorylationin in-solution assays ................................... 79
2.2.26 Protection against dephosphorylation using immunoprecipitated AMPK .............. 80
2.2.27 Nucleotide measurements ....................................................................................... 80
2.2.28 Hydrogen peroxide measurement in cell medium .................................................. 81
2.2.29 Data analysis ............................................................................................................ 81
Chapter 3: AMPK is activated by oxidative stress mainly through increases in cellular AMP/ATP
ratios ........................................................................................................................................... 82
3.1 Introduction ...................................................................................................................... 82
3.1.1 Oxidative stress .......................................................................................................... 82
3.1.2 Hydrogen peroxide .................................................................................................... 86
3.1.3 AMPK and hydrogen peroxide: previous studies ....................................................... 87
3.2 Results ............................................................................................................................... 89
3.2.1 AMPK activation correlated with changes in cellular nucleotides when H O was
2 2
generated using glucose oxidase ........................................................................................ 89
3.2.2 Effect of glucose oxidase in cells expressing an AMP-insensitive mutant of AMPK .. 92
3.2.3 AMPK activation by H O is prevented by catalase ................................................... 96
2 2
3.2.4 Pre-treatment with STO609 affects AMPK activation by glucose oxidase in HeLa and
G361 cells but not in HEK-293 cells..................................................................................... 98
3.2.5 Hydrogen peroxide treatment inhibits Thr172 dephosphorylation in intact cells .. 100
3.3 Discussion ........................................................................................................................ 103
Chapter 4: A novel regulatory site on AMPK ............................................................................ 109
4.1 Introduction .................................................................................................................... 109
4.1.1 New insights into AMPK structure and regulation .................................................. 111
4.2 Results ....................................................................................................................... 114
4.2.1 Purification and phosphorylation of bacterial AMPK ....................................... 114
4.2.2 Mutation of Lys40 and Lys42 inhibits activation of bacterial AMPK by A769662
118
4.2.3 Dephosphorylation of bacterial AMPK cannot be prevented by A769662,
salicylate or AMP .............................................................................................................. 119
4.2.4 Use of unphosphorylated bacterial AMPK to study the effect of direct activators
122
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4.2.5 Mutation of Lys40 and Lys42 in α1 and Lys29 and Lys31 in α2 prevents allosteric
stimulation of AMPK by direct activators in transfected cells .......................................... 126
4.2.6 Dephosphorylation protection by direct activators is prevented by mutation of
Lys29 and Lys31 ................................................................................................................ 129
4.2.7 Basal AMPK activity is severely impaired in stable cell lines expressing a mutated
α2 subunit ......................................................................................................................... 133
4.3 Discussion ...................................................................................................................... 137
Chapter 5: Conclusions and future perspectives ...................................................................... 143
5.1 Introduction .................................................................................................................... 143
5.2 Activation of AMPK by oxidative stress .......................................................................... 143
5.3 Studies of the A769662-binding pocket .......................................................................... 145
References ................................................................................................................................ 148

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List of figures
Figure 1.1. The α,β and γ subunits of AMPK
Figure 1.2. Regulation of AMPK.
Figure 1.3. Activators of AMPK.
Figure 1.4. Downstream effects of AMPK
Figure 3.1. Measurement of H O concentration after incubation with glucose oxidase
2 2
Figure 3.2. AMPK activation by incubation with glucose oxidase
Figure 3.3 Estimated contents of ATP, ADP and AMP at various times after addition of
glucose oxidase
Figure 3.4. Activation of AMPK by glucose oxidase is greatly reduced but not
completely eliminated, in RG cells
Figure 3.5. Effect of H O on WT and RG cells
2 2
Figure 3.6. H O measurements after either H O treatment or incubation with glucose
2 2 2 2
oxidase.
Figure 3.7. Catalase reverts the effect of both H O and glucose oxidase
2 2
Figure 3.8. ADP:ATP ratio increase lasts for less than 60 minutes
Figure 3.9. AMPK activation by H O is not CaMKKβ-dependent
2 2
Figure 3.10. CaMKKβ mediates AMPK activation by H O
2 2
Figure 3.11. Protocol for assays to monitor Thr172 dephosphorylation in intact cells
Figure 3.12. H O promotes Thr172 phosphorylation by inhibiting dephosphorylation.
2 2
Figure 3.13. Effect of H O and A23187 on ADP/ATP ratio in HeLa cells.
2 2
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Figure 4.1 Crystal structure of the human α1β2γ1 AMPK
Figure 4.2 Purification of α1β1γ1 heterotrimer
Figure 4.3 Phosphorylation of α1β1γ1 by CaMKKβ
Figure 4.4 Normalization of wild type and mutated α1β1γ1 complex
Figure 4.5 A769662 dose response curve with wild type and mutated α1β1γ1 complex
Figure 4.6 Titration with PP2Cα
Figure 4.7 Dephosphorylation protection assay
Figure 4.8 Purification and normalization of unphosphorylated α1β1γ1.
Figure 4.9 K40A/K42A mutant is allosterically stimulated only by AMP.
Figure 4.10 Synergistic effect of A769662 and AMP is affected by K40A/K42A mutation.
Figure 4.11 The K40A/K42A mutation prevents activation by A769662 and salicylate in
transiently transfected cells
Figure 4.12 K29A/K31A α2 subunit does not respond to any activator
Figure 4.13 Titration of immunoprecipitated AMPK with PP2Cα
Figure 4.14 AMP does not protect the K29A/K31A mutant from dephosphorylation by
PP2Cα
Figure 4.15 AMPK activators cannot protect the K29A/K31A mutant against
dephosphorylation
Figure 4.16 Establishment of stable cell lines expressing α2-AMPK either wild type or
mutated
Figure 4.17 The basal activity of the K29A/K31A α2 mutant is lower than the wild type
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Figure 4.18 Characterization of α1/α2 knockout HEK-293 cells

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List of tables

Table 2.1. Plasmids used in this thesis
Table2.2. Primers used in this thesis
Table 2.3. AMPK mutants created as part of this thesis
Table 2.4. Peptides used in this thesis
Table 2.5. Commercial antibodies used in this thesis
Table 2.6. Non-commercial antibodies used in this thesis

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