Abstract
Cancer is characterized in large part by aberrant cell
survival. Other diseases, such as Parkinson's disease, Huntington's
disease, and other neurodegenerative disorders, are characterized
by excessive cell death. Regulation of the balance between cell
survival and cell death is critical for the proper functioning of
every cell in the body. Many proteins function together to maintain
this balance, including the apoptosis signal-regulating kinase 1
(ASK1) and its binding partners. Here we describe two distinct
mechanisms by which ASK1 function is regulated. ASK1 interacts with
ASK2 via distinct regions in the C-termini of both proteins and
disruption of this interaction leads to decreased ASK1-mediated
signaling. Additionally, ASK2 can act to inhibit ASK1 by promoting
ASK1 interaction with 14-3-3 proteins. Upon inhibitory
phosphorylation, ASK2 can facilitate ASK1/14-3-3 interaction, and
knockdown of ASK2 reduces ASK1 binding to 14-3-3.
ASK1 is also regulated by Inhibitor of κB kinase
(IKK) via a unique region of its NEMO binding domain.
Overexpression or activation of IKK reduces ASK1 mediated
signaling, and disruption of ASK1/IKK interaction, as well as
inhibition of ASK1 phosphorylation by IKK, increases ASK1-mediated
apoptosis. Taken together, these results shed light on the
intricate regulation of cell death and survival signaling mediated
by ASK1. Understanding these protein-protein interactions may serve
as a starting point for the development of ASK1-directed
therapeutics.
Table of Contents
Chapter 1:
Introduction.......................................................................................................
1
1.1. Protein-Protein
Interactions......................................................................................
2
1.2. Mitogen-Activated Protein Kinase Signaling
.......................................................... 3
1.3 ASK1 Function and Regulation
................................................................................
5
1.3.1 ASK1-mediated Apoptosis
.................................................................................
8
1.3.2 Non-apoptotic functions of
ASK1....................................................................
12
1.3.3 ASK1 Regulation by protein-protein
interactions............................................ 13
1.3.4 ASK1 Regulation by
phosphorylation..............................................................
14
1.3.5 ASK1/ASK2
interaction...................................................................................
16
1.3.6 ASK1 Regulation by Ser967 Phosphorylation and 14-3-3
binding ................. 17
1.4 Disease Relevance to
ASK1....................................................................................
18
1.4.1 ASK1 in neurodegenerative disease
.................................................................
21
1.4.2 ASK1 in cardiovascular disease, diabetes, and
inflammatory diseases ........... 23
1.4.3 ASK1 in
cancer.................................................................................................
24
1.5 Small molecule inhibitors of ASK1
........................................................................
25
1.6 IKK Signaling
.........................................................................................................
26
1.6.1 NF-B
Signaling...............................................................................................
26
1.6.2 Regulation of IKK by NEMO
..........................................................................
32
1.6.3 IKKε/TBK1
......................................................................................................
33
1.6.4 IKK Signaling outside NF-κB and Disease Relevance
.................................... 34
1.7 Crosstalk between ASK1 and IKK pathways
......................................................... 37
1.8 Scope of Dissertation
..............................................................................................
38
Chapter 2: ASK2 promotes ASK1 inhibition or activation through
specific protein-protein
interactions............................................................................................................
40
Chapter 2.1: Dual engagement of 14-3-3 proteins controls
signal relay from ASK2 to the ASK1 signalosome.
.................................................................................................
41
Abstract......................................................................................................................
42
Introduction
...............................................................................................................
43
Methods
.....................................................................................................................
45
Results
.......................................................................................................................
47
ASK2 specifically interacts with 14-3-3
proteins................................................. 47
ASK2 requires phosphorylation for 14-3-3 binding
............................................. 50
14-3-3 binds ASK2 through a novel S964-mediated motif
.................................. 51
14-3-3 is present in a ternary complex with ASK1 and
ASK2............................. 59
ASK2/14-3-3 interaction dictates ASK1/14-3-3
interaction................................. 63
Discussion..................................................................................................................
70
Chapter 2.2: Distinct Regions within ASK1 and ASK2 facilitate
kinase interaction... 72
Abstract......................................................................................................................
73
Introduction
...............................................................................................................
74
Materials and Methods
..............................................................................................
75
Results
.......................................................................................................................
77
ASK1 and ASK2 interact via distinct regions within their
C-termini .................. 77
Expression of an ASK2 peptide reduces ASK1
signaling.................................... 81
Discussion
.....................................................................................................................
86
Chapter 3: IKKβ inhibits ASK1 activity through
phosphorylation and protein-protein
interactions.......................................................................................................................
88
Chapter 3.1: Integration of the Apoptosis signal-regulating
kinase 1-mediated stress signaling with the Akt/PKB-IκB kinase
cascade. .........................................................
89
Abstract......................................................................................................................
90
Introduction
...............................................................................................................
91
Materials and Methods
..............................................................................................
92
Results
.......................................................................................................................
95
Diverse growth factor-initiated pathways impinge on ASK1 at
Ser967............... 95
Akt/PKB is an upstream kinase for Ser967 of
ASK1........................................... 98
IKK mediates growth factor and Akt-induced phosphorylation of
ASK1 ......... 102
IKKβ directly interacts with ASK1
....................................................................
105
The IGF-1/Akt/IKK signaling antagonizes H2O2 -induced ASK1
activation.... 106
IKK inhibits ASK1-mediated apoptosis in a Ser967 dependent
manner............ 106
Discussion................................................................................................................
109
Chapter 3.2: The NEMO binding domain of IKK mediates ASK1/IKK
interaction without disruption of IKK/NEMO
binding.................................................................
113
Abstract....................................................................................................................
114
Introduction
.............................................................................................................
115
Methods
...................................................................................................................
116
Results
.....................................................................................................................
119
Interaction does not require Ser967 phosphorylation or kinase
activation......... 119
IKKβ interacts with the C-term of ASK1
...........................................................
119
ASK1 interacts with the kinase and NEMO binding domains of
IKKβ ............. 122
NEMO and ASK1 form a complex with IKK
.................................................... 127
Disruption of NEMO binding site does not alter ASK1
interaction................... 127
IKK suppresses ASK1-mediated neurite
outgrowth........................................... 130
Inhibition of interaction promotes apoptosis
...................................................... 133
Discussion................................................................................................................
133
Chapter 4: Discussion
.....................................................................................................
136
4.1 General properties of ASK1 interaction with ASK2 and
IKK.......................... 137
4.2 IKK regulation of ASK1 could explain JNK/NF-κB
crosstalk ......................... 137
4.3 Therapeutic significance of ASK1 protein-protein
interactions........................ 138
4.4 Some NBD peptide functions may be due to disruption of
ASK1/IKK interaction
................................................................................................................................
140
4.5 Conclusions and Future
Directions....................................................................
141
Chapter 5: References
.....................................................................................................
146
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