Mechanisms Controlling the Apoptosis Signal-Regulating Kinases (ASK) 1 and 2 Signalosomes Open Access

Cockrell, Lisa Montoya (2009)

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Mechanisms Controlling the
Apoptosis Signal-Regulating Kinases (ASK) 1 and 2 Signalosomes

By Lisa Montoya Cockrell
Mitogen-activated protein kinase (MAPK) cascades transmit biological signals to
elicit cellular responses. Apoptosis Signal-Regulating Kinase (ASK) 1 is a tightly
regulated MAP3K whose activation results in stress responses, including cell death. The
ASK1 signalosome represents the collection of proteins which both regulate and direct
ASK1 activation and suppression. ASK2 is a newly defined member of the ASK1
signalosome. In this dissertation, it was discovered that (i) ASK2 directs the suppression
of ASK1 through engagement of the cell regulatory protein 14-3-3; and (ii) ASK2
interaction with ASK1 is required for 14-3-3-mediated suppression. It was shown that
ASK2 directly binds to 14-3-3, through a phosphorylation-regulated interaction mapped
to serine 964 of ASK2. ASK1, ASK2, and 14-3-3 exist in a specific ternary complex
dynamically controlled by phosphorylation. A 14-3-3 binding defective mutation in
ASK2 greatly diminished the amount of ASK1 in the 14-3-3 complex. Through
dissociation of 14-3-3, ASK1 becomes activated, evidenced both by a decrease in
phosphorylation of serine 967 and an increase in downstream activation of the c-Jun N-
terminal kinase. Inhibition of the ASK1/ASK2 interaction dramatically decreased the
amount of ASK1 in the 14-3-3 complex, confirming the importance of ASK2 in directing
14-3-3-mediated ASK1 suppression. This research has identified a novel mechanism
controlling signal relay between the two MAP3Ks ASK1 and ASK2, and suggests a
model whereby upstream signaling events couple ASK2 serine 964 phosphorylation to
the ASK1 signalosome, through dual engagement of 14-3-3 proteins.

Table of Contents


Chapter 1. Introduction and Background 1
I.) Cell Fate, a Critical Balance Between Survival and Death 2
i.) Apoptosis - Traditional Programmed Cell Death 2
ii.) Novel Mechanisms of Programmed Cell Death 6
II.) ASK1, an Important Mediator of Stress-Induced Apoptosis 7
i.) Discovery 7
ii.) Evolutionary Conservation 8
iii.) Structure 9
iv.) Targets and Downstream Signaling Pathways 11
v.) Substrate Consensus Phosphorylation Sequence 12
vi.) Mechanisms of ASK1 Regulation 14
a.) Modulation of ASK1 Levels 14
b.) Phosphorylation of ASK1 21
c.) ASK1 Protein-Protein Interactions 28
1.) 14-3-3 28
2.) AIP1 35
3.) p21Cip1/WAF1 36
4.) Daxx 37
5.) Raf-1 38
d.) ASK1 Oligomerization 39
e.) ASK1 Signalosome 39
vii.) Mechanisms of ASK1 Activation 40

a.) Oxidative Stress 41
b.) Endoplasmic Reticulum Stress 42
c.) Death Receptors 43
d.) Chemotherapeutics 43
viii.) Consequences of ASK1 Activation 45
a.) Apoptosis 45
b.) Differentiation 46
c.) Innate Immunity 46
ix.) ASK1 Involvement in Disease 47
a.) Neurodegenerative Disorders 47
b.) Cardiovascular Diseases 48
c.) Infectious Disease 49
d.) Diabetes 50

III.) ASK2, a Novel Component of the ASK1 Signalosome 50

i.) Discovery 50
ii.) Gene Structure and Putative Isoforms 51
iii.) Expression 51
iv.) Functions of ASK2 52
a.) Enzymatic 52
b.) Heteromeric Complex with ASK1 56
c.) Apoptotic Activity of ASK2 57
IV.) Biology of 14-3-3 Proteins 58

i.) Description 58
ii.) Crystal Structure Analysis 60

a.) Dimerization 61
b.) Amphipathic Groove 64
c.) C-terminal Loop 66
iii.) 14-3-3 Interactions with Client Proteins 67
iv.) Regulation of 14-3-3 Proteins 70
v.) Consequences of 14-3-3 Binding 71
vi.) Inhibiting 14-3-3 Client Protein Interactions 75
V.) Scope of this Dissertation 77

Chapter 2. Materials and Methods 79

I.) General Methods 80
i.) Reagents 80
ii.) Cell Culture and DNA Transfection 81
iii.) Plasmids and Generation of Mutants 81
iv.) SDS-PAGE and Immunoblotting 87
II.) Interaction Assays 88
i.) Hexahistidine (6xHis) Pulldown 88
ii.) Glutathione S-Transferase (GST) Pulldown 89
iii.) Co-Immunoprecipitation (Co-IP) Assay 89
III.) Enzymatic Assays 90
i.) Phosphatase Assay 90
ii.) Kinase Assay 90
Chapter 3. Dual Engagement of 14-3-3 Proteins Controls Signal Relay from ASK2 to the ASK1 Signalosome 92
I.) Introduction 93

II.) Results 95
i.) ASK2 Specifically Interacts with 14-3-3 Proteins 95
ii.) ASK2 Interacts with 14-3-3 Through the Amphipathic Groove 98
iii.) ASK2 Requires Phosphorylation for 14-3-3 Binding 99
iv.) 14-3-3 Binds ASK2 Through a Novel Ser 964-Mediated Motif 102
v.) 14-3-3 is Present in a Ternary Complex with ASK1 and ASK2 108
vi.) ASK2 Interaction with 14-3-3 Dictates ASK1 Presence in the 14-3-3 Complex 108
vii.) ASK2 Interaction with 14-3-3 Controls ASK1 Function 112
III. Discussion 115

Chapter 4. Structure-Function Relationships Within the ASK1/ASK2 Heteromeric Complex 123
I.) Introduction 123

II.) Results 125
i.) ASK1 Interacts with ASK2 125
ii.) ASK1/ASK2 Interaction is Independent of 14-3-3 Binding 126
iii.) Phosphorylation Does Not Mediate ASK1/ASK2 Binding 129
iv.) The C-terminus of ASK1 is Essential for ASK2 Interaction 132
v.) The C-terminus of ASK2 is Essential for ASK1 Interaction 135
vi.) Development of an ASK2 Peptide Capable of Binding ASK1 138
vii.) Inhibition of the ASK1/ASK2 Complex Diminishes the Presence of 14-3-3 in the ASK1 Signalosome 143
III.) Discussion 144

Chapter 5. Conclusions and Future Directions 150

I.) Conclusions 151

II.) Future Directions 159

Chapter 6. References 164

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