Protein-Protein Interactions Regulating ASK1 Function Public

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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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Pharmacology
Mot-clé	apoptosis ASK1 IKK protein-protein interaction
Committee Chair / Thesis Advisor	Fu, Haian, Emory University
Committee Members	Hepler, John R, Emory University Nahta, Rita, Emory University Khuri, Fadlo, Emory University Griendling, Kathy, Emory University

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