Aurora Kinase A Promotes Oncogenic Signaling Through Novel Protein-Protein Interactions Open Access
Umstead, MaKendra (2016)
Abstract
Cancer is a collection of diseases driven by genomic changes that alter normal protein-protein interactions, induce aberrant cellular signaling, and drive cellular transformation. Aurora Kinase A (Aurora A), a mitotic kinase that is amplified in several cancer types, has emerged as a compelling target for cancer therapy. While increased expression of Aurora A correlates to a worse prognosis for cancer patients, the impact of Aurora A overexpression on protein-protein interactions, oncogenic signaling, and cancer development remains unclear. This work describes the discovery and characterization of the novel interaction of Aurora A with two important mediators of cancer growth and development: H-Ras and Forkhead box transcription factor, FOXO1. The Ras-mitogen activated protein kinase (MAPK) signaling cascade is a critical pathway for sustained cell growth and proliferation in cancer. We validated the interaction of Aurora A and H-Ras and determined that the kinase domain of Aurora A and the N-terminal Switch I and II domains of H-Ras are involved in binding. Aurora A positively regulates this pathway by forming a protein complex with H-Ras and Raf-1, the Ras effector that mediates MAPK signaling. Aurora A stabilizes the H-Ras/Raf-1 protein complex and enhances MAPK signaling in a H-Ras-dependent manner. We also determined that the kinase activity of Raf-1 also functions to enhance binding of the Aurora A/H-Ras/Raf-1 protein complex. Aurora A also promotes oncogenic signaling through negative regulation of the tumor suppressor, FOXO1. In response to cell stress, FOXO1 localizes to the nucleus to initiate transcription of pro-apoptotic genes. Aurora A was found to interact with FOXO1, promoting exclusion of FOXO1 from the nucleus and inhibition of cell death. Overall, this work demonstrates that through novel protein-protein interactions, Aurora A functions as a positive regulator of oncogenic Ras-MAPK signaling and as a negative regulator of the tumor suppressive activity of FOXO1. This provides two potential therapeutic protein-protein interaction targets for cancers with Aurora A overexpression, as inhibition of either the Aurora A/H-Ras or Aurora A/FOXO1 interactions may reduce pro-growth signaling and induce cell death. Ultimately, understanding the role of Aurora A in cellular signaling will provide new opportunities to develop targeted therapies for cancer.
Table of Contents
Chapter 1: Introduction 1
1.1 Cancer and its characteristics 2
1.2.1 The role of protein-protein interactions (PPIs) in cancer 3
1.2.2 PPI network mapping to unravel tumor biology 7
1.3.1 Aurora kinases 7
1.3.2 Aurora A protein structure 8
1.3.3 Aurora A regulation 11
1.3.4 Aurora A in tumorigenesis 12
1.4.1 Non-canonical functions of Aurora A 13
1.4.1.i Aurora A PPIs with GTPases and their regulators 13
1.4.1.ii Aurora A PPIs with transcription factors 14
1.5.1 Novel Aurora A interaction partners 14
1.5.2 Ras 16
1.5.2.i Ras protein structure 16
1.5.2.ii Ras regulation 18
1.5.2.iii Ras in tumorigenesis 20
1.6 Forkhead box protein of the O class (FOXO1) 22
1.8.1 Aurora A and MAPK in cancer 25
1.8.2 Aurora A and FOXO1 in cancer 25
1.9 Scope of the dissertation 25
Chapter 2: Aurora Kinase A interacts with H-Ras and potentiates Ras-MAPK signaling 27
2.1 Abstract 28
2.2 Introduction 28
2.3 Materials and methods 30
2.4 Results 36
2.4.1 Aurora A is a novel H-Ras binding partner 36
2.4.2 Aurora A interacts with H-Ras through the switch I and II regions 40
2.4.3 The kinase domain of Aurora A mediates the H-Ras interaction 40
2.4.4 Aurora A enhances ERK phosphorylation 44
2.4.5 Aurora A-induced ERK phosphorylation requires Ras-MAPK signaling 45
2.4.6 Aurora A forms a protein complex with H-Ras and Raf-1 and acts through H-Ras to enhance MAPK signaling 49
2.5 Discussion 54
Chapter 3: The impact of kinase activity on the Aurora A/H-Ras/Raf-1 protein complex 57
3.1 Introduction 58
3.2 Materials and methods 59
3.3 Results 63
3.3.1 The kinase domains of Aurora A and Raf-1 mediate their interaction 63
3.3.2 The Aurora A/H-Ras interaction is phosphorylation independent 67
3.3.3 Constitutively active Raf-1 enhances Aurora A/Raf-1 binding 69
3.3.4 Kinase activity is required for Raf-1 to enhance the Aurora A/H-Ras binding 70
3.3.5 Sorafenib attenuates the Aurora A/H-Ras interaction 70
3.4 Discussion 74
Chapter 4: Aurora Kinase A interacts with FOXO1 and inhibits FOXO1-induced apoptosis 78
4.1 Introduction 79
4.2 Materials and methods 81
4.3 Results 85
4.3.1 Validation of the Aurora A/FOXO1 interaction 85
4.3.2 Aurora A interacts with FOXO1 independently of AKT phosphorylation 85
4.3.3 Aurora A binds nuclear and cytoplasmic FOXO1 in a distinct pattern 88
4.3.4 Aurora A inhibits FOXO1-induced apoptosis 91
4.3.4 Aurora A suppresses FOXO1-induced apoptosis by inhibiting the nuclear localization of FOXO1 93
4.4 Discussion 99
Chapter 5: Discussion 103
5.1 Novel functions for Aurora Kinase A (Aurora A) are revealed through protein-protein interactions 104
5.2 Aurora A interacts with H-Ras, forming a positive feedback loop that sustains enhanced oncogenic MAPK signaling 107
5.3 Structural domain characterization provides insight into functional outcomes of Aurora A interactions 109
5.3.1 Aurora A domains 109
5.3.2 H-Ras domains 110
5.4 Dual roles for the Aurora A/Raf-1 interaction 111
5.5.1 Functional impacts of the Aurora A/FOXO1 interaction 112
5.5.2 Postulated mechanisms for FOXO1 deregulation by Aurora A 113
5.6 Future directions and translational implications 113
References 116
List of Figures
Figure 1-1. Protein-protein interactions and cancer. 6
Figure 1-2. The structure of Aurora A. 10
Figure 1-3. Aurora A signaling in cancer. 15
Figure 1-4. The structure of Ras proteins. 17
Figure 1-5. Ras activation by receptor tyrosine kinases (RTKs). 19
Figure 1-6. The mitogen-activated protein kinase (MAPK) signaling pathway. 21
Figure 1-7. FOXO1 signaling in cancer. 24
Figure 2-1. Detection of the Aurora A/H-Ras interaction. 38
Figure 2-2. Interactions between Aurora and Ras proteins are mediated through conserved domains. 42
Figure 2-3. Aurora A potentiates ERK activation via H-Ras. 47
Figure 2-4. Aurora A forms a complex with H-Ras and Raf-1, acting through H-Ras to enhance ERK activation. 51
Figure 2-5. Proposed model for the role of Aurora A in the Aurora A/H-Ras/Raf-1 oncogenic signaling complex. 53
Figure 3-1. The kinase domains of Aurora A and Raf-1 are involved in the interaction. 65
Figure 3-2. The Aurora A/Raf-1 interaction is phospho-independent. 68
Figure 3-3. The role of Raf-1 in the Aurora A/H-Ras/Raf-1 protein complex. 72
Figure 3-4. Proposed model for the role of Raf-1 in the Aurora A/H-Ras/Raf-1 signaling complex. 77
Figure 4-1. Validation of the Aurora A/FOXO1 interaction. 87
Figure 4-2. Localization of the Aurora A/FOXO1 interaction. 90
Figure 4-3. Aurora A inhibits FOXO1-induced cell death in non-transformed cells. 92
Figure 4-4a. Aurora A inhibits FOXO1 nuclear translocation and rescues FOXO1-induced cell death. 96
Figure 4-4b. Aurora A inhibits FOXO1 nuclear translocation. 98
Figure 4-5. Potential mechanisms for negative regulation of FOXO1 nuclear translocation and inhibition by Aurora A protein-protein interactions. 102
Figure 5-1. Novel Aurora A interactions and their function in cancer. 106
Figure A-1. Schematic representation of TR-FRET and Venus PCA assays. 129
Figure A-2. The Aurora A/H-Ras interaction localizes to the plasma membrane and possibly the Golgi apparatus in HEK 293T and Cos7 cells. 130
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