PRAS40 and mTOR Signaling: A Paradigm for Crosstalk Among Proliferation, Growth, and Stress Signaling Open Access
Havel, Jonathan Joseph (2013)
Published
Abstract
The Proline-Rich Akt Substrate of 40 kDA (PRAS40) has recently been identified as a binding partner and inhibitor of the mechanistic Target of Rapamycin Complex 1 (mTORC1), a growth factor- and nutrient-sensitive kinase whose activity promotes protein synthesis and cell growth. Despite its inhibitory effect on mTORC1, PRAS40 has been shown to promote cell survival in rodent models of spinal cord injury and tumorigenesis. PRAS40 levels have also been found to correlate with poor prognosis in lung cancer patients, an effect not readily explained by mTORC1 inhibition. Here we demonstrate that in addition to its known cytoplasmic role in inhibiting mTORC1, PRAS40 dynamically shuttles to the nucleus, where it exists in a high-molecular weight complex void of mTORC1 components. Mass spectrometry and immunoprecipitation analyses identify ribosomal protein L11 (RPL11) as a nuclear-specific PRAS40-associated protein. This association is dependent upon both mTORC1- and Akt-mediated phosphorylation of PRAS40 residues S221 and T246, respectively. In addition to its canonical role as a member of the ribosome, RPL11 is known to stabilize the tumor suppressor p53 in response to nucleolar stress by binding and inhibiting the p53-directed E3 ubiquitin ligase HDM2. Interestingly, knock-down (KD) of PRAS40 induces p53 protein stabilization and transcriptional activation in an RPL11-dependent manner. As demonstrated by increased senescence-associated beta-galactosidase activity, PRAS40 KD also induces cellular senescence in a p53-dependent manner. In summary, PRAS40 is identified as a novel effector of Akt and mTORC1 signaling that regulates the RPL11-HDM2-p53 nucleolar stress response pathway to suppress the induction of cellular senescence. These findings may help to explain the pro-tumorigenic effects of PRAS40 and identify the PRAS40- and RPL11-containing complex as a promising target for p53-restorative anti-cancer drug discovery.
Table of Contents
Chapter 1 - General Introduction
1.1 Overview of Signaling Crosstalk and Scope of Dissertation 2
1.2 Proliferation and Survival Signaling - The PI3K-Akt Pathway 3
1.2.1 Pathway overview and regulation 3
1.2.2 Downstream functions 4
i.) Survival 4
ii.) Proliferation 5
1.2.3 PI3K-Akt pathway in cancer 6
1.3 Growth Signaling - mTOR Pathway 7
1.3.1 Pathway overview - mTORC1 and mTORC2 7
1.3.2 Regulation of mTORC1 8
i.) Amino acids 8
ii.) Energy/glucose 9
iii.) Growth factor signaling 10
1.3.5 Downstream functions of mTORC1 11
1.3.6 Regulation and function of mTORC2 11
1.3.7 mTOR signaling pathway in cancer 12
1.4 Stress Signaling - p53 and the Nucleolar Stress Response Pathway 14
1.5 PRAS40 - at the Crossroads of Akt and mTORC1 Signaling 15
1.5.1 Discovery of PRAS40 15
1.5.2 Regulation and molecular function of PRAS40 16
1.5.3 PRAS40 function in disease models 16
Chapter 2 - Development of Novel Technology for the Study of Ternary Protein Complex Dynamics in Living Cells
2.1 Introduction 26
2.1.1 Significance
2.1.2. Limitations of established methodologies
2.1.3 Protein-fragment complementation assay (PCA)
2.1.4 Bioluminescent resonance energy transfer (BRET)
2.2 Materials and Methods 28
2.3 Results 31
2.3.1. A combined PCA - BRET technique detects environmentally-cued dissociation of PRAS40 from intact 14-3-3 dimers in living cells 31
2.4 Conclusions and Discussion 32
Chapter 3 - Development of a Time-Resolved Fluorescence Resonance Energy Transfer Assay for the Discovery of mTORC2-specific Inhibitors
3.1 Introduction 37
3.1.1. mTOR pharmacology 37
3.1.2. Time-resolved Fluorescence Resonance Energy Transfer 37
3.2 Material and Methods 40
3.3 Results 44
3.3.1. Optimization of mTOR-Rictor TR-FRET signal 44
3.3.2. Identification of the Rictor-binding domain of mTOR 44
3.3.3. TR-FRET is capable of detecting mTOR-fragment mediated disruption of the mTOR-Rictor interaction 45
3.4 Discussion 45
Chapter 4 - Nuclear PRAS40 Links the Akt-mTORC1 Signaling Axis to the RPL11-HDM2-p53 Nucleolar Stress Response Pathway to Suppress Cellular Senescence
4.1 Introduction 59
4.2 Materials and Methods 60
4.3 Results 72
4.3.1 PRAS40 dynamically shuttles between the cytoplasm and the nucleus 72
4.3.2 PRAS40 residues 218-227 serve as a Nuclear Export Signal (NES) Sequence 73
4.3.3 PRAS40 is a member of a nuclear-specific RPL11-containing complex
74
4.3.4 The nuclear PRAS40- and RPL11-containing complex is distinct from mTORC1 76
4.3.5 The nuclear-specific PRAS40- and RPL11-containing complex requires PRAS40 residues S221 and T246 and is phosphorylation-dependent 76
4.3.6 The nuclear-specific PRAS40- and RPl11-containing complex is controlled by amino acids and serum factors through the kinase activities of mTORC1 and Akt 78
4.3.7 PRAS40 negatively regulates p53 protein stability and activity in an RPL11-dependent manner 80
4.3.8 PRAS40 KD induces p53 upregulation through a mechanism similar to low concentration Actinomycin D 82
4.3.9 PRAS40 suppresses induction of cellular senescence in a p53-dependent manner 82
4.4 Conclusions and Discussion - Interpretation of Results and Working Model 125
Chapter 5 - General Discussion and Implications of Findings
5.1 Implications for the role of PRAS40 in regulation of the RPL11-HDM2-p53 pathway under homeostatic conditions 131
5.2 A potential molecular mechanism for the observed pro-tumorigenic function of PRAS40 136
5.3 An alternative interpretation 139
5.4 Therapeutic Implications and Summary 140
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