Tumor Suppressor Par-4: Structural Features, Molecular Mechanisms and Function

Preface
Contents
Discovery and Overview of Par-4
	1 Discovery of Par-4
	2 Identification and Chromosome Localization of Human Par-4/PAWR Gene
	3 Ubiquitous Expression of Par-4 in Vertebrate Tissues
	4 Functional Domains of Par-4
		4.1 Nuclear Localization Sequences and Nuclear Export Sequence of Par-4
		4.2 Leucine Zipper (LZ) Domain
		4.3 The SAC Domain of Par-4
		4.4 The PAF Domain of Par-4
	5 Function of Par-4 in Apoptosis
	6 Mechanism of Par-4 Secretion and Apoptosis of Cancer Cells
	7 Par-4 as a Tumor Suppressor
	8 Role of Par-4 in Cancer Stem Cells
	9 Role of Par-4 in Epithelial Mesenchymal Transition and Inflammation
	10 Role of Par-4 in the Neurons, Heart, and Eye
	11 Future Perspectives and Conclusion
	References
Par-4 in Cell Cycle Regulation
	1 Introduction
	2 Cell Cycle
		2.1 Cellular Events During Cell Cycle
		2.2 The Regulation of the Cell Cycle
			2.2.1 p53 Transcriptionally Activates p21 Expression and Arrests the Cell Cycle at G1 Stage
			2.2.2 NF-κB Activates the Transcription of BCL-2
	3 The Role of Par-4 Underlying Regulation of the Cell Cycle
		3.1 The Role of Par-4 in G1 and S Phases
			3.1.1 Cytosolic Par-4 Inhibits Nuclear Localization of NF-κB and Decreases Bcl-2 Expression, Leading to G1 Arrest
				Par-4 Directly Interacts with NF-κB and Blocks Its Nuclear Localization
				Par-4 Represses ζPKC Activity to Indirectly Inhibit NF-κB Pathway
				Par-4 Inhibits p65-Dependent DROSHA Expression and Regulates miRNA
				Par-4 Is a Direct Target of NF-κB
			3.1.2 Nuclear Par-4 Functions as Co-transcriptional Factor of THPA for CCAR1 on the p53/p21 Axis
				Par-4 Elevates p53 Level
				Par-4 Interacts with THAP1 to Promote Transcription of CCAR1
			3.1.3 Par-4 Interacts and Inhibits WT-1 on Bcl-2 Promoter
		3.2 The Role of Par-4 in S Phase: Par-4 Inhibits TOP1 Activity
		3.3 The Role of Par-4 in M Phase
			3.3.1 Par-4 Regulates p-p21 in a p53-Independent Manner and Causes Mitosis Arrest
			3.3.2 The Distribution of Par-4 in Mitotic Cells Determines the Fate of Daughter Cells
	4 PLK1 Function in Mitosis
		4.1 Regulating PLK1 in Cell Cycle
			4.1.1 Transcriptional Regulation
			4.1.2 Post-translational Regulation
		4.2 PLK1’s Biological Function
			4.2.1 PLK1 in Cell Cycle
				PLK1 with CDK1/Cyclin B1
				PLK1 at Centrosomes
				PLK1 at Kinetochores
				PLK1 in Spindle Formation and Chromosome Segregation
				PLK1 in Cytokinesis
			4.2.2 PLK1 in DNA Damage
			4.2.3 PLK1 Is Correlated with NF-κB Signaling
	5 Conclusion and Future Perspectives
		5.1 Preliminary Data Available to Support the Working Model
		5.2 Future Research Needed to Further Test the Working Model
	References
Conformational Studies of the Par-4 C-Terminal Domain
	1 Introduction
		1.1 Par-4 Discovery and Function
		1.2 Rat vs. Human cl-Par-4 and Domain Structure
		1.3 Brief Summary of Structural Work
	2 Overview of Techniques Utilized
	3 Rat LZ (Amino Acids 286–332)
		3.1 Helicity as a Function of pH, Salt, and Temperature
		3.2 Site-Directed Mutagenesis (E310K, E310Q, D305K, and D305N)
		3.3 Hydrodynamic Studies
		3.4 NMR Studies
	4 Rat FL, ΔLZ, and SAC Domain Constructs
		4.1 Structural Analysis of the SAC Domain
		4.2 Hydrodynamic Analysis of FL and ΔLZ Par-4
		4.3 CD Spectroscopy of FL and ΔLZ Par-4
		4.4 NMR Studies of FL and ΔLZ Par-4
	5 The Human cl-Par-4 Fragment
		5.1 The Effects of pH (Coiled Coil Dimer)
		5.2 The Effects of Sodium (Non-coiled Coil Tetramer)
	6 Discussion
		6.1 Folding of the Isolated LZ
		6.2 Other Par-4 Constructs
		6.3 Conclusions and Future Perspectives
	References
Structural Analysis of Par-4 and Crystallographic Analysis of the Regulatory Domain
	1 Introduction
	2 Sample Preparation and Characterization
		2.1 Cloning
		2.2 Overexpression and Purification
		2.3 Sample Analysis by CD and NMR
		2.4 Protein Crystallization
	3 Crystallographic Analysis
		3.1 Data Collection and Analysis
		3.2 Model Building and Refinement
		3.3 Structure of the Homodimer
	4 Functional Implications
		4.1 Regulatory Interactions of Par-4CC
		4.2 Trigger Motif for Homodimerization
		4.3 Preference for Heterodimerization in Par-4LZ Region
		4.4 Regulation of Complex Formation by Par-4CC
		4.5 Masking of the Par-4NES by Dimerization
	5 Conclusions and Future Perspectives
	References
Regulation of Par-4 by Ubiquitinases
	1 The Ubiquitin-Proteasome System (UPS)
	2 Ubiquitin-Activating Enzyme (E1)
	3 Ubiquitin-Conjugating Enzyme (E2)
	4 E3 Ubiquitin Ligases
		4.1 HECT E3 ligases
		4.2 U-Box Ligases
		4.3 RING E3 Ligases
	5 F-Box Proteins
		5.1 F-Box Proteins in Cancer
		5.2 Fbxo45
	6 Ubiquitin-Proteasome System and Tumor Suppressor Proteins
		6.1 Role of Ubiquitination in Prostate Apoptosis Response-4 (Par-4) Regulation
		6.2 Structure and Domains of Par-4
		6.3 Fbxo45-Mediated Degradation of Par-4
		6.4 Rescue of Par-4 Degradation by the Par-4 Amino-Terminal Fragment (PAF)
		6.5 Regulation of Par-4 by Other E3 Ligases
	7 Conclusions and Future Perspectives
	References
Regulation of Par-4 Function by Phosphorylation
	1 Introduction
	2 Positive Regulation of Par-4 Pro-apoptotic Activity
		2.1 Phosphorylation by PKA
		2.2 Phosphorylation by ZIP Kinase
	3 Negative Regulation of Par-4 Pro-apoptotic Activity
		3.1 Par-4 and AKT Interplay
		3.2 Regulation of Par-4 by the Protein Kinase CK2
	4 Conclusion
	Bibliography
Role of Par-4 in GRP78 Translocation
	1 Introduction
		1.1 GRP78 Structure
	2 Role of GRP78 in UPR Regulation Under ER Stress
	3 GRP78 Translocates to the Surface
	4 Endoplasmic Reticulum Transmembrane GRP78 Origin
	5 KDEL Motif-Related Scape of GRP78 from the ER
	6 Secreted GRP78
	7 Translocation from the ER Together with Client Proteins
	8 Par-4
	9 GRP78/Par-4 Interaction
	References
PAR-4 in the Regulation of Stem Cell Death and Embryo Development
	1 Stem Cell Differentiation and the Role of PAR-4 in the Elimination of Unwanted Cells in Embryogenesis
	2 PAR-4 and the Regulation of the Cytoskeleton in Dissociation-Induced Apoptosis of Stem Cells
	3 Ceramide Metabolism and Asymmetric Distribution of PAR-4 During Stem Cell Differentiation
	4 PAR-4 in Fetal Alcohol Syndrome and Dysregulated Stem Cell Apoptosis
	5 Ceramide and PAR-4-Mediated Apoptosis in Stem Cell-Derived Tumor Cells
	6 Conclusions and Future Directions
	References
RASSF2 and the PAR-4 Connection
	1 RASSF2
		1.1 Gene Structure
		1.2 RASSF2 Protein Structure and Subcellular Localization
		1.3 RASSF2 Function
	2 PAR-4
		2.1 The RASSF2 and PAR-4 Interaction
	3 Conclusions and Future Perspectives
	References
Regulation of Tumor Suppressor Par-4 by Ceramide
	1 Introduction
	2 Apoptosis
	3 Autophagy
		3.1 Molecular Mechanisms of Autophagy
		3.2 Autophagy as a Tumor Suppressor Mechanism
	4 Prostate Apoptosis Response-4 (Par-4)
		4.1 Domains of Par-4
		4.2 Establishment of Par-4 as Tumor Suppressor Protein
		4.3 Apoptosis-Inducing Functions of Par-4
		4.4 Role of Par-4 in Autophagy
	5 Ceramide
		5.1 Ceramide Metabolism
		5.2 Tumor-Suppressive Functions of Ceramide
	6 Par-4 and Ceramide Connection
	7 Conclusion and Future Perspectives
	References
PAWR as a Direct SRC-1/HOXC11 Suppression Target
	1 Steroid Receptor Co-activator 1 (SRC-1) and HOXC11 in Cancer
		1.1 SRC-1
		1.2 The HOMEOBOX Family
		1.3 The Role of HOX Genes in the Normal Mammary Gland
		1.4 The Role of HOX Proteins in Breast Cancer
		1.5 SRC-1 and HOXC11 in Transcriptional Activation
	2 Gene Regulation Through Epigenetic Modification
		2.1 DNA Methylation
		2.2 Post-Translational Modifications
		2.3 Epigenetic Regulation of Cancer
		2.4 SRC-1 and Transcriptional Repression
	3 SRC-1 and HOX Transcriptional Repression of PAWR
	4 Conclusions
	References
Index