Protein Tyrosine Phosphatases: Methods and Protocols (Methods in Molecular Biology, 2743)

Preface
Contents
Contributors
Chapter 1: Induction of Translational Readthrough on Protein Tyrosine Phosphatases Targeted by Premature Termination Codon Mut...
	1 Introduction
	2 Materials
		2.1 In Silico Analysis of the PTCome Distribution on PTP
		2.2 Assessing the Induction of Translational Readthrough on PTP
	3 Methods
		3.1 In Silico Analysis of the PTCome Distribution on PTP
			3.1.1 Obtaining the Potential PTCome
			3.1.2 Germline-Associated PTCome
			3.1.3 Cancer-Associated PTCome
			3.1.4 PTCome Qualitative Representation: Kernel Density Plot
			3.1.5 PTCome Quantitative Representation: Barplot of PTC Frequency (See Note 11)
		3.2 Assessing the Induction of Translational Readthrough on PTP
	4 Notes
	References
Chapter 2: Understanding Pseudophosphatase Function Through Biochemical Interactions
	1 Introduction
	2 Materials
		2.1 Conversion of a Pseudophosphatase STYX Domain to an Active Signature Motif (HCX5R)
		2.2 Immunoprecipitation
		2.3 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
		2.4 Immunoblotting
		2.5 Knockdown
	3 Methods
		3.1 Creation of an Active PTP from a Pseudophosphatase
		3.2 Identifying Interacting Partners of Pseudophosphatases
		3.3 Investigating the Biological Significance of Pseudophosphatases by Knockdown
		3.4 Validating Knockdown of the Pseudophosphatase
		3.5 Analyzing Potential Interactions of Pseudophosphatases Through Bioinformatic Structural Analysis
	4 Notes
	References
Chapter 3: CRISPR/Cas9-Mediated Modification of PTP Expression
	1 Introduction
	2 Materials
		2.1 Production of Lentiviral Particles for Genome Editing
		2.2 Transduction of Target Cells and Selection of Transduced Cells
		2.3 Characterization of Cell Clones with Altered Gene Expression of PTPRC or PTPRJ
	3 Methods
		3.1 Production of Lentiviral Pseudoparticles Encoding Cas9 and sgRNA
		3.2 Transduction of Target Cells and Selection of Transduced Cells
		3.3 Selection of Cells with Altered Receptor PTP Expression
	4 Notes
	References
Chapter 4: Osteoclast Methods in Protein Phosphatase Research
	1 Introduction
	2 Materials
		2.1 Production of OCLs in Culture
		2.2 Growth of OCLs on Bovine Bone Fragments and Visualization of Resorption  Pits
		2.3 Fluorescence Staining of  OCLs
		2.4 Growth of OCLs on Calcium-Phosphate-Coated Plastic Plates
		2.5 Production of CRISPR-Mediated Knockout RAW 264.7 Cells
	3 Methods
		3.1 Production of OCLs in Culture from Unselected Spleen Cells of  Mice
		3.2 Growth of OCLs on Bovine Bone Fragments and Visualization of Resorption  Pits
		3.3 Fluorescence Staining of OCLs Grown on Glass Coverslips or Bone Slices
		3.4 Growth of OCLs on Calcium-Phosphate-Coated Plastic Plates
		3.5 Production of CRISPR-Mediated Knockout RAW 264.7 Cells
	4 Notes
	References
Chapter 5: OT-I TCR Transgenic Mice to Study the Role of PTPN22 in Anti-cancer Immunity
	1 Introduction
	2 Materials
		2.1 CTL Activation and Differentiation
		2.2 CTL Re-stimulation
		2.3 Flow Cytometry Analysis
		2.4 ID8 Ovarian Cancer Cell Culture
		2.5 In Vivo ID8 Cancer Model
	3 Methods
		3.1 In Vitro Activation and Expansion of OT-I T Cells
		3.2 Re-stimulation of Effector CTLs
		3.3 Cell Staining and Flow Cytometry Analysis
		3.4 Culture of ID8 Ovarian Carcinoma Cells
		3.5 In Vivo ID8 Cancer Model
	4 Notes
	References
Chapter 6: Protein Tyrosine Phosphatase Studies in Zebrafish
	1 Introduction
	2 Materials
		2.1 Fish Husbandry
		2.2 Genotyping by polymerase chain reaction (PCR)
		2.3 Injection
		2.4 Gel Electrophoresis
		2.5 Equipment
	3 Methods
		3.1 Preparation-Design of sgRNA
		3.2 Preparation-Preparation of HDR Template
		3.3 Preparation-Primer Testing
		3.4 Preparation-Cas9 Concentration  Test
		3.5 Injections-Preparation of Injection Mixture and Injections
		3.6 Screening
		3.7 Verification of the Mutation by Subcloning
		3.8 Deriving a Stable  Line
	4 Notes
	References
Chapter 7: Examining Phosphatases Through Immunofluorescent Microscopy
	1 Introduction
	2 Materials
		2.1 Plating Cells of Interest
		2.2 Plasmid Transfection (If Necessary)
		2.3 Preparation of Fixed Cells
	3 Methods
		3.1 Plating Cells of Interest in a 96-Well  Dish
		3.2 Plating Cells of Interest Using Coverslips
		3.3 Transfection with Phosphatase of Interest Plasmid
		3.4 Fixation and Permeabilization of Cells
		3.5 Immunofluorescence Labeling of Phosphatase of Interest
		3.6 Confocal Imaging with Nikon A1R Microscope
		3.7 Image Processing and Analysis
	4 Notes
	References
Chapter 8: Identification of Protein Tyrosine Phosphatase (PTP) Substrates
	1 Introduction
		1.1 PTP Substrate Identification by In Vitro Substrate Trapping with PTP Active Site Mutants
	2 Materials
	3 Method
		3.1 Prepare the GST-Tagged Proteins
		3.2 Prepare the Cell Lysates
		3.3 Combination of GST-Tagged Proteins and Their Substrates
		3.4 Identification of PTP Substrates
		3.5 Substrate Trapping in the Cellular Context
			3.5.1 Method
	4 Notes
	References
Chapter 9: Kinase-Catalyzed Biotinylation to Identify Phosphatase Substrates (K-BIPS)
	1 Introduction
	2 Materials
		2.1 siRNA Knockdown
		2.2 Kinase-Catalyzed Biotinylation
		2.3 Avidin Enrichment
	3 Methods
		3.1 siRNA Knockdown with Verification by Gel Analysis
		3.2 Kinase-Catalyzed Labeling with ATP-Biotin
		3.3 Avidin Enrichment
	4 Notes
	References
Chapter 10: System-Level Analysis of the Effects of RPTPs on Cellular Signaling Networks
	1 Introduction
	2 Materials
		2.1 Cell Culture and Lysis
		2.2 Protein Digestion and Sample Preparation
		2.3 Tandem Mass Tag (TMT) Labeling and Phosphotyrosine Peptide Immunoprecipitation
		2.4 Immobilized Metal Chelate Affinity Chromatography (IMAC) and LC-MS/MS
	3 Methods
		3.1 Cell Culture and EGF Stimulation
		3.2 Preparation of Samples for Phosphotyrosine Peptide Immunoprecipitation
		3.3 TMT Labeling and Phosphotyrosine Peptide Immunoprecipitation
		3.4 Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography
		3.5 Liquid Chromatography-Tandem Mass Spectrometry Analysis (LC-MS/MS)
		3.6 Data Analysis and Validation
	4 Notes
	References
Chapter 11: Detection of Protein Tyrosine Phosphatase Interacting Partners by Mass Spectrometry
	1 Introduction
		1.1 Immunoprecipitation
		1.2 Proximity-Dependent Labeling with an Engineered Ascorbic Acid Peroxidase 2 (APEX2)
	2 Materials
		2.1 Materials for Cell Culture, Protein Isolation, and Fluorescent Labeling
		2.2 Materials for Sample Preparation for Mass Spectrometry
	3 Methods
		3.1 Preparation of Antibody-Beads Coupling
		3.2 Immunoprecipitation
		3.3 Proximity Labeling Using Ascorbic Acid Peroxidase 2 (APEX2)
		3.4 Fluorescent Detection of APEX2 Labeling
		3.5 Sample Preparation for Mass Spectrometry
		3.6 Cysteine Carbamidomethylation
		3.7 SP3 Peptide Extraction
		3.8 SP3-Mediated Peptide Clean-Up
		3.9 Peptide Solubilization
		3.10 Liquid Chromatography-Mass Spectrometry
		3.11 Data Analysis
		3.12 Statistics and Data Visualization
	4 Notes
	References
Chapter 12: Detecting PTP Protein-Protein Interactions by Fluorescent Immunoprecipitation Analysis (FIPA)
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Fluorescent Staining
		2.3 Cell Lysis and Immunoprecipitation
		2.4 SGS-PAGE
		2.5 Mass Spectrometry
	3 Methods
		3.1 Protein Labeling
		3.2 Cell Lysis
		3.3 Sorbent Preparation
		3.4 Immunoprecipitation
		3.5 Protein Electrophoresis
	4 Notes
	References
Chapter 13: Identifying Transmembrane Interactions in Receptor Protein Tyrosine Phosphatase Homodimerization and Heterodimeriz...
	1 Introduction
	2 Materials
		2.1 Subcloning the Transmembrane Domain of Interest into AraTM Plasmids
		2.2 Cotransformation of AraC, AraC*, and Reporter Plasmids into E. coli
		2.3 DN-AraTM Assay and Analysis
		2.4 Confirming Protein Expression Level by Immunoblotting
		2.5 Confirming Chimera Insertion Topology by Maltose Complementation Assay
		2.6 Confirming Chimera Insertion Topology by Spheroplast Protection Assay
	3 Methods
		3.1 Subcloning the Transmembrane Domain of Interest into AraTM Plasmids
		3.2 Cotransformation of AraC and AraC* Plasmids into E. coli
		3.3 DN-AraTM Assay and Analysis
		3.4 Confirming Protein Expression Level by Immunoblotting
		3.5 Confirming Chimera Insertion Topology by Maltose Complementation Assay
		3.6 Confirming Chimera Insertion Topology by Spheroplast Protection Assay
	4 Notes
	References
Chapter 14: Preparation of Oxidized and Reduced PTP4A1 for Structural and Functional Studies
	1 Introduction
	2 Materials
		2.1 Production of PTP4A1
		2.2 Oxidation Reaction
		2.3 Reduction Reaction
		2.4 Equipment
		2.5 Software
	3 Methods
		3.1 Production of PTP4A1
		3.2 Oxidation Reaction
		3.3 Reduction Reaction
		3.4 Thermal Stability Assay
		3.5 Solution NMR Spectroscopy
	4 Notes
	References
Chapter 15: Measuring the Reversible Oxidation of Protein Tyrosine Phosphatases Using a Modified Cysteinyl-Labeling Assay
	1 Introduction
	2 Materials
		2.1 Cell Care, Passage, and Serum Starvation
		2.2 Preparation of Degassed Lysis Buffer and Hypoxic Glove  Box
		2.3 Cell Stimulation with Epidermal Growth Factor (EGF)
		2.4 Modified Cysteinyl-Labeling Assay
			2.4.1 Hypoxic Lysis and Alkylation
			2.4.2 Buffer Exchange and Reduction
			2.4.3 Biotin Labeling
			2.4.4 Enrichment of Biotinylated  PTPs
			2.4.5 Electrophoresis, Transfer, and Visualization
	3 Methods
		3.1 Cell Care, Passage, and Serum Starvation
		3.2 Preparation of Degassed Lysis Buffer and Hypoxic Glove  Box
		3.3 Cell Stimulation with  EGF
		3.4 Modified Cysteinyl-Labeling Assay
			3.4.1 Hypoxic Lysis and Alkylation
			3.4.2 Buffer Exchange and Reduction
			3.4.3 Biotin Labeling
			3.4.4 Enrichment of Biotinylated  PTPs
			3.4.5 Electrophoresis, Transfer, and Visualization
	4 Notes
	References
Chapter 16: Identification and Optimization of Protein Tyrosine Phosphatase Inhibitors Via Fragment Ligation
	1 Introduction
		1.1 The Role of Protein Tyrosine Phosphatases in the Origin of Human Diseases
		1.2 The Importance of Phosphotyrosine Biomimetics for Modern Drug Discovery
		1.3 PTK Catalytic Site-Directed Inhibitors
		1.4 Phosphorus-Containing Biomimetics of Phosphotyrosine
		1.5 Di-Ionic Non-Phosphorus-Containing Biomimetics of Phosphotyrosine
		1.6 Mono-Ionic Biomimetics of Phosphotyrosine
		1.7 Uncharged Mimetics
		1.8 Chemically Reactive pTyr Mimetics
		1.9 The Latest Development in the Rational Design of pTyr Mimetics
	2 Methods for the Fragment-Based Discovery of PTP Inhibitors
		2.1 The Principle of Fragment Ligation and Its Use in Drug Discovery
		2.2 Identification of pTyr Mimetics Via Fragment Ligation
		2.3 Optimization of pTyr Mimetics Via Fragment Ligation
	References
Chapter 17: Targeting Nonconserved and Pathogenic Cysteines of Protein Tyrosine Phosphatases with Small Molecules
	1 Introduction
	2 Materials
		2.1 Electrophilic Compound Libraries
		2.2 PTP Expression and Purification
		2.3 Electrophile-Containing Compound Screening
	3 Methods
		3.1 Expression and Purification of PTP Domains
		3.2 Screen for Target-Cysteine-Directed Inhibitors Using pNPP as Substrate
		3.3 Counter-Screen for Target-Cysteine-Directed Inhibitors Using pNPP as Substrate
		3.4 Screen for Target-Cysteine-Directed Inhibitors Using DiFMUP as Substrate (See Note 11)
		3.5 Counter-Screen for Target-Cysteine-Directed Inhibitors Using DiFMUP as Substrate
	4 Notes
	References
Chapter 18: In Vitro Phosphatase Assays for the Eya2 Tyrosine Phosphatase
	1 Introduction
	2 Materials
		2.1 Express and Purify EYA2 ED from E. coli
		2.2 Analyze the Eya2 Phosphatase Kinetics Using an OMFP-Based Fluorescent Phosphatase Assay
		2.3 Evaluate Small Molecule Inhibitors of the Eya2 Phosphatase Using an OMFP-Based Fluorescent Phosphatase Assay
		2.4 Evaluate Small Molecule Inhibitors of the EYA2 Phosphatase Using a pH2AX-Based Malachite Green Assay
	3 Methods
		3.1 Express and Purify EYA2 ED from E. coli
		3.2 Analyze the EYA2 Phosphatase Kinetics Using an OMFP-Based Fluorescent Phosphatase Assay
		3.3 Evaluate Small Molecule Inhibitors of the EYA2 Phosphatase Using an OMFP-Based Fluorescent Phosphatase Assay
		3.4 Evaluate Small Molecule Inhibitor of the EYA2 Phosphatase Using a pH2AX-Based Malachite Green Assay
	4 Notes
	References
Chapter 19: High-Throughput Discovery and Characterization of Covalent Inhibitors for Protein Tyrosine Phosphatases
	1 Introduction
	2 Materials
		2.1 High-Throughput Screening
		2.2 Phosphatase Activity Assay
		2.3 GSH Reactivity Assay
	3 Methods
		3.1 High-Throughput Screening
		3.2 Kinetics Characterization
		3.3 Two-Time Point IC50
		3.4 Vanadate Protection
		3.5 Reactivity and Stability Assay
			3.5.1 Relative GSH Reactivity Assay
			3.5.2 Compound Stability Assay
	4 Notes
	References
Index