Protein Tyrosine Phosphatases: Methods and Protocols (Methods in Molecular Biology, 1447)

✍ Scribed by Rafael Pulido (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 405
• Category: Library

✦ Synopsis

This book provides coverage, methodology, and laboratory protocols on the more essential aspects of protein tyrosine phosphatase (PTP) function and regulation, including the use of standardized in vitro functional assays, suitable cell systems, and animal and microorganism models. Chapters covering state-of-the-art technical approaches suitable to decipher the physiologic roles of PTPs, and their involvement in tissue-specific functions, are also included, which will be of utility for both newcomers and experienced researchers in the field of tyrosine- and phosphoinositide- phosphorylation/dephosphorylation. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Protein Tyrosine Phosphatases: Methodsand Protocols aims to aid researchers in better defining the common and individual features of the PTP family members and translating this knowledge into PTP-based therapy for human disease.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: The Extended Family of Protein Tyrosine Phosphatases
1 Tyr Phosphatases: Positive and Negative Protein Regulators of Cell Signaling
2 Classification of Tyr Phosphatases
3 Class I Cys-Based Phosphatases
3.1 Classical PTPs
3.2 VH1-Like/Dual-Specificity Phosphatases (DUSPs)
3.3 SAC Phosphatases
3.4 Paladin
3.5 INPP4 and TMEM55 Phosphatases
4 Class II Cys-Based Phosphatases
5 Class III Cys-Based Phosphatases
6 Asp-Based Phosphatases
7 His-Based Phosphatases
8 Catalytic Mechanism of Tyr Phosphatases
9 The PTPome in Model Organisms
10 Conclusion and Future Perspectives
References
Chapter 2: Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome
1 Introduction
2 Materials
2.1 Analysis of Human PTPome mRNA Expression by Semi-­quantitative RT-PCR
2.2 Global Analysis of Human PTPome mRNA Expression by Quantitative RT-PCR
3 Methods
3.1 Analysis of Human PTPome mRNA Expression by Semi-­quantitative RT-PCR
3.2 Global Analysis of Human PTPome mRNA Expression by Quantitative RT-PCR (RT-qPCR)
4 Notes
References
Chapter 3: Expression, Purification, and Kinetic Analysis of PTP Domains
1 Introduction
2 Materials
2.1 PTP Domains Expression
2.1.1 Prokaryotic Expression of PTP Domains (Exemplified by Expressions of hEya3 and hRPTPμD1)
2.1.2 Eukaryotic Expression of PTP Domains (Exemplified by Expression of the Extracellular Region of PTPBR7–Ecto-­PTPBR7)
2.2 Recombinant Protein Purification
2.2.1 Affinity Chromatography on Glutathione-­Sepharose
2.2.2 Affinity Chromatography on Ni-Sepharose
2.2.3 Anion Exchange Chromatography
2.2.4 Size Exclusion Chromatography
2.3 Analysis of Purified Proteins by SDS-PAGE and Western Blotting
2.4 Mass Spectrometric Analysis
2.5 Tyrosine Phosphatase Assays
3 Methods
3.1 D1 Domain of Human Receptor Protein Tyrosine Phosphatase μ (hRPTPμD1)
3.1.1 Prokaryotic Expression of hRPTPμD1
3.1.2 Purification of hRPTPμD1
3.1.3 Analysis of the Expression and Purification of hRPTP μD1 by SDS-PAGE
3.1.4 Detection of hRPTPμ by Western Blotting
3.2 Human Eya3 (hEya3) Tyrosine Phosphatase
3.2.1 Prokaryotic Expression of hEya3 Tyrosine Phosphatase
3.2.2 Purification of hEya3
3.2.3 Analysis of the Expression and Purification of hEya3 by SDS-PAGE
3.2.4 Mass Spectrometric Identification of hEya3 Using Bottom-Up Approach
3.2.5 hEya3 Tyrosine Phosphatase Assays
3.2.6 Determination of Kinetic Parameters of hEya3 Phosphatase
3.3 Extracellular Domain of PTPBR7 (Ecto-PTPBR7)
3.3.1 Cell Culture
3.3.2 Large-Scale Transfection
3.3.3 Protein Purification
3.3.4 Deglycosylation of Protein
4 Notes
References
Chapter 4: Peptide Microarrays for Real-Time Kinetic Profiling of Tyrosine Phosphatase Activity of Recombinant Phosphatases and Phosphatases in Lysates of Cells or Tissue Samples
1 Introduction
2 Materials
2.1 Preparation of Lysates for Analysis of Tyrosine Phosphatase Activity
2.2 Analysis of the Tyrosine Phosphatase Activity of a Recombinant or Purified Phosphatase or of a Cell Lysate
3 Methods
3.1 Preparation of Lysates for Analysis of Tyrosine Phosphatase Activity
3.2 Analysis of the Tyrosine Phosphatase Activity of a Recombinant or Purified Phosphatase or of a Cell Lysate
4 Notes
References
Chapter 5: Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-­Directed Mutagenesis of PTEN and PTPRZ-B
1 Introduction
2 Materials
2.1 Site-Directed Mutagenesis of PTPs
3 Methods
3.1 Site-Directed Mutagenesis of PTPs
4 Notes
References
Chapter 6: Assays to Measure PTEN Lipid Phosphatase Activity In Vitro from Purified Enzyme or Immunoprecipitates
1 Introduction
2 Materials
2.1 Production of 3-(33P)-PtdIns (3,4,5)P3
2.2 In Vitro PTEN Lipid Phosphatase Assay Components
2.3 Immuno-precipitated Cellular PTEN Lipid Phosphatase Assay Components
3 Methods
3.1 Production of 3-(33P)-PtdIns(3,4,5)P3 Substrate by Labeling at the D3 Position
3.2 In Vitro PTEN Lipid Phosphatase Assay
3.3 In Cell PTEN Lipid Phosphatase Assay
4 Notes
References
Chapter 7: Assessing the Biological Activity of the Glucan Phosphatase Laforin
1 Introduction
2 Materials
2.1 pNPP/OMFP Phosphatase Assay
2.2 Glycogen Phosphatase Assay
3 Methods
3.1 pNPP/OMFP Phosphatase Assay
3.2 Glycogen Phosphatase Assay
4 Notes
References
Chapter 8: Discovery and Evaluation of PRL Trimer Disruptors for Novel Anticancer Agents
1 Introduction
2 Materials
2.1 Identification of Small Molecules Targeting PRL1 Trimer Interface by Virtual Screening
2.2 Validation of PRL1 De-trimerization Compounds by In Vitro Cross-linking Assay
2.3 Validation of PRL1 De-trimerization Compounds by In Vivo Cross-linking Assay
2.4 Evaluation of the Cellular Activity of PRL1 De-trimerization Compounds
3 Methods
3.1 Identification of Small Molecules Binding at PRL1 Trimer Interface by Virtual Screening
3.2 Validation of PRL1 De-trimerization Compounds by In Vitro Cross-linking Assay
3.3 Validation of PRL1 De-trimerization Compounds by In Vivo Cross-linking Assay
3.4 Evaluation the Cellular Activity of PRL1 De-trimerization Compounds
4 Notes
References
Chapter 9: Analyzing Pseudophosphatase Function
1 Introduction
2 Materials
2.1 Conversion of a Pseudophos-phatase STYX Domain to an Active Signature Motif (HCX5R)
2.2 Immuno-precipitation and Immunoblotting
2.3 Knockdown
3 Methods
3.1 Creation of an Active PTP from a Pseudo-phosphatase
3.2 Identifying Interacting Partners of Pseudo-phosphatases
3.3 Investigating the Biological Significance of Pseudo-phosphatases by Knockdown
4 Notes
References
Chapter 10: Crystallization of PTP Domains
1 Introduction
2 Materials
2.1 Se-Methionine Protein Production
2.2 Protein Crystallogenesis
2.3 Crystal Freezing and Mounting
3 Methods
3.1 Construct Design and Sequence Analysis
3.2 Expression Systems Used to Produce PTP Proteins for Crystallographic Analyses
3.3 Preparing Se-Methionine Derivatized Protein
3.4 Assessing the Quality of Your Sample
3.5 Assessing the Stability of Your Sample by Thermofluor
3.6 Crystallogenesis
3.6.1 Pre-­crystallization Test
3.6.2 Crystallization Screening
3.6.3 Rapid Hit Optimization
3.7 Ligand Inhibitor Complexation
3.8 Cryoprotection and Crystal Freezing
3.8.1 Cryofreezing Using Glycerol or PEG
3.8.2 Cryofreezing in Oils
3.9 Online Tools and Resources
3.9.1 Online Links and Useful Websites
3.9.2 Databases Containing Information on Crystallization Conditions for Protein Phosphatases
3.9.3 Crystallography Textbooks
4 Notes
References
Chapter 11: NMR Spectroscopy to Study MAP Kinase Binding to MAP Kinase Phosphatases
1 Introduction
2 Materials
3 Methods
3.1 Production of p38α and Regulatory Protein Tyrosine Phosphatases for NMR Studies
3.1.1 Fresh Transformation and Colony Expression Screening
3.1.2 D2O Adaption (see Table 2 and Fig. 1)
3.1.3 D2O Large Scale Expression
3.1.4 D2O Recycling
3.2 Interaction with Kinase Interaction Motif Protein Tyrosine Phosphatases (KIM-PTPs) and MAPK Protein Tyrosine Phosphatases
3.3 NMR Spectroscopy
3.3.1 NMR Sample Preparation
3.3.2 NMR Experiments, Sequence Specific Backbone Assignment
3.3.3 NMR Experiments, Protein–Protein Interactions
3.4 Global Structures
4 Notes
References
Chapter 12: Visualizing and Quantitating the Spatiotemporal Regulation of Ras/ERK Signaling by Dual-Specificity Mitogen-Activated Protein Phosphatases (MKPs)
1 Introduction
2 Materials
2.1 MEF Cell Culture
2.2 Adenoviral Generation and Purification
2.3 Immunofluo-­rescence Staining of ERK1/2, p-ERK1/2, and Myc
2.4 Subcellular Fractionation and Immunoblot Analysis
3 Methods
3.1 Generation of Recombinant Adenovirus
3.2 Large-Scale Amplification and Purification of Recombinant Adenovirus
3.3 Culture, Adenoviral Infection, and Staining of MEFs for ERK1/2, p-ERK1/2, and Myc
3.4 High Content Microscopy and Analysis
3.5 Culture, Adenoviral Infection, and Subcellular Fractionation of MEFs for Immunoblot
4 Notes
References
Chapter 13: In Situ Proximity Ligation Assay (In Situ PLA) to Assess PTP-Protein Interactions
1 Introduction
2 Materials
2.1 In Situ PLA Detection of DEP-1/PTPRJ Interaction with Its Substrate Protein FLT3 in Hematopoietic Cells
2.2 In Situ PLA Detection of VE-PTP/PTPRB Interaction with VEGFR2 or Tie2 in Endothelial Cells
3 Methods
3.1 In Situ PLA Detection of DEP-1/PTPRJ Interaction with Its Substrate Protein FLT3 in Hematopoietic Cells
3.2 In Situ PLA Detection of VE-PTP/PTPRB Interaction with VEGFR2 or Tie2 in Endothelial Cells
4 Notes
References
Chapter 14: Use of Dominant-Negative/Substrate Trapping PTP Mutations to Search for PTP Interactors/Substrates
1 Introduction
2 Materials
3 Methods
3.1 In Vitro Substrate Trapping
3.2 Identification of Proteins and Substrates That Interact with PTP in the Cellular Context
3.3 In-Gel Digestion and Preparation of Peptides for Mass Spectrometry (MS)
3.4 Other Methods
3.5 Validation
3.5.1 In Vitro Dephosphorylation
3.5.2 In Vivo Dephosphorylation
3.6 Limitations and Cautions
3.7 Future Perspectives
4 Notes
References
Chapter 15: Detection and Identification of Ligands for Mammalian RPTP Extracellular Domains
1 Introduction
2 Materials
2.1 Generating PLAP Fusion Proteins
2.2 Performing RAP Assays and Affinity Chromatography
2.3 Performing Hippocampal-
3 Methods
3.1 Receptor Affinity Probing (RAP): In Situ Detection of Ligands in Cells and Tissues
3.2 Identification of Ligands by Affinity Chromatography
3.3 Validation of RPTP Interactions with Synaptic Partners in Hippocampal Neurons
4 Notes
References
Chapter 16: Production of Osteoclasts for Studying Protein Tyrosine Phosphatase Signaling
1 Introduction
2 Materials
2.1 Production of OCLs in Culture from Unselected Bone Marrow Cells Using Cytokines
2.2 Production of OCLs in Culture from Bone Marrow Macrophages Using Cytokines
2.3 Expressing Foreign Proteins and Knocking Down Endogenous Proteins in OCLs Using Viral Vectors
2.4 Studying Cell Signaling in Cultured OCLs
3 Methods
3.1 Production of OCLs in Culture from Unselected Bone Marrow Cells Using Cytokines
3.2 Production of OCLs in Culture from Bone Marrow Macrophages Using Cytokines
3.3 Expressing Foreign Proteins and Knocking Down Endogenous Proteins in OCLs Using Viral Vectors
3.4 Studying Cell Signaling in Cultured OCLs
4 Notes
References
Chapter 17: Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis
1 Introduction
2 Materials
2.1 Anesthetic Reagents
2.2 Anticoagulants
2.3 Blood Collection
2.4 Platelet Preparation and Aggregation
2.5 Platelet Secretion Using Flow Cytometry
2.6 Platelet Activation, Lysis, Immunoprecipitation (IP), and Immunoblot
2.7 In Vivo Thrombosis Models
3 Methods
3.1 Retro-orbital Mouse Blood Collection
3.2 Isolation of Mouse Washed Platelets
3.3 Mouse Platelet Aggregation Assay
3.4 Platelet Secretion Assay by Flow Cytometry
3.4.1 Mouse Platelet Secretion Assay in Whole Blood
3.4.2 Mouse Platelet Secretion Assay in Washed Platelets
3.5 Platelet Activation, Lysis, IP, and Immunoblot
3.5.1 Platelet Activation and Lysis
3.5.2 Immuno-precipitation
3.6 In Vivo Mouse Thrombosis Models
3.6.1 Ferric Chloride (FeCl3) Carotid Injury Model
Staining of Washed Platelet with CFSE
Preparation of Material and Mouse Surgery
3.6.2 Collagen and Epinephrine-­Induced Pulmonary Embolism Model
3.7 Pharmacological Inhibition of Platelet PTPs
3.7.1 General Considerations
3.7.2 Inhibition of DUSP3 in Human Platelets
3.7.3 Human Blood Collection
3.7.4 Isolation of Human Platelets
3.7.5 Effects of Small-­Molecule Inhibitors on Human Platelet Aggregation
3.7.6 Effects of Small-­Molecule Inhibitors on Human Platelet Secretion
3.7.7 Effects of Small-­Molecule Inhibitors on Human Platelets Using Immunoblot Assays
4 Notes
References
Chapter 18: Functional Analysis of Dual-Specificity Protein Phosphatases in Angiogenesis
1 Introduction
2 Materials
2.1 Basic Required Material
2.2 Cell Culture and Transfection
2.3 Proliferation
2.4 Tube Formation Assay and Time Lapse
2.5 Spheroid Sprouting Assay
2.6 In Vivo Matrigel Plug Assay and LLC Subcutaneous Tumors Model
2.7 Immuno-fluorescence
2.8 Aortic Ring Assay (ARA)
3 Methods
3.1 Cell Culture and Transfection
3.1.1 HUVECs Culture
3.1.2 RNA Interference (RNAi) Transfection in HUVECs
3.2 Endothelial Cells Tube Formation Assay
3.3 Spheroid Sprouting Assay
3.4 HUVECs Proliferation Assay
3.5 Matrigel Plug Assay
3.6 LLC Subcutaneous Tumors
3.6.1 LLC Cells Culture
3.6.2 LLC Subcutaneous Injection
3.7 Anti-CD31 Immunofluorescence
3.8 Aortic Ring Assay (ARA)
4 Notes
References
Chapter 19: Studying Protein-Tyrosine Phosphatases in Zebrafish
1 Introduction
2 Materials
2.1 Microinjection
2.2 Regeneration Assay
2.3 Tissue Lysis for Protein Extraction
3 Methods
3.1 Microinjection
3.2 Regeneration Assay
3.3 Tissue Lysis for Protein Extraction
4 Notes
References
Chapter 20: Live Staining of Drosophila Embryos with RPTP Fusion Proteins to Detect and Characterize Expression of Cell-­Surface RPTP Ligands
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Flies and Embryo Collection
3.2 Aging Embryos, Sorting, and Staging
3.3 Embryo Dechorionation and Preparation for Dissection
3.4 Embryo Live Dissection: See Ref. [7] for a Video Demonstration of the Live Embryo Dissection Process
3.5 Fixation and Immunohisto-chemistry
4 Notes
References
Chapter 21: Methods to Study Protein Tyrosine Phosphatases Acting on Yeast MAPKs
1 Introduction
1.1 Yeast MAPK Signaling and Their Regulation by Tyrosine Phosphatases
1.2 A Strategy to Detect Yeast MAPK Regulation by a Specific PTP
2 Materials
2.1 Yeast Plasmids and Strains
2.2 Culture Media and Solutions
2.3 Transformation of Yeast Cells
2.4 Preparation of Cell Extracts
2.5 SDS-
2.6 Western Blotting
2.7 Data Analysis
2.8 Flow Cytometry GFP Fluorescence Quantification
3 Methods
3.1 Overexpression of PTPs in Saccharomyces cerevisiae
3.1.1 Transformation of Yeast Cells
3.1.2 Yeast Cells Growth, Expression of PTPs, Pathway Activation, and Cell Collection
3.2 Analysis of the Effect of PTPs Overexpression on Yeast MAPK Phosphorylation
3.2.1 Preparation of Yeast Cell Extracts
3.2.2 SDS-PAGE and Western Blotting
3.2.3 Data Analysis (Quantification/Ratios/Statistics)
3.3 Analysis of the Effect of PTPs Overexpression on Yeast MAPK-Mediated Transcriptional Induction
3.3.1 Flow Cytometry GFP Quantification
3.3.2 Data Analysis (Ratios/Statistics)
4 Notes
References
Index