The development of photo-crosslinkable trapping mutants as tools to investigate the interactions of protein tyrosine phosphatases

24 Chapter 1: Introduction.26 1.1 Cell signaling and protein phosphorylation.26 1.2 PPs.27 Figure 1: PPs and PKs maintain homeostasis of protein phosphorylation. 1.3 PTPs.29 Figure 2: Catalytic mechanism of PTPs. Figure 3: Principle of regulation of PTPs through oxidation. 1.3.1 The Class I of PTPs.35 1.3.1.1 DUSPs.36 Figure 4: Classification of PTPs. 1.3.1.2 MKPs.38 1.3.1.3 Atypical DUSPs.40 1.4 VHR.41 1.4. 1 General properties of VHR.41 1.4. 2 Structural characteristics of VHR.43 Figure 5: Comparing structure of VHR with classical PTPs PTP1B and Yersinia PTP. 1.4. 3 Physiological substrates of VHR.46 Figure 6: Summary of known Physiological substrates of VHR. 1.4. 4 Regulation of VHR.50 1.4. 5 VHR in cancer.51 4 1.5 The PRLs.54 1.5.1 Prenylation of the PRLs.55 1.5.2 Expression of the PRLs.56 1.5.3 Subcellular localisation of the PRLs.58 1.5.4 Structural characteristics of the PRLs.60 Figure 7: Structure-based multiple sequence alignment of all three PRLs and the structurally most closely-related PTPs. Figure 8: Solution structure of PRL-3 (PDB: 1R6H). 1.5.5 The PRLs in cancer.69 1.5.6 Regulation of the PRLs by oxidation.74 1.5.7 Other regulatory mechanisms of the PRLs: PRL-1 and PRL-2.76 1.5.8 Interacting proteins and substrates of PRL-1 and PRL-2.78 1.5.9 The PRLs in signaling: PRL-1 and PRL-2.80 1.5.10 Regulation of PRL-3.84 1.5.11 Interacting proteins and substrates of PRL-3.88 1.5.12 PRL-3 in signaling.95 Figure 9: Summary of the signaling pathways affectd by the PRLs and the outcome in cell migration and proliferation. 1.6 Challenges in identifying PTP substrates and interacting partners.102 1.7 Expanded genetic code methodology.103 Figure 10: A general principle of expanded genetic code methodology. Figure 11: Difference in the expression of prokaryotic and eukaryotic tRNAs. 1.7. 1 pBPA.112 Figure 12: Principle of photo-induced cross-linking with pBPA. 1.8 Aims.113 Chapter 2: Materials and Methods.115 2.1 Materials and methods for Chapters 3.1-3.3.115 2.1.1 Reagents.115 2.1.2 Materials.116 2.1.3 Instruments.118 2.1.4 Miscellaneous.119 2.1.5 Buffers, solutions and media.120 5 2.1.5.1 TAE.120 2.1.5.2 SDS-PAGE running buffer.120 2.1.5.3 SDS-PAGE sample buffer.120 2.1.5.4 SDS (10%) stock solution). 121 2.1.5.5 Stacking buffer (4 x) ). 121 2.1.5.6 Resolving buffer (4 x) ). 121 2.1.5.7 Ammonium persulfate (10%)). 121 2.1.5.8 Tris-HCl (1M, pH 7.5). 121 2.1.5.9 Phosphate buffer saline (PBS) . 122 2.1.5.10 EDTA (0.5 M, pH 8.0) . 122 2.1.5.11 Tris-buffered saline (TBS) (10 x) . 122 2.1.5.12 TBS-Tween20 (TBS-T) . 122 2.1.5.13 Staining solutions for protein gels.122 2.1.5.14 Staining nitrocellulose membranes.123 2.1.5.15 Protein transfer.123 2.1.5.16 Stripping buffer for immunoblots.123 2.1.5.17 Striping buffer for His Trap HP columns.123 2.1.5.18 Recharging buffer for His Trap HP columns.123 2.1.5.19 Eluting buffer for GSTrap FF columns.124 2.1.5.10 SOC medium.124 2.1.5.21 Media for cultivation of bacterial cultures.124 2.1.5.22 Lysis buffers for mammalian cell lines.125 2.1.6 DNA amplification by PCR.125 2.1.7 DNA ligation.126 2.1.8 Procedure for DNA isolation.126 2.1. 9 Procedure for DNA propagation.126 2.1.10 Procedure for transformation of chemically competent strains of Escherichia coli (E. coli) . 127 2.1.11 Transformation procedure for electrocompetent E. coli strains.127 2.1.12 Antibodies (ab) and Western blotting.128 2.1.13 Maintenance of mammalian cell lines.130 2.1.14 Removal of His6-tag by GST-TEV . 130 2.1.15 Intact protein sample analysis by LC-MS.131 2.1.16 Circular dichroism (CD) measurements.132 6 2.2 Materials and methods for Chapter 3.1.133 2.2.1 Engineering of the PRL-3: D72A, I141Y, N142Y, N142A, Q145Y.133 Figure 13: Plasmid map for the pET15b vector. 2.2.2 Large-scale bacterial expression of the PRL-3 variants from 2.2.1.135 2.2. 3 Large-scale purification of the PRL-3 variants from 2.2.2.135 2.2. 4 Characterisation of the catalytic activity for the PRL-3 variants I141Y, N142Y, N142A and Q145Y.136 Figure 14: Principle of EnzCheck phosphatase assay kit. 2.2. 5 Generating the PRL-3 variants for small-scale optimisation of conditions for expression with an unnatural amino acid: wtPRL-3, D72TAG, I141TAG, N142TAG.138 Figure 15: Plasmid map for the pTXB3 vector. 2.2. 6 Optimising expression and purification conditions with wtPRL-3 on a small scale.140 2.2. 7 Small-scale test-expression with pACF.141 Figure 16: Chemical structure of pACF. 2.2. 8 Small-scale test-expression with pBPA.142 2.2. 9 Generating pETM20/6His-3Flag-D72TAG, I141TAG and N142TAG.142 Figure 17: Plasmid map for the pCMV-3Flag-1A vector. Figure 18: Plasmid map for the pETM20 vector. Figure 19: Summary o