DEVELOPING SMALL-MOLECULE CHEMICAL BIOLOGY TOOLS FOR STUDYING CELLULAR PHOSPHORYLATION EVENTS IN VIVO CANDY LU HEOK SIEW A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements: The precious experience I gained and learnt in Yao lab for the past years is beyond expression I would like to express my deepest gratitude to people who have helped and guided me through this memorable journey I would like to first express my deepest gratitude to Prof Yao Shao Qin, my mentor and supervisor, the first person who provided me the opportunity to explore proteomics and introduced me into the field of chemical biology His strict guidance and untiring high spirited enthusiasm have significantly impacted and shaped me into a responsible scientific researcher His logical scientific thinking, great insights in chemical biology and acute problem solving approach helped me overcome my superficiality in understanding science I shall miss his intellectual scientific discussion and streaming new ideas after leaving the Yao group I would not be able to endure this journey without the support of my beloved husband Kim, my adorable Bobby, my little boy Tay Hwan and also my family in Malaysia I would like to sincerely thank them for their understanding, concern and constant loving support I must say that I am blessed to have them walking through with me the course of this degree I would like to further extent my gratitude to Souvik, who has been a great supportive friend and a dedicated mentor in microscope and cell culture techniques; Mahesh, who has been a great teacher in teaching me microarray techniques, and intellectual scientific reasoning in solving problems Special thanks to Hong Yan and Jun Qi, who have been such great collaborators; Liu Kai, who has always guided me on special research techniques and scientific knowledge; Lay Pheng who helped and guided me a lot when I first joined the lab We have learned so much from one another both at the professional level and personal level i Sincere thanks are due to all my other colleagues Li Qian, Wu Hao, Farhana, Grace, Kitty, Wei Lin, Shen Yuan, Jing Yan, Pengyu, Haibin, Raja, Kalesh and all the members of the chemistry lab Thank you for their help and simply being there for me for the past years, providing me a nice and warm second home- Yao lab Last but not the least I would like to thank NUS for financial support through the research scholarship ii Table of Contents Chapter 1: Introduction 1.1 Summary 1.2 Proteomics – An Overview 1.3 The Role of Phosphorylation 1.4 Small Molecule Chemical Biology Tools for Studying Cellular Phosphorylation Events In vivo 1.4.1 Small-Molecule Mimetics of Phosphorylated Amino Acids 1.4.2 Activity Based Probes for Protein Phosphatases and Kinases 13 1.4.3 Synthetic Peptide-based Biosensors 20 1.4.4 Chelation-Enhanced Biosensors 22 1.4.5 Small-Molecule Environment-Sensitive Biosensors 26 1.4.6 Genetically Encoded Protein Biosensors for measurement of 30 In vivo Phosphorylation Event 1.4.7 Small-Molecule Microarray for High-Throughput Screening 35 of Cellular Phosphorylation 1.5 High-Throughput Imaging in System Biology 39 iii Chapter 2: PTP1B-ABDF Substrate Trapping Mutant as Protein Biosensor in Detecting Catalytic Activity of C-Src Kinase 2.1 Summary 43 2.2 Introduction 45 2.3 Result and Discussion 2.3.1 Microplate experiment using ABDF labeled PTP1B- 51 STM and PTP1B -DM against PTP1B binding peptide 2.3.2 Microplate investigation on the binding of PTP1B-ABDF 54 against c-Src catalytic domains 2.3.3 Microplate investigation on the binding of PTP1B-ABDF 59 against pRSET c-Src, a catalytically impaired full length c-Src 2.3.4 Microplate investigation on the binding of PTP1B-ABDF 62 against full length active recombinant c-Src and CSK stimulation 2.3.5 In vivo imaging using PTP1B-ABDF against endogenous 65 c-Src Protein Tyrosine Kinase 2.3.6 The use of PTP1B-ABDF to monitor pY416 67 phosphorylation in MCF7 cells induced by PDGF stimulation 2.3.7 In vivo imaging using PTP1B-ABDF against c-Src kinase 69 transfected cells with H2O2 stimulation 2.4 Conclusion 73 iv Chapter 3: High-throughput Imaging of Phsphatase Localization 3.1 Summary 75 3.2 Introduction 76 3.3 Result ad Discussion 3.3.1 LR reaction and colony PCR of Human ORFeome 79 phosphatase plasmids 3.3.2 Subcellular localization of Human ORFeome 83 phosphatase in pDEST47 & pDEST53 expression host 3.4 Conclusion 87 Chapter 4: Experimental Procedures 88 Chapter 5: References 103 Chapter 6: Appendix 6.1 Supplemental Tables 114 6.2 Supplemental Figures 122 v Summary Amongst all the cellular signaling pathways, perhaps the most ubiquitous posttranslational modification used to regulate protein activity is protein phosphorylation Aberrant regulation of the participants of the phosphoproteome network has been implicated in a number of cancer-related diseases, making them the second most important group of drug targets in medicinal research today A major challenge in understanding protein phosphorylation is the sheer complexity of the phosphoproteome network based on the fact that precise timing and spatial aspects of protein phosphorylation are crucial to cell functioning This thesis highlights on the small-molecule based chemical biology tools used to study protein phosphorylation Chapter focuses on the use of smallmolecule mimetics of phosphorylated amino acids and how their use has aided in the study of transient phosphorylation events with both spatial and temporal precision The development of small-molecule biosensors and how it has in the last few years reached the stage where they may be deployed as viable alternatives to traditional protein-based biosensors will be discussed The development of novel smallmolecule tools, such as kinase crosslinkers to identify upstream kinase that is currently untenable using traditional techniques will be highlighted Finally, the use of small-molecules microarrays, high-throughput screening tool and high-throughput imaging that can be used to map out the complex network of the phosphoproteome will be discussed These applications will be highlighted with novel strategies and designs of protein biosensor in the later chapters (Chapter & 3) for the explicit understanding on protein localizations and its functions, with the hope to ultimately lead to protein substrate identification which could potentially serves as drug target appraisal vi List of Publications (2008-2011) Journals: Lu, C.H.S.; Liu, K ; Yao, S.Q (2011) Current small-molecule chemical biology tools for studying cellular phosphorylation events Chem.–Eur J (Submitted) Uttamchandani, M.; Lu, C.H.S.; Yao, S.Q (2009) Next Generation Chemical Proteomic Tools for Rapid Enzyme Profiling Acc Chem Res., 42, 1183-1192 Lu, C.H.S.; Sun, H.; Bakar, F.B.A.; Uttamchandani, M.; Zhou, W.; Liou, Y.-C.; Yao, S.Q (2008) Rapid Affinity-Based Fingerprinting of 14-3-3 Isoforms Using A Combinatorial Peptide Microarray Angew Chem Intl Ed., 47, 7438-7441 Sun, H.; Lu, C.H.S.; Uttamchandani, M.; Xia, Y.; Liou, Y.-C.; Yao, S.Q (2008) Peptide Microarray for High-throughput Determination of Phosphatase Specificity and Biology Angew Chem Intl Ed., 47, 1698-1702 Sun, H.; Lu, C.H.S.; Shi, H.; Gao, L.; Yao, S.Q (2008) Peptide Microarrays for High-throughput Studies of Ser/Thr Phosphatases Nat Protocols, 3, 1485-1493 vii List of Abbreviations 2DE Two-dimensional electrophoresis AA Amino acid ABP Activity base probe ABPP Activity based protein profiling AfBP Affinity Based Probe Ala Alanine Amp Ampicillin BSA Bovine serum albumin CaCl2 Calcium chloride CBD Chitin Binding Domain CFP Cyan fluorescent protein C-terminus Carboxyl terminus Cys Cystein Da Dalton DNA Deoxyribonucleic acid DNTP Deoxy Nucleotide Tri Phosphate E coli Escherichia coli EDTA Ethylenediamine tetraacetic acid EGFP Enhanced Green Fluorescent Protein EPL Expressed Protein Ligation FP Fluorescent Protein FRET Förster resonance energy transfer GFP Green fluorescent protein Gly Glycine viii GSH Glutathione GST Glutathione-S-transferase HCl Hydrochloric acid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performance liquid chromatography HTS High-throughput screening KRD Kinase recognition domain LB Luria-bertani LC Liquid chromatography MALDI Matrix-assisted laser desorption ionization NaCl Sodium chloride NCL Native Chemical Ligation Ni-NTA Nickel- Nitrilo Tri Acetic acid OD Optical Density ORF Open Reading Frame PAGE Polyacrylamide gel electrophoresis PBD Phospho-binding domain PBS Phosphate buffered saline pH Negative logarithm of the hydroxonium ion concentration phTyr PhosphoTyrosine PPI Protein-protein interaction PTK Protein tyrosine kinase RE Restriction Enzyme ix 70 a) M D Allen, L M DiPilato, M Rahdar, Y R Ren, C Chong, J O Liu, J Zhang, ACS Chem Biol., 2006, 1, 371-376; b) M D Allen, J Zhang, Biochem Biophys Res Commun., 2006, 348, 716-721; c) J Zhang, C J Hupfeld, S S Taylor, J M Olefsky, R Y Tsien, Nature, 2005, 437, 569-573; d) J Zhang, Y Ma, S S Taylor, R Y Tsien, Proc Natl Acad Sci USA, 2001, 98, 1499715002 71 K J Herbst, Q Ni, J Zhang, IUBMB Life, 2009, 61, 902-908 72 a) K L Dodge-Kafka, J Soughayer, G C Pare, J J Carlisle Michel, L K Langeberg, M S Kapiloff, J D Scott, Nature, 2005, 437, 574-578; b) X Gao, J Zhang, Mol Biol Cell., 2008, 19, 4366-4373 73 M Offterdinger, V Georget, A Girod, P I Bastiaens, J Biol Chem., 2004, 279, 36972-36981 74 J Zhang, M D Allen, Mol BioSyst., 2007, 3, 759-765 75 Y Kawai, M Sato, Y Umezawa, Anal Chem., 2004, 76, 6144-6149 76 C T Chan, R Paulmurugan, R E Reeves, D Solow-Cordero, S S Gambhir, Mol Imaging Biol., 2009, 11, 144-158 77 K J Herbst, M D Allen, J Zhang, J Am Chem Soc., 2011, 133, 5676-5679 78 I A Yudushkin, A Schleifenbaum, A Kinkhabwala, B G Neel, C Schultz, P I Bastiaens, Science, 2007, 315, 115-119 79 S Ray-Saha, A Schepartz, ChemBioChem., 2010, 11, 2089-2091 80 a) H J Carlson, R E Campbell, Curr Opin Biotechnol., 2009, 20, 19-27; b) A Ibraheem, R E Campbell, Curr Opin Chem Biol., 2010, 14, 30-36; c) A E Palmer, Y Qin, J G Park, J E McCombs, Trends Biotechnol., 2011, 29, 144- 109 152 81 S Q Yao, M Uttamchandani and C H S Lu, Accounts of Chemical Research, 2009, 42, 1183-1192 82 M Uttamchandani, S Q Yao, Curr Pharm Des., 2008, 14, 2428-2438 83 M Schutkowski, U Reineke, U Reimer, ChemBioChem, 2005, 6, 513-521 84 M Kohn, M Gutierrez-Rodriguez, P Jonkheijm, S Wetzel, R Wacker, H Schroeder, H Prinz, C M Niemeyer, R Breinbauer, S E Szedlacsek, H Waldmann, Angew Chem Int Ed., 2007, 46, 7700-7703 85 K Martin, T H Steinberg, L A Cooley, K R Gee, J M Beechem, W F Patton, Proteomics, 2003, 3, 1244-1255 86 H Sun, C H Lu, H Shi, L Gao, S Q Yao, Nat Protocols, 2008, 3, 1485-1493 87 H Sun, C H Lu, M Uttamchandani, Y Xia, Y C Liou, S Q Yao, Angew Chem Int Ed., 2008, 47, 1698-1702 88 a) L Gao, H Sun, S Q Yao, Biopolymers, 2010, 94, 810-819; b) H Sun, L P Tan, L Gao, S Q Yao, Chem Commun., 2009, 677-679 89 L Meng, G A Michaud, J S Merkel, F Zhou, J Huang, D R Mattoon, B Schweitzer, BMC Biotechnol., 2008, 8, 22 90 C H Lu, H Sun, F B Abu Bakar, M Uttamchandani, W Zhou, Y C Liou, S Q Yao, Angew Chem Int Ed., 2008, 47, 7438-7441 91 M Uttamchandani, W L Lee, J Wang, S Q Yao, J Am Chem Soc., 2007, 129, 13110-13117 92 H Wu, J Ge, S Q Yao, Angew Chem Int Ed., 2010, 49, 6528-6532 93 S Wiemann, D Arlt, W Huber, Genome Res., 2004, 14, 2136-2144 110 94 G M Süel, J Garcia-Ojalvo, L M Liberman, M B Elowitz, Nature, 2006, 440, 545-550 95 E Poustelnikova, A Pisarev, M Blagov, M Samsonova, J Reinitz, Bioinformatics, 2004, 20, 2212–2221 96 R Pepperkok, J Ellenberg, Nat Rev Mol Cell Biol., 2006, 7, 690–696 97 A Sigal and R Milo, et al Nature Method, 2006, 3, 525 – 531 98 A A Cohen, et al Science, 2008, 322, 1511-1516 99 G Manning, D B Whyte, R Martinez, T Hunter and S Sudarsanam, Science, 298, 1912–1934 100 O Alper1 and E T Bowden, Current Pharmaceutical Design, 2005, 11, 11191130 101 A B Reynolds, J Vila, T J Lansing, W M Potts, M J Weber, J T Parsons, EMBO J., 1987, 6, 2359-64 102 P Yaciuk, J K Choi, D Shalloway, Mol Cell Biol 1989, 9, 2453-63 103 T Takeya and H Hanafusa, Cell, 1983, 32, 881–890 104 J Schlessinger and A Ullrich Neuron, 1992, 9, 383–91 105 G A Rodrigues and M Park Curr Opin Genet Dev., 1994, 4, 15–24 106 T Hunter Cell, 1997, 88, 333– 46 107 a) W Xu, S C Harrison, M J Eck, Nature, 1997, 385, 595-602; b) J C Williams, et al J Mol Biol., 1997, 274, 757-75; c) W Xu, A Doshi, M Lei M, M J Eck, S C Harrison, Mol Cell.,1999, 3, 629-38 108 M T Brown and J A Cooper, Biochim Biophys Acta., 1996, 1287, 121–149 109 a) C O Arregui, J Balsamo, J Lilien, J Cell Biol., 1998, 143, 861–73; b) A 111 Cheng, G S Bal, B P Kennedy, M L Tremblay, J Biol Chem., 2001, 276, 25848–55; c) F Liang, S Y Lee, J Liang, D S Lawrence,Z Y Zhang, J Biol Chem., 2005, 280, 24857–63 110 S Dadke and J Chernoff, J Biol Chem., 2003, 278, 40607–11 111 S Zhu, J D Bjorge and D J Fujita, Cancer Res., 2007, 67, 10129-10137 112 X L Guo, Y Z Zhong, Yeshiva University, 2008, AAT 3318743 113 J D Bjorge, A Pang, D J Fujita, J Biol Chem., 2000, 275, 41439–41446 114 A J Flint, T Tiganis, D, Barford and N K Tonks, Proc Natl Acad Sci., USA, 1997, 94,1680–1685 115 S K Hansen, M T Cancilla, T P Shiau, J Kung, T Chen and Daniel, Biochemistry, 2005, 44, 7704–7712 116 F Sicheri, I Moarefi and J Kuriyan, 1997 Nature, 385, 602–609 117 R M Kypta, Y Goldberg, E T Ulug, S A Courtneidge, Cell, 1990, 62, 481– 492 118 S M Thomas_and J S Brugge, Annu Rev Cell Dev Biol., 1997, 13, 513–609 119 S Zhougang and R G Schnellmann, Am J Physiol Renal Physiol., 2004, 286, F858-F865 120 A MacAuley, M Okada, S Nada, H Nakagawa, J A Cooper, Oncogene, 1993, 8, 117–124 121 J D Bjorge, C Bellagamba, H C Chengi, A Tanaka, J H Wang, D J Fujita, J Biol Chem., 1995, 270, 24222–24228 122 S Dadke, J Chernoff, Biochem J., 2002, 364, 377-383 123 D J Kemble, Y H Wang, G Sun, Biochemistry, 2006, 45, 14749-14754 112 124 P Cohen P Trends Biochem Sci., 2000, 25, 96–601 125 Z-Y Zhang Curr Opin Chem Biol., 2001, 5, 416–23 126 M Elchebly, P Payette, E Michaliszyn, W Cromlish, S Collins, et al Science, 1999, 283, 1544– 48 127 L D Klaman, O Boss, O D Peroni, J K Kim, J L Martino, et al Mol Cell Biol., 2000, 20, 5479–89 113 Chapter Appendix 6.1 Supplemental Tables No Clone id ORF 96-well Plate Localization (Alternate) Description 8622 10147 246 435 A1 A2 Cytoplasm Nucleus Nucleus Cytoplasm 2039 654 A3 Cytoplasm Dual specificity phosphatase 22 Dual specificity phosphatase (vaccinia virus phosphatase VH1-related) Dual specificity phosphatase 19 8591 678 A4 Nucleus 1089 930 A5 Cytoplasm Nucleus 3745 1200 A6 Cytoplasm 6062 1179 A7 Cytoplasm Plsma membrane Nucelus 6082 1254 A8 Cytoplasm 641 2115 A9 Cytoplasm 10 9013 267 A10 ER Golgi, Nucleus, Plasma membrane Entry Clone Miniprep Miniprep LR Rxn GFP C-terminal Miniprep Miniprep LR Rxn GFP N-terminal Miniprep Miniprep Miniprep Miniprep Miniprep Protein phosphatase 2A 48 kDa regulatory subunit Protein phosphatase (formerly 2A), catalytic subunit, beta isoform Protein tyrosine phosphatase, non-receptor type 7, transcript variant Integrin-linked kinase-associated serine/threonine phosphatase 2C, transcript variant Hypothetical protein FLJ22405 Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Myotubularin related protein Miniprep Miniprep Miniprep Epilepsy, progressive myoclonus type 2A, Lafora disease (laforin) Miniprep Miniprep Miniprep 114 11 7420 318 A11 Mitochondria Cytochrome c, somatic Miniprep Miniprep Miniprep 12 3382 447 A12 Plasma membrane Protein tyrosine phosphatase type IVA, member 3, transcript variant Miniprep Miniprep Miniprep 13 9048 537 B1 Cytoplasm Slingshot Miniprep Miniprep Miniprep 14 15 6314 4071 597 930 B2 B3 Cytoplasm Cytoplasm Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep 1155 B4 Chromosome Miniprep Miniprep Miniprep 3508 1641 B5 Nucleoplasm Cytoplasm Dual specificity phosphatase 13 Protein phosphatase (formerly 2A), catalytic subunit, alpha isoform Protein phosphatase 2, regulatory subunit B (B56), gamma isoform, transcript variant Protein phosphatase 1G (formerly 2C), magnesium-dependent, gamma isoform, transcript variant 16 7603 17 Miniprep Miniprep Miniprep 18 3670 453 B6 Cytoplasm Nucleulos, Cytosol Hypothetical protein FLJ20442 Miniprep Miniprep Miniprep 19 8740 579 B7 Cyoplam Miniprep Miniprep Miniprep 20 8800 531 B8 Nucleus Protein phosphatase 1B (formerly 2C), magnesium-dependent, beta isoform, transcript variant Protein phosphatase 2A 48 kDa regulatory subunit Miniprep Miniprep Miniprep 21 3126 597 B9 Cytoplasm Nucleus Dual specificity phosphatase 14 Miniprep Miniprep Miniprep 22 3671 636 B10 Golgi, Nucleus Hypothetical protein MGC1136 Miniprep Miniprep Miniprep 23 6324 954 B11 Cytoplasm Miniprep Miniprep Miniprep 24 6656 936 B12 Miniprep Miniprep Miniprep 25 7076 972 C1 Plasma membrane Nucleus Miniprep Miniprep Miniprep 26 7862 963 C2 Cytoplasm Protein phosphatase 1, regulatory (inhibitor) subunit 3C Phosphatidic acid phosphatase type 2B, transcript variant Protein phosphatase 1, catalytic subunit, gamma isoform Dual specificity phosphatase Miniprep Miniprep Miniprep Cytoplasm, Cytosol Cytoplasm, nucleus, endosome Chromosome Cytoplasm, nucleolus 115 27 28 882 7554 1086 1062 C3 C4 Cytoplasm ER 29 5095 1146 C5 Cytoplasm 30 5861 1770 C6 Cytoplasm 31 1551 2004 C7 Cytoplasm 32 2100 1962 C8 33 5606 318 C9 Mitochondria 34 35 8056 8703 378 387 C10 C11 36 8316 513 C12 Cytoplasm Plasma membrane Cytoplasm 37 159 744 D1 Nucleus 38 4210 867 D2 Integral to membrane 39 6415 867 D3 40 5533 918 D4 Nucleus 41 7929 924 D5 Nucleus 42 88 957 D6 Nucleus 43 3788 993 D7 Cytosketelon Nucleus, Cytoplasm Nucleus Chromosome, membrane fraction Nucleus, membrane fraction Cytoplasm, Cytosol Nucleus, plasma membrane Plasma membrane Cytoplasm, ER Centrosome, Cytoplasm Nucleolus Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Protein phosphatase (formerly 2A), regulatory subunit A (PR 65), beta isoform, transcript variant Miniprep Miniprep Miniprep Protein phosphatase, EF hand calcium-binding domain 1, transcript variant Miniprep Miniprep Miniprep Cytochrome c, somatic Miniprep Miniprep Miniprep Phosphohistidine phosphatase Protein tyrosine phosphatase, receptor type, S Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Protein phosphatase (formerly 2B), regulatory subunit B, 19kDa, alpha isoform (calcineurin B, type I) Likely ortholog of mouse protein phosphatase 2C eta Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Phosphatidic acid phosphatase type 2C, transcript variant Protein tyrosine phosphatase-like (proline instead of catalytic arginine), member a Protein phosphatase 6, catalytic subunit Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Protein phosphatase (formerly X), catalytic subunit Hypothetical protein MGC10067 Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Protein phosphatase 1, catalytic subunit, alpha isoform Miniprep Miniprep Miniprep Protein tyrosine phosphatase, non-receptor type 2, transcript variant Dual specificity phosphatase 6, transcript variant Protein phosphatase (formerly 2A), regulatory subunit A (PR 65), alpha isoform 116 44 4085 984 D8 Nucleolus Protein phosphatase 1, catalytic subunit, beta isoform Dual specificity phosphatase 11 (RNA/RNP complex 1-interacting) Slingshot Miniprep Miniprep Miniprep 45 4668 993 D9 Nucleus Nucleolus Miniprep Miniprep Miniprep 46 4719 1026 D10 Cytoplasm Nucleus Miniprep Miniprep Miniprep 47 48 4859 2025 1023 1110 D11 D12 Nucleus Plasma membrane Cytoplasm Dual specificity phosphatase 12 Phosphatase and tensin homolog (mutated in multiple advanced cancers 1), pseudogene Hypothetical protein DKFZp761G058 Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep 49 9757 1119 E1 Miniprep Miniprep Miniprep 50 2502 1149 E2 Protein phosphatase 1A (formerly 2C), magnesium-dependent, alpha isoform, transcript variant Dual specificity phosphatase 6, transcript variant Miniprep Miniprep Miniprep 51 3809 1146 E3 Miniprep Miniprep Miniprep 52 5798 1161 E4 Acid phosphatase, prostate Miniprep Miniprep Miniprep 53 4057 1185 E5 Miniprep Miniprep Miniprep 1776 1293 E6 Miniprep Miniprep Miniprep 55 2939 1308 E7 Miniprep Miniprep Miniprep 56 8117 1332 E8 Miniprep Miniprep Miniprep 57 2863 1383 E9 Dual specificity phosphatase 4, transcript variant Protein phosphatase (formerly 2A), regulatory subunit B (PR 52), gamma isoform Protein tyrosine phosphatase, non-receptor type Protein phosphatase (formerly 2A), regulatory subunit B (PR 52), beta isoform, transcript variant Protein tyrosine phosphatase, non-receptor type 11 (Noonan syndrome 1) 54 Miniprep Miniprep Miniprep 58 7841 1422 E10 Cell division cycle 25C, transcript variant Miniprep Miniprep Miniprep 59 8448 1449 E11 Miniprep Miniprep Miniprep 60 8407 1536 E12 Dual specificity phosphatase 10, transcript variant Protein phosphatase (formerly 2B), catalytic subunit, alpha isoform (calcineurin A alpha) Miniprep Miniprep Miniprep 117 61 4501 1575 F1 Cell division cycle 25A Miniprep 62 63 64 N/A 4946 2573 N/A 1776 2382 N/A F3 F4 N/A Miniprep Miniprep F5 N/A Protein tyrosine phosphatase, receptor type, N Protein tyrosine phosphatase, receptor type, A, transcript variant DKFZP566K0524 protein 65 2020 474 66 4644 477 F6 Acid phosphatase 1, soluble, transcript variant Miniprep 67 4051 558 F7 Miniprep 68 966 1620 F8 Dual specificity phosphatase (vaccinia virus phosphatase VH1-related) Lymphocyte-specific protein tyrosine kinase 69 4334 1539 F9 Protein phosphatase (formerly 2B), catalytic subunit, gamma isoform (calcineurin A gamma) Miniprep 70 4872 1545 F10 Eyes absent homolog (Drosophila), transcript variant Miniprep 71 9578 1590 F11 Miniprep 72 95 1782 F12 73 845 1794 G1 Pyruvate dehydrogenase phosphatase isoenzyme Protein tyrosine phosphatase, non-receptor type RNA guanylyltransferase and 5'-phosphatase 74 1447 1812 G2 Myotubular myopathy Miniprep 75 4543 1794 G3 Miniprep 76 10203 1953 G4 Protein tyrosine phosphatase, non-receptor type 6, transcript variant Hypothetical protein FLJ22405 77 2960 2781 G5 Miniprep 78 3032 2541 G6 Protein tyrosine phosphatase, non-receptor type (megakaryocyte) Discs, large homolog (Drosophila) 79 5976 2541 G7 Discs, large homolog (Drosophila) Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep Miniprep 118 80 81 9933 10144 3588 3555 G8 G9 Miniprep Miniprep G12 Myotubularin related protein Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase Protein phosphatase 1D magnesium-dependent, delta isoform Transmembrane phosphatase with tensin homology, transcript variant Myotubularin related protein 82 838 1818 G10 83 2024 1602 G11 84 10312 1866 85 86 87 88 10667 11227 10846 11351 2103 1611 2802 1989 H1 H2 H3 H4 Protein tyrosine phosphatase, receptor type, E Eyes absent homolog (Drosophila) Protein phosphatase 4, regulatory subunit Dual specificity phosphatase 16 Miniprep Miniprep Miniprep Miniprep 89 11706 1515 H5 Miniprep 90 11764 1797 H6 Mitogen-activated protein kinase interacting protein 1, transcript variant alpha Hypothetical protein FLJ22405 91 11976 708 H7 Dual specificity phosphatase-like 15, transcript variant Miniprep 92 12272 816 H8 93 12358 519 H9 94 12005 1698 H10 95 12408 522 H11 96 12482 555 H12 97 98 13025 12889 213 540 A1 A2 99 13237 1794 A3 100 13368 339 A4 Miniprep Miniprep Miniprep Miniprep Miniprep Cyclin-dependent kinase inhibitor (CDK2associated dual specificity phosphatase) Protein tyrosine phosphatase, non-receptor type (striatum-enriched) Protein tyrosine phosphatase type IVA, member Dual specificity phosphatase 22 Miniprep Miniprep Miniprep Miniprep Acid phosphatase 1, soluble Protein tyrosine phosphatase, non-receptor type 22 (lymphoid) Protein tyrosine phosphatase, receptor type, O 119 101 102 13687 13625 531 1056 A5 A6 Hypothetical protein MGC5987 Protein tyrosine phosphatase, non-receptor type 18 (brain-derived) Protein phosphatase (formerly 2A), regulatory subunit B (PR 52), alpha isoform Eyes absent homolog (Drosophila) 103 13596 1344 A7 104 13613 1851 A8 105 106 13659 15047 2316 1164 A9 A10 107 14140 1362 A11 108 14165 1062 A12 109 14212 234 B1 cDNA clone MGC: 75444 IMAGE:4753885, complete cds 110 14329 1449 B2 111 14428 1752 B3 112 14725 1044 B4 113 14960 423 B5 Dual specificity phosphatase 10, transcript variant 1, mRNA (cDNA clone MGC: 74610 IMAGE: 6178330), complete cds Protein tyrosine phosphatase, non-receptor type 9, mRNA (cDNA clone MGC: 87169 IMAGE: 5276190), complete cds Protein tyrosine phosphatase, receptor type, F, mRNA (cDNA clone IMAGE: 4661274), complete cds cDNA clone MGC: 88825 IMAGE: 4896064, complete cds 114 14912 753 B6 Protein tyrosine phosphatase, non-receptor type 2, transcript variant 2, mRNA (cDNA clone MGC: 9389 IMAGE:3872164), complete cds Protein phosphatase 2, regulatory subunit B, delta isoform, transcript variant 1, mRNA (cDNA clone MGC: 51327 IMAGE: 5267510), complete cds Protein tyrosine phosphatase, receptor type, F, mRNA (cDNA clone IMAGE: 5303842), complete cds Tensin-like SH2 domain containing 1, mRNA (cDNA clone IMAGE: 4611842), complete cds 120 115 14941 504 B7 Protein tyrosine phosphatase type IVA, member 2, transcript variant 1, mRNA (cDNA clone MGC: 88163 IMAGE: 4686913), complete cds 116 14954 1251 B9 117 2715 1500 B10 Tensin, mRNA (cDNA clone IMAGE: 4878104), complete cds Protein phosphatase 5, catalytic subunit 118 119 7390 4352 1161 1734 B11 B12 Acid phosphatase, prostate Protein tyrosine phosphatase, non-receptor type 23 121 6.2 Supplemental Figures 6.2.1 Vector Information- pcDNA-DEST47 122 6.2.1 Vector Information- pcDNA-DEST53 123 ... transduction pathways in eukaryotes 1.4 Small Molecule Chemical Biology Tools for Studying Cellular Phosphorylation Events in vivo 1.4.1 Small- Molecule Mimetics of Phosphorylated Amino Acids The elucidation... Molecule Chemical Biology Tools for Studying Cellular Phosphorylation Events In vivo 1.4.1 Small- Molecule Mimetics of Phosphorylated Amino Acids 1.4.2 Activity Based Probes for Protein Phosphatases... Current small- molecule chemical biology tools for studying cellular phosphorylation events Chem.–Eur J (Submitted) Uttamchandani, M.; Lu, C.H.S.; Yao, S.Q (2009) Next Generation Chemical Proteomic Tools