Determination of the Sequence Specificity and Protein Substrates of Protein Phosphatases

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University

Rinrada Luechapanichkul

Graduate Program in Chemistry

The Ohio State University

2014

Dissertation Committee:

Professor Dehua Pei, Advisor

Professor Ross E. Dalbey

Professor James A. Cowan

Rinrada Luechapanichkul

2014

Abstract

Protein phosphorylation is a post-translational modification controlled by two counteracting enzyme families, protein kinases and phosphatases. Protein phosphatases have been demonstrated to regulate many physiological pathways and exhibit distinct specificity in vivo. However, the factors determining their specificity are not well understood. In this study, the intrinsic specificity of various families of phosphatases has been explored utilizing combinatorial peptide library screening.

The sequence specificity of eight classical-protein tyrosine phosphatases (PTPs)

(PTPRA, PTPRB, PTPRD, PTPRO, PTP-PEST, PTP1B, SHP-1, and SHP-2) was determined. While PTPRA showed no selectivity, the other PTPs exhibited similar preferences for peptides containing acidic residues but disfavored basic ones. However, the enzymes differed in their level of selectivity and catalytic activity. Most of the classical-PTPs screened in this study also contains substrate recruiting/regulatory domains; it is likely that the in vivo PTP specificity is enhanced by the recruiting/regulatory domains.

The sequence specificity of atypical dual-specificity phosphatases (DUSPs), which only contain a single catalytic domain, was examined to determine whether they exhibit more stringent specificity than the classical-PTPs. The screening of Vaccinia

VH1-related (VHR) DUSP against pY-peptide libraries revealed two distinct classes of substrates. While class I peptide substrates are similar to the pY motifs derived from reported VHR protein substrates, the novel class II peptide substrates of the consensus

(V/A)P(I/L/M/V/F)X1-6pY exhibit an alternative-binding mode to VHR, as suggested by site-directed mutagenesis and molecular modeling. ROBO1 and LASP1, which contain the class II consensus motif, were demonstrated to be VHR substrates in vitro.

The haloacid dehydrogenase superfamily (HADSF) phosphatases have been shown to dephosphorylate a wide range of substrates including sequence with pY, phosphoserine (pS), and phosphothreonine (pT). The study utilized pS/pT library screening approach for comprehensive analysis of phosphatase specificity to advance the identification of pS/pT protein substrates. The screening of human small C-terminal domain phosphatase 1 (SCP1) revealed its preference for pS-containing peptides with proline residues at the pS+1 position and hydrophobic residues, especially at the pS-1 to pS-3 positions. The tumor suppressor p53 protein, which contains the consensus sequence obtained from screening, was discovered to be a putative SCP1 substrate. This result was validated by in vitro dephosphorylation of p53-pS33 by SCP1 and modulation the phosphorylation of pS33 level in HEK293 cells.

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Dedication

This document is dedicated to my family.

Acknowledgments

I would like to thank my advisor, Dr. Dehua Pei, who always provides valuable guidance, and continuous support for my research. His passion and dedication truly inspire me and brighten up my scientific journey. His persistence has encouraged me throughout difficult times in my research. I am grateful to Dr. Ross E. Dalbey and Dr.

James A. Cowan for their suggestions and serving as my candidacy and dissertation committees. I would like to thank Dr. Thomas J. Magliery for his wonderful combinatorial chemistry class and Dr. Yusen Liu for his assistance and discussion about phosphorylation.

I would also like to thank Dr. Xianwen Chen, Dr. Nicholas G. Selner, Tiffany W.

Meyer, Dr. Qing Xiao, Dr. Amit Thakkar and Dr. Tao Liu for their mentorship and friendship. I would also like to thank Dr. Lalintip Horechareon, Dr. Supranee

Chaiwatpongsakorn, Dr. Pattavipha Songkumarn, Dr. Yasuro Sugimot and Yaowalak

Pratumyot for their generous advice in mammalian cell culture. I would like to thank all former and current Pei lab members, especially Andrew Kunys and Thi B. Trinh, for their friendship and useful discussion.

Finally, I am very fortunate to have my family and my boyfriend to be part of my life. I am grateful to their love and support.

Vita

2006...... B.Sc. Chemistry (First Class Honors), Mahidol University, Thailand

2006...... Visiting Scholar, Department of Chemistry, University of Wisconsin

2009-2014 ...... Graduate Teaching and Research Associate, Department of Chemistry, The Ohio State University

Publications

1. Selner, N.G., Luechapanichkul, R., Chen. X., Neels, B., Zhang, Z. Y., Knapp, S., Bell, C., Pei, D. Diverse Levels of Sequence Selectivity and Catalytic Efficiency of Protein- Tyrosine Phosphatases. Biochemistry, 2013, 53, 397-412.

2. Xiao, Q., Luechapanichkul, R., Zhai, Y., Pei, D. Specificity Profiling of Protein Phosphatases toward Phosphoseryl and Phosphothreonyl Peptides. Journal of the American Chemical Society, 2013, 135, 9760-976.

3. Luechapanichkul, R., Chen, X., Taha, H. A., Vyas, S., Guan, X., Freitas, M. A., Hadad, C. M., Pei, D. Specificity Profiling of Dual Specificity Phosphatase Vaccinia VH1-related (VHR) Reveals Two Distinct Substrate Binding Modes. The Journal of Biological Chemistry, 2013, 288, 6498-6510.

4. Ren, L,, Chen, X., Luechapanichkul, R., Selner, N. G., Meyer, T. M., Wavreille, A. S., Chan, R., Iorio, C., Zhou, X., Neel, B. G., Pei, D. Substrate Specificity of Protein Tyrosine Phosphatases 1B, RPTPα, SHP-1, and SHP-2. Biochemistry, 2011, 50, 2339- 23563.

5. Fowler, S. A., Luechapanichkul, R., Blackwell, H. E. Synthesis and Characterization of Nitroaromatic Peptoids: Fine Tuning Peptoid Secondary Structure through Monomer Position and Functionality. The Journal of Organic Chemistry, 2009, 74, 1440-1449. vi

Fields of Study

Major Field: Chemistry

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Table of Contents

Abstract ...... ii Acknowledgments...... v Vita ...... vi Table of Contents ...... viii List of Tables ...... xi List of Figures ...... xiv

Chapter 1 INTRODUCTION ...... 1 1.1 Protein Phosphatases ...... 1 1.2. Protein Tyrosine Phosphatases (PTPs) ...... 2 1.2.1. Structure and Catalytic Mechanism ...... 2 1.2.2. Functions of PTPs and related Diseases ...... 3 1.2.3. PTP specificity ...... 4 1.3. The Haloacid Dehydrogenase Superfamily Phosphatases (HADSF) ...... 6 1.3.1. Structure and Catalytic Mechanism ...... 6 1.3.2. Functions and related Diseases ...... 7 1.3.3. HADSF specificity ...... 8 1.4. Approaches to Discover New Phosphatase Substrates ...... 9 1.4.1. Conventional approaches ...... 9 1.4.2. Reverse Interactomics Approach ...... 10 1.5. The Screening of the Combinatorial Peptide Libraries ...... 12 1.5.1. Phosphotyrosine Peptide Library screening ...... 12 1.5.2. Phosphoserine/threonine Peptide Library screening ...... 13

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Chapter 2 SPECIFICITY PROFILEING OF CLASSICAL PROTEIN-TYROSINE PHOSPHATASES ...... 18 2.1. Introduction ...... 18 2.2.1. Materials ...... 22 2.2.2. Purification of PTPs...... 23 2.2.3. Library Synthesis ...... 23 2.2.4. Library Screening ...... 24 2.2.5. Synthesis of Selected Peptides...... 25 2.2.6. PTP Assays ...... 26 2.3. Results ...... 27 2.3.1. Peptide Library Design and Screening ...... 27 2.3.2. Substrate Specificity of PTPs ...... 28 2.3.3. Kinetic Properties of Selected Peptides toward PTPRA ...... 33 2.3.4. Positional insensitivity of Acidic Residues ...... 34 2.4. Discussion ...... 34

Chapter 3 SPECIFICITY PROFILEING OF DUAL SPECIFICITY PHOSPHATASE VACCINIA VH1-RELATED (VHR) ...... 51 3.1 Introduction ...... 51 3.2. Experimental Procedures...... 54 3.2.1. Materials ...... 54 3.2.2. VHR Purification and Library Screening ...... 54 3.2.3. Synthesis of Selected Peptides and Enzymatic Assays ...... 55 3.2.4. Molecular Modeling ...... 55 3.2.5. In Vitro Protein Dephosphorylation by VHR ...... 56 3.2.6. In-solution Digestion ...... 57 3.2.7. Mass Spectrometry ...... 57 3.3. Results ...... 58 3.3.1. VHR Substrate Specificity (N-terminal to pY) ...... 58 3.3.2. Identification of Other Class II Motifs and Optimal Distance from pY ...... 60 3.3.3. VHR Substrate Specificity (C-terminal to pY)...... 62 3.3.4. Kinetic Properties of Selected Peptides ...... 63

3.3.5. Class II Substrates Bind VHR in an Alternative Mode ...... 65 3.3.6. Molecular Modeling ...... 67 3.3.7. In Vitro Dephosphorylation of Class II Protein Substrates by VHR ...... 69 3.4. Discussion ...... 70 3.5. Specific Contributions ...... 74

Chapter 4 SPECIFICITY PROFILEING OF SMALL C-TERMINAL DOMAIN ...... 96 PHOSPHATASE 1 (SCP1)...... 96 4.1 Introduction ...... 96 4.2. Experimental Procedures...... 98 4.2.1. Materials ...... 98 4.2.2. Screening of Peptide Libraries ...... 98 4.2.3. Synthesis of Selected peptides ...... 99 4.2.4. Enzymatic Assays ...... 100 4.2.5. Recombinant GST-Jnk1 expression and purification ...... 101 4.2.6. In vitro Jnk1 Dephosphorylation by SCP1 ...... 102 4.2.7. In vitro p53 Dephophorylation by SCP1 ...... 102 4.2.8. In vivo p53 dephosphorylation by SCP1 ...... 103 4.3. Results ...... 104 4.3.1. Phosphoserine/phosphothreonine substrate specificity of SCP1 ...... 104 4.3.2. SCP1 is active toward pY substrates ...... 106 4.3.3. Kinetic Properties of SCP1 toward Selected Peptide Substrates...... 107 4.3.4. Dephosphorylation of Protein Substrates by SCP1...... 110 4.4. Discussion ...... 111 4.5. Specific Contributions ...... 114

REFERENCES ...... 129

List of Tables

Table 2.1 Peptide libraries employed in this work ...... 38

Table 2.2. Peptide Library Screening Conditions ...... 39

Table 2.3. Most Preferred PTPRA Substrates Selected from Library I

(ASX5pYAABBRM) ...... 40

Table 2.4. Most Preferred PTPRA Substrates Selected from Library III

(ASX5pYAABBRM, with reduced Arg and Lys contents) ...... 40

Table 2.5. Most Preferred PTPRA Substrates Selected from Library IV

(AX5pYX5NNBBRM) ...... 41

Table 2.6. PTPRB Substrates Selected from Library III (ASX5pYAABBRM, with reduced Arg and Lys contents) ...... 42

Table 2.7. PTPRB Substrates Selected from Library IV (AX5pYX5NNBBRM) ...... 42

Table 2.8. PTPRD Substrates Selected from Library II (AX8pYAABBRM) ...... 43

Table 2.9. PTPRO Substrates Selected from Library III (ASX5pYAABBRM, with reduced Arg and Lys contents) ...... 43

Table 2.10. PTPRO Substrates Selected From Library IV (AX5pYX5NNBBRM) ...... 44

Table 2.11. PTP-PEST Substrates Selected from Libraries I and III (ASX5pYAABBRM and ASX5pYAABBRM, with reduced Arg and Lys contents) ...... 45

Table 2.12. PTP-PEST Substrates Selected From Library IV (AX5pYX5NNBBRM) ..... 45

Table 2.13. Preferred PTP1B Substrates Selected from Libraries IV

(AX5pYX5NNBBRM) and library V (AX5pYX5EEBBRM) ...... 46

Table 2.14. Most Preferred SHP-1 and SHP-2 Substrates Selected from Library IV

(AX5pYX5NNBBRM) ...... 47

Table 2.15. Kinetic Properties of PTPRA against pY Peptides ...... 48

Table 2.16. Kinetic Properties of PTP1B against pY Peptides ...... 49

Table 3.1. Composition of peptide libraries used in this work ...... 75

Table 3.2.Most preferred VHR substrates selected from peptide library I ...... 76

Table 3.3. Most preferred VHR substrates selected from libraries II–IX ...... 77

Table 3.4. Preferred VHR Substrates Selected from Peptide Library X ...... 78

Table 3.5. Preferred Substrates Selected from Peptide Library XI...... 79

Table 3.6. VHR activity against selected peptide and protein substrates ...... 80

Table 3.7. Effect of N-terminal amine on WT VHR activity ...... 81

Table 3.8. Activity of WT and mutant VHR against pY peptides ...... 82 pNPP, p-nitrophenyl phosphate...... 82

Table 3.9. D164–V(peptide) H-bonding interaction occupancies in MD ensemble ...... 82

Table 3.10. MMBSA and MMGBSA energies of peptides 21 and 22 bound in VHR ..... 84

Table 3.11. Potential Class II VHR Substrates from Database Search ...... 85

Table 3.12. MS/MS data for ROBO1 proteolytic fragment ...... 86

Table 4.1. Most preferred SCP1 substrates selected from peptide library I ...... 115

Table 4.2. Most preferred SCP1 substrates selected from peptide library II ...... 116

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Table 4.3. Reported SCP1 substrates...... 117

Table 4.4. Most preferred SCP1 substrates selected from peptide library III ...... 118

Table 4.5. Most preferred SCP1 substrates selected from peptide library IV ...... 119

Table 4.6. Catalytic Activity of SCP1 toward pSer/pThr/pTyr peptides ...... 120

Table 4.7. Potential SCP1 substrates from database search...... 121

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List of Figures

Figure 1.1. Catalytic Mechanism of Cys-based PTPs (the amino acid numbering PTP1B).

...... 15

Figure 1.2. Mechanism of Asp-based HADSFs. (amino acid in the human mitochondrial deoxyribonucleotidase)...... 15

Figure 1.3. (A) The screening of PTPs against pY peptide library. (B) A portion of the peptide library after tyrosinase/MBTH treatment...... 16

Figure 1.4. (A) The screening of phosphatases against pS/pT peptide library. (B) A portion of the peptide library after NBDH treatment...... 17

Figure 2.1. Histograms showing the sequence specificity of PTPRA (A), PTPRB (B),

PTPRD (C), PTPRO (D), and PTP-PEST (E) on the N-terminal side of pY...... 50

Figure 3.1. Histogram showing the pY sequence specificity of VHR ...... 87

Figure 3.2. Preliminary Docking Investigations ...... 90

Figure 3.3. Comparison of the canonical and reverse binding modes of pY peptides to

VHR...... 90

Figure 3.4. Representation of H-bonds present between Asp-164 and the N-terminal valine residue of peptide 22...... 91

Figure 3.5. RMSD of the protein backbone of the D164N mutant of VHR...... 92

xiv

Figure 3.6. In vitro dephosphorylation of ROBO1 and LASP1 by VHR...... 93

Figure 3.7. Representative V vs [S] Plots for VHR against pY Substrates...... 94

Figure 3.8. Representative reaction progress curves and data fitting...... 95

Figure 4.1. Histogram showing the sequence specificity of SCP1 on pSer/pThr peptide libraries...... 124

Figure 4.3. In vitro dephosphorylation of SCP1 potential substrates...... 127

Figure 4.4. SCP1 dephosphorylated p53 at pSer33 site...... 128

Chapter 1

INTRODUCTION

1.1 Protein Phosphatases

Protein phosphorylation is a widespread and versatile mechanism that orchestrates many processes in living organisms including cell growth, differentiation, secretion and apoptosis [2]. Major protein phosphorylation sites in human proteins occur on Ser, Thr and Tyr residues, which account for 86.4 %, 11.8 % and 1.8% respectively [3]. Cellular phosphorylation levels are tightly governed by two counteracting enzyme families, protein kinases and phosphatases.

Protein phosphatases have been classified into four major categories based on their structures, catalytic mechanisms and the preference of dephosphorylation sites. The first group, protein tyrosine phosphatases (PTPs), utilizes an active site Cys as a nucleophile. This group can be further divided into three subgroups: the classical protein tyrosine phosphatases (classical-PTPs) which can solely dephosphorylate phosphotyrosine (pY) residue, dual-specificity phosphatases (DUSPs), which can dephosphorylated phosphoserine (pS), phosphothreonine (pT) and phosphotyrosine residues, and other phosphatases including cell division cycle 25 (cdc25) and low molecular weight PTPs (LMWPTP) [3, 4]. The remaining groups include phosphoprotein

Ser/Thr phosphatases (PPPs), metal-dependent Ser/Thr phosphatases (PPMs) and haloacid dehydrogenase superfamily (HADSF) phosphatases. However, additional information is required to understand the regulation of phosphatase activity and specificity of PPP and PPM [5, 6]. PPP phosphatases generally have multi-subunit complexes of a catalytic subunit and a variety of regulatory subunits. To date, no obvious consensus of the pS/pT peptide substrates has been identified [5]. Conversely, classical

PTPs, DUSPs and HADSFs typically contain a single chain of multi domains.

Phosphatases have been shown to display stringent specificity and tight cellular regulation in vivo. This project is focused on Classical-PTPs, DUSPs, and HADSFs.

1.2. Protein Tyrosine Phosphatases (PTPs)

1.2.1. Structure and Catalytic Mechanism

The PTP superfamily is characterized by having a unique signature motif HCX5R situated in the active site cleft of the PTP catalytic domain. All PTPs contain a highly conserved active site and have very similar overall structures [7]. Most PTPs consist of a combination of modular domains: the catalytic domain and the recruiting domain, e.g.

Src homology 2 (SH2), fibronectin type III-like domain (FN), kinase-interacting motif

(KIM). The PTP catalytic domain is composed of approximately 280 amino acids that form an / fold structure [8-10]. The minimal catalytic core of PTPs features three distinct motifs including the PTP signature motif, WPD loop, and Q loop. The PTP signature motif contains a nucleophilic Cys and a transition state stabilizer Arg residue

[7]. The mobile Trp-Pro-Asp “WPD” loop forms the sides of the PTP active site cleft.

Aspartic acid in the WPD loop functions as a general acid/base during catalysis. The Q-

2 loop accommodates Gln to mediate the phosphate release by coordinating water during hydrolysis step [11].

All PTPs catalyze dephosphorylation reaction via a common mechanism (Figure

1.1). The reaction starts with the nucleophilic attack of phosphotyrosine substrate by the

Cys215 (number according to Protein Tyrosine Phosphatase 1B (PTP1B)) to generate an enzyme intermediate. During this step, Asp181 functions as a general acid to protonate the phenoxide leaving group. In the second step, the hydrolysis of the phosphoenzyme intermediate mediated by Gln262, which coordinates a water molecule, and Asp181, which acts as a general base, releases inorganic phosphate and active enzyme [12]. The selectivity of PTPs for phosphotyrosine substrates has been attributed to the phosphotyrosine recognition loop (Tyr46 in PTP1B), which restricts access to phosphoserine and phosphothreonine residues and determines the depth of the active site.

The depth of the active site crevice was determined to be about 9 Å for classical PTPs and 6 Å for DUSPs [13]. The co-crystal structure of PTPs with pY peptides have shown that PTPs usually recognize three to five amino acids on each side of pY [14-17]. The amino acid residues surrounding the active site are poorly conserved substrate-binding surfaces, which also contribute to the distinct in vivo substrate specificity of PTPs.

1.2.2. Functions of PTPs and related Diseases

PTPs control a broad spectrum of fundamental physiological processes. The variety of PTP functions has been attributed to their structural differences. PTPs have been shown to function both as negative and positive regulators in cell signaling transduction [10]. A link between PTP1B and diabetes and obesity has been established.

PTP1B has been shown to be a major negative regulator of both insulin and leptin signaling, and PTP1B-deficient mice show increased insulin sensitivity and obesity resistance [18, 19]. Conversely, PTP1B has functioned as a positive regulator in HER2 signaling. Over-expression of PTP1B has been demonstrated to correlate with the expression of HER2. PTP1B-knockout mice delayed breast tumor development. These studies suggest that a PTP1B is an important drug target and inhibition of PTP1B potentially enhances the insulin resistance and attenuates malignancy in HER2-positive breast cancer [20, 21]. Several studies have also shown that PTPs with highly structure similarity exhibits different functions. SHP-1 and SHP2 are SH2 domain containing phosphatases that share 61% sequence identity in their catalytic domains and 55% overall sequence identity [22]. While SHP-1 loss-of-function has demonstrated a severe combined immunodeficiency syndrome in mice [23], SHP-2 positively regulates the

Ras/Erk pathway, which implicates the role of SHP2 in the development of cancer [24].

1.2.3. PTP specificity

Early studies perceived PTPs as “housekeeping enzymes” playing a non-specific role against PTKs. However, later studies have demonstrated that at least some PTPs exhibit stringent specificity in vivo and generate distinct physiological responses. Three major factors that exemplified the substrate selectivity include PTP recruiting domains, posttranslational modifications and the intrinsic PTP domain specificity [25].

PTP recruiting domains are diverse modular domains locating outside the active site [26]. These non-catalytic domains endow the diverse PTP specificity by directing the enzymes to specific subcellular localizations and/or to their physiological substrates and

4 in some cases simultaneously regulating the PTP activities. Several studies have been performed on the SH2 domains, which direct the proteins to dock to the phosphorylated tyrosine residues. SHP-1 has been identified to associate with p130, Grb2 [27] and - chain of interleukin-3 (IL-3) receptor via its SH2 domain [28]. Klingmuller et. al. has shown that the SHP-1 was recruited to the erythropoietin receptor via its SH2 domain leading to inactivation of JAK2 and termination of proliferative signals [29].

PTPs are governed by various post-translational modifications including phosphorylation and proteolysis. Tyrosine phosphorylation of PTPs resulted in the stimulation of phosphatase activity for many PTPs such as SHP-1, and SHP-2 [30].

Conversely, PTP-PEST activity was inhibited by phosphorylation due to its reduced substrate binding affinity by the cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) phosphorylated Ser39 [31]. Additionally, phosphorylation not only modulates the PTP activity but also provides docking sites for protein-protein interactions. For example, the phosphorylation of Tyr789 of a receptor phosphatase

PTPRA potentially served as a binding site for an adaptor protein Grb-2. This study suggested that PTPRA play a role in the regulation of Grb-2-mediated signaling [32].

PTP activity has also been shown to be affected by proteolysis. In several receptor

PTPs, the limited proteolysis can facilitate the interactions between RPTP extracellular domains and their ligands. Proteolysis has also influenced the activities of PTPs. Non- transmembrane PTPs (i.e. in PTP1B, PTP-MEG and SHP-1) are activated by calpain- induced cleavage of their negative regulatory domains [33-35].

The PTP catalytic domain itself also contributes to the PTP substrate specificity.

Many studies demonstrated that interactions between a PTP with two different pY

5 peptides were found to be greatly different by 4-5 ordersof magnitude [36]. The different amino acids and surface electrostatic potential surrounding the active sites in PTPs have contributed to substrate specificity. For instance, the crystal structure of the inactive mutant PTP1B/C215S with the EGF receptor peptide, DADEpYL-NH2, revealed the key interaction between carboxylate side chain of Glu and Asp at P-1 and P-2 with the side chain of Arg47 in PTP1B [14]. In addition, swapping of two highly similar PTP domains also failed to produce PTPs that can target the same substrate. Interestingly, SHP-2/SHP-

1 chimeras partially rescued the basic fibroblast growth factor-induced mesoderm induction; the correct PTP domain is strictly required to restore wild type signaling [37].

Furthermore, different phenotypes have been observed in the knockout of PTP1B and

TC-PTP (72% sequence identity). Mice lacking PTP1B have shown the resistance to the diet-induced obesity and enhanced insulin sensitivity; however, mice lacking TCPTP exhibit defects in the hematopoietic system leading to the early death [38].

1.3. The Haloacid Dehydrogenase Superfamily Phosphatases (HADSF)

1.3.1. Structure and Catalytic Mechanism

HADSFs can be divided into three subfamilies: RNA polymerase C-terminal domain phosphatases (CTDPs/FCPs), Eya Absent phosphatases (EYAs) [39] and HAD phosphatases [5, 40]. These enzymes typically have low sequence similarity (<15%), but all contain a characteristic Rossman-like fold and four signature motifs [41]. The signature motifs are spatially arranged around the active site of HADSFs. Motif I contains a consensus sequence “DXDX(T/V)(L/V)”, where  is hydrophobic residues, X is any proteinogenic amino acids. The first Asp in this motif functions as a

6 nucleophile, while, the second Asp acts as a general acid/base and an obligatory Mg2+ coordinator. The second motif, (S/T), offers a Ser/Thr residue to mediate the substrate orientation by hydrogen bonding stabilization. Less conserved motif III situates

18-30 amino acids apart from motif IV. A conserved leucine residue in the motif III facilitates the motif IV to stabilize the reaction intermediates. Motif IV with a consensus sequence (G/S)(D/S)X3-4(D/E) or (G/S)DD has been shown to mediate the Asp residue in motif I to coordinate Mg2+ [42].

The dephosphorylation reaction catalyzed by HADSF proceeds via a two-step mechanism (Figure 1.2). The first step is a nucleophilic attack on the phosphoryl group of the substrate by motif I Asp41 (in the human mitochondrial deoxyribonucleotidase) to form the phosphoaspartyl intermediate. Subsequently, the phosphoenzyme intermediate undergoes hydrolysis to release inorganic phosphate and the catalytic Asp41 residue. Mg2+ is an essential cofactor for correctly positioning the substrate in the active site close to the nucleophile, and for an electrostatic stabilization of the transition states. The Asp43,

Asp175, and Asp176 residues in motif I and IV participate in the stabilization of the reaction and the coordination of Mg2+ [42-45].

1.3.2. Functions and related Diseases

HADSF phosphatases are recently discovered enzymes [42, 46-48]. Their functions and substrate specificities largely remain to be explored. Although, the current studies of FCPs and EYAs subfamilies have demonstrated crucial cellular functions.

TFIIF-associated C-terminal domain phosphatase (FCP1) is essential for the recycling of

RNAPII during the transcription cycle [49]. The congenital cataracts facial dysmorphism

7 neuropathy syndrome (CCFDN) has been shown to result from the silencing of FCP1; however, the mechanism remains to be determined [50]. Small C-terminal domain phosphatases (SCPs) also play important role in regulation of transcription. The mutation of SCP3 has been observed in small cell lung cancer, renal cell carcinoma and breast cancer. Kashuba et. al. demonstrated that SCP3 acts as a tumor suppressor gene, which functions in the regulation of cell growth and differentiation [46]. EYAs are transcription regulators involved in organogenesis and exhibits both tyrosine and threonine phosphatase activities [47, 51-53]. EYA depletion causes defects or complete loss of the

Drosophila compound eye. In human umbilical vein endothelial cells, knockdown of

EYA3 inhibits tubulogenesis. Similar results were also observed with Benzbromarone and

Benzarone, potent EYA inhibitors [54, 55].

1.3.3. HADSF specificity

HADSFs can dephosphorylate a variety of substrates, such as, metabolites, DNA, pS/pT/pY substrates [56-58]. However, little information is known about the protein substrate specificity of HADSFs [42]. Several studies have suggested strategies that regulate HADSF specificity. Recently, Kestler et. al. have investigated how dimerization effects the catalytic activity and specificity of chronophin, which is known to dephosphorylate pS/pT proteins and pyridoxal-5’-phosphate (PLP). Despite unaltered active site structure, the monomeric chronophin has impaired catalytic activity and effected the substrate coordination presumably due to the tilted of the -hairpin/substrate specificity loop [59]. However, more studies are needed to understand how dimerization controls HADSF activity and specificity.

The active site residues of HADSF have been shown to confer the specificity by acting as a molecular gatekeeper. FCP1 and SCP1 are both RNAPII C-terminal domain

(CTD) phosphatases but prefer different phosphorylation sites [48, 60]. It was previously suggested that the FCP1 substrate specificity is due to a C-terminal breast cancer protein- related carboxy-terminal (BRCT) domain that is only present in FCP1. However, Ghosh et. al. has demonstrated that the residues lining the canyon close to the FCP1 active site potentially determine the substrate specificity. The mutations in the BRCT phosphopeptide binding pocket minimally decreased the FCP1 activity (33%) against the

CTD peptide substrate. However, the BRCT mutants have no effect on FCP1 function in vivo [61].

1.4. Approaches to Discover New Phosphatase Substrates

1.4.1. Conventional approaches

Although the significance of phosphatases in physiological events has been clearly demonstrated, their cellular mechanisms of action are mostly unknown. The identification of substrates and their functional relationship with phosphatases still remain a challenging task in this field. Two conventional methods are commonly used to identify the potential substrates: the substrate trapping and the modulation of the expression of phosphatases.

The substrate trapping method utilizes an inactive phosphatase that is capable of forming a stable complex with its substrate, but cannot perform catalysis. The inactive

PTP has been generated by a single or double mutation of the nucleophile and/or general acid/base residues, which are essential in the dephosphorylation reaction (i.e. C215S,

D181A, Q262A or C215A/D181A in PTP1B). The PTP-substrate complex is usually isolated by immunoprecipitation or affinity purification, separated by SDS-PAGE and subsequently characterized by Western blots or LC-MS/MS. This method has been widely used to identify several PTP substrates; however, information of the related pathways or proteins involved with the PTP of interested is required to select the appropriate antibodies for Western blots. Preliminary knowledge of the potential substrate identity may not be necessary for direct MS method. However, a highly abundant protein of interest in the sample is required to generate the reliable identification. In general, this approach only detects the tight substrate-PTP binding complex, whereas, a real PTP substrate may or may not bind to the PTP with a high affinity.

The alteration of the expression of phosphatases has been performed by two complementary techniques: the overexpression of phosphatases and loss-of-function method. In principal, the overexpression of phosphatases generates the hypophosphorylated substrate; while, the knockdown of phosphatases suppresses its expression and leads to an increased phosphorylation level of the substrate. Both experiments are usually performed to ensure that the results are not obtained from an off- target effect knockdown experiment or an artifact of overexpression of the phosphatase in the cell.

1.4.2. Reverse Interactomics Approach

Reverse interactomics approach combines techniques in chemistry, bioinformatics and biochemistry to identify the binding partners or the enzyme substrates [62-64]. In the

10 first step, the protein is screened against a peptide library to determine a consensus motif.

Then the selected motif is used to search protein databases to identify the putative binding partner or the enzyme substrates. Finally, the identified protein partner or substrate is individually confirmed by the conventional approaches (section 1.4.1). The reverse approach has successfully been used to determine several binding partners of SH2

[64-67], SH3 [68], PDZ [69], and BIR [70] and potential substrates of kinases [71-

74],and proteases [75, 76].

Several methodologies have been utilized to understand the determinants of phosphatase substrate specificity, For example, PTP1B crystal structures and kinetics studies reveal that residues flanking the pY are essential for PTP1B efficient substrate recognition [14, 36, 77]. The PTP specificity was performed by utilizing individual assay with synthetic peptides derived from known PTP substrates [36, 78-81] and pY peptides microarray [82, 83]. However, these approaches require the information of reported specific phosphorylation sites and are relatively low throughput.

Early studies have revealed that PTPs recognize three to five residues on each sides of pY [14-17]. To obtain the complete specificity profile, the use of peptide libraries containing large number of peptides (206 – 2010) may be more suitable. Due to the absence of stable association between the active PTP and a pY peptide, various combinatorial methods were employed using nonhydrolyzable pY mimics (i.e. phosphonodifluoromethyl phenylalanine and malonyltyrosine) or inactive PTP mutants

[84-87]. Consequently, the specificity information is derived from the binding not catalysis studies. However, a high-affinity peptide may not bind to the PTP active site in a productive manner to mediate efficient catalysis. Other library screenings employed to

11 study PTPs include inverse alanine scanning [88], phage display [87], ECLIPSE [89], and

SPOT [90]. Nonetheless, those techniques are unable to systematically determine the sequence specificity of PTPs.

To overcome those shortfalls, the Pei lab has developed a general methodology to systematically profile phosphatase specificity against both pY and pS/pT peptide libraries. The phosphatase specificity profile would be beneficial to identify the physiological substrates, understand related cellular pathways and design selective phosphatase inhibitors.

1.5. The Screening of the Combinatorial Peptide Libraries

1.5.1. Phosphotyrosine Peptide Library screening

The one-bead-one-compound (OBOC) pY peptide library was constructed by using the split and pool technique [91]. Solid phase synthesis of peptides enables the convenient incorporation of unnatural amino acids to the library, for example, 3,5- difluotyrosine (F2Y), which is utilized as a Tyr mimic [92], and Norleucine (Nle), serves as a Met surrogate. The library is then subjected to the limited dephosphorylation reaction by the PTP of interest. The library screening was designed to overcome the difficulty in differentiating the unreacted pY and the Tyr product from the dephosphorylation reaction

(Figure 1.3). This method utilizes the high selectivity of the tyrosinase enzyme that only oxidizes Tyr (but not pY) to give an orthoquinone, which is subsequently derivatized with 3-methyl-2-benzothianolinonehydrazone (MBTH) and oxidized to form a red pigment on bead [92, 93]. The positive beads were separated from the library and

12 individually sequenced by partial Edman degradation-mass spectrometry (PED-MS), a high throughput and relatively inexpensive peptide sequencing technique [94, 95].

1.5.2. Phosphoserine/threonine Peptide Library screening

Phosphoserine/phosphothreonine peptide libraries were prepared by split and pool technique and treated with a minimum amount of phosphatase for a limited time to only permit the detection of the most efficient substrates. The C-terminal library (Figure 1.4

A) was then subjected to the removal of the alloc protecting group and the oxidation reaction. The generated glyoxyl group can be selectively labeled with 4-hydrazino-7- nitro-2,1,3-benzoxadiazole (NBDH) to give green fluorescent beads. Then, the positive beads were manually isolated under a fluorescent microscope and individually sequenced by the PED-MS method. The screening of the N-terminal library (Figure 1.4 B) was similarly performed but by using the beads containing peptides with two orthogonal N- terminal protecting groups. After the enzymatic treatment, the Fmoc-protected peptide portion was treated with piperidine to generate the N-terminal amine for Edman degradation reaction. This procedure removed the residues to expose the N-terminal

Ser/Thr and pS/pT residues. The Ser/Thr on the positive beads can be similarly oxidized by NBDH. The remaining peptides with the N-terminal Alloc protecting group is utilized for the peptide sequence identification by PED-MS [96].

In this study, I have applied the screening methods to investigate the sequence specificity of the phosphatases including classical PTPs (both cytosolic and

13 transmembrane PTPs) (Chapter 2), a DUSP (Chapter3) and a HAD phosphatase (Chapter

4) to demonstrate the general implication of these methods that could be used with any phosphatases selective for pY or pS/pT residues.

Figure 1.1. Catalytic Mechanism of Cys-based PTPs (the amino acid numbering PTP1B).

Figure 1.2. Mechanism of Asp-based HADSFs. (amino acid in the human mitochondrial deoxyribonucleotidase).

Figure 1.3. (A) The screening of PTPs against pY peptide library. (B) A portion of the peptide library after tyrosinase/MBTH treatment. The positive beads show an intense red color. From ref. [92].

Figure 1.4. (A) The screening of phosphatases against pS/pT peptide library. (B) A portion of the peptide library after NBDH treatment. The positive beads show a green fluorescent ring (viewed under a fluorescent microscope). From ref. [96].

Chapter 2

SPECIFICITY PROFILEING OF CLASSICAL PROTEIN-TYROSINE

PHOSPHATASES 1

2.1. Introduction

How PTPs recognize and distinguish their substrates remains considerably undefined. The sequence specificity profile of PTPs would be beneficial to identify their physiological substrates, understand signaling pathways and design selective PTP inhibitors. Previously, our group has developed a methodology to determine the sequence specificity of PTPs [92, 93]. In this study, eight classical PTPs were investigated including PTPRA, PTPRB, PTPRD, PTPRO, PTP-PEST, PTP1B, SHP-1 and SHP-2.

1 This work was reproduced in part with permission from

Ren, Lige, Chen, Xianwen, Luechapanichkul, Rinrada, Selner, Nicholas G., Meyer, Tiffany M., Wavreille, Anne-Sophie, Chan, Richard, Iorio, Caterina, Zhou, Xiang, Neel, Benjamin G., Pei, Dehua (2011). Substrate Specificity of Protein Tyrosine Phosphatases 1B, RPTPα, SHP-1, and SHP-2. Biochemistry, 50(12), 2339-2356. doi: 10.1021/bi1014453. Copyright 2011 American Chemical Society.

Selner, Nicholas G., Luechapanichkul, Rinrada, Chen, Xianwen, Neel, Benjamin G., Zhang, Zhong-Yin, Knapp, Stefan, Bell, Charles E., Pei, Dehua. (2013). Diverse Levels of Sequence Selectivity and Catalytic Efficiency of Protein-Tyrosine Phosphatases. Biochemistry, 53(2), 397-412. doi: 10.1021/bi401223r. Copyright 2013 American Chemical Society. 18

PTPRA is a receptor PTP that contains two tandem PTP domains in the cytoplasm. Both PTP domains of PTPRA are catalytically active, but the membrane- proximal domain (D1) has activities against pY peptides that are generally four to five orders of magnitude higher than that of the membrane-distal domain (D2) [81, 97, 98].

The absence of two critical catalytic residues in the D2 domain – the tyrosine from the pY recognition loop (also known as the KNRY motif) and the aspartic acid from the

WPD loop (which acts as a general acid/base during catalysis) – was shown to be responsible for the decreased activity. The existing data suggest that PTPRA-D1 is responsible for the in vivo phosphatase activity, whereas the D2 domain regulates the activity/specificity of the D1 domain through protein-protein interactions. PTPRA forms catalytically inactive homodimers under physiolgical conditions, and the D2 domain has been shown to be critical for stable dimer formation [99]. PTPRA -D2 has also been shown to directly bind to Src kinase and is required for the dephosphorylation and activation of Src by PTPRA [100]. The catalytic activity and substrate specificity of

PTPRA have been examined using a small panel of pY peptides [81, 101]. These studies revealed that PTPRA is substantially less active than most of the other PTPs and exhibits only modest sequence specificity.

Receptor protein tyrosine phosphatase B (PTPRB) contains an intracellular catalytic domain (D1) and transmembrane fibronectin type III-like domains [102].

Several studies have demonstrated that PTPRB play important role in angiogenesis by regulating the angiopoietin-signaling pathway in human endothelial cells, but no mechanism of action has been elucidated [102]. Recently, Xu et. al. has shown that pY1356 of hepatocyte growth factor receptor was dephosphorylated by PTPRB

19 demonstrated by using substrate trapping, in vitro dephosphorylation, and alteration of expression experiments [103]. The dephosphorylation subsequently inhibited downstream

MEK1/2 and ERK activation [103].

Receptor protein tyrosine phosphatase D (PTPRD) is highly expressed in the central nervous system and serves as cell adhesion molecule and axon guidance regulator

[104]. Nonetheless, mutation or deletion of the PTPRD gene was observed in various types of cancer suggesting the role of PTPRD as a tumor suppressor [105, 106]. Several proteins involved in tumor development have been reported as PTPRD substrates.

PTPRD has been shown to inactivate oncogenic signal transducers and activators of transcription (STAT3) by dephosphorylating STAT3-pY705 [106]. In addition, aurora kinase has been demonstrated to be a PTPRD substrate [107]; however, the phosphorylation site remains to be identified.

Receptor protein tyrosine phosphatase O (PTPRO), initially identified as a renal glomerular podocyte protein (GLEPP1) [108], is a transmembrane PTP. Truncated form of PTPRO (PTPROt) expressed predominantly in hematopoietic cells has shown to reduce phosphorylation of Lyn-pY397. The inactivation of Lyn kinase mediated the action of Pazopanib, an anticancer drug, in B-cells receptor signaling [109]. Several PTPRO substrates have also been identified including Syk [39], Zap70 [109] and ErbB2/HER2

[110], which implicates PTPRO in cell signaling and morphogenesis [39, 109, 110].

Protein tyrosine phosphatase PTP-PEST is a non-transmembrane protein containing Pro, Glu, Ser, Thr (PEST) -rich sequences [111]. Several PTP-PEST substrates have been identified, such as, proto-oncogene protein 2 (Vav2) [112], cellular

Abelson (c-Abl) tyrosine kinase [113], Proline-rich tyrosine kinase 2 (Pyk2) [114],

Wiskott-Aldrich syndrome protein (WASP) [115], and the 130 kDa retinoblastoma- associated protein (p130) [111, 116], which all suggest roles of PTP-PEST in cytoskeletal rearrangement. The p130 protein has been shown to associate to both catalytic domain

[116] and PEST rich-sequence of PTP-PEST [111]. The Pro337Ala mutation of PTP-

PEST has shown to disrupt its association with p130 [111]. More identified substrates are still needed for the growing list of PTP-PEST substrates to understand how PTP-

PEST regulates cellular processes.

PTP1B was the first PTP identified and is one of the most studied PTPs. It is ubiquitously expressed and localized on the cytoplasmic face of the endoplasmic reticulum [117]. PTP1B acts as an important regulator of metabolic signaling. Mice lacking PTP1B exhibit enhanced insulin, leptin, and growth hormone sensitivity and resistance to diet-induced diabetes and obesity [18]. PTP1B is also required for Neu- induced breast cancer [20, 118]. Accordingly, PTP1B is a promising target for treatment of type II diabetes, obesity, and cancer. A large number of phosphoproteins, including receptors, non-receptor protein tyrosine kinases, and other cytoplasmic proteins have been identified as PTP1B substrates in vivo [119]. Examination of the peptide sequences flanking the pY sites dephosphorylated by PTP1B in these proteins, however, does not reveal any simple consensus sequence. This raises the question of whether the catalytic domain of PTP1B has any sequence specificity and how in vivo substrate specificity is achieved.

SHP-1 and SHP-2 are non-transmembrane, Src homology 2 (SH2) domain- containing PTPs, which play critical roles in many signaling pathways and disease processes [120, 121]. The two enzymes share >55% sequence identity, a similar 3D

21 structure, and a common regulatory mechanism [122, 123]. However, they often perform opposite cellular functions, with SHP-1 typically acting as a negative regulator of signaling, whereas SHP-2 is a positive regulator. Some of the functional differences may be attributed to the SH2 domains, which display overlapping but not identical binding specificities and may therefore direct their respective PTP domains to different pY proteins [65]. However, chimeras in which the PTP domains of SHP-1 and SHP-2 were swapped failed to exhibit the same biological activity as the wild-type enzymes [37, 124].

These and other observations have led to the hypothesis that the catalytic domains of

SHP-1 and SHP-2 may have different sequence specificities.

Prior to our work, none of the classical PTPs has been systematically profiled for substrate specificity. In this work, the active site specificity of eight PTPs were determined. We found that while no two PTPs share exactly the same specificity profile , they have broad and overlapping peptide specificity. Most PTPs in this study contain catalytic domains and recruiting domains. Our results suggest that in vivo PTP specificity may be enhanced by substrate recruitment.

2.2. Experimental Procedures

2.2.1. Materials

Reagents for peptide synthesis were purchased from Advanced ChemTech

(Louisville, KY), AAPPTec (Louisville, KY), Chem-Impex (Wood Dale, IL), or Peptides

International (Louisville, KY). N-(9-Fluorenylmethoxycarbonyloxy)succinimide was purchased from Advanced ChemTech. α-Cyano-4-hydroxycinnamic acid, phenyl isothiocyanate, and 3-methyl-2-benzothiazolinonehydrazone (MBTH) were obtained

α from Sigma-Aldrich. N -Fmoc-O-t-butyl-3,5-difluorotyrosine (F2Y) was synthesized as described previously [92]. Tyrosinase from Streptomyces antibioticus was expressed in

E.coli and purified as described previously [125].

2.2.2. Purification of PTPs

The catalytic domains of wild-type a PTP1B (amino acids 1-321) and SHP-1 (aa

205-595) were expressed in E. coli and purified as previously described [16, 125-128].

PTPRA (aa 224-802), containing an N-terminal histidine tag, and SHP-2 (aa 199-593), containing a C-terminal histidine tag, were expressed in E. coli and purified as described

[126]. PTP-PEST (aa 5-300), PTPRB (aa 1686-1971), PTPRD (aa 1299-1899), and

PTPRO PTP domain were expressed in E. coli as fusion proteins with glutathione-S- transferase [129] and purified as described previously [109, 126]. Protein concentrations were determined by the Bradford method, using bovine serum albumin (BSA) as the standard.

2.2.3. Library Synthesis

Peptide libraries I-V (Table 2.1) were synthesized on 5 g of polyethylene glycol acrylamide resin (PEGA resin) (0.4 mmol/g; ~3 × 106 beads/g of dry resin, 150–300 µm diameter in water). All manipulations were performed at room temperature unless otherwise noted. The invariant positions (pYAABBRM and AS) were synthesized with 4 equivalents of Fmoc-amino acids using HBTU/HOBt/N-methylmorpholine (NMM) as the coupling reagents, and the coupling reaction was terminated after negative ninhydrin tests. For the synthesis of random residues, the resin was split into 19 equal portions and each portion was coupled twice, each with 5 equivalents of a different Fmoc-amino

23 acid/HBTU/HOBt/NMM for 1 h (except for F2Y, for which 2.5 equivalents were used and coupled only once). To facilitate sequence determination by mass spectrometry, 5%

(mol/mol) CD3CO2D was added to the coupling reactions of leucine and lysine, whereas

5% CH3CD2CO2D was added to the coupling reaction of Nle [95]. The resin-bound library was washed with dichloromethane and deprotected using a modified reagent K

(7.5% phenol, 5% water, 5% thioanisole, 2.5% ethanedithiol and 1% anisole in trifluoroacetic acid (TFA)) at room temperature for 2 h. The library was washed exhaustively with TFA, DCM and DMF and stored in DMF at -20 °C until use. Libraries

II–V were synthesized in a similar manner. Library III were similarly prepared but with the following modifications. To construct the random sequence region of library III

(which contains reduced Arg and Lys contents), the resin was split into 19 aliquots; two aliquots each contained 0.58% of the total resin mass and were coupled with Fmoc-Arg or Fmoc-Lys, while the remaining 17 aliquots each contained 5.8% of the resin and were coupled to the other 17 Fmoc-amino acids.

2.2.4. Library Screening

In a typical screening experiment, 15 mg of the appropriate peptide library (dry weight, ~45,000 beads) was placed in a plastic micro-BioSpin column (2 mL, Bio-Rad) and extensively washed with DMF and ddH2O. The resin was blocked for 1 h with blocking buffer (30 mM Hepes, pH 7.4, 150 mM NaCl, 0.01% Tween 20, and 0.1% gelatin). The library was then incubated with a given PTP (final concentration 0.5−800 nM) in blocking buffer containing 5 mM tris(carboxyethyl)phosphine (TCEP) (total volume 0.8 mL) at room temperature for 5–30 min with gentle mixing. The resin was

24 drained, washed with 0.1 M KH2PO4 (pH 6.8), and resuspended in 1.6 mL of 0.1 M

KH2PO4 (pH 6.8) containing 1.2 µM tyrosinase and 6 mM 3-methyl-2- benzothiazolinonehydrazone (MBTH). The resulting mixture was incubated at room temperature with gentle mixing and exposure to air. Intense pink/red color typically developed on positive beads after 20−60 min. Positive beads were retrieved from the library using a micropipette under a dissecting microscope, and sequenced by the partial

Edman degradation-mass spectrometry (PED-MS) method [95]. Control experiments without PTPs produced no red beads under otherwise identical conditions. Detailed conditions for individual screening experiments are provided in Table 2.2.

2.2.5. Synthesis of Selected Peptides.

Individual peptides were synthesized on 100 mg of Cross-Linked Ethoxylate

Acrylate (CLEAR) amide resin using standard Fmoc/HBTU/HOBt chemistry. For coupling of pY, 2.0 equivalents of Fmoc-Tyr(PO3H2)-OH were employed, whereas 4.0 equivalents were used for all other amino acids. Resin-bound peptides were washed with

DCM, cleaved from the resin, and deprotected using modified reagent K (79:7.5:5:5:2.5:1

(v/v) trifluoroacetic acid/phenol/H2O/thioanisole/ethanedithiol/anisole) at room temperature for 2 h. After evaporation of solvents, the mixture was triturated three times with 20 volumes of cold Et2O. Precipitates were collected and dried under vacuum, and crude peptides were purified by reversed-phase HPLC on a semi-preparative C18 column

(solvent system: H2O and CH3CN with 0.05% TFA, gradient elution generally from 0 to

60% CH3CN). The identity of each peptide was confirmed by MALDI-TOF mass spectrometric analysis.

2.2.6. PTP Assays

PTP activities were determined by two different methods. For method A, assay reactions were performed in a quartz microcuvette (total volume 120 L) containing 100 mM

HEPES, pH 7.4, 50 mM NaCl, 2 mM EDTA, 1 mM TCEP, 0.1 mg/mL BSA, and 0−1.4 mM pY peptide. The reaction was initiated by the addition of PTP (final concentration

0.5−20000 nM) and monitored continuously at 282 nm (ε = 1102 M-1 cm-1) on a UV-

VIS spectrophotometer. Initial rates were calculated from the early regions of the reaction progress curves (typically <60 s). Data fitting against the Michaelis-Menten equation V =

Vmax · [S]/(KM + [S]) or the simplified equation V = kcat[E][S]/KM (when KM >> [S]) gave the kinetic constants kcat, KM, and/or kcat/KM. For method B, reactions were performed similarly, but reaction progress was monitored continuously at 282 nm until the substrate was depleted. The kinetic constants were obtained by directly fitting the reaction progress curves against equation:

( ) ( )

where t is time, p is the product concentration at time t, [E] is the total enzyme

concentration, and p∞ is the product concentration at infinity [130].

2.3. Results

2.3.1. Peptide Library Design and Screening

Five different peptide libraries were employed in this work (Table 2.1). Library I contains five random residues N-terminal to pY, NH2-ASX5pYAABBRM [where B is - alanine and X is F2Y (used as a Tyr surrogate), norleucine (Nle or M, used as a replacement of Met), or any of the 19 proteinogenic amino acids except for Tyr, Met, or

Cys]. Library II, which is similar to library I but contains eight random residues N- terminal to pY, was designed to test whether residues beyond pY-5 (relative to pY, which is defined as position 0) affect PTP activity and/or specificity. Library III has 10-fold reduced Arg and Lys content, but is otherwise identical to library I, to minimize the bias probably caused by multidentate, electrostatic interactions between the positively charged peptide sequences and negatively charged surface patches on the PTPs [131].

Additionally, library IV was designed to determine the C-terminal specificity and to test whether a PTP requires specific sequences on both N- and C-terminal sides of pY for activity and/or exhibits covariance between the N- and C-terminal sequences. Libraries I and III have a theoretical diversity of ~2.5 x 106, whereas libraries II and IV have theoretical diversities of 1.7 x 1010 and 6.1 x 1012, respectively. All peptide libraries were synthesized in the one-bead-one-compound (OBOC) format on PEGA resin. Library screening involved treatment of a portion of the library beads (typically 15 mg of dry resin in each reaction) with a limited amount of PTP so that only beads that display the most efficient substrates underwent significant dephosphorylation. The optimal PTP concentration and reaction time were determined by trial and error and varied greatly depending on the catalytic efficiency of the PTP (Table 2.2). After termination of the PTP

27 reaction (by washing the library beads with a buffer), the exposed Tyr side chain was

oxidized into an orthoquinone by incubation with tyrosinase in the presence of O2 [132].

The orthoquinone was captured in situ by MBTH to form a reddish pigment, which remained covalently attached to the beads. Both tyrosinase and MBTH were used in excess to ensure that the coloration reaction went to completion. The tyrosinase exhibits no sequence selectivity [93]. Positive beads were isolated manually from the library with a micropipette and were sequenced individually by using the PED-MS method [95].

2.3.2. Substrate Specificity of PTPs

Initial screening of PTPRA against library I resulted in almost exclusively positively charged sequences (Table 2.3). To determine whether this selectivity is an intrinsic property of the enzyme or was the result of preferential enzyme recruitment/resin swelling, PTPRA was screened against library III (Alloc-

ASX5pYAABBRM) (Table 2.4), which is structurally similar to library I but features a

10-fold reduction in the Arg and Lys content. This reduces the probability of multiple

Arg and/or Lys residues in a sequence and therefore the effect of preferential enzyme recruitment/bead swelling on library screening. Library III (total 60 mg) was screened against PTPRA (5 nM) and the most intensely colored beads were sequenced to give 104 sequences (Table 2.4). PTPRA showed only very weak N-terminal sequence selectivity, with some preference for Thr and Ser at the pY−2 and pY−5 positions and a slight preference for acidic residues at pY−2 to −4 positions. Other amino acids were selected with essentially equal frequencies at all of the random positions (Figure 2.1 A). Arg and

Lys were underrepresented at all five positions. However, the number of Arg/Lys

28 selected (4 total) was similar to the expected value of the unselected library [104 × 5 ×

2/(19 × 10) = 5.5 Arg and/or Lys expected]. These observations suggest that PTPRA readily tolerates, but has no special preference for, the positively charged residues and that the Arg- and Lys-rich sequences selected from library I was the result of preferential enzyme recruitment/bead swelling.

To assess whether PTPRA is selective on the C-terminal side of pY and investigate the potential sequence covariance between the N- and C-terminal sequences, we screened the enzyme against library IV, Alloc-AX5pYX5NNBBRM (where X is the same set of amino acids but no F2Y), which is randomized at positions pY−5 to pY+5

(Table 2.1). Note that library IV has a theoretical diversity of 6.1 × 1012; the number of sequences screened (~2.1 × 105 total) represented only a tiny fraction of the sequence space. Nevertheless, screening of library IV confirmed the results obtained from libraries

I, III and provided important new insights. Under normal salt conditions (150 mM NaCl), the selected sequences were overwhelmingly positively charged on both sides of pY

(Table 2.5). Increasing the salt concentration to 300 mM had no significant effect, whereas at 500 mM NaCl, the selected sequences showed a decrease in the Arg and Lys content. The selected peptides were highly diverse in sequence (though there was an overrepresentation of hydrophobic residues at the pY−1 position) and no obvious consensus sequence could be derived. On the basis of these observations and solution- phase kinetic studies (vide infra), we conclude that PTPRA also lacks significant selectivity on the C-terminal side of pY.

The sequence selectivity of the other three receptor PTPs, PTPRB, PTPRD and

PTPRO were examined with library II, III and IV (Table 2.1). These enzymes showed

29 broad specificity profiles (Table 2.6-2.10, Figure 2.1 B-D). They all have moderate to weak preference for acidic and aromatic hydrophobic residues. PTPRB is unique in that it prefers an Asp (but not Glu) residue at the pY-1 position and tolerates small hydrophilic

(e.g., Ser, Thr, Asn) but not basic (Arg, Lys, and His) residues at this position. It has moderate preference for acidic residues at pY-2 and pY-3 and aromatic hydrophobic residues at the pY-4 and pY-5 positions. PTPRD appears to be somewhat more tolerant to basic residues than the other 3 receptor PTPs, as indicated by the larger number of Arg and Lys residues selected.

Screening of the PTP-PEST catalytic domain against libraries I and III produced

96 sequences (Table 2.11). PTP-PEST strongly prefers acidic residues, Asn, or Ser at the pY-2 and pY-3 positions (Figure 2.1 E). It prefers a hydrophobic residue (e.g., Trp, Ile,

Phe) at the pY-1 position and either acidic or hydrophobic residues at the pY-4 and pY-5 positions. To determine the C-terminal specificity, PTP-PEST was also screened against library IV, which features five random residues on each side of pY. Again, PTP-PEST exhibited overwhelming preference for acidic sequences; each of the 39 selected sequences contained two or more acidic residues, which occurred most frequently at the pY+2, pY+1, pY-3, and pY-2 positions (Table 2.12). Only 7/135 sequences selected from the two libraries contained an Arg, Lys, and/or His residue, and these residues usually occupied less critical positions (e.g., pY-5 and pY+5).

The sequence specificity of PTP1B was previously examined by Dr. Ren in our group by screening against library I and other libraries containing the preferred sequences of PTP1B on the N-terminus of pY with randomized amino acids on the C-terminus of pY ([D/E][D/E]VpYX4LNBBRM and WA4pYX5NNBBRM) [93]. The studies revealed

30 the strong influence of the N-terminal sequence on the C-terminal sequences selected suggesting potential sequence covariance between the N- and C-terminal sequences. To further investigate that possibility, in this work, PTP1B were screened against library IV,

Alloc-AX5pYX5NNBBRM (Table 2.13). When screening was carried out under normal salt conditions (150 mM NaCl), the sequences selected by PTP1B were overwhelmingly rich in basic residues (class II in Table 2). Screening at 500 mM NaCl greatly decreased the number of class II sequences and increased the number of acidic (class I) peptides

(Table 2.13). The class I peptides showed an overrepresentation of Asp, Glu, and other small hydrophilic residues at the pY-2 position and Ser, Thr, Glu, and Asp at the pY-4 position, which is in good agreement with previous study on PTP1B against N-terminal library (NH2-SAX5pYAABBRM) [93]. The dramatic effect of salt concentration on the nature of sequences suggests that the Arg and Lys-containing sequences were selected because of better accessibility of PTP1B to the beads carrying the Arg- and Lys-rich peptides. Presumably, the positive charges on the peptides repel each other and increase the distances between adjacent peptides. This in turn improves the swelling of these beads and makes the peptides more accessible to the enzyme. The positively charged beads may also recruit more PTP1B (pI 6.8) to their surfaces than beads carrying neutral or acidic peptides [131]. To test this hypothesis, we screened PTP1B against library V,

Alloc-AX5pYX5EEBBRM (where X is the same set of amino acids but no F2Y) (Table

2.13). This library is identical library IV, except that the Asn-Asn motif C-terminal to the random region was replaced by Glu-Glu. Screening of library V (30 mg) against PTP1B resulted in exclusively class I sequences (Table 2.13). It is likely that the two Glu residues increased the accessibility of the beads carrying acidic peptides through charge-

31 charge repulsion and decreased the accessibility of beads with Arg- and Lys-rich peptides

(by neutralizing the positive charges). Interestingly, PTP1B strongly disfavors a proline at the pY+1 position. Out of the 108 peptides selected from libraries IV and V (Table

2.13), only one sequence (PTLGDpYPRVAQ) contained a proline at position pY+1.

Vetter et al. reported a similar finding [88]. Taken together, these results suggest that

PTP1B has broad specificity C-terminal to pY as well, with a modest preference for acidic and hydrophobic residues.

Previous studies also suggested that substrate specificity of SHP1 and SHP2 exhibited a significant degree of covariance between the N- and C-terminal sequences and peptide sequences on both sides of pY contribute to overall catalytic efficiency [93].

Therefore, SHP-1 and SHP-2 were screened against library IV (Alloc-

AX5pYX5NNBBRM) that contained randomized residues on both N- and C-terminal sides of pY. Investigation of the selected sequences revealed that most of the preferred substrates contained acidic residues on both sides of pY. A few peptides contained no or only one acidic residue (though they often contain one or more Asn, Gln, Ser, and/or Thr) on the N-terminal side of pY, but then their C-terminal sequences had two or more acidic residues (e.g., SWIDFpYEDQVA and VWNNLpYDPFED in Table 2.14). Thus, acidic sequences on the C-terminal side can also render a peptide a good SHP-1/2 substrate.

Some of the selected substrates had an occasional Arg and/or Lys on either side of pY

(e.g., AHDRVpYDWDDF and DDSMLpYEFDRV), suggesting that an Arg- and/or Lys- containing peptide can still be a good substrate of SHP-1/2 as long as it contains acidic residues (four or more) to compensate for the positive charge associated with the Arg and/or Lys. On the basis of the results, together with previous screening results [93], we

32 conclude that SHP-1 and SHP-2 are more selective than PTP1B, and an efficient substrate of SHP-1 or SHP-2 should have a total of three or four acidic residues, preferably with two or three of them N-terminal to pY and one or two on the C-terminal side. Finally, the result also revealed a subtle and yet recurring difference between SHP-1 and SHP-2, i.e., SHP-1 always selected a higher percentage of acidic residues than SHP-2 at the pY+1 position (Table 2.14).

2.3.3. Kinetic Properties of Selected Peptides toward PTPRA

To further evaluate the library screening result, resynthesized representative peptides were first tested against PTPRA, which showed insensitivity to the substrate sequences. Eight pY peptides were chosen for assay against PTPRA to assess the library screening results (Table 2.5, entries 1–8). These peptides contained acidic, neutral, or basic residues on either side of pY. Three of the peptides (Ac-EADTApYAA-NH2, Ac-

ASSDDpYAA-NH2, and Ac-WDEDFpYSASA-NH2) are similar to some of the sequences selected against PTPRA (EIDTApY, TSFDDpY, and WDFDFpY in Table 2.4, respectively). Remarkably, despite their very different sequences, all eight peptides have

4 −1 similar activities toward PTPRA, with kcat/KM values in the range of 1.2–8.0 × 10 M s−1. These activities are approximately two orders of magnitude lower than those of

PTP1B, SHP-1, and SHP-2 (Table 2.16, 2.17), indicating that PTPRA is intrinsically less active. The lower catalytic activity is caused by high KM values, which were ~1 mM for

−1 the peptide substrates tested, while the kcat values (~30 s ) were similar to those of

PTP1B, SHP-1, and SHP-2 (Table 2.16 and 2.17). The kinetic data generally agree with our results obtained from the library screening.

2.3.4. Positional insensitivity of Acidic Residues

Kinetic analysis was performed to confirm the library screening results that PTPs prefer acidic and hydrophobic residues but are insensitive to their positions relative to pY. The kinetic activities of PTP1B were examined because its intermediate level of preference for acidic sequences is more representative of the classical PTP subfamily [16,

133]. To this end, we resynthesized a peptide selected from previous screening (Table

2.16, peptide 1-2, 8-9) [93], and its alanine-scan peptides (Table 2.16, peptide 4-6) containing acidic residues at different positions against PTP1B. Again, all acidic peptides were highly active toward PTP1B and had similar kinetic activities (with kcat/KM values of 1.1 - 3.5 x 107 M-1 s-1 for N-terminal peptides and 4.8 – 5.8 x 107 M-1 s-1 for peptides containing both N- and C-terminal acidic residues). On the basis of these kinetic and specificity profiling data, together with reported catalytic efficiencies of more acidic peptides against PTP1B, SHP1, and SHP2 [93], we propose that the general preference for acidic sequences that lack a specific consensus motif may be a common feature of all

PTPs.

2.4. Discussion

In this study, we applied the peptide library screening method to systematically profile the sequence selectivity of eight classical human PTPs. Interestingly, except

PTPRA displaying no sequence selectivity, the other seven PTPs exhibit similar specificity profiles in that they all prefer acidic and large hydrophobic amino acids and disfavor basic residues between the pY-8 and pY+5 positions. Unlike proteases and

34 kinases, no peptide consensus sequences were observed. However, some PTPs exhibit remarkable selectivity in vivo which can be achieved by several mechanisms including their intrinsic catalytic domain selectivity, subcellular localization, expression pattern, posttranslational modification, and protein-protein interaction.

The lack of sequence selectivity of the PTPRA catalytic domain observed in this study predicts that (1) the in vivo substrate specificity of PTPRA is dictated by substrate recruitment and (2) PTPRA may be capable of hydrolyzing any pY motif recruited to its vicinity, including highly positively charged sequences. Therefore, due to its low intrinsic catalytic activity and inability to hydrolyze pY proteins in the absence of substrate recruitment, PTPRA is expected to function as highly specific enzymes in vivo. This model is in excellent agreement with the results of previous studies. For example,

PTPRA selectively dephosphorylates SRC at pY527 [134]. It was proposed that the SH2 domain of SRC binds to the pY789 of PTPRA and recruits it to the active site of PTPRA.

Other PTPRA substrates, such as LYN and FYN kinases, contain SH2 domains and are likely recruited via the same mechanism [135, 136].

The other receptor PTPs (PTPRB, PTPRD, PTPRO) examined in this study exhibit broad sequence specificity. These enzymes potentially utilize membrane localization to achieve in vivo substrate specificity. Membrane-associated proteins or proteins can be transiently recruited to the membrane to facilitate the dephosphorylation reaction during cell signaling. Some of them contain a second, catalytically inactive PTP domain, which can affect the physiological function of the active PTP domain [137, 138].

Dimerization provides another mechanism for regulating the activity of receptor PTPs

[139, 140]. Receptor PTPs may also be regulated by redox signals [141]. Finally, their

35 large extracellular domains may respond to environmental signals and regulate the activity of the PTP domain.

The library screening of PTP-PEST resulted in a greater number of acidic residues on the C-terminal side than the N-terminal side of pY (Table 2.2) suggesting that the C- terminal sequence is the primary determinant of PTP-PEST substrate specificity. This notion was supported by kinetics assay of PTP-PEST against peptides containing acidic residues on either the N- or C-terminal side of pY performed by Dr. Nicholas G. Selner

[142]. Several PTP-PEST substrates have been identified and generally contained negatively charged residues surrounding the pY site; for example, SRC

(IEDNEpY424TARQ)[143], FAK (SEIDDpY397AEIID), PYK2 (IESDIpY402AEIPD and

IEDEDpY579pY580KASVT) [114], WASP (TSKLIpY291DFIED) [144], and PSTPIP1

(RNELVpY344ASIEV) [145]. The dephosphorylation of substrates containing acidic residues only on the N-terminal side of pY is likely assisted by the recruiting motifs/domains on substrates and/or PTP-PEST. However, for highly negatively charge substrates, substrate recruitment many not be necessary to achieve efficiently dephosphorylation. The intrinsic sequence selectivity of their PTP domains likely plays a key role in controlling their in vivo substrate specificity.

PTP1B has very high intrinsic catalytic efficiency and yet broad specificity, suggesting that it is capable of dephosphorylating a wide variety of proteins. PTP1B is localized to the ER membrane [117, 146]; this localization undoubtedly leads to some preference for a subset of pY proteins such as those localized on the ER membrane.

Previous studies have shown that calpain-mediated cleavage of PTP1B from the ER membrane in platelets allows it to access different substrates [34, 147]. However, this

“compartmentalization” may not be sufficient to endow PTP1B with “exquisite” substrate specificity in vivo. A recent crystal structure has shown that PTP1B can form a complex with the insulin receptor at a site different from its site of dephosphorylation [148].

Although the physiological relevance of these observations remains to be established,

PTP1B might employ additional substrate recruiting strategies via either its catalytic or non-catalytic C-terminal domains. Notably, the C-terminal domain of PTP1B has proline- rich sequences capable of binding to SH3 domain-containing proteins and also has several phosphorylation sites within this region. Moreover, PTP1B activity is regulated spatially within cells [149], possibly as a consequence of oxidation by locally produced reactive oxygen species [141].

Similar to PTP-PEST, the in vivo specificity SHP-1 and SHP-2 is likely to be controlled primarily by their intrinsic sequence selectivity due to their selectivity for acidic sequences demonstrated in this study and high catalytic activities previously reported [93]. However, the in vivo specificity of these enzymes has shown to be controlled by several mechanisms. The auto-inhibitory mechanism of SHP-1 and SHP-2, in which the N-terminal SH2 domain binds to and inactivates the PTP active site in an intramolecular manner, has also enhanced the in vivo specificity [122, 123]. The PTP domain remains inactive until the N-terminal SH2 domain is engaged with a specific target protein. SHP1 and SHP2 contain SH2 domains that can bind specific pY proteins and target the PTP domain to the pY proteins and other protein(s) associated with them.

Previous studies have shown that SH2 domains of SHP-1 and SHP-2 have the overlapping but not identical specificities [65]. The SH2 domains of SHP-1 and SHP-2 may bind to different pY proteins, different pY motifs on the same protein, or the same

37 pY protein with different affinities. Once bound to the target protein(s), the intrinsic sequence specificity of the PTP domain likely dictates which pY residue to dephosphorylate and how fast the reaction takes place. This combinatorial control of substrate specificity is consistent with the previous observation that replacement of the

SHP-1 PTP domain with that of SHP-2 or vice versa did not produce the same phenotype as the respective wild-type enzymes [37, 124].

Previously, comparison of different PTPs with regard to their catalytic efficiencies was previously not possible, because the optimal substrates of PTPs were not known. The availability of the substrate specificity profiles of the eight PTPs from this study allows us to compare their catalytic efficiencies. The finding in this study leads to an intriguing question. Do most PTPs in this study prefer acidic and large hydrophobic residues to the same extent? To answer this question, Dr. Nicholas G. Selner continued the study by evaluating the preference and disfavor using three model peptides (acidic, neutral, and basic) [142]. In contrast to the similar specificity profiles of most PTPs in this study, his study showed that the intrinsic catalytic efficiencies and the degree of preference/disfavor for acidic/basic sequences display differences by up to 2 x 105 –fold

[142].

In conclusion, the eight PTPs examined in this study show similar specificity profiles: they all prefer acidic and large hydrophobic amino acids and disfavor basic residues. The only exception is PTPRA, which exhibits little sequence selectivity.

Surprisingly, none of the eight PTPs exhibit any consensus sequence(s); the preferred acidic and hydrophobic residues were found at essentially any of the positions proximal to the pY residue (pY-8 to pY+5).

Table 2.1 Peptide libraries employed in this work

Library Library Design Amino Acid Composition at Random No. Positions (X) I NH2-ASXXXXXpYAABBRM Ala, Arg, Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val II Alloc-AXXXXXXXXpYAABBRM Ala, Arg, Asn, Asp, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val III Alloc-ASXXXXXpYAABBRM Ala, Arg (10-fold reduced content), Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys (10-fold reduced content), Nle, Phe, Pro, Ser, Thr, Trp, Val

IV Alloc-AXXXXXpYXXXXXNNBBRM Ala, Arg, Asn, Asp, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val V Alloc-AXXXXXpYXXXXXEEBBRM Ala, Arg, Asn, Asp, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

Table 2.2. Peptide Library Screening Conditions

PTP Peptide Library [PTP] PTP Reaction Time [45] (Min) PTPRA I 5 15 III 5 15 IV 20-100 15 PTPRB III 5 - 10 15 IV 20 15 PTPRD II 100 15 PTPRO III 0.5 - 1 5 – 15 IV 2 - 10 15 PEST I 1 15 III 1 15 IV 1 15 PTP1B IV 10 15 SHP1 IV 1 15 SHP2 IV 2 15

Table 2.3. Most Preferred PTPRA Substrates Selected from Library I a (ASX5pYAABBRM)

RRKPF KSRFF KRXXX VKQWR NKRFF IVRRW RRAXX FRPKF RHXXX FWKKN FHRKF MRWRN KNWRR RSRIF RKQWR KKFNW WRAQR RIKAW RFRNI a M, Nle; X, unidentified amino acids; Y, F2Y.

Table 2.4. Most Preferred PTPRA Substrates Selected from Library III a (ASX5pYAABBRM, with reduced Arg and Lys contents)

ALSID VSGEA TVDHT SGNSQ IEWTG TGDTM TWDAG IFIFP TWGHE STNSE IEWTG TNGTG DWYDI TWDFT TWAHD SWDSV LEPTM TNGTG ETGDA WMEFD NWDIF TEQSL LHETI TTITA FNLDW ITSGI WEAID TWTSA LNVTA VHPTD LWEDI MVTGP IIEKY WAFSD MEITA VMETE MWSDF NISGP MLEKY WIESG MVSTA VMETM QEGDW NQWGM EMELF AFETG MVATA VPHTA TFHDI TFGGH LESLF AVETA MWETA DDYWY TGLDS TNAGS AEINW DIHTA NFITA DFYWE TSFDD TYTGD IWDNV EIDTA NTHTA DWDWW TSWDV WLTGP MYENI EMETI PFVTP ESHWQ WDFDF YMYGD TSYNN ENDTA QIATA SDMWS ARSEL NTYHR TAEQA EVPTA SDHTA SFSWP ESMEL SAIHD TLGQM FEETM SDGTS FDWEW SDSHS FESSM FPETG SEVTI SGTEP TDAHM FMSSA HQVTA SEWTL TGIEM TDQHF GELSF HYITT SHSTG a M, Nle; X, unidentified amino acids; Y, F2Y.

Table 2.5. Most Preferred PTPRA Substrates Selected from Library IV a (AX5pYX5NNBBRM)

At 150 mM NaCl

LTIAQpYRMRKR QTKRNpYARRRQ XXXXWpYQFKHK AKIGHpYFARFR NWMKKpYGRRMN KREFWpYRSPNR MHRHIpYHRTRM VPWQNpYANRFR MRNGApYFRRRK XXXXXpYIKINN XXXXXpYRNTXX KRRHMpYLKGRL IMTNFpYKNNRR QMPSFpYRMRRM TKFAEpYRRRIP NPKVApYKRFLR IDDRMpYKRNRF XXXXIpYRRKIR PRHNWpYGAHRM MRIRTpYRGPKA WQGPSpYLRTRK RSTFIpYRRKDR ITDLLpYNQGGL FHNWKpYGRLRK RHRNWpYQKSIR XXXXXpYMRRHR KKQIWpYQSQKR FRANApYRRTXX PFPGLpYRHRHR XXXXXpYKRDKK IQTIHpYRNRRM KRFPLpYLNKRF XXXXXpYMKKTR XXXXIpYRRKRI XXXXXpYRKTRR KRRNWpYGGERF RFNQWpYQKKGW QLKPFpYAMKRF KTANIpYRFSRV RIMGQpYRRPIR RVSHMpYHFKRA PNNIKpYRSRRR

At 300 mM NaCl

FQRQKpYRRRXX XXXXNpYVPWRK NRKKWpYGIRQA ARRPFpYKARKR GNVIWpYRNQGP MPKPRpYMMVXX TARFKpYAKRRH GHNDVpYDKRVK XXKKFpYRKGAK RLRRFpYGIANR ERRKFpYRWRGT STHHKpYRRRFL HPRLMpYKRNFV XDKRVpYRHKNR

At 500 mM NaCl

PKRLMpYRISQK NRISLpYRENRR DTRAFpYGWPRK XXTTRpYGVNKR RVKRFpYARTNK PWNNMpYAPNWT KRQQVpYESRRW VPNRVpYXXXWQ XXRPLpYXXQHK XXXXXpYSPKNR RPNIRpYQSRRF WGGQMpYRRQIS XXXXIpYKTKRR NSMGIpYKDRWK

Table 2.6. PTPRB Substrates Selected from Library III (ASX5pYAABBRM, with reduced Arg and Lys contents) a

FWEED DWFFD WDYEA EFDLF NHDRI DPWXX FYFED DWPWD SDWEM DWELF DDYWT FGWGW WLYED HSLFD DWVEW EWEWN WESSW RYLFX FIWDE WXXPD DWYDI DWDWG WDNVF INYVS WWQDD TSGTD LGYDW GYDFY WDNVF SNFSA YWIED YYDEF TSWDA YWDTT LEFWP MPWNI WIHDD WQEEW FGIDV YWDTM PDFTG ITFXX WEEWD WIDDF SSIES WFDXX WDIXX FRSNV WYDYD YWEES YDDFW FYDPI WDLXX WNTXX WWDYD FWDEM FEDTW SWDIS NEVTA VNTNS FFDFD WSDDS WEDSW GFDTA SDSVG NIIST GMEWD ITDDI FDDNF GWDXX QDXXI HPITS WMEID ITDDI FDDIW FIDGM DYYPF ISNIS WDGFD NEYDW IEDXX ITDNA EMWYP SHQVX WEWID IDWEW DWDFY ASEAV DWWPQ a M, Nle; Y, F2Y

a Table 2.7. PTPRB Substrates Selected from Library IV (AX5pYX5NNBBRM)

XXKWMpYKRNVK AMAPIpYGRFKQ RRNFTpYRGGIM QSNRFpYAKRRI VPNAMpYRMKKR ATNNLpYGRMIR KPNITpYARRGR NPEADpYMKRPR QLTEMpYRGKRM MHKDMpYAGKRF RFFPNpYARLKR XXXXXpYGKRFK GTQGMpYRQRWD ASPNMpYSNKPK RREGNpYXXKWQ XXXXXpYGRKWP QRPTMpYRTRXX IKRRVpYGMRGK RGKSTpYVFNQR XXXXXpYXKKGM KGNKApYRGARF IPPGMpYGFRGQ MRXXXpYRHRRI RKMRPpYIRKQG FSADVpYRXXXX ASSHVpYNAVRR LXXXXpYRVHRS NSXXXpYAQPRR MPDNIpYTRIRR RPFSIpYGXXXX RFATDpYRQRPK RRQPXpYXXXXX XXXQVpYMRPRT KLRGSpYRQTHF XXXXXpYRIKLK aM, Nle

a Table 2.8. PTPRD Substrates Selected from Library II (AX8pYAABBRM) Class I Class II Intensely Colored Lightly Colored

XDWIDWEN AWFDETQW VWDLMWEE AIEKWWDM XKRWWKNN ARNRKRGN NDFFLGDW WLDPDATW EWDWMEID AWTEMDRW PKSRFRQQ SKKRQRNI DFVTWVDF SGTDSWSW WDWGAAFD MTFWSDSI IKRWPTRL QVGRRXXX DSWWXXDW AFMRKKMD PADWDPMM FRSHQQRR AARKKRLI TWLETDLF XXIDFKDI PDDFNWNF RRLRSRVN aM, Nle

Table 2.9. PTPRO Substrates Selected from Library III (ASX5pYAABBRM, with reduced Arg and Lys contents) a

FFEWD LWEDF ESWDM EPDWI DDFWI EHVTA WNDFD VWDDF QMWDF ETEKV EETWM TFQTW NIDFD FMDEF WSSDF WNDMF DDAXX ITAVF NQDVE WYEDV WATDW ITDNF WEPYF GTGYL EDWWD YWDDI AMGDF GSEGF NDSWY GTGYI EEWWD TWDDL NYWEP TWEWT NEWWT XXIWP FEWYD QWDDI IWYDI TWEFS WDWAV NWFAI FDLWE WIEDT PFFDI FFDNS SDFHI PWWXX FDIWE WEYEF WAFES FFEHT WEHSP HTWQI FEWGD VEWDW FMWDV YFEQV SDINH ISYGI MEWTD WDMDF SGWDM WFDXX SEHTI MWGTA WDVLD WEIDW FFTDT PFDTI EFWFF SWTXX DWWND IDMDW DDEFW VWDTA DPFYY WNSNR EWYSD WEWDI DDDWF FIDTV EWTNF FGNTA EWNSE WEWDI YDDFW WIDSI DFWWS WNSXX FNIWD IEWDM FDDYM FNDXX ESWWN WSGXX WYYND ESWDW FDDHS FPEXX DFIWS TITTM FVWPD DVWEF WDDTM IGEAS DFAWS NPQTI ATWGE EIWDW WDEXX IGETG DTIWN GATTA TWVND EWPEW EWEFF MVETA ETWGT GITSM IWXXD DWHEF EFDWF DENFW DAWTA HTTGG WIHSD EWWES DFEWN EEPFW ETWGM KNHAP NVIGD DWYES EWEYI DEWNF DFASP ASGGM WEDEF EWWEI EWEFI DDWMF DFHXX TKGAI WEDEF DWYDV EWDWG DEWWA DIVNT a M, Nle; Y, F2Y

a Table 2.10. PTPRO Substrates Selected From Library IV (AX5pYX5NNBBRM) Class I Class II

FFEQApYFEDNH NNQXXpYMFDEE TQXXXpYDMSWE RQFRKpYGGRKE GLNFGpYWDDFG EWFDSpYAWDIS XXXXTpYEFGQD RPTNFpYRQPQR XXXXXpYWDSEE XXXXXpYMWLDD DFWWDpYEVWPS DQWDEpYSPWFS PIXXDpYWDVDT PFEDFpYMPWWG EXXWDpYEPMWN RGIQDpYGQFFR FENEApYWEVDF FSQEIpYVSWAN AWQDMpYDWQPF XXXXXpYGIQTW IENDMpYFDSEL AWGNFpYEEEWS AISDTpYEAWWN LTXXXpYGLKTW FQMFDpYFEPPE TNXXXpYEDWEI ASVDSpYDFASF DSNSIpYQVRKR NEEVFpYWELVE SEXXEpYDEIFD TTSNWpYDMSPV VNGXXpYKANKK XEAATpYWELFD TNPAWpYEDWLE XXWDMpYQEDGK RQTXXpYGRPRS SDWGIpYWDQQE XXXXXpYEDPND IAFFEpYQDMEW RGNALpYRSHKM GMXXDpYWENMW DAWWDpYEDLFI VAWDNpYSEWDV RFKTSpYQSRQR ARNDFpYWDMSV FWSNApYDDFWQ XXXDKpYSEMEW RRSAVpYRGGHK VSWWWpYWENPI SWSVPpYDDWPI WGNXXpYNDWDV XXXXXpYGKKRM FPFIPpYWNDED AELFPpYEEWIW VDFWGpYQWEDD SIGXXpYRRFKQ SGPXXpYWWDEV GEWMFpYEEGFQ EWNTFpYSWDES SNRRApYGRLMK TPSVQpYWSEEM EPWTWpYEDWGM QWSEDpYTFEWE RRFASpYNSKRM TWDTNpYWVEVD TWQAVpYDDAWF XXXXXpYQPEQE TRKGMpYGSRAK XXXRApYFMEFE XXXXXpYDWEDE PFTDWpYTFETT XXXXXpYXKSAK APFFDpYWIEPP WLNSPpYDWDEI GRDMFpYTFFEE GMXXXpYGRVRW LITDIpYWSDFS SXXXPpYENEDT NNRNIpYSYSED XXTVPpYGRKIK EIDPWpYWSEGF DADVWpYDFDLE LWEDVpYSWGDH LQRGPpYKRGRR IQDPTpYFWEPT DPIEIpYEIDWF XDDWFpYSTLEN VKSGQpYNFRRI TQFNDpYWFMEE VXXXXpYDWIEE SXXXXpYTPWEQ KADGIpYTRRRF FPIEWpYWPGED WNVFDpYEFAET XXXXXpYSWAEN KHRGVpYAHRKF MEDLFpYFGSEW PEFDWpYDNAEF ISGEWpYNNWNE DERQApYVRGRK IXXXXpYWGFEV PADGFpYDSSDW WGDDFpYSIPPW ARNFRpYATGWG PSDWDpYFNFLD EWAQApYDWFEN TFDEMpYTTTPN RKTGApYGFKSR QTEEFpYFWNPE XXXXXpYDGSDF QHDXXpYGDWKD ATKQApYRITRR VEMDPpYFWQWD DVWNWpYDRFDQ NRMPQpYGEFMW NXXXXpYGWKPR ESXXXpYWQPWD XXXXXpYDSWEW IFDVDpYGREFW XXXXXpYRKAQR QGXXXpYWMPWE IRXXXpYEVFDW LSWQDpYGGWFD XXXXXpYRRKKW FINEFpYMDLDW NVFASpYDWPWD FWDSTpYGPRVV IRLRRpYGQKMK IDWVApYAEWMD IEWIFpYDQWAD WLDDEpYRVWDP XXRMMpYRQAKR QDFESpYMDWSS WEWSVpYDVNWD IRNXXpYRDDDF RVHAFpYGKKVW XXXXXpYADTTA SWXXXpYDPMAD aM, Nle

Table 2.11. PTP-PEST Substrates Selected from Libraries I and III (ASX5pYAABBRM a and ASX5pYAABBRM, with reduced Arg and Lys contents)

WEEDE DWYQD VVDDW WEYDQ FEDNW GWDNM MWDEE DWWGD FFEEL WDYDS WDDNI LFDAP WDYED EKFID SWDEI WDYDA WEDSM LNDGG EWWDD ETHNE SFDDL EMWDW WDDNP EDWNY PWFDE TWGFD WSEDI DWSDF WDENV DEFAW WGMDE EDDDW FTDDI EAWDT WEDVM DDNWT DWDLE XEDEF FNEEP ELNEM DYDWW DDLFS DWDLE WDDEV VSDDS NWFDI DYDWW EDMWT WFEYD TDEEA VSDDS VFNDP DMDWW EDWNI WFDID EVDDW MPEES VFNDP DWDWI WDTNL WDYFD EWDDA EEYDW SWPDV DWDSM DLFSI WENYE GYDEW EEWDW FPGEI EWDSA DMWGI WEIGD WQDDF DDNEF QVSDV DWDSM EWQSI WDGGD FQDEF EEMDF EEDTV SWDFS WSTNS ENWWD WSEEW QDWDY NDDWW NIDYS TTNSS DFFNE WIDDF GEWDF MDDYW GWDNM MVRSS a M, Nle; Y, F2Y

a Table 2.12. PTP-PEST Substrates Selected From Library IV (AX5pYX5NNBBRM)

DDWWQpYDALDF PLDLDpYEFFWD DSDDFpYAPDFW QWDWFpYVDEPI DAIDTpYDWQAP WWDAFpYDNEMN DVDMWpYSEVEP PWEPVpYWDTDF ELWWEpYDIQPP PFDIWpYENWDD DSWSDpYNIDDF NFDSFpYSEFFN EWQLQpYEELVF GHKFEpYDDWWD EFSFApYNDNDW WIPDDpYIDNDM DLWWSpYEEVGV NFITDpYEFWDI ADDEDpYGWAGI SWQDMpYWEXXX EQRTWpYDDWGP TTQLIpYEEDWF IEMDDpYMNFPW WPWENpYGGDFM PEDWLpYEIWPL RAISFpYDDTWF WEPDIpYGWTFE MWQDIpYGWPFD WDVDFpYEDMGQ SQATFpYDDLWN WDPEMpYSVFFE INWSEpYLDLWE RMDDMpYDMWPQ SNLTGpYEDFFV GWDEVpYTDWDP NNQLWpYWEDDK AFEFEpYEEPWM EEDWIpYGWFEP XADVDpYWEAEG aM, Nle

Table 2.13. Preferred PTP1B Substrates Selected from Libraries IV a (AX5pYX5NNBBRM) and library V (AX5pYX5EEBBRM) Library IV Library IV Library V (150 mM NaCl) (500 mM NaCl) (150 mM NaCl)

Class I class I SSVIDpYFRKNT class I GTDHApYHDNLL NRSRMpYQRGLF WNGMDpYDNFVG NGFDSpYNRFTR NFISQpYSQWDT ADVFQpYKRNRW NHQQApYRVRRI IQSNDpYGIIHG PTLGDpYPRVAQ QTEPMpYSDKWD IINSMpYRRGHH PARXXpYSRIRR MEIEFpYGDAIP EMQSMpYNRSFG FFGEWpYGNDRS FTQNTpYTRRKH QRGQNpYRRAVR AEWDPpYGLDFI GSLTVpYEPMWE IISNQpYRFARR RSNLRpYSRRIM ESLELpYTEMSE class II FMGFEpYTIPEQ RRRMTpYGSFGT STWDMpYFGTAP EENHIpYRRSRQ TQLGEpYGGWHN class II RRFSKpYRVDGR HSSDTpYNEDSM KERQTpYVRHHS FNAPMpYEEWVG RRRXXpYNTKMM RRNXXpYFLKRA QDDWIpYFDTFQ LRDKLpYMQRKR SGWAFpYGSPER ANRRFpYHRRRR RKIGLpYRHLRM FFEXXpYEVGWG XESKPpYDFKRR EQTFSpYQDMWF AIAGIpYNRRKA RKFGNpYRRHVS FEVWApYQSDXX DSREGpYESHNR PESDApYSDWFQ DGNXXpYRGTRR RHAGFpYINRRS MQSWIpYDDAWV XIFGApYSRRRV LIILSpYGWNDN FQRFFpYRQSQR RVGLGpYQKFAR ASFSPpYNXXEL VTLRApYNRRNI WEMDVpYQAHAH FKNRIpYGARRA STNGRpYFEIAQ AQWTXpYEFWEP KSWRFpYRTKPQ NMWFWpYATPNE GFAGGpYRRRHG TAKNRpYSRNRR KVDTDpYDTRDG XKHRGpYKKRWH PDGTPpYQWNED HFPASpYTIRRK TRQQGpYRVKRR MWETDpYFDKGF RTNSGpYMTRKK KVEDWpYGESFP INRNIpYSRRXX VRRAFpYRTIGH PTHDDpYLQHRR RRAVGpYSNRRH EFEAFpYGQFEH IERRLpYKRRVH VNASRpYRRHKL WFSSEpYSMNDR KASAIpYRRFAX MGQDGpYGWNFE IGHXXpYGRRKK XXRNVpYRRRGF NTGDHpYITTFK PKRAMpYSMIRR AWEAVpYGEPED MPRSApYMRRIK XXKGLpYSRAVR KDANTpYIHSGR VRRRMpYKNTWM NDMXXpYEWKVD NSSRSpYIQRKR XNSAFpYTRQKW FETHVpYGNDSK IRFGNpYGARKR VAWMNpYGNQLD KLDNApYIDGFG AFSWPpYKSRRR XVNMVpYSMDFN KKDSApYNHLNM RMRGQpYRRKVH FFDGEpYTWDHT ASWSNpYMKHRH IKRRRpYGSNRK REENLpYTQHWE NSFTSpYTRAGW GQIASpYRIRWR VFENMpYRRHKR MRAPVpYRRART APQTVpYRKDWD RSFXVpYRVRXX aM, Nle

a Table 2.14. Most Preferred SHP-1 and SHP-2 Substrates Selected from Library IV (AX5pYX5NNBBRM) SHP-1 SHP-2

Q N S D E pY E W E T F E F E N F pY T W A E N N I S D D pY W S D M V T I D V W pY D M D G N S F W E D pY T D D V A V D I W L pY D E W T P W E E V D pY Q E G F L E I D W L pY R V E D G P Q W E D pY F T D A G N D W Q N pY D D D V D I S E F D pY M E A Q I A D I A V pY E G E W D V R Q D D pY M W E N V R D K I F pY D I D E E I I E I D pY M E D E P F E Q L T pY E S F E I D N E A E pY A E D W D P E N T F pY E F D W N P A E T D pY W A V V D D D S M L pY E F D R V W G D V D pY F S L G D I E P P W pY W D E T G Q F D S E pY L D M D Q W E M V S pY M E Q D E N E V A E pY E L D W E E D N W A pY W A E D Q E D L M E pY E T W E I H E S W F pY E V D E E D D T Q E pY V E W P V E G N G W pY D D W V V Q E S I D pY N V A W E E F P W F pY A L E E G T N I W D pY D D F W Q D T I N W pY D M D F Q T D F W D pY A D A S W I F N I M pY D I D D K N I W T D pY D Q E M E G S I Q F pY D E W D A D F F F E pY E P E E L F I I N T pY E D W S E D I N W D pY A T F F D P F T T W pY D E G D G D F I I E pY Q D N D D A F W N A pY Q D W Q E F I E D V pY D S G L W V W N N L pY D P F E D E P N G D pY W M D P I - N F F E pY G F E G I E A E E L pY A I P I F V T N P F pY W D E A E E N F I D pY E D I L I - D F E M pY D S F L I P E A D S pY D Q E E F N S W Q M pY T D D M F E S W P D pY Q Q E D T D X X X X pY D D L D L I D G D S pY S F Q W E I Q S N T pY N D G T D Q S V G D pY A M W D G X X X X X pY D D L V I N G D D M pY D S F W V X X X X X pY D E X X A D W I D P pY N E P W V Q I F X X pY D E W D T D T D D M pY W E I P L E X X X X pY D P D L E

47 S W I D F pY E D Q V A T X X X X pY D E D W I W Q E D Q pY M A L E S I X X X X pY D M W D E G V G E A pY D W M D F X X T E I pY E D W E R Q E F D L pY G V V E D T X X X X pY D P P S G F W A D G pY D M A N E P X X X X pY E D T W A E D F E W pY T T Q D N X X D S N pY W D D E R G W T D M pY A D D L S X X X X X pY D D S Q D W E P E V pY N V A F N D I H X X pY W E E W P V P N E T pY W D D L Q X X X X N pY E L E P H D W T D I pY Q L R E E X X G X X pY W D D V V I F Q D S pY Q E F E E X X X X X pY E N D A W F N Q D W pY S F D H T X X X X X pY M E D A V G P L E M pY S F D I E D N X X X pY D V W E V W N V D G pY E A D V W X X X X X pY Q E D M - D D E W L pY E V P N E X X X X D pY D I A Q I P F W D M pY Q S D Q N H A X X X pY I D F E D V D E W F pY A A D E Q X X X X X pY E T W W D V G S E T pY D D P W M I X X X X pY M E M D D A I D Q A pY E E G W F X X X X X pY W D V G - D E D I W pY A V N E S R X X X X pY L D F S F V I D I I pY E D N M W X X X X X pY A E T W D F D D F F pY E M A E M X X X X X pY S D N A I A H D R V pY D W D D F X X X X X pY V T D M W V E E F F pY N V N E T X X X X R pY S D V I L W F E G P pY D M D D T X X X X X pY A T D I I G F E A S pY F F D D I T X X X X pY W K D E E F T D P W pY E M E N D X X X X X pY X X X R W F T D S W pY T W G S E Q X X X X pY S A E E H P S D I M pY S I D T E I G S X X pY N X X X V N P D G W pY M Q W E E X X X X X pY S H F X X aM, Nle; X, residues that could not be unambiguously determined. Acidic, basic, and aromatic residues are shaded in red, blue, and gray, respectively.

Table 2.15. Kinetic Properties of PTPRA against pY Peptides

Entry Peptide Sequence kcat KM kcat/KM No. (s-1) (mM) (mM-1 s-1)

1 Ac-EADTApYAA-NH2 26 ± 1 1.4 ± 0.1 19

2 Ac-ASSDDpYAA-NH2 ND ND 13

3 Ac-WDEDFpYSASA-NH2 ND ND 69

4 Ac-ARKRIpYAA-NH2 39 ± 1 0.49 ± 0.03 80

5 Ac-RRISTpYAA-NH2 49 ± 3 1.2 ± 0.1 41

6 Ac-SASASpYSASA-NH2 23 ± 4 0.53 ± 0.15 43

7 Ac-AAAAApYEEVH-NH2 ND ND 39

8 Ac-AAAAApYRHRR-NH2 30 ± 2 2.6 ± 0.2 12

a 9 Ac-TATEPQpYQPGENL-NH2 10 1.07 9.35

a 10 Ac-TSTEPQpYQPGENL-NH2 9 1.16 7.76

a 11 Ac-LIEDNEpYTARQGA-NH2 9 0.77 11.69

12 Phenyl phosphate ND ND 0.020

13 pNPP 0.54 ± 0.28 2.7 ± 0.3 0.19 aData from ref. [101]. Data reported represented the mean ± SD from three or more independent sets of experiments.

Table 2.16. Kinetic Properties of PTP1B against pY Peptides

Entry Peptide Sequence kcat KM kcat/KM No. (s-1) (M) (M-1 s-1)

1 Ac-ASSDDpYAA-NH2 33 ± 1 18 ± 1 1.8

2 Ac-DTADVpYAA-NH2 31 ± 3 8.9 ± 1 3.5

3 Ac-DTADApYAA-NH2 38 ± 2 35 ± 2 1.1

4 Ac-DTAAVpYAA-NH2 32 ± 1 18 ± 0.14 1.8

5 Ac-DAADVpYAA-NH2 32 ± 4 10 ± 1 3.1

6 Ac-ATADVpYAA-NH2 31 ± 1 12 ± 2 2.5

a 7 Ac-AAAApYAAAA-NH2 - - 1.3

8 Ac-DTADVpYDWEF-NH2 29 ± 5 5.2 ± 1 5.8

9 Ac-DTADVpYGEFTI-NH2 33 ± 3 7.0 ± 1 4.8 aData from ref. [88]. Data reported represented the mean ± SD from three or more independent sets of experiments.

Figure 2.1. Histograms showing the sequence specificity of PTPRA (A), PTPRB (B), PTPRD (C), PTPRO (D), and PTP-PEST (E) on the N-terminal side of pY. The y axis represents the percentage of selected peptides that contained a particular amino acid (x axis) at a given position within the peptide (pY-8 to pY-1, on the z axis). Data shown were from libraries I and III for PTPRA and PTP-PEST, library II for PTPRD, and library III for PTPRB and PTPRO. M, Nle; Y, F2Y.

Chapter 3

SPECIFICITY PROFILEING OF DUAL SPECIFICITY PHOSPHATASE VACCINIA

VH1-RELATED (VHR) b

3.1 Introduction

The studies of PTPs substrate specificity [36, 87, 88, 93] together with my work in Chapter 2 revealed that although no two PTPs share exactly the same substrate specificity profile, they generally have broad and overlapping sequence specificity toward peptide substrates. However, most of these PTPs also contain other domains (e.g.

SH2 domains and membrane localization motifs) in addition to the catalytic domain, it is likely that the in vivo substrate specificity of these enzymes is enhanced by their

“recruiting” domains. In this regard, the substrate specificity of the subgroup of atypical dual specificity phosphatases raises intriguing questions [150]. Because most members of this subgroup contain only the catalytic domain and do not have any recognizable

b This research was originally published in journal of biological chemistry.

Luechapanichkul, Rinrada, Chen, Xianwen, Taha, Hashem A., Vyas, Shubham, Guan, Xiaoyan, Freitas, Michael A., Hadad, Christopher M., Pei, Dehua. Specificity Profiling of Dual Specificity Phosphatase VHR Reveals Two Distinct Substrate-Binding Modes. Journal of Biological Chemistry. 2013; 288: 6498-6510. © the American Society for Biochemistry and Molecular Biology. 51 substrate-recruiting domains or surfaces, do their PTP active sites have more stringent sequence selectivity compared with the classical PTPs? Or alternatively, do they simply act as relatively nonspecific phosphatases in vivo?

The 21-kDa vaccinia VH1-related (VHR) phosphatase is a member of the atypical dual specificity phosphatase subgroup and one of the first dual specificity phosphatases identified [151, 152]. Other than the conserved PTP signature motif, HCXXGXXR, VHR has little sequence similarity to the classical PTPs. Knockdown of VHR by siRNA resulted in cell cycle arrest in G1/S and G2/M, indicating a critical role for VHR in cell cycle progression [153]. A link between VHR and several types of cancers has recently been established [154-156]. Despite the importance of VHR in cell cycle regulation and cell signaling pathways, only a few physiological VHR substrates have been identified.

VHR has been shown to inactivate mitogen-activated protein kinases (MAPKs) Erk and

Jnk in vivo by dephosphorylating both tyrosine and threonine phosphorylation sites within the Thr-X-Tyr motif of their activation loop [157-159]. However, purified Erk

4 proved to be a relatively poor in vitro substrate of recombinant VHR (kcat/KM ~ 10

M-1s-1) compared with its highly efficient dephosphorylation by other MAPK

6 -1 -1 phosphatases (kcat/KM ~ 10 M s ) [158, 160]. This raises the question whether dephosphorylation of the MAPKs by VHR in vivo is aided by any recruiting strategy.

Other than the MAPKs, signal transducers and activators of transcription 5 (STAT5) with a single pY site, epidermal growth factor receptor, and ErbB2 have also been reported as

VHR substrates [161, 162]. Two recent studies showed that phosphorylation of VHR at

Tyr138 is required for its efficient dephosphorylation of MAPKs and STAT5 in vivo [161,

163]. In the case of STAT5, it was suggested that the pY138 motif binds to one of the SH2 domains of the STAT5 homodimer, thereby recruiting VHR to the substrate protein [161,

164]. In addition, this interaction displaces the STAT5 pY694 peptide from the SH2 domain and renders it available for dephosphorylation by the VHR active site. How

Tyr138 phosphorylation increases its activity toward Erk and Jnk is currently unknown.

Kang and Kim [165] recently reported that the vaccinia-related kinase 3 binds to and activates VHR and that this direct interaction regulates Erk signaling, but again the molecular mechanism of activation remains a mystery. By using synthetic peptides derived from mitogen activated protein kinases p38 and Jnk1, Dixon and co-workers

[166] showed that VHR is highly active against pY peptides but has only very weak activity toward pS and pT substrates. It was reported that VHR has a moderate preference

(by 2–3-fold) for bisphosphorylated peptides (e.g. pTXpY where pT is phosphothreonine and pY is phosphotyrosine) over monophosphorylated pY peptides (e.g. TXpY) [158,

167].

To our knowledge, there has been no systematic study of the VHR substrate specificity. In this work, we profiled the sequence specificity of VHR by screening combinatorial peptide libraries. VHR recognizes two distinct classes of peptide sequences. Although the first class of substrates are similar to the pY motifs derived from known VHR protein substrates, the class II substrates contain a (V/A)PI motif and bind to the VHR active site in an opposite orientation relative to the class I substrates. Our results suggest that the sequence specificity of the VHR active site is a key determinant of its in vivo substrate specificity.

3.2. Experimental Procedures

3.2.1. Materials

DNA primers for site-directed mutagenesis experiment were purchased from

Integrated DNA Technologies (Coralville, IO). Pfu Turbo polymerase and DpnI enzymes were from Stratagene (Santa Clara, CA). GST-LASP1 (LIM and SH3 domain protein1) was obtained from Novus Biologicals (Littleton, CO), and roundabout homolog 1

(ROBO1)-Myc-DDK was from Origene (Rockville, MD). Abl protein-tyrosine kinase was from New England Biolabs (Ipswich, MA). Anti-ROBO1 (clone 7E3.1) and anti-pY

(clone 4G10) antibodies were obtained from Millipore (Temecula, CA). Sequencing grade trypsin was from Promega (Madison, WI). Other reagents were the same as described in Chapter 2.2.1.

3.2.2. VHR Purification and Library Screening

Wild-type and mutants human VHR and was expressed in E. coli and purified as described previously [166]. VHR mutants were generated by the QuikChange mutagenesis method using the following primers: D18N, cgacctgctctcgaacggcagcggctgctac; D47N, gtctgtggctcagaacatccccaagctg; E159Q, gtgaggcagaaccgtcagatcggccccaacga; and D164N, gagatcggccccaacaatggcttcctgg. All mutations were verified by sequencing the entire coding region. The screening was similarly performed as previously described in Chapter 2.2.3. The library beads were

54 incubated with VHR (final concentration 200 nM) for 15 min in 800 l in blocking buffer

(30 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% Tween 20, and 0.1% gelatin).

3.2.3. Synthesis of Selected Peptides and Enzymatic Assays

Peptides were prepared using Fmoc solid phase synthesis as in Chapter 2.2.4. The

VHR enzymatic assays were performed by two techniques described in Chapter 2.2.5.

The measurement of absorbance change at 282 nm was performed against peptides with acetylated N-terminus; while the reaction progress curve technique was determined against peptide substrates containing free N-terminus.

3.2.4. Molecular Modeling

We utilized the available crystal structure of the C124S mutant of VHR bound with a pY peptide in the active site (Protein Data Bank code 1J4X) [167]. The peptides studied herein were built using the backbone atoms of the native ligand in the crystal structure, keeping the pY residue unmodified. Additionally, the C124S mutation was inverted using UCSF Chimera [168]. The protonation states of titratable amino acids were identified using pdb2pqr utility [169] for pH 7. To prepare these systems for molecular dynamics (MD) simulations, the structures were solvated in an octahedron box of TIP3P water molecules with counter ions to make the system neutral in terms of charge [170, 171]. The MD simulations involved a three-step minimization before the production MD simulations. In the first step, only water molecules were allowed to relax, whereas the protein and ligand were fixed. In the subsequent steps, the entire system was allowed to minimize followed by a gradual increase of the temperature of the system

55 from 0 to 300 K. The resulting minimized system was used for production MD simulations using the isothermal–isobaric (NPT) ensemble. These MD simulations were performed using the AMBER ff03 force field as implemented in the AMBER 10.0 software package [172]. The MD trajectories were then utilized to determine relative free energies of binding of peptides using the Poisson-Boltzmann (MM-PBSA) [173-175] and the generalized Born (MM-GBSA) methodologies [176] as implemented in AMBER.

3.2.5. In Vitro Protein Dephosphorylation by VHR

ROBO1 (1.0 g) was phosphorylated with Abl (100 units) in the presence of ATP

(10 mM) in NEBuffer for kinase (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM

EGTA, 2mM dithiothreitol, and 0.01% Brij 35) overnight at 37 °C (total reaction volume,

20 l). The kinase reaction was terminated by the addition of 50 mM EDTA, pH 7.0

(final concentration). Subsequent dephosphorylation of the protein was carried out by adding 0.25 ng of VHR to the above solution followed by incubation at 25 °C. At various time points (0–50 min), 8-l aliquots were withdrawn and mixed with an equal volume of

2× SDS-PAGE loading buffer. The samples were boiled for 10 min, separated by 8%

SDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-pY antibody.

The membrane was washed in 200 mM glycine, pH 2.2, 3.5 mM SDS, and 1% Tween 20 and reprobed with anti-ROBO1 antibody following the manufacturer’s recommendations.

LASP1 was phosphorylated by Abl and evaluated as a potential VHR substrate in a similar manner.

3.2.6. In-solution Digestion

Abl-phosphorylated ROBO1 or LASP1 from above (1.0 g) was reduced with 10 mM dithiothreitol at 25 °C for 30 min in 100 mM NH4HCO3 and alkylated with 40 mM iodoacetamide in 100 mM NH4HCO3 at 25 °C for 30 min. Excess iodoacetamide was quenched with 40 mM dithiothreitol at 25 °C for 30 min. LASP1 was digested at a 1:20 enzyme/substrate ratio in 100 mM NH4HCO3, pH 7.6 with Glu-C (sequencing grade;

Promega) at 37 °C for 16 h followed by trypsin digestion at 37 °C for 5 h. ROBO1 was digested with trypsin overnight at 37 °C. The resulting peptides were desalted with a C18

ZipTip and eluted with 50% acetonitrile containing 0.1% trifluoroacetic acid.

3.2.7. Mass Spectrometry

The peptides from the above proteolytic digests were separated by reversed-phase

HPLC (Dionex Ultimate 3000 capillary/nano-HPLC system, Dionex, Sunnyvale, CA) and analyzed on a Thermo Fisher LTQ Orbitrap XL (Thermo Finnigan, San Jose, CA).

The peptides were separated on a 0.2 × 150-mm C18 column (3 m, 200 Å; Michrom

Bioresources Inc., Auburn, CA) at a flow rate of 2 ml/min with mobile phase A containing H2O and 0.1% formic acid and mobile phase B containing acetonitrile and

0.1% formic acid. A blank run was carried out in between each sample injection. Protein and peptide sequences were determined by the MassMatrix database search engine

(MassMatrix 2.4.2, February 22, 2012).

3.3. Results

3.3.1. VHR Substrate Specificity (N-terminal to pY)

Dr. Xianwen Chen, a former group member, previously examined the specificity of VHR by performing preliminarily library screening and kinetics measurement. Her specific contribution is indicated in Chapter 3.5. Because PTPs, which have been characterized previously, generally show stronger specificity on the N-terminal side of pY [36, 87, 88, 93], we first screened VHR against a peptide library containing five random residues on the N-terminal side of pY, NH2-ASXXXXXpYAABBRM-resin

(library I; where B is -alanine and X is 3,5-difluorotyrosine (F2Y), norleucine, or any of the 17 proteinogenic amino acids excluding Met, Cys, and Tyr) (Table 3.1). Library screening involved treatment of a portion of the one bead-one compound library (40,000 beads/reaction) with a small amount of VHR (200 nM final concentration) for a short time (15 min) so that only the most preferred sequences would undergo significant dephosphorylation. The reaction product (i.e. exposed tyrosine side chain) was then selectively oxidized by tyrosinase into an orthoquinone, which was incubated with 3- methyl-2-benzothiazolinonehydrazone to form a reddish pigment that remained covalently bound to the positive beads [132, 177]. The colored beads were removed from the library and individually sequenced by the PED-MS [95]. The N-terminal dipeptide

Ala-Ser was added to minimize any potential bias on the library screening by the positive charge associated with the free N terminus, whereas the BBRM motif facilitated peptide

58 release (cleavage after the Met by CNBr) and MS analysis (Arg provides a fixed positive charge).

Screening of a total of 160,000 library beads (in four separate experiments) produced 98 positive sequences, which can be separated into two different classes on the basis of sequence similarity (Table 3.2). Inspection of the class I substrates (92 sequences) revealed that VHR has strong sequence selectivity immediately N-terminal to pY (Figure 3.1A). At the pY-1 position, VHR strongly prefers a Pro residue (in 52% of all class I peptides) followed by Ile, Nle, and other amino acids of small side chains (Ser,

Ala, Val, and Thr). At the pY-2 position, the most preferred residues are Asp, Ser, Asn, and Thr. Six of the selected peptides also contained the (S/T)(D/E)pY motif. Thus, the screening result is in agreement with the previously reported VHR recognition motif - pTXpY- (where X is Glu, Gly, or Pro) [166, 167]. Although pS and pT were not included in the library, Asp and Glu can often serve as effective pS and pT mimetics, respectively.

Therefore, the strong selectivity for Asp/Glu at the pY-2 position suggests that pS/pT` would be preferred at this position. Furthermore, the ability of VHR to tolerate both S/T and pS/pT at this position implies that VHR should be able to dephosphorylate the pY residue of the mitogen-activated protein kinases even when the TXY motif is not stoichiometrically phosphorylated on both tyrosine and threonine residues. VHR shows a modest preference for acidic (Glu and Asp) and hydrophobic residues (Phe, Trp, and Ile) at pY-3 to pY-5 positions but tolerates most of the other amino acids (Figure 3.1A). The class II substrates, although present in only six of the selected sequences, have a tight consensus; they all contain an API (or APL) motif (Table 3.2).

To test whether VHR has any specificity for amino acid residues beyond the pY-5 position, we repeated the screening experiment against library II, NH2-

AXXXXXXXXpYAABBRM (80,000 beads) (Table 3.1), which contains eight random residues N-terminal to pY. Surprisingly, of the 16 positive sequences, seven contained an

N-terminal API or APF motif (Table 3.3). The two groups of peptides clearly have different sequences. Although the class I peptides are rich in either acidic or basic residues throughout their sequences, the API-containing peptides (class II) typically have acidic residue(s) close to pY and hydrophobic amino acids between the acidic residues and the N-terminal API motif. Because the class II peptides all have the API motif at the

N-terminus, we tested the importance of a free N-terminal amine for VHR recognition by screening it against library III, Alloc-AXXXXXXXXpYAABBRM (Table 3.1), which contained an N-terminal allyloxycarbonyl (Alloc) group but was otherwise identical to library II. Screening of library III (80,000 beads) produced 49 positive sequences (Table

3.3). Remarkably, none of the selected peptides contained an API motif; they all have sequences similar to those of the class I peptides selected from libraries I and II. The drastically different screening results between libraries II and III suggest that VHR contains a specific binding site for the N-terminal API motif.

3.3.2. Identification of Other Class II Motifs and Optimal Distance from pY

Because library II contained a fixed Ala at the N-terminus that might have biased the library screening toward the N-terminal API motif, we asked whether VHR could recognize sequences other than API. To answer this question, we screened VHR against

60 libraries IV (NH2-XXXXXpYAABBRM) and V (NH2-XXXXXXXXpYAABBRM)

(80,000 beads for each library) (Table 3.1). Both libraries had completely random sequences at the N-terminus. Among the 17 sequences selected from library IV, 14 (82%) contained an N-terminal NH2-(V/A)PX motif where X is often a -branched amino acid

(e.g. Thr, Ile, and Val) (Table 3.3). The more frequent selection of an N-terminal Val than Ala suggests that Val is the preferred N-terminal residue for VHR. The remaining three peptides contained an N-terminal Thr, which is structurally similar to Val.

Screening of library V did not result in an overwhelming selection of peptides with N- terminal (V/A)PX motifs (Table 3.3). Of the 27 selected sequences, only two peptides contained N-terminal VPT and APQ sequences. However, the selected sequences clearly showed an over-representation of Val at the N-terminus (12 of the 27 peptides). A possible explanation for the above observations is that the NH2-(V/A)PX motif needs to be situated at an appropriate distance from the pY residue for VHR to engage in optimal interactions with both the NH2-(V/A)PX and pY motifs; either increasing or decreasing the distance would diminish its binding affinity for VHR.

To determine the optimal distance between the (V/A)PX motif and pY, we modified library IV by adding Ala, Val, Ala-Pro, or Ala-Pro-Ile to its N-terminus to give libraries VI–IX, respectively (Table 3.1). The four libraries (120,000 beads each) were screened against VHR under the same conditions. We noted that the positive beads from libraries VI and VII developed reddish color faster and of a greater intensity than those in libraries VIII and IX, although the former had a smaller number of colored beads due to their lower probability of having sequences containing the (V/A)PI combination. With

61 either Ala or Val at the pY-6 position, the sequences selected from libraries VI and VII contained predominantly Pro at the pY-5 position and hydrophobic residues, most frequently Ile, at the pY-4 position (Table 3.3). Similarly, most of the peptides selected from library VIII had Ile or other hydrophobic residues (e.g. Nle, Leu, Val, and Phe) at the pY-5 position. On the other hand, the positive sequences selected from library IX did not show any obvious trend except for the frequent selection of Asp, Glu, Ser, and Thr at the pY-2 position. The selection of charged residues throughout the random region is reminiscent of the class I sequences derived from libraries I and II (Table 3.2). Taken together, the library screening results suggest that VHR contains a second binding site that specifically recognizes the peptide motif NH2-(V/A)P(I/L/M/V/F), and for optimal binding to VHR, this motif should be separated from the pY residue by one to six amino acid residues.

3.3.3. VHR Substrate Specificity (C-terminal to pY)

To determine the substrate specificity of VHR on the C-terminal side of pY, we first screened it against library X, IDFD(D/P)pYXXXXXNNBBRM (Table 3.1), which contains a preferred class I sequence of VHR on the N-terminal side of pY. Analysis of the selected sequences revealed several features. First, the results confirmed the preference of VHR for a Pro residue at the pY-1 position; 39 of the 43 peptides (91%) had a Pro at this position (Table 3.4 and Figure1B), whereas Asp and Pro were equally populated in the original library X (50% each). Second, VHR prefers a small (Gly, Ala, and Ser) or to a lesser extent a hydrophilic residue (Gln, Glu, and Arg) at the pY+1

62 position. It does not show significant selectivity beyond the pY+1 position, although basic residues are dramatically under-represented (only one Lys in 43 sequences). To test whether the preferred N-terminal sequence biased the C-terminal sequences, we also screened VHR against a library containing five random residues on either side of pY,

NH2-AXXXXXpYXXXXXNNBBRM (library XI; Table 3.1). The data confirmed the preference of VHR for an N-terminal AP(I/V/L) motif and broad specificity on the C- terminal side of pY (Table 3.5).

3.3.4. Kinetic Properties of Selected Peptides

To confirm the library screening results, representative peptides selected from the peptide libraries were individually synthesized, and their kinetic properties against VHR were determined in solution and compared with those of previously reported peptide substrates. Peptide Ac-VSEDPpYAASAS (Table 3.6, peptide 1), a class I sequence

-1 selected from library I, has a kcat value of 4.5 s , a KM value of 117 M, and a kcat/KM value of 3.8 × 104 M-1 s-1. Consistent with the screening data, substitution of Ser for Asp at the pY-2 position of peptide 1 had minimal effect on the kinetic activity, whereas phosphorylation of the pY-2 Ser reduced the activity by 3-fold (Table 3.6, compare peptides 1–3). The latter observation is in contrast to previous reports that bisphosphorylated MAPK peptides (pTXpY) were 2–3-fold more active than the monophosphorylated peptides (TXpY) [158, 167]. Given the relatively small effect upon phosphorylation of the pY-2 residue, we believe that VHR readily accepts both

(pS/pT)XpY and (S/T)XpYproteins as substrates, and the effect of phosphorylation is

63 sequence-dependent. The importance of Pro at the pY-1 position is highlighted by the

2.5-fold decrease in activity when the Pro was replaced by Ile, the second most frequently selected amino acid at this position, and the 24-fold activity reduction upon replacement by Lys (which was not selected during screening) (Table 3.6, compares peptides 1, 4, and

5). Replacement of the C-terminal AASAS sequence of peptide 1 with acidic motifs

(YDFAS and DFEAS) resulted in a slight reduction in activity (Table 3.6, peptides 6 and

7) in agreement with the preference of VHR for a small, neutral residue at the pY+1 position. The class II peptide tested, Ac-FAPISpYAASAS (peptide 8), was also highly

4 -1 -1 active with a kcat/KM value of 6.0 × 10 M s . In comparison, control peptides Ac-

SASASpYSASA and Ac-ARKRIpYAA, which were not selected from the library, showed 100–500-fold lower activity (Table 3.6, compare peptides 1, 9, and 10).

4 -1 -1 Phosphorylated Erk and Jnk have a kcat/KM value of 4 × 10 M s [158, 159], whereas the pY694 peptide of STAT5 (peptide 11) has a second-order rate constant of 6.2 × 103

M-1 s-1. These results demonstrate that both class I and II peptides are efficient VHR substrates and that the active site of VHR displays a high level of sequence selectivity.

The structural determinants of class II substrates were further assessed by synthesizing and assaying peptides 12–24, which contained a free versus acetylated N-terminus, Val versus Ala as the N-terminal residue, and different numbers of intervening residues between the (V/A)PI motif and pY (Table 3.7). The results show that VHR strongly prefers a (V/A)PI motif with a free N-terminal amine; acetylation of the N terminus reduces the catalytic efficiency by 10–15-fold (compare peptides 12 and 13; peptides 14,

15, and 16; and peptides 17, 18, and 19). An N-terminal Val generally gives slightly

64 higher activity than the corresponding APX peptides (1.5–2-fold). Finally, the kinetic activity increased as the number of intervening residues between the (V/A)PI motif and pY decreased, reaching the maximal activity at two residues (compare peptides 20–24).

Further reduction in the distance decreased the activity. To our knowledge, peptide 22 is

6 -1 -1 the most active VHR substrate reported to date (kcat/KM value of 1.0 × 10 M s ).

3.3.5. Class II Substrates Bind VHR in an Alternative Mode

The fact that VHR is able to recognize class II substrates containing a varying number of intervening residues between the (V/A)PI and pY motifs suggests that VHR has a separate binding site for the (V/A)PI motif. To locate the secondary binding site, we initially attempted to co-crystallize the catalytically inactive C124S mutant VHR with several class II substrates. Unfortunately, all of our efforts were unsuccessful. We therefore resorted to site-directed mutagenesis to map the (V/A)PI-binding site on VHR.

Because a free N terminus increases the reactivity by 10–15-fold primarily through improved binding (lower KM value), we reasoned that the secondary binding site may contain at least one acidic residue that engages in charge-charge interactions with the positively charged N terminus of the substrate. Inspection of the co-crystal structure of

VHR bound with peptide DDE(Nle)pTGpYVATR [167] identified four acidic residues within a 24-Å distance from the active site. Among these acidic residues, Asp18, Asp47, and Glu159 residues are located near the binding surfaces for the N-terminal portion of the peptide substrate, whereas Asp164 interacts with the C-terminal fragment by forming a hydrogen bond with the Thr residue at the pY + 3 position. These four residues were

65 individually mutated to the corresponding Asn or Gln, and the kinetic properties of the resulting VHR mutants were assessed against p-nitrophenyl phosphate and class II substrates containing both free and acetylated N-termini.

The D47N, E159Q, and D164N mutants had essentially the same catalytic activity as WT-VHR toward p-nitrophenyl phosphate, whereas the D18N mutant was 2-fold less active due to a lower kcat value (Table 3.8). Thus, mutation of residue 47, 159, or 164 does not significantly affect the active site structure of VHR. When peptide NH2-

APINDIpYAA (peptide 18, which contains a free N-terminus) was used as the substrate,

WT-VHR and the D18N, D47N, and E159Q mutants had similar activities with KM

5 -1 -1 values of 15–30 M and kcat/KM values of 1.2–5.0 × 10 M s . In contrast, the D164N mutant was 8.8-fold less active due to its increased KM value (170 M instead of 23 M for WT). After peptide 18 was acetylated at the N-terminus (Ac-APINDIpYAA), all five

4 -1 -1 enzyme variants had similar kcat/KM values (0.47–1.9 × 10 M s ). Note that N- acetylation reduced the activity toward WT, D18N, D47N, and E159Q VHR by 15–55- fold but only by 3.8-fold for the D164N mutant. The same observation was made with another class II substrate, NH2-APFPQDIpYAA (peptide 15). These data indicate that in the ES complex, the N-terminus of the class II substrates is near Asp164; to make this possible, the peptide substrate must bind to VHR in an orientation opposite to what has been reported for class I substrates [167]. To further illustrate the different binding modes of the two classes of substrates, we tested WT and D164N VHR against a class I peptide that bears a free N-terminus, NH2-VSEDPpYAA-NH2 (peptide 25). Unlike the class II substrates, the D164N mutation had no significant effect on the kinetic activity of this

66 class I peptide (Table 3.8). Interestingly, the presence of a free N-terminal amine on the class I peptide reduced its activity toward WT-VHR by 4-fold (compare peptides 1 and

25). Presumably, binding of this substrate in the canonical mode [167] places the N- terminal amine near a positively charged surface distal from Asp164. Repulsive interaction with the positively charged VHR surface decreases its binding affinity for the enzyme.

3.3.6. Molecular Modeling

To gain structural insight into the alternate binding mode by class II substrates, we collaborated with Dr. Hashem A. Taha and Dr. Shubham Vyas in the Hadad

Laboratory (The Ohio State University) to conduct the molecular modeling of peptides 21

(NH2-VPISVTpYAA) and 22 (NH2-VPIVTpYAA) binding to VHR. Initial attempts to dock the peptides onto the protein using Autodock 4.0 [178] were unsuccessful. As a result, we adopted the backbone atoms of the peptide resolved in the crystal structure to morph peptides 21 and 22 but kept the pY constant. Because the crystal structure indicates a classical mode of binding (i.e. with the C-terminus near the Asp164 residue), we rotated/ inverted the studied peptides to explore other possible binding orientations.

Furthermore, both peptides 21 and 22 contain a proline residue, which can adopt a cis or trans configuration. Thus, eight peptide protein complexes were subjected to the MD simulations and binding energy calculations as described under “Experimental

Procedures.” MD simulations were completed through 28 ns to eliminate any potential bias in the initial structures of the complexes.

It is clear by the root mean square deviation plots of the complexes that no unusual structural fluctuations were observed in any of the performed simulations (Figure

3.2A and 3.2B). The relaxed structures of peptide 22 bound to VHR in both of the canonical and reverse binding modes and their comparison with the VHR co-crystal structure are shown in Figure 3.3. In addition, atomic interactions involved in the active site of VHR are presented in Figure 3.3D. These snapshots demonstrate that the key interactions between the peptides and protein exist primarily at the active site pocket with some conformations showing H-bonding interactions with the Asp164 residue (Figure

3.4). This observation holds true only for ligands in the reverse orientation and for approximately 12–22% of the conformational ensembles (Table 3.9). In other words, binding is not significantly affected by other amino acids in the peptide. This is consistent with the experimental kinetic studies where the KM values of peptides 13, 15, 16, and 18–

24 remained remarkably unaltered (Table 3.7).

Computed MM-PBSA and MM-GBSA energies for different binding modes are summarized in Table 3.10. The binding energies of peptides 21 and 22 are more favorable in the reverse mode (N terminus pointed toward Asp164) than the canonical binding mode (when the Pro residue is in trans configuration), which is in agreement with the experimental observations.

Furthermore, MD simulations show that both peptides prefer the trans configuration of the Pro residue over a cis proline. Although both peptides prefer the reverse binding mode, modeling results do not favor one peptide over the other in agreement with the experimental observation that peptides 21 and 22 have rather similar

KM values (Table 3.7). In addition to the MD simulations with wild-type VHR, we also carried out MD simulations on the D164N mutant. The mutation was performed using the

UCSF Chimera software suite and was subjected to the same simulation protocol as that used for the wild-type enzyme. There was no unusual structural change upon D164N mutation as shown in the root mean square deviation plot (Figure 3.5). This suggests that there are no significant deviations in the binding modes of the ligands upon D164N mutation.

3.3.7. In Vitro Dephosphorylation of Class II Protein Substrates by VHR

To examine the physiological relevance of the class II substrates, we searched the human proteome for proteins with a free N-terminal (V/A)P(I/L/M/V/F) motif and a pY residue near the N terminus. According to the PhosphoSite database, no human protein contains such an N-terminal sequence as most of the eukaryotic proteins are N-terminally acetylated [179]. We next searched the database for proteins containing internal

(V/A)P(I/L/M/V/F)X0–6pY motifs as potential VHR substrates. Table 3.11 lists some of the potential substrates, which have been experimentally demonstrated to be phosphorylated at the putative class II VHR sites in vitro and/or in vivo. Two of the proteins, LASP1 (LIM and SH3 domain protein 1) and ROBO1, were selected for further study because their putative class II VHR sites (TSAPVpY171QQPQQ and

VAPVQpY1114NIVEQ, respectively) are known to be phosphorylated by Abl kinase [180,

181], which is commercially available. Indeed, treatment of recombinant ROBO1 and

LASP1 with Abl resulted in efficient tyrosyl phosphorylation of both proteins (Figure

3.6, A and B). We collaborated with Dr. Xiaoyan Guan in Freitas Laboratory (The Ohio

State University) to identify the phosphorylation of ROBO1 and LASP1 by mass spectrometry. Protease digestion of the resulting phospho-ROBO1 followed by LC-

MS/MS analysis confirmed the phosphorylation of Tyr1114 (Figure 3.6C and Table 3.12).

Due to lack of proper protease cleavage sites near LASP1 Tyr171, confirmation of Tyr171 phosphorylation by LC-MS/MS was unsuccessful. Western blot analysis with anti-pY antibody showed that both ROBO1 and LASP1 were efficiently and completely dephosphorylated by VHR (Figure 3.6, A and B). Thus, VHR is able to dephosphorylate class II motifs in intact proteins. Consistent with this observation, peptides corresponding

171 1114 to the pY and pY sites were efficient VHR substrates with kcat/KM values of 4.1 ×

103 and 1.9 × 103 M-1 s-1, respectively (Table 3.6).

3.4. Discussion

The lack of any recognizable substrate-recruiting domain/surface by the atypical dual specificity phosphatases implies that their PTP active site must possess substantial sequence selectivity to selectively dephosphorylate their cognate substrates. Screening of combinatorial peptide libraries against VHR, a representative member of the subgroup, reveals that VHR indeed has significantly narrower sequence specificity than the classical

PTPs [36, 87, 88, 93]. The selected sequences, which should represent the most reactive substrates of VHR, exhibit remarkable sequence covariance. On the basis of sequence similarity, the peptide substrates can be divided into two major classes (classes I and II).

Within the class I substrates, the peptides can be further separated into two subclasses

70 with the class IA and IB substrates having sequences of

(E/D/)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) and (E/D/)(T/S)(D/E)pY(G/A/S/Q)

(where  is a hydrophobic residue), respectively. To date, five protein substrates have been reported for VHR, and the pY sites dephosphorylated by VHR contain the following sequences: Erk, GFLpTEpYVAT [159]; Jnk, FMMpTPpYVVT [158]; STAT5,

KAVDGpYVKP [161]; epidermal growth factor receptor, VDADEpYLIP [162]; and

ErbB2, IDETEpYAAD [175]. These in vivo VHR substrate motifs largely match the consensus sequences derived from our library screening. Two of the proteins, phosphorylated Erk and Jnk, have been purified to homogeneity and assayed against recombinant VHR in vitro [158-160]. Both protein substrates had a kcat/KM value of 4 ×

104 M-1 s-1. Short peptides corresponding to the pTXpY motifs of these two proteins gave similar kcat/KM values [166, 167], suggesting that most of the interactions between VHR and these proteins are mediated through the VHR active site. On the other hand, the

694 STAT5 pY peptide is a substantially poorer substrate in vitro (kcat/KM value of 6.2 ×

103 M-1 s-1). It has been proposed that the activity of VHR toward STAT5 in vivo is enhanced by additional interactions between pY138 of VHR and the STAT5 SH2 domain

[161, 164]. Note that the class I peptides selected from the libraries generally have kcat/KM values of 1–4 × 104 M-1 s-1 (Table 3.6), which is in the same range as the protein substrates. These data suggest that the sequence specificity of the VHR catalytic site is a key determinant of its substrate specificity in vivo. The relatively low intrinsic catalytic efficiency (defined as the kcat/KM value of a PTP active site toward its optimal peptide substrate) of VHR toward class I substrates as compared with other PTPs (e.g. PTP1B,

SHP-1, and SHP-2 have intrinsic catalytic efficiencies of >107 M-1 s-1 [93]) suggests that the activity of VHR toward its physiological substrates is likely enhanced by substrate recruiting strategies. Nevertheless, the specificity profile of VHR should be useful for identifying additional protein substrates of VHR.

Our screening result (class I substrates) is consistent with a previous structural study of VHR bound with a class I substrate, DDE(Nle)pTGpYVATR [167]. In the canonical binding mode, the pY side chain occupies the relatively shallow active site pocket, making interactions with Arg130 and the amide nitrogens of residues 125–130

(Figure 3.3A). The pT side chain of the pTXpY motif points into a nearby basic pocket containing Arg158. It is expected that the frequently selected Asp at the pY-2 position would bind to this pocket and electrostatically interact with Arg158. The pY+1 Val stacks over VHR residue Tyr128, providing an explanation for the preference of the enzyme for a small residue (i.e. Gly, Ala, or Ser) at the pY+1 position as larger side chains would create steric clashes with Tyr128. The acidic residues at pY-3 to pY-5 positions make contacts with the positively charged protein surfaces, consistent with the selection of acidic and hydrophobic residues at these positions (Figure 1). Finally, in the canonical mode, Asp164 of VHR forms a hydrogen bond with the side chain of Thr at the pY+3 position.

A surprising finding of this study is that VHR also recognizes a second class of pY peptides (class II). The class II substrates have entirely different sequences, and all contain a (V/A)P(I/L/M/V/F) motif, preferably at the N terminus of the peptide.

Mutagenesis and modeling studies suggest that the class II peptides bind to VHR in an

72 orientation opposite to that of the class I substrates (Figure 3.3C). In this alternative binding mode, the pY side chain binds to the active site pocket, presumably making similar contacts with the active site residues as in the canonical mode (Figure 3.3A). The

VPI motif binds to a shallow pocket near Asp164, engaging in charge-charge interactions between the peptide N-terminal ammonium ion and the carboxylate side chain of Asp164.

The bipartite binding mode permits the intervening residues to form a loop, which may or may not directly contact the protein surface. This scenario explains how VHR can accommodate intervening sequences of varying amino acid composition and lengths (at least one to six amino acids). It is remarkable that the class II substrates are generally 1–2 orders of magnitude more reactive than the class I peptides. It should be noted that although our database search did not identify any intact protein containing the N-terminal class II motifs it is possible for some proteins to undergo proteolytic processing at internal positions to expose an N-terminal NH2-(V/A)P(I/L/M/V/F) motif and subsequently serve as VHR substrates [1, 182-184]. The ROBO1 and LASP1 results suggest that proteins containing internal (V/A)P(I/L/M/V/F)X0–6pY motifs may also act as in vivo VHR substrates as N-terminally acylated class II peptides are still efficient

VHR substrates, often more reactive than the most efficient class I peptides (e.g. peptide

8 in Table 3.6).

In summary, the substrate specificity of VHR has been determined by using one- bead-one-compound (OBOC) phosphotyrosine peptide libraries. The library screening reveals two distinct types of peptide substrates, which have been confirmed by solution- phase kinetic analysis. The class I substrates are similar to the reported VHR substrates.

The class II substrates contained (V/A)P(I/L/M/V/F) motif. Mutagenesis studies and molecular dynamics simulation suggest the opposite binding orientation of class II peptide relative to the canonical binding mode of the class I peptides. ROBO1 and

LASP1 proteins containing the class II motifs are efficient VHR substrates in vitro. The specificity profile may be useful to predict their physiological substrates in signaling pathways.

3.5. Specific Contributions

Dr. Xianwen Chen performed the screening of library II, III, VI, VIII, IX and kinetics measurement of peptide 1-9. Dr. Hashem A. Taha and Dr. Shubham Vyas (from Hadad

Lab, OSU) performed peptide molecular modeling (Chapter 3.2.6). Dr. Xiaoyan Guan

(from Freitas Lab, OSU) performed mass spectrometry experiment (Chapter 3.2.9).

Table 3.1. Composition of peptide libraries used in this work

Library No. Library design Amino acid composition at random positions (X)

I NH2-ASXXXXXpYAABBRM Ala, Arg, Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

II NH2-AXXXXXXXXpYAABBRM Ala, Arg, Asn, Asp, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val III Alloc-AXXXXXXXXpYAABBRM Ala, Arg, Asn, Asp, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

IV NH2-XXXXXpYAABBRM Ala, Arg, Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

V NH2-XXXXXXXXpYAABBRM Ala, Arg, Asn, Asp, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

VI NH2-AXXXXXpYAABBRM Ala, Arg, Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

VII NH2-VXXXXXpYAABBRM Ala, Arg, Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

VIII NH2-APXXXXXpYAABBRM Ala, Arg, Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

IX NH2-APIXXXXXpYAABBRM Ala, Arg, Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

X NH2-IDFD(D/P)pYXXXXXNNBBRM Ala, Arg, Asn, Asp, F2Y, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

XI NH2-AXXXXXpYXXXXXNNBBRM Ala, Arg, Asn, Asp, Gly, Gln, Glu, His, Ile, Leu, Lys, Nle, Phe, Pro, Ser, Thr, Trp, Val

a Table 3.2.Most preferred VHR substrates selected from peptide library I (NH2-ASX5pY)

Class IA Class IB

IWDDA EVWDM RRFDV EPMDP MFQNP TSEND WQDDA PWDDM DAFDW FGNDP SFKNP FDEPD WWDEA ADWDM GWEXX FRVDP FKINP ALESD WIENA EWQEM WEEAP IQMDP WQDPP XEYTD SEEWA XWNEM ADWDP SRRDP FEFSP DEGTD RKFNF WSDEM WEPDP VSEDP XXESP XXXTD WTDDI MQEGM EQWDP NFDDP RRPSP EIWSE NDFDI KSFNM IETDP EPWDP DFDSP DDWDI GWDTM LLRDP FRVDP WEQSP Class II NDNDI QFFDS ATIDP PKWDP WQESP GAPIQ WSEDI FREFS PENDP IDFDP QVISP RAPIQ IIRDI IHQSS FITDP WIDEP IKESP FAPIS MNKFI PIISS FTIDP EWSFP QFFSP FAPIV FPEGI XTSWS EWQDP TIIGP FRQSP APLMN PNSNI MWEDT WVTDP FDVNP RKPSP XXIIG EFSSI TESWT GLFDP WPNNP ESITP FEDTI VNFDV WASDP WIKNP EAPTP a M, norleucine; Y, F2Y; X, residues that could not be unambiguously determined. The peptides selected for further kinetic analysis were underlined.

Table 3.3. Most preferred VHR substrates selected from libraries II–IX a

NH2AX8pY AllocAX8pY NH2X5 NH2X8pY NH2AX5 NH2VX5 NH2APX5 NH2APIX5 (library II) (library III) pY (library V) pY pY pY pY (library (library (library (library IV) VI) VII) VIII) (library IX)

AFFIRRKDA ANDDLEDA APXXX WLIEETDE APAWDA VPIITT APIMEDF APIAWEDA ARDTTNEHA AVAEWIVDA APQRI ANLKRWDI APFFNA VPIAWG APMPNDI APIITMDD AXXXXSSWP ARWTWAGEA APTIS APQPWRFP APFNKI VPINHD APFPQDI APIVPMEI ARRVHNWWL AEDFEEVNA APTHI DDMDWDNE APIWRY VPINLP APIRRDI APIIDEEM AIISESWDQ AGVPSWEQA APFVT DEQDDWEW APINDI VPITHF APIVNEW APIATWGS AEWEDELEM AXXXKKDRA VPIDT EAMWDFDI APIADW VPISVT APIPINI APIAWDNI AVDWWNEDW AGWDDMDWA VPVNA FPDFDDNF APINPF VPIFTN APLQRNI APIMSEWD AMFFEDSW AFEWWELSD VPIVD INRSWVED APIIPM VPIWQP APIAHNI APIIFPWI APIVTDMA AEDWDDDWE VPTAA KEWEDEDF APIFQD VPVWHT APLMDNI APIRTXXX APIWIWPED ATTWENDDF VPSMF MFEEQWFD APLNPF VPQSWG APAPWQI APIREDXX APIGWQFSE AMWETEFDF VPSSW VVSDVWED APLFAG VPQHAF APSIEWI APIMXXXX APIQVIEEI AGWDWDLDF VPGHD VSWEWEVD APMPDF VPSWQV APFPRAF APIMEADD APIARNNLM ASEWGRADI VPFDF VQAVEWDV APVRDI VPTFQP APIRVDF APIDVFDF APIMWWDDV AIDLEWQDI VPQNV VPTQNWDM APVTT VALWQS APMYPDI APIYPDDW APFGPSWGP ADSWEWLEI TAGMG VEEDQEWS APRFLN VVRWQT APQFNDP APILNFED APRFTKLSA AWDQDMWEI TDAGH VRRSDPAA ARFQWQ VXXWAM APLITDS APIMAQEF AWSFFDDEI TGGSD VVQRGFSL AIIWXX VFRVNP APIEWDT APIVPPEI AFRKRNFRI VKMWAPTS AKRRFR VQPHAP APLQNEF APIMDAES AENEELAWI VAFRVRNM VTEHTG APLITEF APIFKNGI AEKDDWDWI VQRLAPNS VSDFIG APMNEEI APIVGFNP APNANWEDL VDRGRSFL VTEFMG APIFDEI APIPWDSD AEWIDNNDM VDRRFKT VTEFHT APIISEI APIIWDSE AFSVDNQDM WIEDRXXX VTIGAG APINTEI APIRSMSN AWDRFEFDN XEIFSFDD VSDDEG APMLSEW APIIPVSW ARRIRKNFN XFWGEIDP APRPRFW APIMDPTI AKGNRFKFN XINNRIWS APIFPGG APIISPWI AWKSRLKWN XVPIATGF APIFPGG APIQEEWI AIEMFWDDP APIWEGI APIIDXXW ANMADEMDP APRRWIN APIPTXXX AWNQETDP APMNPNI APIRSNXX ALDPNIWDP APISWNI APIPNXXX AWSEMNEDP APTAWXX AITLDNADP APWKRNI AFLDMSFDP APVAWQR AIRWRRKFP APRPPRI ADSWDSINP APQINSI AEDSEFTNP APTKWSP ARRWRNNSP APRPRTI AFANDEWWP APSRRWD ASDFWFDWQ APFMDWE AXXNTRKMS APIMNWF ATNTWNFQS APSMIWR ADSWEQFDT APVNXXI AWFDNDLDV APKRWXX AFFDGETDW ADVQGIEDW AEEAEPWEW AWLEMGAEW ADVEDWWMW a M, norleucine; Y, F2Y; X, residues that could not be unambiguously determined.

Table 3.4. Preferred VHR Substrates Selected from Peptide Library Xa

DyGFVNX PyAMTMX PyASPHV PyGIEXX PyGSXXX PyAPIXX DyGSXXX PyGWSWD PyGTXXX PyQAVPI PyAPXXX PyQDNXX DyGISWE PyEPDWT PyALSWN PyRHDNX PyEVIDF PyGQAXX DyGMXXX PyGDWIE PyGQNWN PyGESII PyQTIDD PySIEXX PyAITPX PySNGSX PyQETQV PyGFIDD PySPXXX PyRSPXX PySAISF PyGAWDA PyANSFP PyASWDD PyGMXXX PyQPXXX PySPIEA PyTVTSP PyDGHTK PyEQTWE PyADHII PyAEDWI PySTIET a M, norleucine; Y, F2Y; X, residues that could not be unambiguously determined; y, pY.

a Table 3.5. Preferred Substrates Selected from Peptide Library XI (NH2AX5pYX5)

APAFGpYSNMHS AIVWSIpYQPLDQ APITGFpYSFDEN ASGSEVpYEALEF AAPIMXpYXQKVG AIWDLApYGSIPM APITFApYDDHLT ASMGNApYGLEED APTINpYVEEAW NLGVNApYRRKML APIVDSpYVRGWA ASSAGApYSDFFM AAPWWEpYGEILH AMINTRpYTWKNN APMFGApYEEQTI ASTGDWpYTMDGF AQQNIpYAPLKW ANIIDIpYDTWWE APMVDVpYRRGWQ ASTNDVpYKMEAP AATARLpYERLEE ANSLSNpYNTELV APMVNEpYSVTRK ATSKDVpYQTQNP ADSEFMpYFWELD ANSNNQpYHMFHE APQIGSpYVEWTP ATSMVDpYTARQD AEISGApYKRRIF ANSNGSpYWSIPE APRIDIpYGKSH ATTNDFpYGGQSR NETENSpYMGFEK ANSVSNpYAEKTD APSASSpYGWDFE AVIDNTpYGETFF AETNDQpYGEQIW ANWNSMpYGIFPE APSFLDpYDVPPR AVISGEpYGQPQS AFDAMApYGQIPF APDRQIpYGRRIT APSINIpYDVWPT AVSLSLpYELGGK AFGPKFpYVRNTR APFHTApYTKQMF APSIDMpYQLFEQ AVTRNVpYEEDWG AFHKFApYQTRKT APFTNLpYGELRT APTAGIpYALRPI AWESDVpYGRVQI AFMGDLpYEGSFN APIAGDpYFDFSI APTFPDpYAPPFD AWXXXSpYTQPSD AFSEGMpYRTDHI APIENLpYGFMSG APTQWTpYTEMTG AXGATApYVDDIW AGSQADpYIDFPD APIFEApYNTKGP APVLNGpYFEDDF AXGWDApYFDEWD AHXXXIpYGNMFE APIFDGpYRNGPP APVLNGpYFEDDF AXINAApYSWTQD AIAKTMpYGQVNF APIIGWpYQTAMP APVMDFpYKRFDT AXLWGGpYMNGSR AIDGDApYDSWMT APIITTpYNTPQG APVNAFpYSHDKH AXWAHGpYGRKVA AIENDLpYGFFTN APIPAVpYEQDIM APVVVDpYGTMNT AXXDFFpYDNTPV AIETGSpYMGGIG APIPVDpYTDXXX APXXGLpYAQEPV AXXWTNpYSVFTD AISSXXpYEDTEF APIPIApYGATRV APXXXXpYGEGQA AXXEDPpYLQWEF AISTDDpYEVSAH APISMSpYIQPSF AQDAGApYHIRRR AXXFSMpYQEISA AISTTNpYGEVFM APISNVpYFDSTW AQFTNApYRLTHR AXXMFSpYGDTLE AISTGLpYETPQD APISKGpYSRTTW AQSLNDpYWEPDL AXXNHIpYSRTGR AITHNApYNDDIQ APITTNpYELADG ARVDQIpYKRFHV

aAcidic residues are shaded in red, basic residues are in blue, hydrophobic residues are in yellow, Pro is colored in green, while pY is bolded and colored in gray. M, norleucine; X, residues that could not be unambiguously determined.

Table 3.6. VHR activity against selected peptide and protein substrates

kcat/KM Peptide Sequence k (s-1) K (μM) cat M (x 103 M-1 s-1)

1 Ac-VSEDPpYAASAS-NH2 4.5 ± 0.3 117 ± 22 38

2 Ac-VSESPpYAASAS-NH2 4.3 ± 0.4 96 ± 22 44

3 Ac-VSEpSPpYAASAS-NH2 2.9 ± 0.1 203 ± 18 14

4 Ac-VSEDIpYAASAS-NH2 6.0 ± 1.2 386 ± 110 15

5 Ac-VSEDKpYAASAS-NH2 3.3 ± 0.5 2,000 ± 490 1.6

6 Ac-VSEDPpYYDFAS-NH2 5.0 ± 0.1 174 ± 3 29

7 Ac-VSEDPpYDFEAS-NH2 2.1 ± 0.1 110 ± 17 19

8 Ac-FAPISpYAASAS-NH2 5.1 ± 0.4 85 ± 18 60

9 Ac-SASASpYSASA-NH2 0.08

10 Ac-ARKRIpYAA-NH2 0.40

11 Ac-KAVDGpYVKPQI-NH2 2.2 ± 0.2 337 ± 50 6.2

Ac-TSAPVpYQQ-NH2 (ROBO1 pY1114) 4.1

Ac-VAPVQpYNI-NH2 (LASP1 pY171) 1.9 Erk ~40a Jnk ~40b

Table 3.7. Effect of N-terminal amine on WT VHR activity

kcat/KM Peptide Sequence k (s-1) K (μM) cat M (x 103 M-1 s-1)

12 Ac-APIAWEDApYAA-NH2 6.2

13 NH2-APIAWEDApYAA-NH2 5.3 ± 0.2 91 ± 8 58

14 Ac-APFPQDIpYAA-NH2 7.6 ± 0.4 360 ± 40 21

15 NH2-APFPQDIpYAA-NH2 8.6 ± 0.6 35 ± 1 270

16 NH2-VPFPQDIpYAA-NH2 7.1 ± 0.1 17 ± 3 410

17 Ac-APINDIpYAA-NH2 8.2 ± 0.1 420 ± 8 19

18 NH2-APINDIpYAA-NH2 6.3 ± 0.4 23 ± 0.4 290

19 NH2-VPINDIpYAA-NH2 6.5 ± 0.7 17 ± 1 380

20 NH2-VPISSVTpYAA-NH2 7.0 ± 0.6 25 ± 1 290

21 NH2-VPISVTpYAA-NH2 7.0 ± 0.1 17 ± 1 640

22 NH2-VPIVTpYAA-NH2 6.8 ± 0.3 7 ± 1 1000

23 NH2-VPITpYAA-NH2 5.9 ± 0.4 28 ± 5 210

24 NH2-APIQpYAA-NH2 5.5 ± 0.8 17 ± 1 340

Table 3.8. Activity of WT and mutant VHR against pY peptides pNPP, p-nitrophenyl phosphate.

kcat/KM -1 Sequence VHR kcat (s ) Peptide K (M) -1 M (x 103 M s-1)

WT 1.7 ± 0.1 5200 ± 300 0.32

D18N 1.0 ± 0.1 6700 ± 400 0.15 pNPP D47N 2.3 ± 0.1 5600 ± 400 0.40

E159Q 2.1 ± 0.1 7300 ± 500 0.29

D164N 1.8 ± 0.1 5600 ± 400 0.31 WT 6.3 ± 0.4 23 ± 0.4 290 D18N 2.6 ± 0.1 20 ± 0.1 120

18 NH2-APINDIpYAA-NH2 D47N 7.3 ± 1.0 15 ± 1.9 500 E159Q 4.9 ± 0.4 30 ± 5.3 170

D164N 5.5 ± 1.1 170 ± 34 33

WT 19 8.2 ± 0.1 420 ± 8 D18N > 100 4.7

17 Ac-APINDIpYAA-NH2 D47N > 100 9.0

E159Q > 100 5.8

D164N 4.8 ± 0.4 550 ± 80 8.7 WT 8.6 ± 0.6 35 ± 0.1 270 15 NH2-APFPQDIpYAA-NH2 D164N 3.0 ± 0.2 220 ± 27 14

14 Ac-APFPQDIpYAA-NH2 WT 7.6 ± 0.4 360 ± 40 21

WT 6.0 ± 0.2 630 ± 48 9.5 24 NH2-VSEDPpYAA-NH2 D164N 4.9 ± 0.1 770 ± 50 6.5

Table 3.9. D164–V(peptide) H-bonding interaction occupancies in MD ensemble

b a H-bond 21 22 Donor Acceptor

V@H1 6.94 2.83 D164@OD1 V@H2 6.70 2.71 V@H3 3.60 2.64 V@H1 0.92 1.55 D164@OD2 V@H2 1.85 1.53 V@H3 1.73 1.57 Total 21.7 12.8 aIn reverse orientation with cis-Pro; bReverse orientation with trans-Pro.

Table 3.10. MMBSA and MMGBSA energies of peptides 21 and 22 bound in VHR in a canonical (C) orientation and reverse (R) orientation.

Peptide PBSA (kcal/mol) GBSA (kcal/mol)

C R C R cis-Pro –89.8 ± 8.4 –88.5 ± 8.9 –91.0 ± 8.9 –88.6 ± 9.5 21 trans-Pro –85.0 ± 7.4 –89.4 ± 9.0 –82.6 ± 7.9 –95.5 ± 8.9

cis-Pro –89.3 ± 8.3 –74.9 ± 10.6 –85.8 ± 8.8 –77.9 ± 8.3 22 trans-Pro –93.3 ± 9.1 –93.3 ± 6.8 –86.0 ± 6.8 –95.7 ± 7.8

Table 3.11. Potential Class II VHR Substrates from Database Search

Protein Phosphorylation Site CacyBP calcyclin binding protein VAPITTGpY71TVKIS TGM3 transglutaminase E ASAPIGRpY111TMALQ MOSPD3 unknown function protein VAPIPSHpY106DVQDR CRIK Citron Rho-interacting kinase PTQVPLQpY1258NELKL complement receptor of the immunoglobulin VSIG4 superfamily LDTVPLDpY388EFLAT CDK3 cyclin-dependent kinase 3 FGVPLRTpY159THEVV ALMS1 Alstrom syndrome protein 1 PTVPLSYpY1940SRREK MHC class I region proline-rich protein PRR3 CAT56 PRVPLPHpY53PPNPA MINK Misshapen-like kinase 1 HRVPLKPpY592AAPVP AKAP12 A kinase (PRKA) anchor protein 12 AVVPLSEpY852DAVER JMJD7 unknown function protein CVPLAVPpY31LDKPP REPS2 RalBP1-interacting protein 2 RVPLSHGpY212SKLRS ZO1 Zona occludens protein 1 EEPAPLSpY1140DSRPR RFWD3 ring finger and WD repeat domain 3 LRAPLDApY181FQVSR IL27RA Interleukin-27 receptor subunit alpha TAPLDSGpY613EKHFL ARHGEF7 iso1 guanine nucleotide exchange factor 7 PAPLTPApY442HTLPH ARHGEF7 guanine nucleotide exchange factor 7 PAPLTPApY620HTLPH GRK6 G protein-coupled receptor kinase 6 SVAPFADpY161LDSIY FGFR3 Fibroblast growth factor receptor 3 LSAPFEQpY770SPGGQ FGD1 Faciogenital dysplasia 1 protein LAPFLKMpY467GEYVK FRK FYN-related kinase PAPFDLSpY221KTVDQ PPA1 pyrophosphatase 1 AAPFSLEpY17RVFLK LASP1 LIM and SH3 domain protein 1 IPTSAPVpY171QQPQQ ROBO1 roundabout homolog 1 QEVAPVQpY1114NIVEQ

Table 3.12. MS/MS data for ROBO1 proteolytic fragment QEVAPVQpY1114NIVEQNK

Figure 3.1. Histogram showing the pY sequence specificity of VHR (A) Histogram showing the sequence specificity of VHR on the N-terminal side of pY (constructed with the 92 class I sequences selected from library I). The y axis represents the percentage of selected peptides that contained a particular amino acid (x axis) at a given position within the peptide (pY-5 to pY-1 on the z axis). M, Nle; Y, F2Y. (B) Sequence specificity of VHR on the C-terminal side of pY (plotted with sequences selected from library X). The y axis represents the percentage of selected peptides that contained a particular amino acid (x axis) at a given position within the peptide (pY+1 to pY+5 on the z axis). M, Nle; Y, F2Y.

Figure 3.2. Preliminary Docking Investigations. Our initial attempts at docking peptides 21 and 22 into the binding site failed to reproduce the crystal structure binding mode. Docking experiments were carried out using AutoDock4 in combination with the AutoDock-Tools GUI. A gridbox (56 × 76 × 60 points with a grid spacing of 0.375 Å) was placed over the binding site of the VHR protein and centered on the crystal ligand (which was then removed). The Lamarckian genetic algorithm with a maximum of 20 million energy evaluations in combination with a local search algorithm was used for each of the 100 individual docking runs. All other parameters were left to the preset values in AutoDock-Tools. A number of docking simulations were carried out including native ligand docking, MD-ensemble docking, docking of only the pY residue (where the gridbox was centered on the pY-binding pocket), flexible docking (where residues in the binding pockets were allowed to rotate in the docking simulations). All these efforts resulted in binding poses of with no valuable information. (continued)

Figure 3.2. (Continued) (A) RMSD of the VHR protein backbone when complexed with peptides 21 (left) and 22 (right). Different complexes are indicated by different colors (black: trans-Pro, canonical orientation; red: cis-Pro, canonical orientation; blue: trans-Pro, reverse orientation; green: cis-Pro, reverse orientation). (B) RMSD of the peptide backbone of peptides 21 (left) and 22 (right). Different ligands are indicated by different colors (black: trans-Pro, canonical orientation; red: cis-Pro, canonical orientation; blue: trans-Pro, reverse orientation; green: cis- Pro, reverse orientation).

Figure 3.3. Comparison of the canonical and reverse binding modes of pY peptides to VHR.

A, crystal structure of p38 peptide (DDE(Nle)pTGpYVATR) bound to C124S VHR protein (24). B and C, MD relaxed structures of canonical and reverse binding modes of peptide 22 to VHR protein, respectively. In the reverse binding mode, the N-terminus (N) of peptide 22 is close to the side chain of Asp164, whereas the canonical mode has the N- terminus pointing away from Asp164. Key interactions include Thr-Arg125 and Val- Arg125 for the canonical binding mode and Ala-Ser24, Thr-Pro162, and Ile-Asn13 for the reverse mode. D, interactions between the pY side chain and the pY-binding pocket of VHR. The peptides are shown as sticks with carbon, nitrogen, oxygen, hydrogen, and phosphorus atoms colored green, blue, red, white, and orange, respectively. Their N and C termini are labeled with “N” and “C,” respectively. The protein surfaces are colored according to local electrostatic potential with positively charged residues in blue, negatively charged areas in red, and key residues labeled in black. Molecular graphics and analyses were performed with the UCSF Chimera package.

VA L H3 H2 H1 OD2 D164 OD1

Figure 3.4. Representation of H-bonds present between Asp-164 and the N-terminal valine residue of peptide 22. Interactions of all peptides with VHR were present primarily at the active site pocket of VHR. These interactions are present in all VHR–peptide complexes. For some complexes (i.e. those shown in Table S4), H-bonding interactions exist (13–22%) between the N- terminal valine of peptide and Asp-164 of VHR.

Figure 3.5. RMSD of the protein backbone of the D164N mutant of VHR.

Figure 3.6. In vitro dephosphorylation of ROBO1 and LASP1 by VHR. A, tyrosyl phosphorylation of ROBO1 (1 g) by Abl kinase (100 units) and subsequent time-dependent dephosphorylation of ROBO1 by VHR (0.25 ng; 0–50 min at 25 °C). Western blot with anti-ROBO1 indicated similar protein loading in all lanes. B, left panel, complete dephosphorylation of ROBO1 (1 g) with excess VHR (2.5 g; 45 min at 37 °C). Right panel, phosphorylation of LASP1 (1 g) by Abl (40 units; 45 min at 37 C) and subsequent complete dephosphorylation by VHR (2.5 g; 45 min at 37 °C). C, MS/MS spectrum showing the fragmentation pattern of pY1114 of ROBO1 (see Table 3.12 for exact m/z values of all peaks).

Figure 3.7. Representative V vs [S] Plots for VHR against pY Substrates. The initial rates were calculated from the early regions of the reaction progress curves at 282 nm (usually <30 s) and fitted against the Michaelis-Menten equation to obtain the kcat, KM, and kcat/KM values.

Figure 3.8. Representative reaction progress curves and data fitting. Reactions were initiated by the addition of VHR as the last component and monitored continuously at 282 nm. The data were fitted to equation

M ( ) ( ) cat cat by nonlinear least squares regression fitting (ref. [130]).

Chapter 4

SPECIFICITY PROFILEING OF SMALL C-TERMINAL DOMAIN

PHOSPHATASE 1 (SCP1)

4.1 Introduction

Several phosphatase families including PPPs, PPMs, DUSPs, and HADSFs have shown to dephosphorylate pS/pT residues, which are prevalent phosphorylation sites

(98.2%) in human proteome [3]. Their substrate specificities have been determined using in-solution kinetic analysis of small panel of pS/pT peptides [185-188] and analysis of available phosphatase crystal structures [42]. However, current understanding of factors determining the substrate specificity is still limited. Our group has recently developed a methodology that enables the systematic determination of pS/pT specificity profile and facilitate the identification of new pS/pT substrates [96]. The validity of the method has been shown by the study of substrate specificity of VH1, a phosphatase in DUSP family

[96]. To identify novel phosphatase substrates and demonstrate the generality of the method that is applicable to any phosphatases with pS/pT activity, the human Small C- terminal Domain Phosphatase 1 (SCP1), which belongs to FCP subfamily of HADSFs, was investigated in this study.

The SCPs were first identified as transcription regulators and co-repressors in the

REST/NRSF (repressor element1 silencing transcription factor/neuron-restrictive silencer factor) complex to silence the neuronal genes [48]. Interestingly, the expression of SCPs is restricted to non-neuronal tissues and neuroepithelial precursor cells [189]. Although

SCPs play important roles in a range of cellular processes including gene transcription, neuron and osteoblastic differentiation, embryonic development and cancer metastasis

[48, 189], only a few proteins have been identified as the substrates of SCPs including the

C-terminal domain (CTD) of RNAPII [48, 189], Snail protein (the E-cadherin transcriptional repressor [190], Smad1-3 (critical transducers for transforming growth factor -initiated (TGF-)/bone morphogenetic protein (BMP) family of cytokines and morphogens) [191, 192] and pRB1 (tumor suppressor retinoblastoma protein 1). Kinetic analysis of RNAPII peptides showed that SCP1 preferentially dephosphorylated pS5 rather than pS2 on the RNAPII heptad motif (Y1S2P3T4S5P6S7) by 67-fold [45,48, 189]. In addition, peptides that contain higher number of pS5 sites exhibited higher catalytic efficiency, presumably by increased local concentration of the substrate [42].

To our knowledge, there has been no systematic determination of SCPs substrate specificity. In this study, we profiled the sequence specificity of SCP1 by using combinatorial pS/pT peptide library screening to mediate the identification of new SCP1 substrates. We found that SCP1 prefers pS-containing peptides with Pro residues at P+1 position and aromatic and aliphatic hydrophobic residues on both the N-terminal and C- terminal side close to pS, especially at P-1 to P-3 position. On the basis of SCP1 substrate specificity, we identified p53-pS33 as a putative substrate of SCP1 and subsequently

97 confirmed our finding by in vitro dephosphorylation study and overexpression of SCP1 in

HEK293 cells experiment.

4.2. Experimental Procedures

4.2.1. Materials

Reagents were the same as Chapter 2.2.1 with the exception of these following reagents. Etoposide and 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBDH) were obtained from Sigma-Aldrich (St. Louis, MO). GST-p53, GST-p38 (active) and anti- p53 antibody were obtained from SignalChem (Richmond, BC, Canada). Anti-flag antibody was purchased from Sigma. Anti-p53-pSer33, anti-p53-pSer46, anti-GAPDH and anti-Jnk-pT183 were obtained from Cell Signaling (Beverly, MA). Anti--actin was from

Thermo Scientific Pierce (Rockford, IL). SCP1 (amino acid: 77-256, no tag) was obtained from Dr. Yan Zhang (University of Texas at Austin, Austin, TX). Peptide libraries were synthesized on polyethylene glycol acrylamide (PEGA) resin (0.4 mmol/g,

150-300 μm diameter in water) as previously described [96].

4.2.2. Screening of Peptide Libraries

To determine the pS/pT substrate specificity, SCP1 was screened against three peptide libraries: Fmoc/Alloc-XXXXX-pS/pT-AANNBBRM-PEGA (library I, where B is -alanine and X is Nle, L-2-aminobutyrate (Abu or U, used as a Cys analog) or any of the 16 proteinogenic amino acids excluding Met, Cys, Ser, and Thr), Alloc-pS-

XXXXXNNBBRM-PEGA and Alloc-pS-PXXXXXNNBBRM-PEGA (library II and

98 library III, where B is -alanine and X is norleucine (Nle or M, used as a methionine analog) or any of the 18 proteinogenic amino acids excluding Met and Cys). The library synthesis and the screening were similarly carried out as previously described [96], but with minor modifications in the phosphatase reaction buffers and conditions. In a typical screening reaction, 5 mg of library beads (~15,000 beads) were blocked with SCP reaction buffer (50 mM HEPES, pH7.4, 150 mM NaCl, 10 mM MgCl2, 0.1% bovine serum albumin, 0.01% tween 20) for 60 min. After being washed three times with the reaction buffer, the beads were suspended in 800 μl of SCP1 reaction buffer containing

25 nM SCP1 and 5 mM tris(carboxyethyl)phosphine and incubated for 2 h. For pY peptide library, the screening experiment involved the incubation of library IV, Alloc-

ASXXXXX-pY-XXXXXNNBBRM-PEGA (where X is Nle, or any of the 18 proteinogenic amino acids excluding Met, Cys, and Tyr), with 500 nM SCP1 in SCP1 buffer for 15 min. Control experiments without SCP1 produced no colored beads under identical conditions.

4.2.3. Synthesis of Selected peptides

Peptides were synthesized as previously described in Chapter 2.2.4. The coupling reaction of pS and pT employed 2.0 equivalents of Fmoc-amino acid [96]. For hydrophobic peptides (peptide 16-22, Table 4.1), rink resin (0.28 mmole/g) was used instead of CLEAR-amide resin to improve the coupling and cleavage yield. The resin- bound peptides were cleaved from the resin and side-chain deprotected using

69:7.5:5:5:5:2.5:1 (v/v)trifluoroaceticacid/phenol/H2O/thioanisole/1,3-dimethoxybenzene

/ethanedithiol/anisole at room temperature for 2 h. Peptide trituration and purification were performed as described in Chapter 2.2.4.

4.2.4. Enzymatic Assays

SCP1 activity toward pS and pT substrates was detected using an EnzChekTM phosphate assay kit (Molecular Probes, Eugene, OR) following manufacturer’s instructions. The final concentration of SCP in the assay was 0.004 – 4 M in 50 mM

HEPES, pH 7.4, 150 mM NaCl and 10 mM MgCl2 in a quartz microcuvette. Reactions were performed by the preincubating SCP1 with Purine nucleoside phosphorylase (final concentration 0.01U) and MESG (final concentration 200 M) for 10 min and initiated by the addition of peptide (0.001-1.8 mM, total reaction volume was 150 l) as the last component and monitored continuously at 360 nm. The initial velocity (<60 s) was calculated using an extinction coefficient (ε = 6400 M-1 cm-1) derived from a standard curve generate with known concentrations of inorganic phosphate. Data fitting against the

Michaelis-Menten equation V = Vmax · [S]/(KM + [S]) or the simplified equation V = kcat[E][S]/KM (when KM >> [S]) gave the kinetic constants kcat, KM, and/or kcat/KM.

For pY containing peptides, the dephosphorylation reaction was initiated by the addition of SCP1 (final concentration 0.1-2.4 M) and monitored continuously at 282 nm

(for Tyr, Δε = 1102 M-1cm-1) on a UV-Vis spectrophotometer as noted in Chapter 2.

100

4.2.5. Recombinant GST-Jnk1 expression and purification

293T cells, which express large T antigen, were grown in 100- or 150 mm poly- lysine-coated plates in a 37 C humidified atmosphere containing 5% CO2, using

Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum. On the day of transfection, cell density was approximately 70-80% confluent.

The expression vectors were transfected into 293T cells using the FuGENE 6 transfection reagent according to the manufacturer’s specifications. For 150-mm plates, 20 g of plasmid DNA was mixed with 60 l FuGENE 6 transfection reagent in 2 ml of serum- free DMEM. The FuGENE 6 reagent/DNA complex mixture was added evenly into the medium in a drop-wise manner. The complex was left in the medium until cells were harvested. Forty-eight hours later, cells were treated with 400 M sodium arsenite for 90 min, rinsed twice with ice-cold phosphate buffered saline (PBS), then harvested into lysis buffer (20 mM HEPES (pH 7.4), 50 mM -glycerophosphate, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol (DTT), 10 mM NaF, 1 mM

Na3VO4, 20 nM microcystin-LR, 2 g/ml leupeptin, 2 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride at a ratio of 1.5 ml per 150-mm plate. Protein concentration of the lysate was determined using Coomassie protein assay reagent

(Thermo Scientific, PA). The cell lysates were centrifuged at 4C for 30 min at 21,000g.

The supernatant was transferred into a new tube and incubated with glutathione- sepharose beads (at a ratio of 27 l of settled beads per ml of supernatant) at 4C for 1 h.

The beads were washed three times with lysis buffer, three times with washing buffer

(100 mM Tris-HCl, pH 7.6, 500 mM LiCl, 0.1 % Triton X-100, and 1 mM DTT), three 101 times with reaction buffer (20 mM MOPS, pH 7.2, 2 mM EGTA, 10 mM MgCl2, 1 mM

DTT, and 0.1% Triton X-100), and finally eluted reaction buffer containing 10 mM reduced glutathione. The recombinant proteins together with a reference protein bovine serum albumin were resolved through gel electrophoresis using NuPAGE Bis-Tris gel, and stained with Coomassie blue. The purity and yield of the recombinant proteins were estimated by densitometry.

4.2.6. In vitro Jnk1 Dephosphorylation by SCP1

Recombinant GST-JNK1 (75 ng) was incubated at 37C for 60 min with 1-1000 nM recombinant SCP1 in a 50 l reaction containing 1X reaction buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM MgCl2.) The reaction was stopped by mixing 17 l of 4X

LDS sample buffer (Invitrogen) followed by heating at 70C for 10 min. The samples were resolved by electrophoresis using 10% NuPAGE gels. Following electrophoresis, the proteins in the gels were transferred onto a polyvinylidene difluoride (PVDF) membrane. The phosphorylated Jnk1 were detected with anti-Jnk1-pT183, anti-Jnk1- pT183pY185 and anti-pY4G10 following manufacturer’s recommendations.

4.2.7. In vitro p53 Dephophorylation by SCP1

GST-p53 (860 nM, 76 kDa) was phosphorylated with active GST-p38- (1.9 nM) in the presence of ATP (10 mM) in kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM

MgCl2, 1 mM EDTA, 2 mM dithiothreitol, and 0.01% Brij 35) for 90 min at 37°C. The kinase reaction was terminated by buffer exchange into SCP1 buffer (50 mM HEPES, pH

TM 7.4, 150 mM NaCl, 10 mM MgCl2) using Zeba spin desalting column (Thermo, # 102

89882). Subsequent dephosphorylation of GST-p53 was initiated by the addition of

SCP1 (final concentration, 1 nM) to the above solution at 25°C. At various time points

(0-480 min), 5 ul aliquots were withdrawn and mixed with an equal volume of 2X SDS-

PAGE loading buffer. The samples were boiled for 10 min, separated on 10% SDS-

PAGE (200 V, 45 min), transferred to PVDF membrane (100 V, 60 min) and immunoblotted with anti-p53-pS33 site (1:2000 in 2% nonfat dry milk in TBST, Tris- buffered saline plus Tween20, buffer overnight at 4°C). The level of p53 phosphorylation at Ser33 as a function of time was determined using a Typhoon Phosphoimager and

Imagequant software. The catalytic efficiency (kcat/KM) was estimated from the reaction progress curves. The data reported represent the mean ± SD of three independent experiments.

4.2.8. In vivo p53 dephosphorylation by SCP1

The human embryonic kidney HEK293 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified incubator at

5 37 °C with 5% CO2. Approximately 3 × 10 cells were seeded in 6-well culture plates

(BD Falcon) in 1 mL of media and cultured for 24 hours. The pCMV-SCP1 plasmid

(Myc-FLAG-tagged-CTDSP1, Origene, #RC212037) was transiently transfected into cells using Lipofectamine®2000 reagent (0-2 g of plasmid, 4 l of Lipofectamine2000 in 200 l optimem). Twenty-four hours after transfection, the cells were treated with etoposide (final 8 M for 6 hours) [193, 194]. The cells were lysed in lysis buffer (200

l/well, 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1X SigmaFAST Protease

103

Inhibitor Cocktail, 1X Roche Phosphatase Inhibitor cocktail) at 4 °C for 15 min and centrifuged at 15,000 rpm for 5 min. The cell supernatants were separated on SDS-

PAGE and immunoblotted as previously described using anti-flag, anti-p53-pS33, anti- p53, anti-GAPDH, and anti--actin.

4.3. Results

4.3.1. Phosphoserine/phosphothreonine substrate specificity of SCP1

SCP1 (25 nM) was first screened against library I, Fmoc/Alloc-XXXXX-pS/pT-

AANNBBRM-PEGA, in two separated experiments (total ~30 mg dry weight, ~90,000 beads) for 2 h. After dephosphorylation, the beads were treated with Pd(PPh3)4 and N- methylaniline to remove the Alloc group from 50% of each peptides. The peptides were subjected to 5 cycles of Edman degradation reaction to remove the five random residues.

The exposed Ser/Thr product of dephosphorylation was oxidized with sodium periodate and selectively labeled with NBDH to give 153 fluorescent beads. Note that Ser and Thr were omitted from the five random residues in this library. The other 50% of each peptide were treated with piperidine to remove their N-terminal Fmoc protecting group and individually sequenced by the PED-MS method to give 97 complete sequences (Table

4.1). SCP1 selected overwhelmingly pS peptides (94%) although pS and pT were equally populated in the library. Interestingly, SCP1 has a strong preference for hydrophobic residues at the P-1 to P-3 position, especially Phe and Ile (Figure 4.1 A). Additionally, moderate preference for Ile at P-4 position was observed with P-5 position exhibiting no preference.

104

Due to the overwhelming selection of pS-containing peptides from the library I, the C-terminal sequence specificity was then determined by screening library II and III, which only contain pS-peptides. Library II Alloc-pS-XXXXXNNBBRM-PEGA (total

~30 mg dry weight, ~90,000 beads) was incubated with SCP1 (25 nM) for 2 h. The library was then subjected to deallylation and oxidation to give a glyoxyl group, which was selectively reacted with NBDH to give 207 fluorescent beads, which were sequenced to give 144 complete sequences (Table 4.2). SCP1 strongly prefers Phe (50%) or other small hydrophobic residues (Ile, Leu, Nle) (17%) at the P+1 position, followed by other amino acids with small side chains (Thr, Ser, Ala, Gly) (Figure 4.1 B). At the pS+2 position, the most preferred residues are Phe, Ile and Pro. SCP1 has a preference for hydrophobic residues on the P+3 to P+5 but other residues are acceptable. Interestingly, all three reported SCP1 substrates contain Pro at position P+1 (Table 4.3); however, the screening showed only 4 out of 146 sequences containing Pro at this position. The disagreement may be due to our screening method that hindered the possibility to detect peptides containing Pro at this position. The potential bias against proline at P+1 position was potentially due to the cyclization side reaction lowering the free amine available for

NBDH labeling. When the peptides containing the NH2-(S/T)-PX1X2X3 motif undergo oxidation by periodate, the aldehyde product can be rapidly cyclized by the nucleophilic attack by the amide nitrogen of the third amino acid residue of the chain (X1). The cyclization product is stable for days in aqueous solution at room temperature [195]. One- pot oxidation and labeling was first utilized to overcome the bias; however, no hit was observed. The problem therefore addressed by utilizing library III (Alloc-pS-

105

PXXXXXNNBBRM-PEGA) containing fixed Pro at P+1 position. Library III (~35 mg,

105,000 beads) was screened against SCP1 (1-25 nM). The screening resulted in 161 positive beads, which were sequenced to give 94 complete sequences (Table 4.4) showing a general preference for hydrophobic residues from P+2 to P+6, most frequently

Phe and Ile (Figure 4.1 C). At P+6, SCP1 accepted various amino acids with moderate preference of Ile (17%), Pro (15%) and Arg (11%). These results indicate that SCP1 generally prefers both aromatic and aliphatic hydrophobic residues on the peptide substrates both in the presence and absence of Pro residue at P+1 position. Overall,

SCP1 substrates have the consensus sequences of XX-pS-PXXXXX (where X is any amino acid and is a hydrophobic amino acid).

4.3.2. SCP1 is active toward pY substrates

For SCP1, the majority of the studies were focused on pS/pT substrates and small substrates (eg. 6,8-difluoro-4-mehtylumbelliferyl phosphate (DiFMUP), 4-nitrophenyl phosphate (pNPP)). Previous literature reported that Arabidopsis SCP1-like small phosphatase (SSP) had no activity when assayed against a Src derived pY-containing peptide (RRLIEDAE-pY419-AARG) [196]. To test whether SCP1 is active toward any pY substrates. We screened SCP1 (500 nM) against a pY library IV Alloc-ASXXXXX-pY-

XXXXXNNBBRM-PEGA (50 mg, 150,000 beads) in SCP1 buffer for 15 min. The beads were subjected to selective oxidation by tyrosinase and labeling reaction with MBTH to form a red pigment on the positive beads. Screening of library IV resulted in 70 red beads, which were sequenced to give 53 complete sequences (Table 4.5). SCP1 exhibited

106 a general preference for acidic and hydrophobic residues on the N-terminus (Figure 4.2

A), except at P-1 position where most of the selected sequences contained hydrophobic residue (94%) (Phe, Trp, and Nle). On the C-terminus side of pY, SCP1 prefers acidic and hydrophilic residues at the positions close to pY (P+1 to P+3) and has broad specificity at P+4 to P+5 position (Figure 4.2 B).

4.3.3. Kinetic Properties of SCP1 toward Selected Peptide Substrates.

To confirm the library screening results, we resynthesized, and assayed SCP1 against the several pS containing peptides (Table 4.6, peptides 1-9). Peptide 1 and 2 (Ac-YEIMMF- pS-PFN-NH2 and Ac-YNUFFF-pS-PFN-NH2) were excellent SCP1 substrates, with

6 -1 -1 kcat/KM values of 1.7 - 3.6 x 10 M s , in agreement with the overwhelming hydrophobicity of selected residues from the screening of library I. Nonetheless, a selected peptide 3 (Ac-YDFHQE-pS-PFN-NH2), showed 100-200 folds lower in the activity. Peptide 3, which contains acidic residues on the N-terminus was obtained from the screening as the acidic residues has shown to increase the aqueous solubility rendering the enzyme accessibility to the beads [96]. Peptides 4-7 (Table 4.5), with hydrophobic residues at P+1, have relatively low activities (with a kcat/KM value of 4 x

102 to 1.5 x 103 M-1s-1). The significance of Pro residue was confirmed by the ~10-fold higher in activity of peptide 9 (Ac-YA-pS-PFFTI-NH2) compared to peptide 7 (Ac-YA- pS-FIFPD-NH2). Consistent with the screening data, alteration of hydrophobic residues on the C-terminus with positive charges reduced the activity by ~ 3 folds (compared peptide 9, Ac-YA-pS-PFFTI-NH2, and peptide 8, Ac-YA-pS-PIRIR-NH2). The most

6 -1 -1 active peptide (peptide 1, with kcat/KM value of 3.6 x 10 M s ) selected from the N-

107 terminal library screening has kcat/KM 200 folds larger than that selected from C-terminal

6 -1 -1 library screening (peptide 9, with kcat/KM value of 1.7 x 10 M s ). This, together with the 1600-fold higher in the catalytic activity in the presence of N-terminal residues

(compared peptide 10, Ac-MI-pS-PSKKRTIL-NH2 and peptide 11, Ac-pS-PSKKRTIL-

NH2), indicated that the primary specificity determinant of SCP1 resides on the N- terminal side of pS.

Representative pY peptides selected from library IV and artificially designed substrates were employed to determine the SCP1 catalytic activity (Table 4.5, peptide 23-

27). Selected pY Peptides containing acidic and hydrophobic residues are moderately

4 -1 -1 reactive, with kcat/KM of (0.4 - 4.6) x 10 M s . Peptides containing basic and neutral residues (Table 4.5, peptide 26 and 27) have significantly lower activity, with kcat/KM of

10-50 M-1s-1).

The specificity information from SCP1 screening and kinetics analysis was utilized to predict the SCP1 protein substrates as shown in figure 4.3. Initially, the

PhosphoSite database (http://www.phosphosite.org) was searched against the peptide motif XXX-[pS/pT]-PXXXX (where X is any amino acid and  is a hydrophobic amino acid) resulting in 154 phosphoserine and 100 phosphothreonine containing peptides. Next, the proteins were filtered for the basis of nuclear localization to give 99 potential substrates listed in Table 4.7. The crystal structures of potential substrates were investigated for the accessibility of the phosphorylation sites. The proteins JNK1 and p53 were selected for further studies due to their phosphorylation sites residing on loop regions. Additionally, Peptide 15 (STAT1 peptide) was also tested. This peptide was

108 already available in our laboratory and contained the consensus sequence at every position except the Pro residue at P-2 position. A control peptide 12 (Ac-SYSPT-pS-PS-NH2) derived from RNAPII, a known SCP1 substrate, has

3 -1 -1 only moderate activity, with kcat/KM of 6.7 x 10 M s , which is in the same range of the

4 - reported value of a similar peptide, (Ac-SYSPT-pS-PS-NH2, with kcat/KM of 1.0 x 10 M

1s-1) [45]. All four potential peptide substrates displayed high catalytic activity, with the

5 -1 -1 kcat/KM values in the range of (0.4-4.5) x 10 M s . Interestingly, phosphothreonine

Jnk1-pT183 peptide has comparable activity to the phosphoserine peptides of other potential substrates (compared peptide 16, with peptide 13-15); whereas, the screening results showed that pT peptides were less preferred than pS peptides (6% vs 94%). To confirm the screening results and assess the contribution of each position to the overall catalytic activity, the substitution of pT with pS and alanine-scanning study were performed. Replacement of pT of Jnk1 peptide with pS increased its activity by ~23-fold

(Table 1, compare peptide 16 and 17). Mutation of the Pro at P+1 position has the largest effect (~ 159-fold); whereas, substitution of Tyr at P+2 position leads to similar catalytic activity (1.5–fold increase). Replacements of N-terminal hydrophobic residues with Ala at P-1 to P-3 position lead to a reduction in the activity from ~4.2-fold to ~100-fold.

More pronounced effect was observed for the position closer to pS site. These results confirm that SCP1 has a strong preference for pS peptides containing Pro at P+1 position and hydrophobic residues on the N terminus. Peptide 13 (p53-pS33 peptide, Ac-

YENNVL-pS-PLPSQ-NH2) is the most active substrate among all four peptides derived from the potential protein substrates.

109

4.3.4. Dephosphorylation of Protein Substrates by SCP1.

To test whether p53 and Jnk1 could serve as in vitro SCP1 substrates, we performed dephosphorylation assays of p38-phosphorylated GST-p53 and collaborated with Dr. Yusen Liu in Center of Perinatal Research, The Research Institute at Nationwide

Children’s Hospital, OH, for the in vitro dephosphorylation of activated GST-Jnk1 purified from 293T cells. The phosphorylated Jnk1 and p53 were treated with SCP1 for varying periods of time. The reactions were monitored by SDS-PAGE and Western blots with specific antibodies. (Figure 4.4 A, B, and C). The catalytic efficiencies were estimated from the amount of phosphorylated proteins quantified by a phosphoimager.

We determined the dephosphorylation of Jnk1 at two specific sites, Thr183 and Tyr185

(Figure 4.4 A and B). Jnk1 has low catalytic activities, with the kcat/KM of 1220 and 660

M-1s-1 for the dephosphorylation of pT183, and the dual dephosphorylation of pT183pY185 respectively. The global anti-pY-4G10 antibody was also used to quantify the phosphorylation of pY; however, SCP1 seems to have relatively low activity toward pY

-1 -1 residues, with kcat/KM of 110 M s . Surprisingly, the activity of Jnk1 protein is

183 inconsistent with the high activity of peptide corresponding to Jnk1-pT , with kcat/KM of

1.1 x 105 M-1s-1. The phosphorylation of p53 was performed by active-p38 kinase reaction [197, 198]. Detection of phosphorylation at pS46 was unsuccessful as a result of either inefficient in vitro phosphorylation of Ser46 by p38 or the quality of anti-p53-pS46 antibody. On the contrary, phosphorylated p53-Ser33 was clearly recognized in Western blot experiment (Figure 4.4 C). SCP1-mediated dephosphorylation of p53 at this site

110

5 -1 -1 showed high catalytic activity, with kcat/KM of 2.9 x 10 M s (Figure 4.4 D), which is

33 similar to the high kcat/KM value of p53-pS peptide.

To further validate whether p53-pS33 is an SCP1 substrate, we tested the effect of

SCP1 overexpression on etoposide-induced p53 phosphorylation in HEK293 cells.

Etoposide, a topoisomerase II inhibitor, was shown to increase the phosphorylation level of p53 [193, 194]. Overexpression of SCP1 inhibited p53 phosphorylation at Ser33 site compared with non-transfected control (Figure 4.5 A). In addition, the decreased Ser33 phosphorylation was observed with increasing amount of pCMV-flag-SCP1 plasmid

(Figure 4.5 B).

4.4. Discussion

In this study, we have systematically profiled the sequence selectivity of SCP1 by screening against combinatorial peptide libraries. The specificity profile of SCP1 was supported by our kinetics experiment and the previously reported crystal structure [45].

From our kinetics measurement, the catalytic efficiency of most active peptides selected from the screening (Peptide 1: Ac-YEIMMF-pS-PFN-NH2, Table 4.6) was 530-fold more than that of the RNAPII derived peptide (Peptide 12: Ac-SYSPT-pS-PS-NH2). The p53-

33 pS peptide (Peptide 13: Ac-YENNVL-pS-PLPSQ-NH2, Table 4.6), which contains the consensus sequence derived from the screening, has approximately 67-fold higher in catalytic efficiency than the RNAPII derived peptide. The p53-pS33 peptide and RNAPII peptide exhibited similar kcat (~2 folds) but drastically different KM (~ 40 folds, with KM of 18.6 M and 726 M for p53-pS33 peptides and RNAPII peptides respectively).

111

The X-ray structure of SCP1 shows a large hydrophobic pocket adjacent to the

SCP1 active site [45]. This pocket has demonstrated in the crystal structure of SCP1 with the RNAPII peptide, PSYSPT-pS-PS (pdb:2GHT) [45]. The hydrophobic amino acids are mostly preferred at P-1 to P-3 presumably due to the favorable interactions with residues in the hydrophobic pocket (Phe106, Val118, Ile120, Val127, and Leu155). From the crystal structure, Phe106 in the SCP1 hydrophobic groove interacts with Pro at P-2 position.

Interestingly, the RNAPII contains hydrophilic residues at P-1 and P-3, Thr and Ser respectively. Thus, to achieve favorable binding interactions, the hydrophilic side chains of Thr at P-1 and Ser at P-3 form an intramolecular hydrogen bond to each other in a - turn conformation. These information, together with the observation that Pro at P+1 of

RNAPII peptide provides no role in SCP1-RNAPII recognition in the crystal structure

[45], prompted us to examine the binding interaction of SCP1 with the hydrophobic p53- pS33 peptide (Ac-YENNVL-pSer-PLPSQ-NH2). The peptide was soaked into SCP1 crystal currently under performed by Dr. Yan Zhang (University of Texas at Austin,

Austin, TX). The crystal structure will help us understand how SCP1 recognizes the hydrophobic substrate. Previous studies have shown that most of known SCP1 substrates contains Ser at P-5 (Table 4.3). In addition, Ser at this position (Ser7) and Ser5 of CTD of

RNAPII can be dually phosphorylated at least by TFIIH. Aspartic and glutamic acids are generally considered as pS and pT mimics. Thus, it is interesting to examine if Glu residue of p53-pS33 peptide accommodate the same binding site as Ser7 of RNAPII.

Based on the results from my study, one of predicted SCP1 substrates is the p53 tumor suppressor protein, which plays essential roles in cell growth, cell division, cell

112 survival and programmed cell death [199]. The interaction of p53 with mdm2 (mouse double minute 2 homolog, an E3 ubiquitin ligase) regulates p53 stability leading to generally low cellular level of p53 [199]. The level of p53 is increased by the p53 activation from the intracellular and extracellular stress signals [197] (i.e. the regulation of cellular oncogenes, ultraviolet, -irradiation and chemotherapeutic drugs). The genotoxic effect leads to various post-translation modifications including phosphorylation at multiple sites (pS-6, 9, 15, 20, 33, 37, 46, 315, 371, 376, 392; pT-18, 55, 81) [200].

From this study, p53-pS33 is predicted as a SCP1 substrate; however, SCP1 may dephosphorylate other phosphorylated residues on p53. Thus, future study will perform by detecting the phosphorylated residues indicated above. The p53-pS46 peptide, which matches the consensus sequences from library screening, may also be SCP1 substrate due to its efficient catalytic activity in vitro (Peptide 14, Table 4.6). However, the p53-pS46 was not detected in the p38-activated p53 and etoposide treated HEK293 using the available anti-p53-pS46. In addition, loss-of-function analysis of SCP1 is currently investigated by Sunghak Kim (Nakano Lab, Department of Neurological Surgery, OSU) to ensure that the decrease in phosphorylation level of p53-pS33 was not from artifact of overexpressed SCP1. Although, several kinases have been identified to phosphorylate p53, only DUSP26 [201] and PP2A [202] have been reported as p53 phosphatases. None of those enzymes specifically dephosphorylates pS33 site.

In summary, SCP1 displays a remarkable selectivity for pS over pT containing peptides, Pro at P+1 position and hydrophobic amino acids on the N- and C-terminus flanking pS residue, especially at pY-1 to pY-3. The in vitro dephosphorylation and

113 overexpression of SCP1 experiments have raised the possibility that p53 is an SCP1 substrate.

4.5. Specific Contributions

Dr. Yusen Liu (from Center for Perinatal Research, The Research Institute at Nationwide

Children’s Hospital, OH) performed the in vitro dephosphorylation of Jnk1 (Chapter

4.3.6).

114

Table 4.1. Most preferred SCP1 substrates selected from peptide library I (97 sequences)

* Sequences selected for kinetic assays.

115

Table 4.2. Most preferred SCP1 substrates selected from peptide library II (144 sequences)

* Sequences selected for kinetic assays.

116

Table 4.3. Reported SCP1 substrates.

117

Table 4.4. Most preferred SCP1 substrates selected from peptide library III (94 sequences)

* Sequences selected for kinetic assays.

118

Table 4.5. Most preferred SCP1 substrates selected from peptide library IV (53 sequences)

* Sequences selected for kinetic assays.

119

Table 4.6. Catalytic Activity of SCP1 toward pSer/pThr/pTyr peptides

Entry Name Sequence kcat KM kcat/KM (s-1) (M) (M-1s-1)

1 Ac-YEIMMF-pS-PFN-NH2 6.4 ± 0.3 1.8 ± 0.4 3600000

2 Ac-YNUFFF-pS-PFN-NH2 13.6 ± 1.0 8.2 ± 2.0 1700000

3 Ac-YDFHQE-pS-PFN-NH2 ND ND 1600

4 Ac-YA-pS-VFPIF-NH2 ND ND 410

5 Ac-YA-pS-GDNII-NH2 ND ND 850

6 Ac-YA-pS-FPIIT-NH2 ND ND 900

7 Ac-YA-pS-FIFPD-NH2 ND ND 1500

8 Ac-YA-pS-PIRIR-NH2 ND ND 6000

9 Ac-YA-pS-PFFTI-NH2 ND ND 17000

10 Ac-MI-pS-PSKKRTIL-NH2 ND ND 1600

11 Ac-pS-PSKKRTIL-NH2 ND ND 1

12 RNAPII Ac-SYSPT-pS-PS-NH2 4.8 ± 0.3 716.0 ± 105.8 6700

13 p53-pSer33 Ac-YENNVL-pS-PLPSQ-NH2 8.4 ± 1.3 18.6 ± 8.0 450000

14 p53-pSer46 Ac-YDDLML-pS-PDDIE-NH2 ND ND 37000

15 STAT1 Ac-NLLPM-pS-PEEFY-NH2 ND ND 120000

16 JNK1 Ac-YTSFMM-pT-PYVVT-NH2 ND ND 110000

17 Ac-YTSFMM-pS-PYVVT-NH2 18.3 ± 1.2 7.3 ± 1.6 2500000

18 Ac-YTSFMM-pT-AYVVT-NH2 ND ND 690

19 Ac-YTSFMM-pT-PAVVT-NH2 9.3 ± 0.6 53.0 ± 11.9 170000

20 Ac-YTSFMA-pT-PYVVT-NH2 ND ND 1100

21 Ac-YTSFAM-pT-PYVVT-NH2 ND ND 12000

22 Ac-YTSAMM-pT-PYVVT-NH2 ND ND 26000

23 Ac-ESNFF-pY-DEDLI-NH2 ND ND 46000

24 Ac-DQNTF-pY-SLNWD-NH2 ND ND 4100

25 Ac-VLQEM-pY-EQFPF-NH2 ND ND 5500

26 Ac-YRKRF-pY-RYKF-NH2 ND ND 49

27 Ac-SASAS-pY-SASA-NH2 ND ND 10

120

Table 4.7. Potential SCP1 substrates from database search

[XXXpS-PXXXX] a

aX is any amino acid and is a hydrophobic amino acid (Continued)

121

Table 4.7. (Continued)

(Continued)

122

Table 4.7. (Continued)

123

Figure 4.1. Histogram showing the sequence specificity of SCP1 on pSer/pThr peptide libraries. (A) Plot of most preferred substrates selected from library I (Fmoc/Alloc- XXXXX(pSer/pThr)AANNBBRM-PEGA) (B) Plot of most preferred substrates selected from library II (Alloc-pSer-XXXXXNNBBRM-PEGA) (C) Plot of most preferred substrates selected from library III (Alloc-pSer-PXXXXXNNBBRM-PEGA). The y-axis represents the percentage of selected peptides that contained a particular amino acid (x-axis)from P-5 to P+6 (z-axis). M, Nle; U, Abu.

124

Figure 4.2. Histogram showing the sequence specificity of SCP1 on the (A) N- terminal and (B) C-terminal side of pTyr peptides selected from library IV (Alloc-ASXXXXX- pTyr-XXXXXNNBBRM-PEGA). The y-axis represents the percentage of selected peptides that contained a particular amino acid (x-axis) from P-5 to P+5 (z-axis). M, Nle.

125

Figure 4.3. The selection process of potential SCP1 substrates.

126

Figure 4.4. In vitro dephosphorylation of SCP1 potential substrates. Table tabel (A) Dephosphorylation of Jnk1 by SCP1 (0-1000 nM) and (B) Time-dependent dephosphorylation of Jnk1 by SCP1. (C) Western blots showing the remaining pSer33 level after different reaction times (0-480 min) of dephosphorylation reaction of GST-p53 (450 nM) by SCP1 (2 nM). (D) Reaction progress curves for SCP1-mediated GST-p53 dephosphorylation at pSer33 site. The y-axis values were derived from (C) by phosphor- imaging analysis and relative to that at reaction time 0 (100%)

127

Figure 4.5. SCP1 dephosphorylated p53 at pSer33 site. (A) HEK293 cells were co-transfected with vectors expressing FLAG-SCP1. After 24h, cells were left treated or untreated with etoposide for 6 h. Cell lysates were subjected to Western blot analysis with indicated antibodies. (B). Phosphorylated p53 at pSer33 was evaluated when transfecting the cells with increasing amount of FLAG-SCP1 vector.

128

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