Sequence Specificity of Src Homology 2 Domains

Home , ABL2, ANKS1A, FER (gene), HSH2D, LRRC8C, SH2 domain

SEQUENCE SPECIFICITY OF SRC HOMOLOGY 2 DOMAINS

DISSERTATION

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

Pauline H. Tan, B. S.

Graduate Program in Chemistry

The Ohio State University

2011

Committee:

Professor Dehua Pei, Advisor

Professor Jennifer J. Ottesen

Professor Karin Musier-Forsyth

Pauline H. Tan

2011

ABSTRACT

Src-homology domains are small modular domains that recognize phosphotyrosine-containing proteins and couple activated protein kinases to intracellular signaling pathways. Since they often have overlapping functions, their SH2 domains often compete for binding to the same pY proteins. Determining their sequence specificities will help identify target proteins. Consequently, this will help understand the molecular basis for their cellular functions. Twenty-six kinase SH2 domains were screened against a phosphotyrosyl (pY) peptide library, and positive beads were sequenced by partial Edman degradation and mass spectrometry. The data revealed that the kinase family SH2 domains selected a class of pY peptides consisting of mostly hydrophilic and hydrophobic residues at the pY+1 and pY+3 positions, respectively.

After validating their binding, the literature was searched to find known SH2 targets and their pY motifs.

Seventeen SH2 domains from several different protein families were also purified, screened, and sequenced to determine their binding motifs. The majority of the

SH2 domains had high selectivity at the pY+3 or pY+1 position with a few selecting for multiple peptide classes. For example Vav1 and Vav2 SH2 domains selected for three classes of peptides. These minor classes of peptides may be motifs of new protein targets that have not been identified yet. Some SH2 domains such as the Grb7 family, HSH2D,

ii and Vav family had high selectivity of Asn at the +2 position but little selectivity at other positions. More subtle differences were observed between protein families at certain positions. For instance, SH2 domains from a family of GTPase signaling proteins preferred Pro at the +3 position, while SH2 domains from the PIK3 family preferred norleucine at the +3 position.

Genetic disorders such as Noonan’s syndrome and hematologic disorders such as juvenile myelomonocytic leukemia are caused by mutations in the SHP2 (PTPN11) gene.

The mutations are mainly located in the SH2 binding cleft or in the NSH2/PTP interface.

Mutations in the SH2 binding cleft may alter sequence specificity, resulting in new potential binding partners. Identifying novel binding partners may help lead to a better understanding of the complex mechanism of these disorders. Five SHP2 SH2 mutants were screened against a combinatorial phosphotyrosyl (pY) peptide library, and positive hits were sequenced. Only the T52S SH2 mutant exhibited a specificity switch from small to large branched hydrophobic residues at the +1 position. The T42A and L43F

SH2 mutants exhibited increased binding affinities. In contrast, the specificities and binding of E76K and E139D SH2 mutants remained unchanged. After examining the crystal structure generated by PyMOL, altered specificity involved a substitution of a bulky Thr with a smaller Ser residue allowing more space for larger branched residues.

The T52S consensus was entered into a protein database to search for new protein targets that bind to the mutant only. Twenty-six potential targets resulting from the database search were identified for the T52S mutant.

iii

DEDICATION

Dedicated to my family

ACKNOWLEDGMENTS

I would like to thank my advisor Dr. Dehua Pei for his constant intellectual guidance and encouragement throughout these years. His dedication to science will serve as guide for me to achieve in my career. My experience in the Pei lab has provided me many opportunities to grow not only as a scientist but also as a person. I also wish to thank my committee members for guiding me during my candidacy and dissertation defense.

I am deeply indebted to my senior labmates Dr. Anne-Sophie Wavreille and Dr.

Yanyan Zhang, for their guidance on the projects and answering many questions. I would also like to thank my colleagues Dr. Qing Xiao, Dr. Amit Thakkar, Dr. Tao Liu,

Dr. Xianwen Chen, Nick Selner, Tiffany Meyer, and Andrew Kunys, and the rest of the

Pei lab members, past and present, for their support, friendship and intellectual discussions at lunchtime.

Finally I wish to thank my parents and sister for their unconditional love, moral support, and encouragement. I would like to thank my parents for teaching me to work hard and achieve to my best ability. I also want to thank my sister Stephanie for her friendship and support throughout these years. Without hard work and family support, I would not be where I am now.

VITA

June 2005 ...... B. S. in Chemistry & Biochemistry

The Ohio State University

2005-2011 ...... Graduate Teaching and Research Associate

The Ohio State University

PUBLICATIONS

1. Chen, X., Tan, P., Zhang, Y. and Pei, D., “On-bead screening of combinatorial libraries: reduction of nonspecific binding by decreasing surface ligand density”, J. Comb. Chem. 2009, 11, 604-611.

FIELDS OF STUDY

Major Field: Chemistry

TABLE OF CONTENTS

Abstract ...... ii

Dedication ...... iv

Acknowledgments...... v

Vita ...... vi

List of Tables ...... xii

List of Figures ...... xv

List of Abbreviations ...... xvii

Chapter 1 Introduction ...... 1

1.1. SH2 Domains ...... 1

1.2. Other Methods to determine the SH2 specificity ...... 5

1.2.1 Solution phase pooled library ...... 5

1.2.2. Microarray library (Oriented peptide array library (OPAL)) ...... 6

1.2.3. One bead one compound library (OBOC) ...... 7

1.2.4. Phage display ...... 12

1.2.5. Two-hybrid systems ...... 13 vii

Chapter 2 Specificity of kinase SH2 domains ...... 15

2.1. Introduction ...... 15

2.2. Experimental Procedures...... 19

2.2.1 Materials ...... 19

2.2.2. Synthesis of the pY library and individual peptides ...... 20

2.2.3. SH2 constructs ...... 22

2.2.4. Protein expression and purification ...... 23

2.2.5. SH2 protein labeling ...... 24

2.2.6. pY library screening ...... 25

2.2.7. Partial Edman degradation...... 26

2.2.8. Synthesis of individual pY peptides ...... 27

2.2.9. Determination of dissociation constants by SPR ...... 28

2.2.10. Determination of dissociation constants by fluorescence polarization ...... 29

2.3. Results ...... 29

2.3.1. pY library synthesis and screening ...... 30

2.3.2. General specificity of the kinase family SH2 domains ...... 31

2.3.3. Sequence specificity of Src family kinase SH2 domains ...... 31

2.3.4. Abl1 and Abl2 SH2 domains ...... 44

2.3.5. Csk and MATK SH2 domains ...... 48

viii

2.3.6. Fes and Fer SH2 domains ...... 51

2.3.7. Tec family kinase SH2 domains ...... 56

2.3.8. Brk family kinase SH2 domains ...... 63

2.3.9. Zap N and C SH2 domains ...... 67

2.3.10. Syk N and C SH2 domains ...... 71

2.3.11. Binding affinities of selected peptides against SFK SH2 domains ...... 74

2.3.12. Binding affinities of selected peptides against selected kinase SH2 domains

...... 76

2.3.13. Database/Literature search for selected kinase SH2 domains ...... 79

2.4. Discussion ...... 83

2.5. Acknowledgments ...... 86

Chapter 3 Specificity of non-kinase SH2 domains ...... 87

3.1. Introduction ...... 87

3.2. Experimental Procedures...... 90

3.2.1. Materials ...... 90

3.2.2. Synthesis of the pY library ...... 91

3.2.3. SH2 constructs ...... 91

3.2.4. Protein expression and purification ...... 91

3.2.5. SH2 protein labeling ...... 91

3.2.6 pY library screening and sequence determination ...... 92

3.3. Results ...... 92

3.3.1. Vav1 and Vav2 SH2 domains ...... 92

3.3.2. Rasa1 NSH2 and Rasa1 CSH2 domains...... 97

3.3.3. Tns2, Tns4, and HSH2D SH2 domains ...... 101

3.3.4. Grb7, Grb10, and Grb14 SH2 domains ...... 107

3.3.5. PIK3R1 NSH2 and PIK3R2 CSH2 domains ...... 113

3.3.6. PIK3R3NSH2 and PIK3R3CSH2 domains ...... 117

3.3.7. SHB, SHD, and SHF SH2 domains ...... 120

3.4. Discussion ...... 127

3.5. Acknowledgments ...... 129

Chapter 4 SHP2 SH2 disease-causing mutants ...... 130

4.1. Introduction ...... 130

4.2. Experimental Procedures...... 132

4.2.1. Materials ...... 132

4.2.2. Construction, expression, purification, and biotinylation of wild type SH2

domain and mutant SH2 domains ...... 132

4.2.3. Library screening and sequencing ...... 133

4.2.4. Synthesis and labeling of the pY peptides ...... 133

4.2.5. Affinity measurement by fluorescence polarization ...... 134

4.3. Results ...... 134

4.3.1. SHP2 NSH2 and CSH2 mutants ...... 134

4.3.2. Binding affinities of selected peptides...... 143

4.3.3. Literature search ...... 145

4.3.4. Structural analysis of the SHP2 NSH2 domain ...... 147

4.4. Discussion ...... 148

4.5. Acknowledgments ...... 151

Chapter 5 Conclusion ...... 152

Bibliography ...... 156

LIST OF TABLES

Table 1. Libraries used for SH2 screenings ...... 21

Table 2. Peptide sequences selected from Src SH2 screening ...... 34

Table 3. Peptide sequences selected from Fyn SH2 screening ...... 35

Table 4. Peptide sequences selected from Yes SH2 screening ...... 36

Table 5. Peptide sequences selected from Fgr SH2 screening ...... 37

Table 6. Peptide sequences selected from Lyn SH2 domain ...... 38

Table 7. Peptide sequences selected from Lck SH2 screening ...... 39

Table 8. Peptide sequences selected from Hck SH2 screening ...... 40

Table 9. Peptide sequences selected from Blk SH2 screening ...... 41

Table 10. Peptide sequences selected from Abl1 SH2 screening ...... 45

Table 11. Peptide sequences selected from Abl2 SH2 screening ...... 46

Table 12. Peptide sequences selected from Csk SH2 screening ...... 49

Table 13. Peptide sequences selected from MATK SH2 screening ...... 50

Table 14. Peptide sequences selected from Fer SH2 screening ...... 53

Table 15. Peptide sequences selected from Fes SH2 screening ...... 54

Table 16. Peptide sequences selected from Bmx SH2 screening ...... 57

Table 17. Peptide sequences selected from Itk SH2 screening ...... 58

Table 18. Peptide sequences selected from Tec SH2 screening ...... 59 xii

Table 19. Peptide sequences selected from Txk SH2 screening ...... 60

Table 20. Peptide sequences selected from Btk SH2 screening ...... 61

Table 21. Peptide sequences selected from Brk SH2 screening ...... 64

Table 22. Peptide sequences selected from Frk SH2 screening ...... 65

Table 23. Peptide sequences selected from SRMS SH2 screening ...... 66

Table 24. Peptide sequences selected from Zap NSH2 screening ...... 69

Table 25. Peptide sequences selected from Zap CSH2 screening ...... 70

Table 26. Peptide sequences selected from Syk NSH2 screening ...... 72

Table 27. Peptide sequences selected from Syk CSH2 screening ...... 73

Table 28. Dissociation constants (Kd, µM) of selected pY peptides binding to SH2 domains determined by SPR and FP ...... 78

Table 29. Known SH2 domain binding partners and binding motifs based on a literature search ...... 81

Table 30. Human proteins with minor class peptide motifs predicted to bind to Abl and

Fes family SH2 domains ...... 82

Table 31. Peptide sequences selected from Vav1 SH2 screening ...... 94

Table 32. Peptide sequences selected from Vav2 SH2 screening ...... 95

Table 33. Peptide sequences selected from Rasa1 NSH2 domain ...... 99

Table 34. Peptide sequences selected from Rasa1 CSH2 domain ...... 100

Table 35. Peptide sequences selected from Tns2 SH2 domain ...... 103

Table 36. Peptide sequences selected from Tns4 SH2 domain ...... 104

Table 37. Peptide sequences selected from HSH2D SH2 domain ...... 105

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Table 38. Peptide sequences selected from Grb7 SH2 screening ...... 109

Table 39. Peptide sequences selected from Grb10 SH2 screening ...... 110

Table 40. Peptide sequences selected from Grb14 SH2 screening ...... 111

Table 41. Peptide sequences selected from PIK3R1 NSH2 screening ...... 114

Table 42. Peptide sequences selected from PIK3R2 CSH2 screening ...... 115

Table 43. Peptide sequences selected from PIK3R3 NSH2 screening ...... 118

Table 44. Peptide sequences selected from PIK3R3 CSH2 screening ...... 119

Table 45. Peptide sequences selected from SHB SH2 screening ...... 122

Table 46. Peptide sequences selected from SHD SH2 screening ...... 123

Table 47. Peptide sequences selected from SHF SH2 screening ...... 124

Table 48. Peptide sequences from SHP2 T42A NSH2 screening ...... 137

Table 49. Peptide sequences from SHP2 E76K NSH2 screening ...... 138

Table 50. Peptide sequences from SHP2 L43F NSH2 screening ...... 139

Table 51. Peptide sequences from SHP2 T52S NSH2 screening ...... 140

Table 52. Peptide sequences from SHP2 E139D CSH2 screening ...... 141

Table 53. Dissociation constants (Kd, µM) of representative pY peptides towards the

SHP2 mutants...... 145

Table 54. Human proteins predicted to have improved binding affinity for SHP2 T52S

NSH2...... 146

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LIST OF FIGURES

Figure 1. Ribbon diagram of the pYEEI-Src SH2 complex structure ...... 3

Figure 2. Principle of solution phase pooled library ...... 6

Figure 3. SH2 domain screening methods ...... 10

Figure 4. Peptide sequencing by partial Edman degradation (PED)-MS ...... 12

Figure 5. Sequences specificities of Src, Yes, Hck, and Fgr SH2 domains...... 42

Figure 6. Sequence specificities of Fyn, Lyn, Lck, and Blk SH2 domains ...... 43

Figure 7. Sequence specificities of Abl1 and Abl2 SH2 domains ...... 47

Figure 8. Sequence specificities of Csk and MATK SH2 domains ...... 51

Figure 9. Sequence specificities of Fer and Fes SH2 domains ...... 55

Figure 10. Sequence specificities of Bmx, Itk, Tec, Txk, and Btk SH2 domains ...... 62

Figure 11. Sequence specificities of Brk, Frk, and SRMS SH2 domains ...... 67

Figure 12. Sequence specificities of Zap N and CSH2 domains ...... 71

Figure 13. Sequence specificities of Syk N and CSH2 domains ...... 74

Figure 14. Sequences specificity of Vav1 SH2 domain ...... 96

Figure 15. Sequences specificity of Vav2 SH2 domain ...... 97

Figure 16. Sequence specificities of Rasa1 N and CSH2 domains...... 101

Figure 17. Sequence specificities of Tn2 and Tns4 SH2 domains ...... 106

Figure 18. Sequence specificity of HSH2D SH2 domain ...... 107 xv

Figure 19. Sequence specificities of Grb7, Grb10, and Grb14 SH2 domains ...... 112

Figure 20. Sequence specificities of PIK3R1 NSH2 and PIK3R2 CSH2 domains ...... 116

Figure 21. Sequence specificities of PIK3R3 N and CSH2 domains ...... 120

Figure 22. Sequence specificities of SHB and SHD SH2 domains ...... 125

Figure 23. Sequence specificity of SHF SH2 domain ...... 126

Figure 24. Sequence specificity of SHP2 NSH2 mutants and its wild type ...... 142

Figure 25. Sequence specificity of SHP2 E139D CSH2 mutant and its wild type ...... 143

Figure 26. SHP2 NSH2 and mutant crystal structures with peptide GEpYVNIEF (in blue stick form) ...... 148

xvi

LIST OF ABBREVIATIONS

Abu α-Aminobutyric acid, or (S)-2-aminobutyric acid

Ac acetyl

α alpha

β beta

γ gamma

BCIP 5-Bromo-4-chloro-3-indolyl Phosphate

BSA Bovine Serum Albumin

CLEAR Cross-Linked Ethoxylate Acrylate Resin

DCM Dichloromethane

DIPEA diisopropylethylamine

DMF N,N-dimethylformamide

DMSO dimethylsulfoxide

DNA Deoxyribonucleic Acid

DTT Dithiothreitol

Fmoc 9-Fluorenylmethoxycarbonyl

Fmoc-OSU N-(9-Fluorenylmethoxycarbonyloxy) succinimide g gram(s)

GST Glutathione S-Transferase xvii h hour(s)

HBTU O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HOBt N-hydrobenzotriazole

HPLC High Pressure Liquid Chromatography

IPTG Isopropyl-β-D-1-thiogalactoside

ITAM immunoreceptor tyrosine-based activation motif

ITIM immunoreceptor tyrosine-based inhibition motif k kilo

L liter(s)

LB Luria-Bertani m milli

MBP maltose-binding protein

μ micro

M moles per liter

MALDI-TOF Matrix Assisted Laser Desorption Ionization-Time of Flight min minute(s)

MS mass spectrometry m/z mass to charge ratio (MS) n nano

NHS N-hydroxysuccinimidyl

Nle Norleucine

xviii

PCR Polymerase Chain Reaction

PED Partial Edman Degradation

PEG Polyethylene Glycol

PITC Phenylisothiocyanate pY Phosphotyrosine rpm revolutions per minute

RU response unit

SA-AP Streptavidin-Alkaline Phosphatase

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

SH2 Src homology 2

SH3 Src homology 3

SPR Surface Plasmon Resonance

RT room temperature

TCEP Tris(2-carboxylethyl)phosphine

Tris Tris(hydroxymethyl)aminomethane

Standard one letter codes are used for deoxynucleotides, and standard one- or three-letter codes are used for amino acids.

xix

CHAPTER 1 INTRODUCTION

1.1. SH2 Domains

Protein-protein interactions, essential to all cellular processes, are mediated frequently by modular domains, which recognize specific binding motifs in their binding partners. Src homology 2 (SH2) domains have been studied extensively for two decades.

They are small modular domains, consisting of about 100 amino acids, identified in numerous proteins with diverse biochemical functions such as growth factors receptors, kinases, phosphatases, and adaptor proteins1. They recognize phosphotyrosine (pY) binding motifs and couple activated protein kinases to intracellular signaling pathways.

Other binding motifs such as the PTB, PDZ, SH3, and WW domains were also discovered 2; 3; 4. The human genome encodes approximately 120 SH2 domains in 110 proteins, ten of which contain dual SH2 domains5. SH2 domains play important roles in signal transduction that is triggered by a variety of surface receptors to downstream molecules, which regulate cellular events, such as cell growth, adhesion, and migration6.

Sequence alignment revealed a conserved motif FLVRES characteristic of SH2 domains.

The SH2 domain consists of seven beta strands flanked by two alpha helices. The secondary structure consists of a combination of βA-αA-βB-βC-βD-βD’-βE-βF-αB-βG7; 8

(Figure 1). The structural features of the SH2 domains with their binding partners have been revealed extensively in the literature9; 10; 11. The two major contact regions,

1 common in most SH2 domains, are the pY-binding pocket and a hydrophobic binding pocket for the C-terminal residues. The main binding interaction is the insertion of the pY side chain into a deep pocket of the SH2 domain where an invariant arginine residue

Arg βB5 forms a bidentate interaction with the pY phosphate group10. For most SH2 domains, sequence specificity is mostly governed by two or three residues on either side of the pY. There may also be selectivity in the pY+4 and pY+5 position12. The hydrophobic binding pocket, consisting of the central β-sheet, αB, EF and BG loops, facilitates the interaction of the residues C-terminal to the pY.

Figure 1. Ribbon diagram of the pYEEI-Src SH2 complex structure PDB ID 1sps drawn with Pymol.

Although all of the SH2 domains share a common structure, there are several different binding modes of the pY-SH2 interaction. First, based on x-ray crystal structures of Src SH2, the pY peptide typically binds in an extended conformation perpendicular to the central beta sheet with its pY residue and third residue C-terminal to the pY (pY+3) making significant contributions to specificity and binding affinity. That mode is called the “two pronged plug” mode10. There are also other types of binding modes such as the “open groove” mode, “β-turn” mode, and “three pronged” mode. In the “open groove” mode, the specificity extends beyond the pY+3 position such as in the

SHP2 NSH2 domain12; 13. The “β-turn” mode is exhibited in the Grb2 SH2 domain. This interaction is promoted by the blockage of the pY+3 position with a bulky Trp residue forcing the peptide backbone to reverse in a beta turn14. The “three pronged” mode involves the pY-2, pY+3, and the pY positions in three binding pockets. SLAM- associated protein (SAP) SH2 domain provides an example of this type of mode15. The three pronged mode interaction has broader substrate specificity since it can employ either all three prongs or use a combination of any of the two for peptide/protein recognition. This flexibility expands its pools of potential binding partners. Identifying the binding modes of the SH2 domains allows the prediction of target proteins and brings important information of protein functions15. The binding patterns and partners of the

SH2 domains have been studied previously by using a variety of methods, each having its advantages and disadvantages. These techniques will be reviewed in the next paragraph followed by a combinatorial method developed in our laboratory used to determine SH2 specificity.

1.2. Other Methods to determine the SH2 specificity

1.2.1 Solution phase pooled library

With the solution phase pooled library method invented by Songyang et al16, degenerate peptides, synthesized and dissolved in appropriate buffers, are passed through an affinity column containing the resin-fused SH2 protein (usually a fusion protein with a

GST or MBP) (Figure 2). After several washing steps, the peptides are eluted off the column and sequenced by Edman degradation16. They identified the specificity of thirteen SH2 domains using this technique17. Since the hits are decoded by pool sequencing, only the average amino acid residue at each position is determined.

Therefore, individual sequences cannot be revealed. Also, covariance cannot be accounted for. Finally, ligands with high affinity for the SH2 domain but in low abundance may not be detected.

Figure 2. Principle of solution phase pooled library GST-tagged fusion protein is bound to a glutathione column by affinity interaction. The immobilized GST protein can used to select binding peptide ligands.

1.2.2. Microarray library (Oriented peptide array library (OPAL))

In another approach peptides are synthesized either directly on solid support by the SPOT method or synthesized and cleaved to be spotted on array surfaces such as gold or glass. The concept of SPOT method is based on solid-phase synthesis and the ability for a droplet of low volatile solvent to form a circular spot on a planar surface of a porous membrane. The small spot containing appropriate reagents forms an open reactor for chemical reactions involving the reacting compounds anchored to the membrane support18; 19 6

Rodriguez et al.19 applied the SPOT technique to synthesize oriented peptide arrays (OPAL) AXXXX[pS/pT]XXXXA, AXXXX[pY]XXXXA, and

AXXXXX[pS/pT][QDPF]XXXA (X represents a random position of one of the 19 natural amino acids (except Cys)) to study the phosphorylation dependent interactions.

The oriented peptide library pools are generated by scanning each degenerate position with any of the 19 natural amino acids (except Cys). The membrane containing the oriented peptide is subjected to antibody blotting assays. By reading the positive spots, the specificity can be determined.

Microarray analysis is a high throughput method that can be used to determine protein binding partners, mapping of antibody epitopes, and profiling major enzyme classes20. An oriented peptide array library (OPAL) has also been used to determine the function of seventy-six SH2 domains21. The advantage of using OPAL is that the results are directly read from the array eliminating peptide sequencing. A large number of arrays can also be synthesized within a short time. There are some disadvantages though.

First, each residue must contribute independently to binding in an additive manner in order for this method to be successful. Also, this neglects the covariance of individual residues failing to produce high resolution data. Second, it suffers from nonspecific binding with negative charged residues (Asp or Glu) as seen from a conspicuous column of dark spots in the corresponding OPAL screen. Third, it is labor intensive and/or costly by a robot to synthesize many oriented peptide libraries.

1.2.3. One bead one compound library (OBOC)

The one bead one compound library (OBOC) approach, pioneered by Lam et al.22, is a technique used to generate libraries of a large size. Each bead will have a unique sequence bound to it22. It uses a “split-and-pool” approach for synthesizing the library. In the “split and pool approach, the beads are divided into “x” number of portions, and “x” coupling reactions are performed on each portion. The beads are combined together, mixed, and then divided again for another round of chemical reactions. There are many advantages of solid phase library synthesis. First, excess reagents are used to drive the reaction to completion, and the excess material can be removed by extensive washing. Second, unnatural amino acids can be incorporated into the library during synthesis. Finally, individual sequences from a selected bead can be sequenced by Edman degradation or tandem mass spectroscopy yielding high resolution screening data.

Recently, with OBOC, Lam et al. developed bead segregation, which allows for encoding a library efficiently23. This technique employs topologically segregated bifunctional beads, which are made by a simple biphasic solvent strategy. The testing molecule is on the surface of the bead and the encoding tag is in the inner layer. The encoding tag will not interfere with screening. We applied this method in our laboratory to generate reduced density libraries where the surface contains 10-fold fewer peptides on the surface than in the core. The core contains more peptides for sequencing. The reduced density library was synthesized to solve the problems of avidity and nonspecific interactions that occur during screenings. To perform the bilayer segregation, the beads are soaked in water overnight followed by quick resuspension in 55:45 (v/v)

8 dichloromethane/diethyl ether containing the amino acid to be coupled on the surface.

This organic layer, immiscible with water, will only penetrate the surface of the bead, leaving the inner layer unreacted24. Synthesis of the reduced density library will be explained in more detail in Chapter 2.2.2.

Colorimetric or fluorescent based screenings are used to identify the beads that carry a specific sequence recognized by the SH2 domain. The SH2 domain is labeled either chemically via a protein fusion or enzymatically via a ybbr tag catalyzed by Sfp.

In colorimetric based screening, the protein is usually labeled with a biotin tag. This recruits streptavidin-alkaline phosphatase (SA-AP), which binds to biotin with very high affinity. Upon addition of substrate BCIP, the SA-AP removes the phosphate to form indole. As a result, dimerization occurs under the presence of oxygen to form indigo precipitating onto the bead surface turning it turquoise25 (Figure 3a.). In fluorescent based screening, the SH2 domain is directly labeled with a fluorescent dye (Texas red, fluorescein, etc.). The binding hits in this type of screening will exhibit a fluorescent ring around the bead. The beads are removed manually under a light or fluorescent microscope with the aid of a pipette and sorted by color intensities of dark, medium, or light. In fluorescent based screening (Figure 3b.) no washing steps are required. By contrast, the washing steps in colorimetric screening may be biased towards slow binders because proteins with fast kon and koff kinetics may be washed off from the bead.

9 a.

Figure 3. SH2 domain screening methods (a) Colorimetric method. (b) Fluorescent based screening method. Details for these screenings will be described in Chapter 2.2.6.

Selected beads are sequenced by Edman degradation or tandem mass spectroscopy yielding high resolution screening data to identify the resulting peptide.

Traditional Edman degradation is costly and only degrades one bead at a time. Mass spectrometry generates too many fragments making interpretation difficult. Finally,

10 isobaric amino acids cannot be differentiated. In our laboratory, we have developed partial Edman degradation (PED) to sequence beads quickly, and at a low cost. PED follows steps similar to conventional Edman degradation with a few differences. The beads containing unique sequences can be pooled together and sequenced. A mixture of capping and degrading agents is added to the beads. A small percentage of the peptides will be N-terminally capped, resistant to TFA cleavage, while the rest of the peptides will lose one amino acid. This cycle is repeated until a sequence specific truncation product

(peptide ladder) is obtained26. The sequence of the full-length peptides is decoded by matrix assisted laser desorption ionization mass spectrometry (MALDI) (Figure 4). This method has been proven to be efficient and inexpensive (at $0.50 per peptide), in comparison to sequencing a few peptides a day by conventional Edman degradation. The important advantage of PED-MALDI is that individual sequences can be obtained27; 28; 29.

With this information, we will be able to determine if a modular domain has one or more distinct binding consensus sequence. Also, a low abundant family of ligands can be identified in screening if it has a high enough affinity to the domain. After obtaining many sequences, the effect of covariance for each domain can be studied12; 29. This method is the basis for determining the SH2 specificity in the next few chapters.

Figure 4. Peptide sequencing by partial Edman degradation (PED)-MS The MALDI spectrum from the bead carrying the sequence AYSpYLQPLNBBRM is shown. Key: M*, homoserine lactone.

1.2.4. Phage display

In 1985, George P. Smith et al30 invented phage display, demonstrating the ability of peptides to be displayed on filamentous phage by fusing the peptide of interest onto gene III of the phage. It is a high throughput screening method for studying protein interactions. In phage display, a peptide or DNA target is initially immobilized on a solid surface. Next, a phage display library is added. Unbound phage is then washed away.

Phage are eluted off with an excess of target, and subjected to another round of screening

(panning) until the best set of binders is enriched on the surface. The DNA in selected phages are collected and allowed to replicate in bacteria to confirm identities. The nucleotide sequence from each phage can be identified by DNA sequencing to determine the peptide that bound to the target30. This technique offers a way to generate a library without the limitations of chemical synthesis because it is synthesized in a living organism. Phage display has a direct physical link between phenotype (the displayed peptide) and genotype (its encoding DNA), rendering easy identification of the selected peptides by DNA sequencing. Moreover, bacteriophages are resistant to harsh conditions, such as low pH and low temperatures, which can be used to dissociate bound phage from a target. The stringency can also be controlled depending on the number of rounds of screening. Along with these advantages, there are also some limitations. First, unnatural amino acids cannot be directly incorporated into the library easily. This is true with post translational modifications such as phosphorylation. Protein kinases can be used either in vivo or in vitro to transfer phosphate groups to tyrosines, but the amount of ligands they phosphorylate may be very small31. Also, the diversity of the library is dependent on the transformation efficiency of the phage (up to 1010 variants).

Nonspecific binding may be high due to the size of the phage. Lastly, the peptide specificity may become too enriched after subsequent rounds of panning so low affinity binders with high specificity may be lost.

1.2.5. Two-hybrid systems

Pioneered by Stanley Fields and Ok Kyu Song in 1989, yeast hybrid systems were originally designed to detect protein–protein interactions using the GAL4 transcriptional

13 activator of the yeast Saccharomyces cerevisiae32. GAL4 is composed of a binding domain and the activating domain. These two domains are modular and like most eukaryotic transcription factors, they can function in close proximity to each other without direct binding. This means that if the transcription factor is divided into two fragments, transcription can still be activated even though the two factors are not connected directly. Since two-hybrid systems are conducted in vivo, protein purification is not required. However, the main limitation of two-hybrid systems is that many false positives and negatives may be generated due to over expression of the proteins in yeast.

This may lead to nonspecific interactions or incorrectly modified proteins in yeast. This may be problematic for posttranslational modifications such as phosphorylation since there is no endogenous tyrosine kinase in the yeast. A tyrosine kinase gene may be coexpressed, but false positives and negatives may occur due to the way the reporter gene is activated making interpretation difficult33.

CHAPTER 2 SPECIFICITY OF KINASE SH2 DOMAINS

2.1. Introduction

The role of a tyrosine kinase is to catalytically transfer phosphate groups from

ATP to proteins inside the cell. It functions as an “on” or “off” switch in many cellular functions. It also has an associated protein tyrosine phosphatase that removes the phosphate group from the protein. Tyrosine kinases function in many signal transduction cascades and are divided into two families containing receptor and nonreceptor kinases34.

There are about 2000 kinases, and more than 90 of them have been found in the human genome35. These phosphorylated sites are recognized by SH2 domains, which then activate other proteins downstream.

Twenty-six out of the 120 SH2 domains from the human genome are in tyrosine kinases. Tyrosine kinases phosphorylate tyrosine residues on proteins leading to several downstream events. Due to the complexity of these cellular events, the specific roles of the tyrosine kinases in signal transduction are not well known. Since the kinase SH2 domains have overlapping functions, they will compete for the same pY proteins.

Therefore, it is essential to determine the sequence specificity of each kinase SH2 domains. After determining the sequence specificity, the consensus may used to predict their binding partners. Determining the binding partners may help understand the

15 functions of these kinase SH2 domains. Twenty-six of the kinase SH2 domains were purified and screened against a pY library to determine their binding motifs.

Src family kinases (SFK), which contain eight proteins Src, Hck, Lck, Lyn, Blk,

Yes, Fgr, and Fyn, are non-receptor tyrosine kinases36. Fyn, Yes, and Src are expressed ubiquitously whereas the remaining members are expressed only in certain cell types.

They share a common structure consisting of a unique N-terminal domain required for membrane attachment, followed by an SH3, SH2, and the kinase domain. SFKs also share an auto regulatory mechanism. Their activity is negatively regulated by phosphorylation of the tyrosine in the C-terminal tail leading to SH2 domain binding and blocking the active site as a result. In contrast, their activity is activated either by a competing phosphorylated protein binding to its SH2 domain or by dephosphorylation of theC-terminal tyrosine. SFKs are activated in response to a wide range of receptors such as tyrosine kinase receptors, integrin receptors, G protein coupled receptors, and by cellular stress. They phosphorylate substrates in the cytosol, at the inner part of the plasma membrane, or at cell-cell adhesions36. SFKs are protooncoproteins required for progression through at least two phases of the cell cycle, so mutations to those proteins may deregulate the cell cycle leading to cancer and other diseases. Identifying the sequence specificity will be important for designing inhibitors for these kinases37.

Abl (Abelson murine leukemia viral oncogene homolog 1) kinase family consists of Abl1 and Abl2 kinases. They are cytoplasmic protein tyrosine kinase implicated in cell division, cell adhesion, differentiation, and stress response. The Abl1 gene located on chromosome 9 can translocate to chromosome 22 into a region known as the

16 breakpoint cluster region (Bcr) gene leading to a fusion gene present in myelogenous leukemia38. The resulting Bcr-Abl fusion protein is an unregulated cytoplasm-targeted tyrosine kinase that promotes cell proliferation in the absence of cytokines38. The SH2 domain of Abl1 was also found to be crucial for the transformation and leukemia 18 development in mice39; 40. Abl1 SH2 is known to bind to PI3K and activate PI3K/Akt signaling pathways41. Therefore, the inhibitors of Abl1 SH2 domain can potentially serve as anti-leukemia drugs. Abl2 is involved in Burkitt’s lymphoma (BL). There is correlation with high Abl2 expression and BL cells42. Abl2 plays a role in apoptosis via phosphorylation of the apoptotic Siva1 protein, control of cell motility, and morphogenesis by modifying F-acting structure42.

Tec kinase family consists of nonreceptor tyrosine kinases Bmx, Btk, Itk, Tec, and Txk kinases. They each contain a pleckstrin homology (PH) domain, Tec homology

(TH) domain, SH3 domain, SH2 domain, and kinase domain43; 44. They lack the C- terminal regulatory tyrosine residue characteristic of SFKs43. They are involved in intracellular signaling by cytokine receptors, lymphocyte surface antigens, heterotrimeric

G-protein coupled receptors, and integrin molecules. Btk is the most commonly studied protein because of its association with X-linked agammaglobulinemia (XLA). XLA assumes a complete block in B-cell maturation45. Itk, Txk, and Tec are activated by T- cell receptor (TCR) signaling leading to induction of their phosphorylation and activation43. The SH2 domain of Bmx interacts with the Src-binding (SB) domain of Cas in a phosphorylation dependent manner and required for integrin-induced Bmx-Cas interaction44.

The Brk kinase family consists of breast tumor kinase (Brk), Fyn-related kinase

(Frk), and Srms kinases. Brk is identified in human metastatic breast tumors. Its SH2 domain also associates with insulin receptor substrate-4 (IRS-4)46. Frk is overexpressed in pancreatic beta cells, which lead to an increased susceptibility to the beta cell toxin streptozotocin and to cytotoxic cytokines. This suggests that Frk may participate in events leading to beta cell destruction47; 48. Frk also positively regulates the tumor suppressor PTEN by associating with and phosphorylating it, leading to subsequent stabilization preventing its ubiquitination by NEDD4-1 in breast cancer cells. There is also a correlation between Frk levels and PTEN levels observed in breast cancer tissue samples48.

Fes kinase family consists of Fes and Fer kinase. Their SH2 domains mediate phosphotyrosine-dependent interactions with putative substrates or regulators as well as possible regulatory or intramolecular or intermolecular interactions49. Recently, overexpression of Fes in transgenic mice revealed an additional role of the kinase in angiogenesis. Fes exhibits a positive regulatory effect on kinase activity and may be involved in cell growth50. Identifying the binding partners of both SH2 domains may help define their functions.

Spleen tyrosine kinase (Syk) and tyrosine kinase 70 kDa ζ-associated protein

(ZAP-70) are in the same family. They both contain tandem SH2 domains, N-terminal to the catalytic domain. Upon antigen-receptor stimulation in B or T cells, Syk or ZAP-70 are recruited to the BCR/TCR through the binding of their tandem SH2 domains to immunoreceptor tyrosine based activation motifs (ITAMs) and subsequently regulate B

18 or T cell developments, respectively51. The inhibition of Syk/ZAP-70 SH2 domains thus has a potential to treat diseases associated with abnormal kinase activities52.

Megakaryocyte-associated protein kinase (MATK) and C-Src kinase (Csk) phosphorylate are negative regulators that inactivate SFKs by phosphorylating the tyrosine on the C-terminus53. Each one consists of an SH3, SH2, and a catalytic domain.

Deletion or mutation of key residues in Csk SH2 abolished activity both in cells and in vitro. Csk SH2 domain binds to phosphorylated paxillin and pp125 FAK which anchors

Csk to the adhesion structures where Src kinase is active54; 55. MATK is known to interact in a specific and SH2-dependent manner with c-Kit. It likely participates in the transduction of growth signals induced by this cytokine56. MATK is also likely to be involved with breast cancer cells by associating its SH2 domain with ErbB-2 following heregulin stimulation57. By determining their specificity, inhibitors can be designed to suppress these tumor cells.

2.2. Experimental Procedures

2.2.1 Materials

Oligonucleotides were purchased from Integrated DNA Technologies (Coralville,

IA). Human spleen marathon cDNA library was from Clontech Laboratories (Palo Alto,

CA). Restriction endonucleases were from New England Biolabs (Beverly, MA). The expression vectors pET22b(+) and pMAL-p2 were purchased from Novagen

(Madison,WI) and New England BioLabs (NEB), respectively. Talon resin and glutathione resin were purchased from Clontech Laboratories (Palo Alto, CA). Fmoc protected L-amino acids were purchased from Advanced Chemtech (Louisville, KY),

Peptides International (Louisville, KY), or NovaBiochem (La Jolla, CA).

OBenzotriazole-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) and 1- hydroxybenzotriazole hydrate (HOBt), were from Peptides International. NHS-PEG4- biotin was obtained from Quanta Biochem. All solvents and other chemical reagents were purchased from Sigma (St. Louis, MO), Fisher Scientific (Pittsburgh, PA), or VWR

(West Chester, PA). Phenyl isothiocyanate (PITC) was purchased in 1-mL sealed ampoules from Sigma-Aldrich and a freshly opened ampoule was used in each experiment. Streptavidin-alkaline phosphatase (SA-AP) conjugate (~1 mg/mL) was purchased from Prozyme (San Leandro,CA). TentaGel S NH2 resin (90 μm, 0.26 mmol/g, and ~100 pmol/bead) was purchased from Peptides International. 5-Bromo-4- chloro-3-indolyl phosphate (BCIP) disodium salt was from Sigma (St. Louis, MO).

Protein concentration was determined by the Bradford method using BSA as a standard.

2.2.2. Synthesis of the pY library and individual peptides

Library I was synthesized on 5 g of 90 µm TentaGel S NH2 resin with a loading capacity of 0.3 mmol/g. The linker LNBBRM was synthesized with four equivalents of

Fmoc-L-amino acid, using HOBt/HBTU/N-methyl morpholine (NMM) in DMF as coupling reagents. The coupling reaction was allowed to proceed for 1 h followed by washing with DMF (3x), DCM (3x), and DMF (3x). The Fmoc group was removed by treatment twice with 20% piperidine (5+15 min.), and the beads were washed exhaustingly with DMF (3x) and DCM (3x), and DMF (3x). To synthesize the random positions, the resin was split into twenty equal portions each containing 250 mg of resin.

Each portion was coupled twice with 5 equivalents of the Fmoc-L-amino acid,

HOBt/HBTU/NMM for 1 h. The random positions are denoted with an X. X is any of the 18 natural amino acids except methionine and cysteine. Methionine and cysteine are substituted with norleucine (Nle) and α-aminobutyric acid (Abu) respectively. To differentiate isobaric amino acids during sequence determination by mass spectrometry,

5% CD3CO2D (mol/mol) was added to the coupling reactions of Leu and Lys, whereas

5% CD3CD2CO2D (mol/mol) was added to the coupling reaction of norleucine.

Reduced density libraries II and III were synthesized in a similar fashion except for the first few coupling steps. To generate the segregated bead layer the TentaGel S

NH2 resin was soaked in water overnight, then drained, and suspended in 55:45 (v/v)

DCM/diethyl ether containing Fmoc-Met-OSU (0.05 equiv.) and Boc Met-OSU (0.45 equiv.) The mixture was incubated on a rotary shaker for 30 min. Then, the resin was washed with 55:45 DCM/diethyl ether and DMF (10x). After washing, the resin was coupled with x equivalents of Fmoc-Met-OH and HOBt/HBTU/NMM in DMF for 1 h.

The Boc group was removed with 70% TFA (v/v), followed by acetyl capping with

Ac2O, catalyzed by DMAP/NMM for 30 min. The remainder of the synthesis was similar to library I. Library III does not contain Abu in the random positions. The pY libraries used are summarized in Table 1.

Table 1. Libraries used for SH2 screenings

Library I AXXpYXXXLNBBRM Library II AXXpYXXXLNBBRM (reduced density) Library III AXXpYXXXXLNBBRM (reduced density)

Side chain deprotection was conducted with reagent K trifluoroacetic acid [(TFA) containing 7.5% phenol, 5% thioanisole, 5% H2O, 2.5% ethanedithiol, and 1% anisole] for 2 h at room temperature. The resin was washed with TFA, DCM, and DCM containing 5% (v/v) DIPEA, and DCM before dessication under vacuum and storing at -

20 °C.

2.2.3. SH2 constructs

The DNA fragments coding for human Hck (amino acids 139-243), Lck (amino acids 117-226), Blk (amino acids 116-225), Fgr (amino acids 140-247), and Fyn (amino acids 144-251) SH2 domains were isolated by polymerase chain reaction (PCR) from the human spleen Marathon-Ready cDNA library with the primers 5’-C GAATTC CATATG

CAG ACA GAG GAG TGG TTT TTC AAG-3’ and 5’-CATCGTA AAGCTT TCA

GTCGAC GGA AGA CAT GCA GGG CAC CGA-3’ for Hck; 5’-C GAATTC

CATATG AAA GCG AAC AGC CTG GAG CC-3’ and 5’-CATCGTG AAGCTT TCA

GTCGAC CTG GGT CTG GCA GGG GCG-3’ for Lck; 5’-C GAATTC CATATG

GAG AGC CTG GAA ATG GAA AGG-3’and 5’-CATCGTC AAGCTT TCA GTCGAC

CTG CGG GGC CGG GCG CAC ACA-3’ for Blk; 5’-C GAATTC CATATG CAA GCT

GAA GAG TGG TAC TTT GGA-3’and 5’-CATCGTG AAGCTT TCA GTCGAC CCT

TGG CAT CCC TTT GTG-3’ for Fyn; 5’-C GAATTC CATATG CAA GCT GAA GAG

TGG TAC TTT GGA-3’and 5’-CATCGTC AAGCTT TCA GTCGAC CTG CGG CTT

CAT GAT GGT GCA-3’ for Fgr; and 5’-C GAATTC CATATG GAT TCC ATT CAG

GCA GAA GAA-3’ and 5’-CATCGTC AAGCTT TCA GTCGAC TTT CAC AGT TGG

ACA CAC AGT-3’ for Yes. The DNA fragment coding for Lyn SH2 (amino acids 125-

245) was isolated by PCR from rat Lyn-pCDM8 plasmid58, a generous gift from Dr. Clay

Marsh at The Ohio State University with the primers 5’-C GAATTC CATATG ACA

GAA GAG TGG TTT TTC AAG GAT AT-3’ and 5’-CATCGTG CTGCAG TCA

GTCGAC CTG TGG CTT GGG ACT AAT ACA-3’. Each of the PCR products were digested with restriction endonucleases NdeI and SalI and ligated into pET22b-ybbr13-

His construct (constructed in our lab). The final protein construct is an isolated SH2 domain with a C-terminal ybbr13 (DSLEFIASKLA) 6-His tag.

To construct MBP fusion Fgr SH2 domain, the PCR product was digested with restriction endonucleases EcoRI and HindIII and ligated into the corresponding sites of the pETmal vector described29. The final protein construct is an MBP fusion protein with an N-terminal 6-His tag.

Lck SH2, Fes SH2, Fer SH2, Bmx SH2, Tec SH2, Txk SH2, Itk SH2, and Abl2

SH2, Abl1 SH2, Frk SH2, Brk SH2, SRMS SH2, Btk SH2, MATK SH2, and Csk SH2 were provided as GST fusion proteins by Dr. S. S.-C. Li (University of Western Ontario).

The identities of the DNA constructs were confirmed by dideoxy sequencing. The vectors were transformed into DH5α or Rosetta BL21(DE3) cells for expression and purification.

2.2.4. Protein expression and purification

E. coli Rosetta BL21 (DE3) cells harboring the DNA plasmid were grown in LB medium to the mid-log phase and induced by the addition of 0.2 mM IPTG (final concentration) for 3 hours at 30 °C. The cells were harvested by centrifugation at 5000 rpm and lysed in the presence of protease inhibitors (1 mM PMSF, 1 µM pepstatin A, and

3 mg/mL trypsin inhibitor), 40 mg/ L cell culture of protamine sulfate, and 30 mg/ L cell culture of lysozyme at 4 °C for 30 min. The cell suspension was briefly subjected to sonication followed by centrifugation at 15,000 rpm to separate the supernatant from the cell debris. The, the supernatant was loaded onto a Talon cobalt affinity column or a

GST column, washed, and eluted according to the manufacturer’s recommended procedure. The proteins were concentrated down with an Amicon concentrator. The

GST SH2 proteins were passed through a G-25 column to remove GSH. The G-25 elution buffer contained 20 mM HEPES, 150 mM NaCl pH 7.5. The proteins were stored either as 30% glycerol stocks or without glycerol, flash frozen in dry ice/isopropanol, and stored at -80 °C.

2.2.5. SH2 protein labeling

Chemical labeling: MBP and GST SH2 proteins were chemically labeled with either NHS-biotin or NHS-chromogenic biotin. SH2 proteins (2 mg/mL in buffer containing 20 mM HEPES, 150 mM NaCl pH 7.5) were incubated with 1 to 2.5 equivalents of N-hydroxysuccinimidyl-biotin (NHS-biotin) or N-hydroxysuccinimidyl- chromogenic biotin (NHS-chromogenic biotin) in 1 M NaHCO3 at room temperature for

40 min. The reaction mixture was incubated with 100 µL of 1M Tris pH 8.0 for 10 min to quench any unreacted NHS-biotin. The labeled protein was passed through a G-25 column to remove any excess biotin. For selected proteins, labeling efficiency was determined by measuring the absorbance of chromogenic-biotinylated proteins at a wavelength of 354 nm. Proteins were concentrated down and stored as 30% glycerol stocks at -80 °C.

Enzymatic labeling with Sfp: Ybbr tagged SH2 protein (final concentration 50

µM) was combined with 14.3 µL of CoA conjugates (final concentration 75 µM) (its synthesis is described in the next paragraph), phosphopantheinyl transferase Sfp (final concentration 5 µM), and labeling buffer (50 mM HEPES, 10 mM MgCl2, pH 7.5 buffer) to obtain a total reaction volume of 1 mL. The mixture was incubated in the dark at 37

°C for 25 min. After the reaction, the labeled protein was passed through a G-25 column to remove excess CoA conjugate. Labeled proteins were added to 30% glycerol, flash frozen in dry ice/isopropanol, and stored at -80 °C.

Synthesis of CoA conjugates. Biotin CoA conjugate was synthesized according to the procedure of Walsh et al59. Li CoA salt (2.8 µmol) was dissolved in 280 µL of 100 mM sodium phosphate buffer pH 7.0. A slight excess of PEG2-biotin was dissolved in 50

µL DMSO. The two solutions were combined together and incubated in the dark at room temperature for 2 h. The reaction was quenched with 4 µL of 2-mercaptoethanol. Sfp enzyme was expressed from the pET22b-Sfp plasmid, a generous gift from the Walsh group, and purified on a Talon cobalt affinity column according to manufactured protocol.

2.2.6. pY library screening

In a micro-BioSpin column (0.8 mL, Bio-Rad), 10-100 mg of the pY library was swollen in DCM and DMF. It was blocked in HBST-gelatin buffer (30 mM HEPES, pH

7.4, 150 mM NaCl, 0.1% gelatin and 0.05% Tween 20) for 1 h to minimize nonspecific interactions. Then, the resin was incubated with different concentrations of a biotinylated

SH2 domain (50-500 nM final concentration) and gently mixed overnight at 4 °C. The

25 protein solution was drained, and beads were treated with SA-AP buffer (30 mM Tris, 1

M NaCl, 20 mM KH2PO4, pH 7.6) with 1 µg/mL streptavidin-conjugated alkaline phosphatase (SA-AP) for 10 min at 4 °C. The SA-AP was drained away, and the beads were quickly washed with SA-AP buffer, HBST-gelatin buffer, and staining buffer (30 mM Tris, 100 mM NaCl, 5 mM MgCl2, and 20 µM ZnCl2, pH 8.5). The beads were transferred to a Petri dish using staining buffer, and fresh BCIP (dissolved in staining buffer) was added to a final concentration of 0.5 mg/mL. The resin was gently mixed at room temperature for about 30 minutes to 1h until turquoise color developed on the beads. The resin was quenched with 1 mL of 2 M HCl for 10 min. Next, the resin was washed extensively with water to remove excess BCIP. The beads were picked manually under a dissecting microscope and sorted by color intensity into dark, medium, and light categories. A total of about 100 mg of pY library was screened against each SH2 domain to obtain approximately 100 positive beads.

2.2.7. Partial Edman degradation

Peptide sequences from the target bead were identified using the PED/MALDI-

26 TOF procedure . Beads were pooled and suspended in 2:1 (v/v) pyridine: H2O with

0.1% triethylamine. Then, a mixture of a 45:1 (12 µL:0.75 mg for seven cycles or less) or a 60:1 (12 µL:0.564 mg for eight cycles) of PITC/Fmoc-OSU was dissolved in 160 µL pyridine, and added to the above suspension. This reaction was allowed to proceed for 6 min followed by washing with DCM. Then, the beads were subjected to two rounds of

TFA treatment 6 min. each, followed by washing with DCM and pyridine. The cycle was repeated n-1 times where n equals the number of residues to be sequenced. After the last

26 cycle, the beads were treated with 20% piperidine twice (5+15 min) to remove the Fmoc group from the degraded peptide ladders. Next, the beads were subjected to a 20 min reduction step (20 µL methyl sulfide, 10 mg ammonium iodide in 500 µL TFA on ice).

Then, the beads were individually transferred to microcentrifuge tubes (1 bead/tube). For peptide cleavage, each bead was incubated with 20 µL of 40 mg/mL CNBr in 70% TFA in the dark overnight. The excess CNBr solution was removed using a Speedvac concentrator connected to a vacuum. The cleaved peptide ladders were then subjected to

MALDI-TOF analysis.

2.2.8. Synthesis of individual pY peptides

Individual peptides were synthesized on 70-100 mg of CLEAR amide resin with a loading capacity of 0.46 mmol/g using standard Fmoc/HOBt/HBTU chemistry. All of the peptides have a common C-terminal linker LNBK-NH2 except HYpYEEI and

LTpYEND, which has a BK and LNK linker, respectively. The N-terminus was acetylated by Ac2O. Peptide cleavage and deprotection were carried out in reagent K

[7.5% phenol, 5% thioanisole, 5% H2O, 2.5% ethanedithiol, and 1% anisole in trifluoroacetic acid (TFA)] for 2 h at room temperature. The peptides were precipitated in cold diethyl ether and lyophilized. The peptides were either labeled with NHS-

(fluorescein-5-carboxyamido) hexanoic acid succinimidyl ester (6(5)-SFX) or with NHS-

PEG4-biotin. For biotinylation, approximately 5 mg of crude peptide was dissolved in a minimal volume of DMSO and reacted with 1.2 eq of NHS-PEG4-biotin dissolved in

DMF. After 1 h incubation at room temperature, the reaction was quenched with 10% piperidine, and the mixture was triturated twice with 3 x 1 mL volumes of Et2O. Then,

27 the precipitate was collected and dried under vacuum. The biotinylated pY peptide was purified by reversed-phase HPLC on a C18 analytical column (Vydac 300-Ǻ pore size, 4.6 x 25 mm). For fluorescein labeled peptides, about 0.5 mg of crude peptide was dissolved in DMSO and reacted with one eq of NHS- 6(5)-SFX for 1 h at RT. Then, the labeled peptide was purified by reverse-phase HPLC on a semipreparative C18 column. The identity of each peptide was confirmed by MALDI-TOF mass spectrometric analysis using α-hydroxycinnamic acid as the matrix. The fluorescent labeled peptide concentration was determined by the absorbance of fluorescein at 495 nm.

2.2.9. Determination of dissociation constants by SPR

Binding affinity of the pY peptides against the Src kinase family SH2 were measured by surface plasmon resonance (SPR) analysis on a BIAcore 3000 instrument used at room temperature. All of the binding studies measured by SPR were performed with isolated SH2 domains with the exception of GST Lck SH2. A sensorchip containing immobilized streptavidin was conditioned with 1 M NaCl in 50 mM NaOH according to the manufacturer’s instructions. The biotinylated pY peptides were immobilized into the sensorchip by flowing 6 μL of ~ 8 μM pY peptide solution in HBS-EP buffer (10 mM

HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% polysorbate 20) until the response rate reaches the plateau value. Data was acquired by passing increasing concentrations of an SH2 protein (0 to 10 µM) over the sensor chip at a flow rate of 15

µL/min for two min. A blank flow cell containing no pY peptide was used as a control to correct for solvent bulk and/or nonspecific binding interactions. Between runs, the sensor chip surface was regenerated by flowing a stripping solution (200 mM NaCl, 10 mM

NaOH, and 0.05% SDS) for 5s to 10s at a flow rate of 100 µL/min. The equilibrium response unit (RUeq) at each concentration was obtained by subtracting the response of the blank flow cell from the response of the sample flow cell. The Kd was obtained from a nonlinear regression fitting of the data against the protein concentration using

KaleidoGraph version 3.6 (Synergy Software, Reading, PA):

RUeq= RUmax[SH2]/(Kd+[SH2]) where RUeq is the response at a certain SH2 concentration and RUmax is the maximum response unit.

2.2.10. Determination of dissociation constants by fluorescence polarization

Fluorescein-labeled peptide (final concentration 100 nM), 0.1% BSA, SH2 protein (final concentration from 0 µM to 256 µM) and phosphate buffer (25 mM sodium phosphate, and 150 mM NaCl at pH 7.3) were combined and incubated at rt for 2.5 h.

Samples in 384-well plates at RT (~20 °C) were analyzed on a SpectraMax M5Multi-

Mode Microplate Reader (Molecular Devices, Sunnyvale, CA). Fluorescence anisotropy values (A) were measured in triplicates and the dissociation constant was determined by nonlinear regression fitting of the data using KaleidoGraph version 3.6 (Synergy

Software, Reading, PA) to the following equation when the peptide concentration L is

100 nM:

2 (Amin+(Amax*Qb/Qf-Amin)*((L+[SH2]+Kd)-sqrt((L+[SH2]+Kd) -

2 4*L*[SH2]))/2/L)/(1+(Qb/Qf-1)*((L+[SH2]+Kd)-sqrt((L+[SH2]+Kd) -4*L*[SH2]))/2/L) where Amin and Amax is the minimum and maximum anisotropy respectively and Qb/Qf is the ratio of the intensities of the bound and free protein.

2.3. Results

2.3.1. pY library synthesis and screening

Library I (AXXpYXXXLNBBRM) was initially synthesized to study the sequence specificity of SH2 domains because several residues on either side of the pY were essential for SH2 binding. X represents norleucine (Nle), aminobutyric acid (Abu), or any of the 18 natural amino acids except for Met and Cys. B represents β-alanine. Nle and Abu substitute for Met and Cys respectively. A linker LNBBRM was added at the

C-terminus to facilitate mass spectrometric analysis (positively charged Arg), to facilitate cleavage from the resin (after Met with CNBr), and to shift peptide signals to >600 m/z to avoid matrix interference.

Library I contained high peptide density on the bead. The high amount of peptide on the bead may cause nonspecific interactions to the protein due to avidity effect. In order to reduce the avidity effect, a reduced density Library II (AXXpYXXXLNBBRM) was synthesized. The important step in the synthesis of the reduced peptide density on the surface was to generate an inner layer with normal peptide density to provide enough material for sequence but with an outer layer with reduced peptide density. This was done using a bead segregation step. In the first step, 45% Boc-Met-OSU and 5% Fmoc-

Met-OSU were coupled, allowing the peptide density in the outer layer to be reduced by

10 fold.

Library III (AXXpYXXXXLNBBRM) does not contain aminobutyric acid in any of the random positions. Library III contained an extra +4 position in the library, and will be used in Chapter 3 for screenings of other SH2 domains that may have selectivity at the

+4 position.

2.3.2. General specificity of the kinase family SH2 domains

Approximately 100 mg of pY library was screened (about 10% of the library) for each SH2 domain. Then, the positive beads were sorted by intensity and sequenced by partial Edman degradation followed by MALDI-TOF. Sequences for each SH2 were sorted and presented in a table and in a 3-dimensional histogram. The positions of each consensus are listed in the y-axis with the histogram of the most N-terminal position closest to the x-axis. In the tables, the bold text represents peptides from intensely colored beads, the italized and underlined text represents the medium colored beads, and the normal text represents the light colored beads from screening. U represents the amino acid Abu while M represents norleucine. The kinase SH2 domains mainly selected a single class of peptides with few covariances. Some SH2 domains, selected for a second minor peptide class though. The SH2 domains exhibited high specificities at the +1 and

+3 positions with the exception of a few SH2 domains which had high selectivity at the

+2 position. As expected, sequence overlap among SH2 domains were present, but with some subtle differences. Mainly hydrophilic or small residues were selected at the +1 position, and hydrophobic residues were selected at the +3 position.

2.3.3. Sequence specificity of Src family kinase SH2 domains

A total of 615 mg (~1.76 x 106 beads) of Library I was screened against Yes (121 sequences/65 mg), Hck (118 sequences/240 mg), Src (131 sequences/200 mg), and Fgr

(135 sequences/110 mg) SH2 domains. Also, 440 mg (~1.26 x 106 beads) of pY library was screened against Fyn (98 sequences), Lyn (136 sequences/90 mg), Blk (86 sequences/60 mg), and Lck (103 sequences/110 mg) SH2 domains. All of the Src kinase

31 family SH2 sequences are summarized from Table 2 to Table 9. From the selected sequences, the Src SH2 consensus was found previously to be

XXpY(A/T/E/N/D/S)X(I/M/V/L) (Figure 5)25. The consensus recognized was

HYpY(A/H/D/E)(Q/E)(M/I/U) for Yes SH2; HYpY(A/D/E/H)X(M/V/I/U) for Hck SH2; and XXpY(A/Y/S/T)X(U/M/I) for Fgr SH2 (Figure 5). The consensus recognized by the other Src family kinase SH2 domains was H(K/R/D/E)pY (E/D/A)X(I/M/U/V/P) for Fyn

SH2; (H/N/Y/A)(Y/F)pY (E/H/D)(E/F/Q)(M/I/U/V) for Lyn SH2;

(H/N/P)(Y/E/F/D)pY(E/D/A)E(I/L/U/M) for Blk SH2; and

(H/N/Y)(F/Y)pY(E/H/A/Y)X(V/U/M/I) for Lck SH2 (Figure 6). Lck SH2 also selected a minor class of sequences with the consensus NPpYAX(T/K). Yes, Hck, Src, and Fgr

SH2 domains frequently selected Ala at the +1 position. Yes, Hck, and Src SH2 domains also selected Asp or Glu at the +1 position. In contrast, Fgr SH2 hardly selected for Asp or Glu. Instead, Fgr SH2 selected smaller residues such as Ser, Abu, and Thr at the +1 position. Selectivity at the +2 and -2 positions was more relaxed even though His was found frequently at the -2 position. Hck, however, almost exclusively selected His at the

-2 position. Fyn, Lyn, Blk, and Lck SH2 exhibited a strong specificity for Glu at the +1 position and hydrophobic residues at the +3 position. Although selectivity at the +2 position was not very strong, Glu was predominantly selected by the Blk and Lyn SH2 domains at that position. Due to its tolerance for small hydrophobic residues such as Pro and Ala, Fyn SH2 was less selective at the +3 position than the other SFK SH2 domains.

By contrast, Blk and Lyn selected mostly branched hydrophobic residues such as Ile and

Leu at the +3 position. From analyzing the individual sequences of each SH2 domain,

32 some sequence covariance was observed. For example, in Hck SH2, when the +1 residue was Ala, Val was frequently found at the +3 position. However, in Yes SH2 the +3 residue was Abu and Nle when the +1 position was Ala.

Table 2. Peptide sequences selected from Src SH2 screening

MYpYAEA HTpYTQI WHpYTEM HRpYDFV VTpYAFA PVpYTQI KYpYAFM HIpYSFV QFpYEND WQpYVQI GYpYTFM VRpYSIV KFpYRTF RLpYYQI GGpYAIM EWpYMLV FSpYMYF HHpYARI HDpYAIM YGpYTLV IGpYVLH TGpYARI NHpYAIM HYpYSMV RLpYHAI HMpYTRI RGpYAIM FMpYAQV RFpYTAI SYpYTRI VVpYAIM TWpYTQV GVpYYAI DMpYTSI MYpYTIM GYpYQVV HMpYNEI QNpYATI HLpYEKM DGpYTVV HLpYDFI QVpYATI AYpYALM LSpYVYV MFpYSFI YYpYQTI DYpYTLM HTpYTFI HYpYTTI HVpYENM GGpYYFI IVpYAVI HVpYMNM HLpYFHI RGpYAVI HMpYDMM RMpYAII VYpYAVI HYpYEMM HYpYNII VYpYDVI IVpYEMM HEpYSII EFpYNVI MLpYEMM HHpYTII FWpYNVI PYpYNMM YGpYTII HApYNVI YMpYNMM HMpYDKI WMpYQVI VFpYSMM HIpYHKI KApYSVI HFpYTMM AHpYNKI YGpYYVI SYpYTMM HMpYQKI SVpYAYI SNpYYMM QRpYSKI FApYFYI HApYSPM RApYTKI YRpYTYI HLpYTQM RKpYTKI VFpYYYI EMpYYQM RGpYQLI HHpYEEL VFpYNSM HQpYTLI ARpYKKL FRpYTSM TFpYTLI RHpYQKL IYpYTSM YDpYYLI SYpYTTL HLpYEVM ERpYAMI NFpYYTL TSpYEVM FGpYAMI HYpYTYL VRpYTVM AIpYAQI FLpYEAM IApYAYM NMpYAQI HLpYTAM TLpYNYM RApYAQI HMpYTAM VVpYYYM RYpYAQI HHpYEDM KYpYKER HHpYDQI IFpYNEM MYpYAMR TYpYDQI PFpYNEM MFpYSMR ALpYHQI HMpYTEM FHpYGRR

Table 3. Peptide sequences selected from Fyn SH2 screening

TYpYALA ARpYQEI DUpYETV HPpYUQA HDpYTSI QRpYGKV HIpYESA HEpYTTI HDpYQEV GEpYHLA GKpYAUL PDpYQQV TQpYQIA NGpYAQL GKpYUKY DPpYRQA KHpYDHL ATpYEIY DYpYSQA DYpYEUL QRpYEYY ARpYYAA EKpYEHL PKpYHPY HDpYAMU KQpYHQL DRpYHRY QNpYDKU AKpYRNL RRpYPGY GIpYDVU ESpYYQL EEpYVTY QNpYDYU PEpYDAM VTpYEEU SRpYDKM Class II HNpYEYU PKpYDQM KKpYDRS DPpYGFU HEpYDRM SUpYESS HKpYGVU NDpYDRM DPpYEYS KSpYHUU QDpYEUM PDpYHES KRpYQQU GGpYEEM KKpYRPS HTpYSKU HTpYEIM TQpYRYS EEpYIDF HTpYEVM NDpYYQS QKpYNRF NIpYPQM SHpYPGF NKpYPYM GGpYPHF QYpYRKM KDpYQSF HPpYSAM IFpYRYF YRpYARP HEpYYEF HMpYDEP HEpYAUI EMpYDRP HEpYAUI PHpYEAP RRpYUKI EApYHHP PPpYDKI AKpYHSP AFpYDTI RGpYQUP HRpYDVI DEpYQDP FDpYEEI HKpYVQP NTpYELI YDpYYUP YRpYENI HEpYAEV PPpYGKI ADpYASV NDpYHAI HTpYEDV HRpYHDI UEpYELV DRpYHKI PTpYEMV LGpYPYI GIpYETV

Table 4. Peptide sequences selected from Yes SH2 screening

HYpYAVA PLpYFQI AUpYDEM LYpYHMA HRpYFQI NYpYDFM FHpYAUU ALpYHUI AMpYEDM SYpYAEU MVpYHEI ELpYEHM LGpYAIU HYpYHHI GHpYELM HRpYAIU SYpYHKI QDpYENM TEpYANU MLpYHQI MApYHEM SQpYAQU AIpYHQI HMpYHLM DPpYATU EGpYHYI DTpYLEM TMpYAVU ITpYNMI NVpYNUM HDpYAYU YApYSUI HApYQQM NHpYUIU ULpYTEI TSpYQQM YIpYDEU NYpYTHI NQpYTLM HDpYDMU TKpYAKL NYpYTNM FYpYEMU HQpYAQL HQpYTRM DWpYEVU HUpYAQL RPpYVGM HTpYHUU HWpYAQL HYpYYQM PFpYHDU HRpYDEL EYpYAVP LFpYHMU HFpYDFL HFpYDSP GGpYHTU EEpYDHL HEpYFEP PMpYHVU SYpYEYL HQpYHUP AYpYQUU HEpYGVL HApYYEP HTpYSTU GYpYHFL DRpYYEP UTpYTTU HEpYMEL GLpYTSQ HFpYYEU URpYAAM HFpYHQS GYpYYMU QLpYAUM HApYELT NYpYEWF KGpYAUM UGpYNTT TEpYAUI RQpYAKM DYpYAVV HDpYAUI LHpYAKM PFpYUQV MYpYAQI EPpYANM NTpYDDV EDpYUUI TFpYAQM VRpYDDV PYpYULI AYpYAQM HNpYEIV SFpYDUI IEpYAQM HKpYENV FApYDQI ERpYARM PFpYEQV YTpYDYI PHpYATM UYpYHEV HLpYEEI NRpYAVM AYpYHRV KQpYEEI QApYAYM QGpYTQV SApYEVI VGpYUEM HGpYTYV EYpYEYI HYpYUHM HHpYVDV HKpYFDI NHpYUVM HNpYAQY KGpYGGY

Table 5. Peptide sequences selected from Fgr SH2 screening

NYpYUMA NVpYYKU HRpYAUM YKpYSVV NVpYHMA RPpYYMU SYpYAIM HUpYVUV SIpYWWA PGpYYSU VUpYAKM TFpYWUV AYpYYKA ASpYULF NKpYANM PYpYNUY SIpYYLA HUpYQQF KGpYUUM FGpYSNY FTpYYRA HIpYQTF SYpYUSM TYpYVNY AYpYAUU HLpYTAF NFpYFQM NLpYYQY PGpYAFU NUpYYQF NMpYHYM HDpYAKU HWpYAGI LYpYNQM Class II SRpYAMU TYpYAII HSpYNVM PYpYFRG PTpYAQU LKpYAMI FFpYPRM HYpYWGK DYpYARU SApYASI NUpYQFM QYpYYAK NLpYARU VSpYAVI SFpYQKM GMpYYNK RGpYAVU PHpYUMI HFpYQRM MYpYUNR AYpYUNU SYpYUQI NYpYQTM UYpYQMR HLpYUNU SYpYUQI NRpYSAM NYpYVIR VLpYUYU VIpYDKI GIpYSMM NYpYVLR QYpYEKU GHpYFUI NMpYSRM GRpYFUU RMpYHHI WNpYSRM PKpYFGU PRpYHII GRpYSTM QLpYHLU AFpYHKI VApYTIM HIpYHRU ULpYQLI HYpYTLM NMpYHYU YTpYSAI AHpYTLM IKpYNYU GHpYSTI VMpYTQM GYpYMHU QTpYSYI MFpYTRM UGpYMLU MQpYSYI HQpYVYM GYpYMTU UIpYTRI NUpYWTM RFpYQNU YNpYYGI PEpYYKM AIpYQRU NMpYYQI LVpYAFP RApYSQU NGpYAFL FVpYFYP FLpYSRU PRpYULL HRpYHMP FYpYSTU RRpYDQL HUpYQMP QRpYTIU RYpYEML YGpYTQP UTpYTNU HFpYHGL AFpYTYP PFpYVHU HEpYRIL WSpYYAP LKpYWVU EYpYSLL QFpYVYT SIpYYUU HFpYTEL IVpYAGV QTpYYIU RVpYTLL GApYHLV HMpYYKU NYpYTVL YRpYQNV NEpYYKU SApYYLL UIpYSKV

Table 6. Peptide sequences selected from Lyn SH2 domain

HEpYAFU NFpYHQI VYpYHHM HYpYTTY NIpYDUU HEpYLEI EHpYHPM HIpYDTU EYpYNEI HFpYHSM Class II HMpYDVU HApYNQI HEpYITM YLpYQWF VYpYDVU GYpYNYI HLpYNQM FFpYRTF HRpYEIU HUpYQKI HIpYQAM HApYRWF QRpYEIU YPpYRFI HFpYSUM YLpYRWF HHpYELU HFpYSEI HVpYTEM VFpYRWF NYpYERU HUpYTEI HKpYTEM HIpYWEF HUpYHFU NRpYYTI NEpYTMM FUpYWHF AFpYHHU HFpYAAL YSpYTPM HSpYYWF EFpYHHU HIpYDFL HNpYTPM HTpYYWF TYpYHQU HIpYDHL NVpYTQM EFpYNHG UFpYHTU HLpYDRL AYpYYAM ETpYFFH TYpYHVU HQpYDTL HVpYTEP HApYLFH HFpYIEU HIpYEUL VYpYPFR YEpYPYH HLpYNQU HLpYHUL HRpYWFT HMpYWYK HLpYQIU YWpYHFL HVpYANV KIpYRFW HFpYSRU NIpYKQL HUpYUDV HFpYTHU NYpYTEL VYpYUDV HHpYYEU HLpYTSL HKpYUEV HLpYHWD HApYALM RYpYDDV HGpYDFF HNpYUAM NQpYDQV HYpYEIF HDpYUNM HRpYDQV WYpYHNF GEpYUTM HFpYDTV EHpYHTF AYpYDAM HFpYDVV YYpYHYF HUpYDEM HMpYEIV YRpYAEI NApYDNM HTpYETV FUpYAQI HVpYDVM HYpYHUV HIpYUEI YApYEDM AYpYHHV UEpYEEI NYpYEEM HLpYHKV IYpYEFI DMpYEEM HYpYHWV HFpYEHI HFpYEFM IHpYKAV HTpYEPI HTpYEFM HYpYSAV AYpYEQI AEpYEGM HHpYYQV QYpYESI PFpYELM HMpYAYY FRpYEVI IRpYENM HUpYEUY NHpYEYI HVpYENM LYpYHFY RLpYHEI HYpYEPM HYpYNYY AFpYHFI NIpYESM HSpYQEY

Table 7. Peptide sequences selected from Lck SH2 screening

NYpYYNA HHpYEDM NPpYAQG NIpYAIU HApYEFM NPpYATK NFpYAVU YYpYEIM NPpYADT HYpYUTU HIpYEIM NFpYEIU HVpYEQM Class III HYpYETU YApYHYM YHpYWVY GEpYHFU HLpYSWM YHpYWVU HUpYHYU HFpYYNM YApYWLG HYpYYQU EFpYHYN YSpYFFH YFpYDLF IUpYNWP MFpYFFH TGpYEYF NIpYTEP SVpYFFH HHpYGWF NYpYYEP MYpYPFH YMpYHFF FEpYYHP UYpYPYH HYpYHFF HFpYEUV YUpYWFH WEpYHGF NFpYEDV DWpYWFH VFpYHWF NYpYEEV LYpYYFH AYpYHYF HLpYEEV FPpYYHH ERpYQFF NFpYEHV YHpYWYQ HEpYRWF DYpYEHV SRpYYLR IFpYSWF HVpYEMV WEpYWHT HFpYYPF HFpYHAV IYpYGFW HMpYAEI HVpYHDV ETpYGFW NVpYAVI HYpYHNV AFpYHFW HLpYAYI HFpYHTV HGpYQEW HVpYDQI NFpYNQV HQpYPFY VYpYEFI HMpYSVV TYpYPYY HFpYEFI NFpYTQV FYpYHPQ VHpYEVI HMpYYDV YYpYEVI HEpYYDV HYpYHNI HIpYAFY YRpYHSI FRpYHUY LYpYHYI YWpYHFY NMpYYEI AYpYHGY HEpYAQL WFpYHHY HVpYETL TPpYRHY HYpYHAL HSpYAVM Class II HVpYDEM NPpYAYT HLpYEUM NPpYAEK NFpYEUM NPpYAHU

Table 8. Peptide sequences selected from Hck SH2 screening

HFpYHQA HIpYENI HMpYHTM HIpYANU NYpYEYI HYpYHVM HUpYANU SYpYHFI ALpYHYM HIpYASU MYpYHYI HEpYMEM HSpYATU HFpYNFI HLpYQUM HEpYUDU HLpYNII HLpYSDM HFpYUEU HFpYSRI HHpYSMM HFpYDLU GFpYYEI HVpYTLM HFpYDQU HYpYYQI HYpYTTM HApYEMU XEpYYTI HHpYTVM THpYFRU AYpYHTK HLpYYEM NFpYHIU HIpYAHL HFpYEVP HYpYHVU NYpYALL HYpYNFP HYpYQIU HYpYASL HIpYHHR HLpYQYU HYpYDEL HFpYAUV HYpYSLU HLpYDFL HVpYADV HSpYWAU HIpYDNL HUpYAHV HLpYYEU THpYDVL HVpYALV HHpYYQU HEpYELL HEpYAQV HYpYEIF HRpYEML LYpYATV HHpYEMF YNpYMSL HYpYATV HYpYEQF MFpYRYL HUpYAVV SLpYFLF HVpYTUL HVpYDDV PFpYHFF HYpYANM HYpYDNV AYpYHMF HApYANM HEpYEUV HFpYQEF HFpYAQM HYpYEEV UDpYFGG HFpYASM HFpYEEV TIpYGFG HLpYUEM HLpYHLV WDpYLFH HYpYDEM HLpYSNV HYpYAAI HEpYDIM HFpYTDV HEpYADI LPpYDQM HVpYTDV HLpYAEI HFpYDRM HRpYYEV HLpYALI HDpYDTM HFpYYEV HFpYATI HUpYDVM HYpYYQV HVpYDLI HVpYDVM HLpYYTV HHpYEUI HYpYEPM HFpYHGW NYpYEII HIpYESM NGpYFYY HSpYELI FYpYEVM HYpYHUY NYpYENI HQpYHFM HVpYENI FRpYHQM 40

Table 9. Peptide sequences selected from Blk SH2 screening

HNpYEEA HFpYSQI NEpYAUV HMpYENA NDpYSYI HMpYDEV HTpYTEA GUpYTEI NLpYTEV HGpYAEU HSpYYEI NYpYQNW HHpYAEU HEpYAEL HQpYEFY HYpYDEU HIpYAQL FIpYSGY HQpYDVU HEpYUKL HEpYEAU HFpYDEL THpYEUU HHpYDEL HEpYEMU HApYDYL HFpYENU HYpYEUL NFpYETU HDpYEEL HFpYHQU NVpYEEL NYpYQEU HKpYEIL HEpYSIU HEpYELL HEpYSQU HKpYEML HEpYTRU TYpYHEL QYpYYDU HEpYRTL HGpYYEU HEpYSEL NYpYYNU PYpYTEL KApYHME HGpYAEM HPpYEEF NUpYAQM HDpYAMI HNpYUQM NIpYAQI PFpYDEM PUpYAQI HYpYDQM HEpYATI HSpYDVM HQpYUDI HTpYEDM HRpYUEI UHpYEEM HUpYDDI EDpYEEM HYpYDEI HEpYEQM HVpYDQI HQpYEVM HYpYDSI PYpYEVM HGpYDSI HDpYFEM HRpYEUI HKpYTEM HSpYEDI MApYTFN NSpYEDI HMpYEIP NDpYENI HTpYEIP IYpYHEI HFpYETP HPpYHEI HQpYTEP HMpYHYI HGpYAUV

Figure 5. Sequences specificities of Src, Yes, Hck, and Fgr SH2 domains. Abu is not present in the library used to screen Src SH2

Figure 6. Sequence specificities of Fyn, Lyn, Lck, and Blk SH2 domains

2.3.4. Abl1 and Abl2 SH2 domains

The Abl family selected a consensus similar to some of the SFK SH2 domains.

Abl1 SH2 was screened against 90 mg of resin to obtain 91 beads, 73 of which were full and partial sequences recovered after PED-MS (Table 10). Abl2 SH2 was screened against 100 mg of Library I or II to obtain 71 full and partial sequences (Table 11). Both

Abl1 and Abl2 SH2 domains selected for Ala and Asp at the +1 position and hydrophobic residues at the +3 position. There was low selectivity at the -2 and -1 positions. Abl1 recognized the consensus XXpY(D/A/H/E)X(V/I/T/S), while Abl2 recognized

XXpY(A/H/D/E)X(I/V/U) as its consensus. Abl1 selected 2 minor classes containing the consensus of φXpY(A/h)(V/u)(T/S) and φφpYpYXNE while Abl2 SH2 preferred a minor class of sequences containing the consensus φφpY(H/T/a)X(T/D/E) where φ denoted hydrophobic residues (Figure 7).

Table 10. Peptide sequences selected from Abl1 SH2 screening

Class I UVpYHQA HYpYDYV HMpYMNA SHpYEAV NFpYALU NFpYENV HIpYANU AYpYENV YHpYDVU PIpYGHV HLpYHNU HFpYHNV HFpYTMU HVpYDNY VHpYGHF YHpYENY PVpYGHF MYpYHPY PYpYAHI IPpYYVY QWpYAKI HUpYETV HSpYANI HDpYDFI Class II HFpYDFI FWpYANE IIpYDLI WWpYDNE VYpYDLI YYpYHNE HLpYDQI HWpYYNE AFpYHNI PFpYAHL Class III HYpYDRL UHpYAUS HFpYDSL WYpYAIS AWpYEHL LFpYAVS WEpYENM IWpYAVS HTpYENM WYpYENS HYpYENN VPpYHIS WDpYDQP IYpYHQS HYpYDQP YYpYHVS HLpYEIP LFpYAUT HYpYHRP LHpYAUT NYpYHYP MFpYAST HHpYAUV DFpYAVT VYpYAHV YHpYUUT PYpYAKV VFpYUVT UUpYAMV HFpYEAT PFpYAVV HYpYFNT WYpYUNV LHpYHUT NIpYDAV NFpYHNH YDpYDUV FFpYGHQ HEpYDFV TYpYDLV

Table 11. Peptide sequences selected from Abl2 SH2 screening

KApYADA ALpYAAM NLpYHNE SEpYAQA PFpYANM ILpYKME PMpYAUU NApYANM LQpYLNE PMpYANU RVpYAYM UHpYRVN DHpYASU PApYDNM EMpYFLQ W+N,Y+HpYATU UVpYGLM YYpYHIQ FKpYATU SDpYGVM AUpYTLQ XXpYAYU UIpYAUP AApYAHT EHpYEKU SYpYAEP AVpYGTT UApYFVU XXpYAQP MHpYHEU PTpYAVP UTpYHNU PYpYUNP YApYHVU PIpYDIP HYpYRVU XXpYDVP AKpYTFU NApYEUP EGpYAYF EEpYSQP KPpYPIF PIpYAEV UYpYAVG IIpYAQV PYpYAUI FFpYUEV XXpYAFI VYpYUMV SYpYAKI FApYUVV HIpYAKI IHpYUWV SYpYALI FSpYDTV DQpYAMI VQpYELV VLpYAMI SPpYHEV NLpYANI FApYHIV SLpYUEI XXpYHMV VHpYDII EGpYHSV PGpYDTI KPpYMVV PYpYEAI EVpYSUV VQpYEEI HQpYANY GFpYEMI FNpYVFI Class II AQpYAUL YQpYHID HGpYADL SIpYHND PTpYAKL AVpYPAD DLpYAQL UUpYRPD QEpYASL PDpYTMD NFpYDTL SNpYEAE AHpYTVL FGpYEFE

Figure 7. Sequence specificities of Abl1 and Abl2 SH2 domains

2.3.5. Csk and MATK SH2 domains

A total of about 120 mg of Library II was screened against Csk SH2 domain (50 full sequences), whereas 100 mg of Library II was screened against MATK (115 full and partial sequences) SH2 domains. The sequences are listed in Table 12 and Table 13 respectively. Csk SH2 recognized the consensus XXpY(A/T)X(I/V/U) and MATK selected for a consensus of XXpY(A/c)X(F/I/L) (Figure 8)24. MATK frequently selected for Lys at the +2 position.

Table 12. Peptide sequences selected from Csk SH2 screening

QDpYURA HGpYAUM TNpYKMA DUpYASM AHpYAUU EEpYUUM AEpYAUU HYpYKQM HQpYAUU HGpYTTM HSpYAEU PGpYAUP HVpYAKU MUpYKYP ULpYARU NLpYQTP KHpYAVU HKpYRVP PNpYKLU PIpYSSP MUpYPPE HVpYTUP YLpYITF HApYULR HQpYAGI YRpYATS DLpYARI TPpYIUS PTpYURI DYpYTAS ATpYHUI HUpYART ENpYSAI TDpYAKV DSpYSUI RTpYAQV HTpYSQI QEpYARV PQpYTEI HKpYURV KQpYTWI KPpYKUV QEpYAVL GNpYRUV UUpYSQL HSpYTLV HKpYTUL DEpYTTV HTpYTTL HMpYTTV

Table 13. Peptide sequences selected from MATK SH2 screening

IApYARA IKpYEII PEpYUIV HQpYUSA DUpYKII TKpYURV TFpYHKA QDpYKMI UQpYURV VApYHLA FQpYKQI UKpYUYV UIpYQYA XXpYLMI HIpYFSV NNpYAAU ISpYQII EMpYKIV NNpYAAU FSpYQKI VLpYQLV MKpYAIU UDpYRUI INpYTKV IWpYAQU NYpYRHI TQpYAKW MLpYATU MGpYSLI ITpYSMW SGpYUMU ALpYAUL LUpYQYY FUpYQKU HApYAKL YTpYSKU PNpYANL Class II UTpYTKU ITpYATL VRpYAUK SMpYTRU NMpYUAL IPpYAFK VYpYYKU TApYUKL SVpYAGK TApYAFF AHpYUVL TVpYAGK ITpYAGF VUpYHKL IIpYGLK RGpYAIF IUpYKKL ILpYHTK INpYAKF UFpYKTL TFpYMYK IRpYAKF MUpYNUL VFpYNLK KpYAKF TVpYRUL UHpYQYK VTpYAKF YVpYRRL ASpYYSK TApYAMF TFpYYTL VLpYYVK TSpYARF VYpYYTL VFpYATQ LEpYAVF PYpYAUM VKpYEKQ HLpYUIF YRpYAKM QVpYKFQ EMpYULF TMpYAQM IMpYQWQ HDpYEKF TTpYARM IVpYSYQ VNpYKKF QVpYASM IRpYUNR QLpYKMF HDpYAVM ARpYUYR MVpYKTF EYpYUKM FApYEKR GSpYVKF IRpYGUM YEpYNAS IGpYKMH THpYKIM YEpYUKT YQpYAUI QRpYQLM SMpYUYT FKpYAKI VFpYQFN YTpYKFT SKpYALI pYUKP IUpYRQT TRpYARI YTpYRTP TTpYUMI UTpYTHP VApYUSI HApYAAV

Figure 8. Sequence specificities of Csk and MATK SH2 domains

2.3.6. Fes and Fer SH2 domains

Approximately 110 mg of Library II was screened against Fer SH2 to obtain a total of 167 full and partial sequences. They both selected for two peptides classes. Fer sequences are listed in Table 14. Approximately 60 mg of resin was screened against Fes

SH2 to obtain 121 full and partial sequences. Sequences for Fes SH2 are listed in Table

15. Unlike the SFK SH2 domains, both of these SH2 domains exhibited a strong preference for Glu at the +1 position (Figure 9). Both of these SH2 domains selected hydrophobic residues at the +3 position with minor differences. Fes SH2 selected for Ile,

Nle, and Leu while Fer SH2 selected for Tyr, Ile, and Phe. Another class of sequences

51 containing the XXpYEN(D/E) consensus was identified in both SH2 domains. This small class of peptides was derived mainly from dark colored beads, which correlated with tight binding. Due to the strong selection of Glu at the +1 position and Asn at the

+2 position, there was little selectivity at the +3 position (Figure 9).

Table 14. Peptide sequences selected from Fer SH2 screening

AUpYEIA NSpYEEI PUpYELM PIpYEVY XXpYEPS IWpYENA AHpYEEI EHpYEDP HDpYEVY LDpYEVS EFpYESA VHpYEFI FEpYEEP THpYEYY EMpYEYS TYpYEVA HLpYEGI HMpYIYP PDpYEYY MGpYNLS UGpYEYA MQpYEII DApYDEV SApYQSY HGpYSYS HMpYEYA KMpYEII NVpYDIV MGpYEFT LEpYIYA NUpYEKI HMpYDNV Class II HSpYENT WQpYEDU RUpYELI DHpYDPV TWpYEDD SWpYEPT NYpYELU AQpYENI LMpYEDV LYpYEED RGpYIMT MHpYENU IYpYEMI EWpYEDV YLpYEID HUpYENU AYpYEPI MGpYEEV LHpYEND AHpYENU PApYEPI MEpYEHV PWpYEND UNpYENU SLpYEPI YQpYELV WNpYEND FQpYENU MLpYESI MSpYELV UKpYLKD NDpYEYU YHpYETI MKpYEPV YUpYUNE FGpYQHU ELpYEVI FEpYEPV NRpYDLE DGpYYYU TPpYEYI RGpYESV FApYDNE MYpYEDU TYpYEYI GHpYETV ALpYDNE YIpYEVU EEpYIUI (T+H)pYEVV PHpYEDE RYpYEDF DUpYEDL NIpYEVV YMpYEFE YFpYEDF FEpYENL EQpYEVV AYpYEGE ERpYEFF MEpYENL TApYEVV PUpYENE QTpYELF VDpYENL XXpYIIV UQpYENE EApYELF FTpYENL ITpYEUY PWpYENE EWpYEPF AYpYEPL MLpYEDY SFpYGQE VRpYEPF NYpYEVL SApYEEY ETpYSYE YQpYEPF HHpYEYL GMpYEFY QFpYTYE DLpYEQF LYpYHFL HEpYEHY SQpYEDG UGpYEVF VFpYWDL HTpYEIY QMpYEDN XXpYEVF XFpYEMM NVpYEIY THpYENN HHpYEVF YSpYEPM LGpYEIY DApYEPN VEpYEYF RLpYEPM MHpYEIY IMpYIDN EHpYQEF XXpYEQM AHpYELY GYpYEGQ HQpYWWF YWpYEQM IIpYELY NDpYEIQ TUpYDNI HYpYERM HQpYEPY FDpYENQ MYpYDNI GQpYEYM YDpYEPY APpYENQ DYpYDYI ENpYEAM SLpYEPY NDpYEPQ HEpYEUI WGpYEEM FRpYEPY QDpYDAS MEpYEUI EApYEFM ELpYERY QLpYEES LApYEDI DYpYEHM DPpYESY DHpYEMS

Table 15. Peptide sequences selected from Fes SH2 screening

YTpYEUA AMpYETI HYpYEWM Class II DPpYENA LQpYETI GPpYEYM AYpYEND MGpYEHU HLpYEVI GYpYEYM NIpYENE PYpYELU HIpYEYI SApYEYM SVpYENE QUpYELU HRpYEYI QYpYEYM QLpYYGE HKpYENU SRpYEYI IQpYHYM WRpYENN NLpYENU MSpYIII MGpYDFP XXpYYIN MPpYEPU PHpYUNL EYpYEVP EYpYYNN NYpYERU YHpYEUL NGpYEDV DGpYELQ VHpYEVU YGpYEEL TUpYEDV QYpYENR YUpYEYU RIpYEFL XVpYEEV VYpYENR SMpYULF ULpYEFL NFpYEFV AIpYDSS IRpYEUF MNpYENL LHpYEIV NEpYEVS SUpYEFF NHpYENL RYpYEIV HTpYEYS EMpYEFF UVpYENL EIpYEIV DYpYIYS IEpYEHF ENpYENL EHpYELV DHpYEAT XGpYENF HLpYENL EVpYEMV YSpYEET LRpYENF AEpYEQL AUpYEMV MTpYEHT YRpYERF SWpYETL MYpYENV HEpYELT IFpYERF HMpYEVL LRpYEPV YQpYENT UHpYETF NDpYEVL RApYEPV PYpYEVT (A+W)MpYEYF GEpYEYL ELpYEQV DLpYEVT TNpYEYF FTpYEYL UKpYEQV FUpYWIT SRpYEYF LYpYRHL AEpYERV IHpYEYF DYpYYYL UHpYESV VMpYEUI SRpYDIM VFpYEVV RApYEDI ULpYEUM YMpYEVV VEpYEDI IGpYEDM YTpYEYV QMpYEEI ELpYEEM XMpYFVV YGpYEII HFpYEEM VYpYYMV EQpYEII MHpYELM RQpYYMV LMpYEII NIpYELM QVpYENW GSpYEMI DRpYENM VUpYEIY HEpYEMI LWpYENM DGpYEIY HSpYEMI MYpYEPM LKpYELY LMpYENI YHpYERM ERpYEMY YTpYEPI QYpYESM GApYENY NUpYESI UUpYESM FUpYESY PTpYESI SSpYETM ETpYETY EFpYETI MFpYEVM EHpYHAY

Figure 9. Sequence specificities of Fer and Fes SH2 domains

2.3.7. Tec family kinase SH2 domains

A total of 480 mg of Library II was screened against the Tec family. Within the

Tec family of SH2 domains, specificities were less similar to each other. Bmx (Table 16)

(78 full and partial sequences) and Itk (Table 17) (117 full and partial sequences) SH2 domains recognized a general consensus of XXpY (A/U/E)N(E/U/P) and

XXpY(E/d/a)NE, respectively (Figure 10). Unlike most kinase SH2 domains, Bmx and

Itk SH2 domains selected predominantly for Asn at the +2 position. However, the selectivity at the +3 position was not very stringent for both of them. Tec (Table 18)

(149 full and partial sequences), Txk (Table 19) (73 full and partial sequences), and Btk

(Table 20) (88 full and partial sequences) SH2 domains selected a consensus of

XXpY(H/D)X(U/V/I), XXpY(E/d)X(V/I/U) , and XXpY(D/E)X(V/U/I) respectively

(Figure 10). Tec SH2 selected for Asp and His at the +1 position while Txk SH2 selected mostly for Glu. Tec and Txk SH2 exhibited preference for small hydrophobic residues such as Val at the +3 position compared to SFK SH2 domains which prefer larger branched hydrophobic residues.

Table 16. Peptide sequences selected from Bmx SH2 screening

PMpYUNA HTpYANG MRpYENA FUpYANG XWpYENA PUpYUNG FFpYHNA XXpYUNG PLpYANU PIpYENG FIpYANU YFpYUEH FYpYANU FUpYUNH PYpYUNU PFpYUNH PLpYUNU AMpYDNH FVpYUNU NEpYENH LRpYDNU FEpYHNL LIpYEFU LFpYYNL WUpYENU XXpYAMN FQpYENU LYpYEUM PFpYENU PRpYEIM FGpYENU PApYANP HMpYHNU PEpYANP YEpYAND PFpYANP PLpYAND STpYUNP PFpYAND GVpYENP PFpYUND XXpYENP YUpYQND PNpYENP SFpYANE PIpYHNP FEpYANE HDpYUNQ FTpYANE PHpYUNQ FFpYANE FFpYENQ YApYUNE SUpYENQ FVpYUNE NHpYANS FRpYUNE PIpYANS PTpYUNE FFpYUNS PVpYUNE PYpYHNS FFpYUQE FFpYANT MMpYDNE PQpYENV SFpYENE PLpYENV SGpYENE PYpYENV PMpYHNE FQpYANW VYpYQNE HWpYENY XXpYQNE XXpYENY PMpYWVE GApYUNF

Table 17. Peptide sequences selected from Itk SH2 screening

DNpYSAA LDpYDEP ETpYENY VNpYUNE LQpYANA SWpYDEP KEpYENY LHpYUNE GGpYENA MDpYDIP HEpYHAY NWpYUNG EEpYEYA YUpYDNP LEpYLAY FEpYYNE FYpYUWA WEpYDVP UEpYANF YQpYEDP Class II Class III TApYEEF MTpYEEP UUpYAND DTpYEFD ALpYENF WEpYEFP SYpYAND EEpYESD EDpYESF HHpYENP WUpYAND EEpYWDD NDpYMSF QEpYEPP NFpYANE QMpYEDE DDpYUDF WDpYEVP SYpYANE LYpYEDE YDpYUNF TFpYHLP VTpYANE WGpYEDE QEpYEII HVpYLHP WEpYANQ NNpYEFE MQpYEII DEpYANU PFpYDND YLpYELE HEpYELI YEpYASU WTpYDNE SWpYELE DEpYEMI SEpYEFU DApYDNE YEpYEME LMpYENI YDpYEIU DGpYDNE FQpYEVE DLpYENI VEpYELU WPpYEND EWpYIQE LEpYSNI YDpYEVU YPpYEND UYpYNLE TSpYWYI LEpYEYU EApYEND DEpYWDE VYpYAEL QEpYHFU EKpYENE LEpYELG WGpYEDL TEpYANV HYpYENG DGpYFHG UEpYENL UApYEEV FLpYENH WNpYEDH AEpYETL LYpYENV YQpYENH DYpYEEH GGpYANM DGpYEYV PEpYENK WEpYEFN AMpYANM NGpYVNV DYpYENN PEpYELQ SEpYNVM EVpYANW NGpYENQ DQpYDDT HEpYVEM TYpYENW FEpYHND WEpYEIT HGpYAEP MEpYQNW ILpYHND NDpYAYP GYpYUNW MQpYHNE

EEpYAYP WEpYANY WNpYUND

Table 18. Peptide sequences selected from Tec SH2 screening

HKpYHLI HTpYDEV RWpYEDQ MRpYHTI YQpYDHV FVpYHNS LLpYHTI PApYDIV EApYYNS WQpYHVI YFpYDLV GMpYNRI QTpYDMV QEpYTLI FVpYDQV HHpYDDL YKpYDSV KQpYDEL LHpYDVV YDpYDFL UPpYEEV PUpYDIL EWpYEEV NUpYDVL TVpYHEV DQpYELL YRpYHEV YQpYHEL HFpYHEV HTpYHEL YKpYHFV NGpYHHL MKpYHGV YLpYHQL LYpYHHV NEpYHSL LKpYHLV GFpYQUL YHpYHLV NDpYUIM LKpYHLV DKpYDEM HQpYHQV YNpYDEM YLpYHRV MNpYDLM AMpYHTV WUpYDMM LYpYHTV FTpYHUM AMpYHVV HGpYSVM UFpYHVV HEpYAVP HYpYNUV ELpYDUP QPpYNEV YEpYDEP QYpYNVV LYpYDTP YDpYTLV DFpYHEP HDpYAET LHpYHIP HSpYDNT RIpYAEV FHpYDVT HKpYAEV YQpYEVT YGpYAIV AMpYWIT HFpYATV GEpYAVV HVpYQYD SQpYUIV HYpYDME KQpYDUV MHpYSFG IHpYDUV TYpYDSH HFpYDEV DDpYGNH 59

Table 19. Peptide sequences selected from Txk SH2 screening

EDpYDFA WSpYAQP XXpYMMH AKpYDGA HEpYDIP TYpYEID ATpYIRA IYpYDQP WWpYURE KPpYAEU XXpYDVP SDpYDNE VEpYAQU DWpYEEP MWpYELE AFpYDIU LYpYEVP TYpYENE YHpYDIU KGpYHFP TYpYAQN LApYXXU IPpYAIV TDpYUHQ LHpYELU VEpYATV TFpYAHS AVpYEMU KQpYAVV UWpYEET TDpYEVU WSpYUNV TNpYYDT FHpYEVU XYpYDIV TSpYWSE UMpYHTU TDpYDLV KEpYMHU TLpYDMV TLpYEFF HQpYDTV LEpYELF KFpYEUV TYpYTIF KLpYEUV TEpYAII WYpYEUV VEpYDLI TEpYEUV HQpYDQI PYpYEFV PYpYDQI PApYEIV TMpYEEI FHpYEIV KUpYEEI UGpYELV HMpYELI TUpYELV WDpYELI KPpYELV ASpYEVI WWpYENV AUpYEVI HUpYESV TMpYGFI VYpYETV WGpYAEL XMpYEVV DUpYUNL PVpYHIV FHpYEEL SYpYHRV DYpYEEL XXpYLQV WEpYEHL YLpYNNV WNpYQVL TTpYYFY TUpYSLL KUpYWNU LYpYEDM WEpYKIU HIpYELM TSpYYQV DEpYEMM TSpYWHW EEpYENM KKpYFVV TGpYEQM TDpYYRL

Table 20. Peptide sequences selected from Btk SH2 screening

YHpYDIA TRpYDYL PKpYEYV DVpYHHA YYpYENL QTpYHLV AVpYHIA EEpYHML WUpYHNV MPpYAFU EPpYIFL GVpYLUV VLpYUUU IN?pYTFL LYpYNEV FEpYDIU XApYADM AYpYNTV TTpYDIU LTpYDDM YUpYTYV MUpYDQU ESpYDLM TDpYAFW YVpYDTU GpYDQM WUpYFAY SVpYDVU DMpYDTM QEpYGMY TYpYDVU IEpYDYM XXpYDVU YVpYEDM TNpYDWU DGpYEVM WUpYAFD NUpYDYU ISpYQDM YIpYIYH USpYHEU YDpYTDM YDpYNWH LEpYHFU UDpYYTM XXpYQYH TDpYHFU NPpYDUP KNpYSSH FEpYHNU WNpYDHP KLpYQFK PFpYHNU WYpYDHP DFpYEDN WWpYQQU EFpYDVP FYpYNKN EUpYTIU EHpYAIV YHpYDWQ AIpYTQU SRpYANV LLpYEWQ UFpYDEI LVpYAYV YUpYAQR HHpYDFI NWpYUEV HPpYEFR NSpYDII THpYDUV YEpYETR QNpYDNI QQpYDEV MMpYYYY TTpYDQI AApYDFV MMpYDSI HIpYDLV EPpYDTI UVpYDMV PQpYDTI QLpYDMV HMpYDYI MYpYDMV TRpYDYI MYpYDMV QIpYEHI YQpYDTV YEpYELI TTpYDTV WYpYHVI TTpYDTV DApYIYI DYpYDYV LYpYAEL YQpYEMV XLpYDEL HMpYEMV HFpYDTL XKpYEVV

Figure 10. Sequence specificities of Bmx, Itk, Tec, Txk, and Btk SH2 domains

2.3.8. Brk family kinase SH2 domains

Andrew Kunys provided the sequences for the Brk family kinase SH2 domains.

A total of about 270 mg of Library II was screened against the Brk family. The Brk family generally selected for hydrophilic residues at the +1 position and hydrophobic residues at the +3 position. The consensus for Brk (155 full & partial sequences), Frk,

(114 full & partial sequences) and Srms (117 full & partial sequences) SH2 domains are

XXpYDX(V/U), XXpYEX(I/L), and XXpY(E/H)X(V/F/I), respectively (Figure 11). Brk and Frk SH2 also selected for a second minor peptide class XXpYEN(D/E) similar to the

Fes and Fer minor class. Their sequences are summarized in Table 21-Table 23. The distinct difference in Brk and Frk SH2 was the selectivity at the +3 position. Brk SH2 selected for small residues such as Val and Abu, while Frk SH2 selected for large branched residues such as Ile and Leu. Brk selected mostly for +1 position Asp while

Frk selected mostly for Glu. SRMS selected for a wider range of hydrophobic residues at the +3 position.

Table 21. Peptide sequences selected from Brk SH2 screening

NYpYDEA HUpYERU ELpYDNP FNpYEDV HDpYEVY UApYDIA NFpYETU MQpYDTP TYpYEDV UQpYTDY NDpYDIA HDpYEVU WRpYDYP LEpYEDV HIpYTQY PMpYDVA HQpYEYU EApYEIP AHpYEEV QMpYDVA YApYGYU FApYEVP NHpYEEV Class II EEpYDVA MMpYPGU QGpYSFP YDpYEIV DIpYDND QPpYEUA DFpYQEU HRpYVHP EWpYELV VUpYEND DDpYEHA EFpYSDU GGpYAMV HVpYENV LYpYEND EEpYETA LEpYSIU UYpYDUV DLpYESV IFpYEFE DMpYFPA ENpYTDU YEpYDUV IYpYESV FLpYENE FEpYYTA HEpYTIU TQpYDDV EHpYETV KQpYENE YSpYAWU TMpYTLU QMpYDDV PYpYEVV FPpYEWE TYpYDEU USpYTQU YRpYDEV PKpYEVV AWpYENN YQpYDEU EVpYTSU TFpYDFV PIpYEVV QDpYEWG DMpYDIU QQpYYIU ARpYDIV SLpYNEV FYpYDYK GGpYDIU IIpYDMF HApYDIV EIpYSTV AKpYGPQ DHpYDLU EMpYDTF EFpYDIV PLpYTEV EYpYDIT VLpYDNU DTpYDVF TYpYDIV EEpYTHV NIpYVLT YPpYDMU YFpYEEF PLpYDIV EApYDAY MSpYYAD TMpYDMU EDpYELF IMpYDIV SGpYDDY VLpYDQU HApYENF HIpYDIV NLpYDDY TApYDTU EDpYQEF IHpYDKV PGpYDEY STpYDVU SEpYDEI YDpYDKV VLpYDIY YYpYDVU QGpYDQI LIpYDNV XYpYDIY YSpYDVU NYpYEAI VPpYDNV VLpYDIY ELpYDWU EQpYEDI NFpYDNV AGpYDIY MLpYDYU AQpYELI DVpYDMV HFpYDKY HFpYEDU DHpYDGL PHpYDQV MPpYDLY HRpYEEU NHpYDNL IYpYDSV MEpYDLY NMpYEEU QSpYEIL LDpYDVV NEpYDMY SQpYEIU FQpYENL HWpYDVV AHpYDPY SEpYEIU NSpYDTM QTpYDVV HDpYDQY VDpYEKU DSpYEIM LHpYDVV QFpYDSY YUpYELU DHpYENM QIpYDVV DEpYDSY ELpYELU DGpYTEM UIpYDYV USpYDVY IDpYENU DYpYAUP MQpYDYV LGpYEIY XMpYEMU DFpYDUP ETpYEAV VSpYENY NSpYEPU GDpYDIP RMpYEUV IApYEQY

Table 22. Peptide sequences selected from Frk SH2 screening

Class I IQpYDNA URpYEKI HVpYTDL Class II HSpYEIA LDpYEMI NFpYTPL HFpYEID HFpYEKA HPpYENI NYpYTTL HFpYELD SWpYELA HLpYENI YTpYYEL MKpYEND HVpYSDA HTpYEQI HKpYYSL NDpYEND AFpYSIA HIpYEQI YApYAEM HYpYEDN HSpYYNA WHpYETI DLpYAKM HQpYAIU PNpYEVI AVpYUEM FPpYNIH NMpYAVU QRpYEVI VQpYDVM HSpYVDK HVpYUGU VVpYEVI WHpYEEM NApYNHQ HGpYDIU PEpYHUI QIpYETM EPpYHVS ALpYEIU NIpYHEI MFpYHNM VRpYWUS EGpYIDU HKpYHQI HFpYTIM TMpYQDT QKpYMLU HMpYHRI HApYUIP NVpYQRD SFpYRYU KFpYHTI WDpYUYP UTpYVWD TSpYTNU HEpYQVI HQpYEVP HApYWWD YLpYWEU PPpYSWI HYpYFIP NFpYHDE HDpYYHU PTpYTLI QGpYSWP IVpYUSN NApYYLU EMpYYNI VYpYTVP URpYYYU HYpYYQI HIpYVQP EFpYAQF NEpYYSI HUpYAQV ERpYEDF NDpYYYI VLpYUEV FWpYEEF VEpYAKL HDpYULV UQpYENF EApYUUL NApYUSV VYpYETF HTpYUNL SVpYEDV GKpYIYF TKpYUQL HLpYEGV EVpYTMF MEpYDML LVpYESV UQpYVDF SDpYEUL NLpYHDV FDpYUAI NKpYEDL NYpYHNV WQpYUUI VYpYEKL DYpYHNV GYpYUHI RUpYENL LHpYTAV QDpYUNI XMpYETL NPpYEUY MUpYDSI EYpYGIL SQpYEUY WVpYEAI PSpYHIL ELpYEQY EWpYEUI NIpYHLL UEpYNSY HSpYEEI AFpYHSL NUpYQSY QPpYEHI HGpYMDL HLpYEHI IGpYQIL HGpYEII WHpYSDL

Table 23. Peptide sequences selected from SRMS SH2 screening

NIpYHFA TTpYAGI DFpYEUM NYpYHQD HFpYALU THpYUMI XWpYEIM RTpYHTD HFpYAMU HTpYDSI AYpYELM RTpYHTD HVpYDHU SVpYEEI HFpYEMM HApYMND SHpYDVU TFpYEII PYpYEYM HFpYNND HMpYEUU DNpYEMI NKpYFVM UWpYSND TIpYEUU WMpYENI KEpYHDM HLpYWME WRpYEDU KIpYETI NVpYHLM KKpYDTG ULpYEFU TMpYETI MGpYHNM KKpYMSK WGpYERU HQpYEVI THpYHVM YQpYHIR EMpYEVU VIpYEVI VHpYYYM PNpYITS VGpYFYU HFpYHNI WWpYUYP NEpYTGS UVpYHUU HTpYHQI GYpYEHP HHpYULT HFpYKRU UHpYHVI URpYELP SHpYHYT NApYNVU PEpYHVI NIpYELP ATpYVQT YNpYNYU PEpYHYI PYpYHYP WWpYGFN WYpYQVU FYpYHYI WRpYAIV TMpYWWU XXpYLKI UTpYULV KIpYDWU WQpYAGL AGpYUWV QHpYUUF YRpYASL AHpYDIV HQpYEDF VHpYUHL DGpYDLV TNpYEHF HNpYUWL HVpYEUV EEpYENF VLpYDFL HHpYEFV LDpYESF TUpYDKL VMpYEIV FEpYEVF TEpYDSL TGpYEMV YApYEYF NPpYEIL WWpYEYV HEpYFEF QHpYEIL SGpYHEV ELpYHDF LYpYEIL HEpYHFV SUpYHEF PVpYEKL UEpYHIV VNpYHEF HYpYELL HQpYHIV GFpYHIF HHpYESL AHpYHPV PYpYHMF KTpYETL EMpYHSV PFpYHNF KUpYGRL KDpYMLV LRpYHNF IYpYIQL HYpYRUV HUpYHQF TDpYUMM VUpYSFV PIpYHSF XEpYUNM IIpYTDV QRpYVWF NEpYDLM QWpYYIV AApYYIF WFpYDQM TQpYHYW TDpYYIF FHpYDSM TLpYAPY

Figure 11. Sequence specificities of Brk, Frk, and SRMS SH2 domains

2.3.9. Zap N and C SH2 domains

Zap N and CSH2 domains were previously screened by Dr. Anne-Sophie

Wavreille as individual domains, but the screening results were poor due to possible misfolding of the isolated domains. To solve the misfolding issue, the tandem ZAP

(tZAP) SH2 domain was constructed with a single point mutation in the binding site of

67 the N or C SH2 by Dr. Yanyan Zhang. 180 mg of Library I or II was screened against

ZAP N and C SH2 domains. tZAP CSH2 mutant (NSH2) (82 full and partial sequences) selected for a consensus of XXpY(A/S)XL, and tZAP NSH2 mutant (CSH2) (88 full and partial sequences) selected for XXpYAXL (Figure 12). Their sequences are summarized in Table 24 and Table 25. Unlike the other kinase SH2 domains, these two SH2 domains exclusively selected for Leu at the +3 position. ZAP NSH2 selected for Ser along with

Ala, resulting in a lower specificity at the +1 position.

Table 24. Peptide sequences selected from Zap NSH2 screening

WTpYARI GApYARL HRpYSVM XFpYRRI GUpYARL WRpYNYM HFpYSRI YFpYARL HYpYDYI UHpYARL HLpYKWF YYpYDYI IHpYARL LYpYRRF HFpYAUL QIpYARL HMpYRYF VHpYAUL PLpYARL RRpYRYF GRpYAUL NMpYARL WYpYKFH HYpYNFL RQpYARL WFpYRYH HKpYSFL FRpYARL MFpYRRN AYpYSFL MRpYARL LRpYLIR RYpYAHL KYpYARL LLpYRFR HFpYDHL YRpYNRL IYpYRWR HFpYSHL PYpYNRL WSpYRYR HMpYSHL NApYSRL HRpYWFR NYpYAIL RFpYSRL XXpYAHY YYpYDIL XMpYSRL WFpYHYK YYpYDIL HRpYSRL WSpYWRK GRpYAKL PRpYSRL WYpYF+P/D+EL TRpYAKL NYpYASL HYpYAKL HVpYDSL HHpYSKL YUpYDTL HIpYSKL HYpYRTL YLpYSKL HHpYSTL URpYSKL HMpYSTL HYpYDLL YMpYSTL HLpYSLL NUpYAVL HApYAML HUpYNVL WFpYHML HKpYSVL YYpYNML HEpYAYL HIpYSML PHpYDYL YYpYANL YSpYYYL YYpYENL QRpYARM YHpYAQL HFpYDRM HKpYAQL HHpYDRM DYpYAQL HFpYHRM TYpYAQL HHpYSRM HRpYDQL WNpYSRM HYpYDQL HFpYYRM HVpYSQL EYpYESM

Table 25. Peptide sequences selected from Zap CSH2 screening

TYpYGHI ANpYUKL PUpYARL WEpYGHM UGpYMRR YRpYAKI YNpYUKL SEpYARL HFpYHYM NGpYMRR YRpYHAL UQpYUKL WIpYARL YHpYUUT NMpYMRR MRpYKAL FRpYUKL AKpYARL RRpYAQT FGpYYGR RKpYQAL FHpYHKL TLpYARL ARpYART HGpYEIW HSpYAUL DWpYMKL DMpYARL WMpYHUW MRpYRUL HApYQKL NRpYARL PWpYFTY UVpYMEL FRpYQKL QYpYARL YRpYARU AYpYHFL WRpYTKL YTpYURL SWpYGHU HHpYAHL RHpYAML WMpYHRL FRpYGHG HQpYAHL RRpYAML GSpYHRL MYpYGHG PMpYEHL FGpYHML NYpYHRL WGpYGHH HPpYMHL PMpYANL DGpYRRL UFpYGHR HYpYDIL PQpYANL GVpYRRL URpYGHR PHpYHIL RRpYANL RYpYSRL GHpYGHR GHpYAKL FSpYANL MGpYUSL YMpYGHR LHpYAKL PFpYFNL GGpYHSL WFpYKYH PHpYAKL UHpYAQL URpYRSL NWpYAKR ENpYAKL TVpYAQL YFpYHTL R+E/V+WpYARR GPpYAKL ARpYHQL HEpYAYL WGpYHTR

Figure 12. Sequence specificities of Zap N and CSH2 domains

2.3.10. Syk N and C SH2 domains

Due to similar folding problems like ZAP SH2, tandem Syk SH2 CSH2 mutant

(NSH2) was constructed by Dr. Y. Zhang and used for screening. 280 mg of Library I or

II was screened against Syk N and C SH2 domains. Syk NSH2 (76 full and partial sequences) recognized the consensus XXpYAX(L/M) and Syk CSH2 (56 full and sequences) selected for XXpY(T/M)XL (Figure 13). Individual sequences are summarized in Table 26 and Table 27. Just like the ZAP SH2 domains, Syk N and C

SH2 domains selected for Leu at the +3 position. Syk CSH2 selected for Thr and Nle instead of Ala at the +1 position.

Table 26. Peptide sequences selected from Syk NSH2 screening

HYpYHUF VApYAKM HUpYAFI KLpYAKM SRpYAKI URpYAKM HUpYANI TVpYAKM IHpYARI VIpYAQM HVpYAYI PMpYARM HUpYULI HUpYAVM SEpYKKI SHpYUKM QVpYRRI AYpYHHM UEpYAKL HLpY(H+E)M HRpYAKL HApYIVM TWpYAKL LRpYKAM YHpYAKL AApYKHM VVpYAKL LVpYKHM AFpYAQL RHpYQVM NApYARL LIpYRIM ULpYARL YUpYRKM FRpYUKL LApYRRM KYpYUKL HSpYRSM MYpYHRL APpYTLM HYpYITL PSpYAGP NIpYKKL HGpYAKV RQpYKKL EUpYATV SIpYKKL HMpYNGV RApYKQL QApYKRL HMpYTRD HQpYLYL XUpYIFF GNpYQKL HTpYAKR RUpYRUL HTpYASR IWpYRHL HKpYAUR IMpYRKL HKpYATR SUpYRKL KNpYAQY HIpYRKL HHpYAQY HUpYRRL ADpYMLY RFpYRTL VPpYRWY HLpYRWL HKpYRYW KTpYRYL XXpYAKL EApYAKM EMpYAKM LEpYAKM

Table 27. Peptide sequences selected from Syk CSH2 screening

XNpYERL XLpYTAL YYpYFQT PRpYERL HHpYTNL ANpYFSF NVpYESL HSpYTPL XYpYFSR XXpYIPI TFpYTQL XXpYFTA XVpYMAL MYpYTQL GHpYFTA GApYMAL XYpYTQL TMpYFTA XXpYMQL ELpYTRL XXpYFTL MGpYMQL GFpYTRL YDpYFTQ ARpYMRL FTpYTRL ATpYFTQ XXpYMRL XXpYTSL VDpYFTR PTpYQAL IRpYTSL ESpYFTR VTpYQEL HHpYTVL HSpYFTR SIpYQRL XXpYTVL RSpYFTS MRpYQRL SGpYTVL SDpYFTY SVpYQSL XXpYTVL SVpYFTY GVpYQSL PMpYKQG PYpYQSM VDpYKRK YRpYSRL XXpYTAL ILpYYQL YTpYTAL XRpYYRL XXpYYTL YMpYYTR

Figure 13. Sequence specificities of Syk N and CSH2 domains

2.3.11. Binding affinities of selected peptides against SFK SH2 domains

The SFK SH2 sequence specificity was further evaluated by determining the equilibrium dissociation constants of several representative peptides using the SPR technique (BIAcore). Eight representative peptides were synthesized (Table 28). Peptide

#1 HYpYEEI, a well-known peptide motif that binds to SFK SH2 domains, acted as a positive control. Within the Src family, the SH2 domains selected mostly for Ala or Glu at the +1 position. To test the importance of the pY+1 position, Glu was mutated to an

Ala in peptide 2. Peptide #2 was expected to bind to all the SFK SH2 domains. Histidine was frequently the tallest peak in the specificity plots even though the selectivity at the -2

74 position with the exception of Hck was low. To test the importance of His at the -2 position, His was mutated to an Ala in peptide #3. In addition, unique sequences identified in the screenings were tested to see if they bound to their corresponding protein well. The ANPpYADT type sequences were only found in Lck SH2 screenings while the sequences PApYQMP and HDpYTIA were found in the preliminary Fgr SH2 screenings.

These unique peptides were tested against all the SFK SH2 domains to see if they bind well to all of them or only to specific SH2 domains. Peptides #14 and #12, ALpYHQI and HHpYTII, respectively, were previously tested with Src SH225. They were also tested against the other SFK SH2 domains for comparison.

A total of 64 equilibrium dissociation constants were determined, and the results are summarized in Table 28a. According to the data, all of the SFK SH2 domains bound to peptide #2 (Kd from 0.2 to 0.75 µM) and peptide #1 (Kd from 0.04 to 0.24 µM) with high affinity. Mutating the -2 position His (peptide #3) caused a slight decrease in binding affinity in some of the SFK SH2 domains. However, only Hck SH2 demonstrated the largest change, a three-fold decrease, in binding affinity. This three- fold decrease indicated that the selection of the -2 position His was likely real. As hypothesized, the SFK SH2 binding affinity for the peptides #10, #11, and #13 varied.

Hck, Lyn, Blk and Fgr SH2 bound poorly to the unique peptides, but Yes and Fyn SH2 domains bound to all of them with dissociation constants ranging from 0.05 to 3.41 µM.

We conclude that Yes and Fyn SH2 were less selective than Hck, Lyn, Blk, and Fgr SH2 domains. Src SH2 bound to all the peptides except peptides #10 and #13 with high affinity. Peptide #14 (Kd s from 0.2 to 5.5 μM) and peptide 12 (Kd s from 0.4 to 5.1 μM)

75 were well tolerated by SFK SH2 domains, but with weaker affinities than peptides #1 and

#2. The dissociation constants for GST fusion Lck SH2 protein were much lower than those measured with non-fusion proteins due to the tendency for GST to dimerize. As a result, the binding affinity was overestimated. However, relative differences in binding could be observed. GST Lck SH2 bound to peptides #1 and #2 very well, but bound to peptide #10 more weakly. These dissociation constants correlate consistently with the screening results.

2.3.12. Binding affinities of selected peptides against selected kinase SH2 domains

Some of the kinase SH2 domains selected unique sets of peptides that SFK SH2 domains seldom select for. For example, Fes and Fer selected exclusively for Glu at the

+1 position, while Tec SH2 selected mainly for Asp at the +1 position. Also, Bmx exhibited strong selectivity towards Asn at the +2 position and lower selectivity at the other positions. From examining the KD s of the SFKs, most of the SH2 domains bound to the tested peptides with high affinity validating our methodology. However, there were occasional unique sequences that were selected during screening. For example,

IWpYAVS was only selected by Abl1 SH2, whereas LTpYEND and NIpYENE were selected during Fes and Fer SH2 screenings. These unique sequences selected in screening may either be real hits or false positives. Therefore, unique sequences, selected by the other kinase SH2 domains, were resynthesized and further evaluated for binding by measuring their equilibrium dissociation constants to their corresponding SH2 domain by fluorescence polarization. Summarized in Table 28b, Fes and Fer SH2 domains bound to peptide #1 with high affinity with Kd s ranging from 3.9 µM to 4.2 µM, but

76 poorly to peptide #2 with a Kd larger than 100 µM. This was consistent with the screening data validating the high selectivity of Glu at the +1 position. Tec SH2 bound five times better to Peptide 4 than to peptides #1 and #2. When the +2 Asn was mutated to Ala the Kd increased 2 fold demonstrating that the +2 Asn contributed tobinding of

Bmx SH2. Bmx SH2 bound 10 times better to Peptide #8 than to Peptide #6, as expected because pYENI type sequences were not selected frequently by Bmx SH2 during screening. However, it often selected for pYENE type sequences. Minor class

IWpYAVS bound to Abl1 and Abl2 SH2 domains at nanomolar concentrations, which may correspond to a new binding motif. This peptide sequence will be entered into the protein database to identify any new potential binding partners containing this peptide motif.

Table 28. Dissociation constants (Kd, µM) of selected pY peptides binding to SH2 domains determined by SPR and FP

(a). From SFK SH2 domains. (b). From selected kinase SH2 domains All peptides were acetylated at the N-terminus and contained a C-terminal linker LNBK-NH2 except HYpYEEI, which contained the linker BK, and LTpYEND, which contained a LNK linker. * asterisk denotes the SH2 protein used was a GST fusion. ND, not determined. All values are in units of µM. The identity of peptides 1-14 is indicated in part b.

Peptide Hck Lyn Blk Yes c-Src Fgr Fyn Lck* 1 0.21±0.02 0.17±0.02 0.24±0.02 0.073±0.005 0.16±0.01 0.19±0.02 0.050±0.01 0.04±0.01 2 0.31±0.01 0.58±0.05 0.37±0.03 0.34±0.1 0.20±0.02 0.75±0.05 0.39± 0.1 0.09±0.03 3 0.92±0.07 0.64±0.02 0.49±0.04 0.19±0.01 0.33±0.06 0.94±0.02 0.23±0.04 0.06±0.01 4 ND ND ND ND ND ND ND ND 5 ND ND 50±2 ND ND ND ND ND 6 ND ND ND ND ND ND ND ND 7 ND ND ND ND ND ND ND ND 8 ND ND 1.9±0.09 ND ND ND ND ND 9 ND ND 6.6±0.6 ND ND ND ND ND 10 46±10 34±12 >100 0.85±0.06 8.3±0.8 >50 3.2±0.6 0.33±0.05 11 27±6 26± 4 >100 0.85±0.1 3.9±0.4 >100 1.2±0.1 0.13±0.02 12 2.9±0.1 4.8±0.5 3.0±0.2 0.50±0.2 0.67±0.06 5.2±0.7 0.40±0.07 0.07±0.01 13 >100 >100 27±5 1.4±0.3 11±4 >100 3.4± 2.0 0.18±0.01 14 3.2±0.2 3.3±0.1 2.1±0.09 0.41±0.07 0.72±0.04 5.3±2 0.26±0.04 0.08±0.01

Peptide Sequence Fer* Fes* Abl1* Abl2* Bmx* Tec* 1 HYpYEEI 3.9±0.3 4.2±0.28 18.3±1.4 2.5±0.2 ND 12±1 2 HYpYAEI >100 >100 30±3 4.5±0.3 ND 12±2 3 AYpYAEI ND ND ND ND ND ND 4 HYpYDEI ND ND 14±1 1.7±0.2 ND 2.6±0.3 5 IWpYAVS ND ND 0.49±0.04 0.35±0.02 ND ND 6 HYpYENI ND ND ND ND 37±3 ND 7 HYpYEAI ND ND ND ND 81±9 ND 8 NIpYENE 5.1±0.5 7.8±0.6 ND ND 8.1±0.6 77±10 9 LTpYEND 12±2 23±4 ND ND ND ND 10 ANPpYADT ND ND ND ND ND ND 11 PApYQMP ND ND ND ND ND ND 12 HHpYTII ND ND ND ND ND ND 13 HDpYTIA ND ND ND ND ND ND 14 ALpYHQI ND ND ND ND ND ND

2.3.13. Database/Literature search for selected kinase SH2 domains

After determining the specificity of the kinase SH2 domains, several peptide sequences were used to manually search the protein database on the internet to identify potential binding proteins. Since it would be tedious to filter out millions of hits for the

SFK SH2 domains, a literature search was initially conducted to search for well- established binding partners instead. A literature search was also conducted for the Tec,

Fer, and Abl families, which all have different sequence specificities from SFKs. The literature search revealed that twenty proteins are known to bind to Src kinase family

SH2 domains. There were other unlisted proteins that at least bind to the kinase portion of the protein, but it was unknown whether they could interact with the SH2 domain as well. At least nineteen proteins also bind to the other kinase SH2 domains from Tec, Fer, and Abl (Table 29). Several of the proteins have known pY binding motifs that bind to their corresponding SH2 domain. These known pY motifs were compared with the sequence consensus to see if they were similar to each other. These pY binding motifs corresponded to the sequence consensus found during screening, validating our method for determining binding partners from sequence specificity. Tec SH2 bound to the consensus XXpY(H/D)X(U/V/I) while Itk SH2 bound to the consensus XXpY(E/d/a)NE.

Interestingly, according to Table 29, LAT and Slp76 are known to associate with Itk and

Btk SH2 domains. Their motifs contained mainly Glu at the +1 position. LAT is known to only interact with Itk with pY motifs containing an Asn at the +2 position. Tec interacted weakly with LAT and Slp7660. Many proteins that bind unknown SH2 domains have pY-containing motifs. Those proteins may be entered into a database on

79 phosphorylation sites. From the search results and the screening data, the binding motif may be predicted. Then, further tests could be performed to validate them.

Table 29. Known SH2 domain binding partners and binding motifs based on a literature search

Protein pY motifs SH2 bound Reference 397 FAK DDpY AEI All SFKs 61; 62 489 β-adducin pY TDP Fyn 63 LMP2A pYEEA Lyn 64 115 StpA pY AEV Lck, Fyn 65 402 Pyk2 DIpY AEI Lck, Src, Fyn 62 571 Slap130 IDpY DSL Fyn 66 348 Syk pY ESP Blk, Lyn, Fyn 67 SH2B of K15 pYEEV Lyn 68 460 Srcasm AIpY EEI Fyn, Hck 69 SLM-1 motif unknown Fyn 70 271 295 SKAP55 DIpY EVL,VDpY ASY Fyn 66 454 833 ANKS1A HPpY ELL and RPpY EEP Lck, Fyn 71 G-CSFR Src, Yes, Fyn, pY561TFI Hck 72 228 246 Rack1 HLpY TLD, and/or NRpY WLC Src 73 Tio motif unknown Lck, Src, Fyn 65 94 451 AFAP-110 pY EEA, pY DYI Src, Lyn, Fyn 61; 72 Cbl motif unknown Fyn, Abl1 74; 75 Sam68 motif unknown Src 72 762 Cas p130 pY DYN Src, Bmx 44; 72 381 409 Cbp PDpY EAI, SDpY ESI Lyn 76 PLCg2 motif unknown Btk 77 EphB2 KIpY605IDP, FTpY611EDP Abl1, Abl2 78 Dok1 IYpY361DEP Abl1, Btk 77; 79 Her-2 motif unknown Abl1 80 PDGFR-beta motif unknown Abl1 80 IRS-1 motif unknown Fer 81 SLP-76 DDpY113ESP, GDpY128ESP ADpY145EPP Itk, Btk, Txk 82; 83 LAT pYENE, PDpYENL,& pYHNP Itk 60 Blnk pYEPP Btk, Itk 60 PLpY36DVP, SHpY121ILE, CApY148CHT, & Rin1 VApY632QDP Abl1 84 IRS-4 pYRAY, pYNND, pYDAE Brk 85 Bcr motif unknown Abl1, Fes 86; 87 Vav1 motif unknown Tec 77; 88 Sam68 pYEDY Brk 89 Paxillin motif unknown Brk 90 Cortactin motif unknown Fer, Abl2 91; 92 Kit ALpY703KNL Fes 93

Table 30. Human proteins with minor class peptide motifs predicted to bind to Abl and Fes family SH2 domains

Protein pY motifs Reference ADI1 DKpYEND BICD1 NKpYENE K10, K13, K15 LKpYENE LAT ASpYENE palmdelphin (PALMD) SVpY218AVS 60S ribosomal protein L4 (RPL4) QPpY52AVS 151 doublecortin (DCX) IVpY AVS 94

Since it would be too tedious to test every single consensus in this tyrosine kinase family, only a few of the interesting SH2 domains, especially minor classes, or those containing unique sequences, were further examined to identify any potential binding partners.

Also, some of the minor class peptides such as pYEN(D/E), identified in Fes and Fer

SH2 screening, and IWpYAVS from Abl1 screening were also employed to search protein databases to identify new potential binding proteins. YEN[DE], and YAVS were entered into a database of phosphorylation sites and kinase specificity (website: http://www.phosphosite.org). Only sequences with phosphorylated tyrosines were considered. 6 pY hits/266 hits, and 3 pY hits/200 hits were identified for YEN[DE] and

YAVS, respectively, listed in Table 30. These new potential binding proteins were entered into a literature database to find if they have been shown to bind to the Abl1 SH2 domain. After conducting the literature search, there were only a few search results on these proteins. Currently, out of all the binding partners identified, doublecortin is the only one known to bind to Abl1. It is unknown whether its interaction is mediated by the

SH2 domain94. Based on the binding data, it is possible that the sequence YAVS could

82 be targeted by Abl1 SH2 domain. This finding could be tested either by performing coimmunoprecipitation or peptide pulldown assays to confirm that the proteins palmdelphin, 60S ribosomal protein, and doublecortin bind.

2.4. Discussion

The specificity of twenty-six kinase SH2 domains was determined using a combinatorial approach. These SH2 domains were screened against a linear pY peptide library and sequenced by PED/ MALDI-TOF. In general, the consensus for the kinase family SH2 domains was more similar than expected especially within SH2 families.

They all shared a general preference for hydrophilic or small hydrophobic residues and hydrophobic amino acid residues at the +1 and +3 positions, respectively. There are several reasons for this finding. First, isolated pure proteins were used during screening.

In the real cellular system, competition exists. Cells need to maintain a low level of cross recognition so they can function as they need to. There are other factors that may contribute to its specificity. It is known that the SH3 domain, recognizing proline-rich motifs, is also involved with protein-protein interactions and likely also contribute to sequence specificity in tandem with the SH2 domain95.

From the SPR studies, the SFK SH2 domains bound to pYEEI with the highest affinity just as been shown previously16. Unexpectedly, the pYEEI motif was not selected frequently or at all in the SFK SH2 screenings. However, there was some selection for pYEEV or pYEEM motifs by some of the SFK SH2 domains. Most of the

SFK SH2 proteins have pIs less than 7 resulting in an overall negative charge.

Consequently, a negatively charged peptide may not be easily selected during screening due to charge repulsion.

Even though the sequence specificity among the kinase SH2 was not significantly different, individual sequences could be obtained for each of the kinase SH2 domains.

Other methods used to determine specificity could not detect covariances and low abundant peptides. From examining the individual sequences, a few low abundant peptide classes and covariances were detected. For example, in the Fes family, a second class of sequences containing pYEN(D/E) was identified. Subtle covariances were exhibited in Hck and Yes SH2 domains, even though they both belonged to the same family. When the +1 position is Ala the +3 residue is Val for Hck SH2, while in Yes

SH2, the +3 residue is Abu and Nle.

By being able to identify individual sequences, subtle differences at each position could be observed. For example, within the Brk family, Brk SH2 selected for small hydrophobic residues, while Frk SH2 selected for large branched residues at the +3 position. The Fes SH2 family exhibited a strong preference for Glu at the +1 position compared to the SFK SH2 domains, which had a broader specificity. One would expect the Bmx family to exhibit similarities in their sequence specificity. However, differences were most prominent within the Bmx family. For example, most of the SH2 domains within the Tec family do not have selectivity at the +2 position. By contrast, Itk and Bmx

SH2 exhibited a strong selection for N at the +2 position similar to Grb2 SH214.

However narrow the sequence specificity of a kinase SH2 domain would be, likely depends on the kinase’s particular function or the location in the signaling pathway. For

84 example, some of the kinase SH2 domains such as Abl, and Src family kinases, MATK, and Csk kinase SH2 domains have a broader consensus with a tendency to interact with many targets. Src family kinase SH2 domains are mainly involved upstream of the signaling pathway regulating cell receptors96, while some of the other kinase SH2 may be further downstream with more defined roles. It is possible that SH2 domains exhibiting a broader specificity may have lesser defined roles in the signaling pathway. This may be further investigated.

A literature search was performed to identify known protein targets and binding motifs for some of the kinase SH2 domains. At this point, 20 known SFK SH2 substrates have been found in the literature. Their pY motifs closely matched the corresponding consensus. Some of the kinase SH2 specificity data from minor classes was also entered into a protein database based on phosphorylation and kinase specificity (websites: http://www.cbs.dtu.dk/databases/PhosphoBase/; http://phospho.elm.eu.org/about.html) to search for potential binding partners. After determining their potential binding partners, the role of each kinase SH2 domain in the signaling pathway may be predicted. Some of these potential substrates may be tested and validated by immunoprecipitation or by peptide pull-down assays. By examining the specificity of the entire kinase family SH2 domains, the subtle differences in specificity may be important in providing a molecular basis for different roles of the tyrosine kinases in cell signaling. Also, this may facilitate the design of inhibitors targeting these SH2 domains. Since the specificity of the kinase

SH2 domains is generally more similar than expected, we want to see if differences may be more obvious between different groups of SH2 domains such as between kinases, G

85 receptor, and adaptors. The specificity of seventeen non-kinase SH2 domains from different families will be determined in Chapter 3.

2.5. Acknowledgments

Dr. A.-S. Wavreille (former Pei lab member) performed the expression, purification, and screening of Src SH2 and Syk CSH2. Shane Glasgow (former lab member) contributed to some of the data for the Bmx SH2 screenings. Andrew Kunys

(current Pei lab member) performed the expression, purification, and screening of Itk,

Btk, Frk, Brk, and SRMS SH2 domains. Dr. Y. Zhang (former Pei lab member) performed the expression, purification, and screening of Abl1, MATK, tandem Syk N, and ZAP N and C SH2 domains. Xianwen Chen (current Pei lab member) performed the expression, purification, and screening of Csk SH2. HYpYEEIBK and LTpYENDLNK were provided by Dr. A.-S. Wavreille and X. Chen respectively.

CHAPTER 3 SPECIFICITY OF NON-KINASE SH2 DOMAINS

3.1. Introduction

Vav1 and Vav2 are guanine nucleotide exchange factors (GEF) for Rho GTPases.

Vav2 is found in most tissues whereas Vav1 is found mainly in hematopoietic cells.

These both contain a dbl homology (DH) region, a pleckstrin homology (PH) domain,

SH2 domain, and SH3 domain, a proline-rich motif that enables binding to SH3- containing proteins, an acidic-rich (Ac) region, and a ‘calponin-homology’(CH) region97.

Vav1 transduces TCR signals to multiple biochemical pathways and to several cytoskeleton-dependent processes. Vav1 may also function as an adaptor98. Vav2 is capable of associating with autophosphorylated growth factor receptors through its SH2 domain99. Since inhibition of Vav1 appears to be a promising target for cancer therapy100, determining the specificity is essential.

Rasa1 (p120-RasGAP) is a down-regulator of downstream signaling with the receptors of PDGF, EGF, ephrin, insulin, and CSF-1. Rasa1 contains an SH3 domain, two SH2 domains, a pleckstrin homology (PH) domain, and a calcium-dependent phospholipid-binding domain (CaLB/C2). Both SH2 domains are capable of binding to p190-RhoGAP, a GAP protein for the Rho family of small GTPases. Previously, Rasa1 has been identified as the essential negative regulator of the Ras-signaling pathway. It acts as a transducer in other signal transduction pathways and plays a role in

87 proliferation, migration, and anti- and pro-apoptosis that is independent of its GAP activity101.

Tensin (Tns) 2 and Tns4 are involved in cytoskeletal regulation. They bind to several structural and signaling molecules such as vinculin, paxillin, Src, and actin102.

Tns2 is localized in the cytoplasmic tail of integrin focal adhesion molecule102. Tns4 may have oncogenic and tumor suppressor functions and has been shown to be involved in colorectal cancer correlating to changed cell morphology and increased cell motility102.

Tns4 has also been found to be down-regulated in prostate cancer compared to normal.

Its SH2 domain may possibly modulate signal transduction cascades, which then activates a pathway leading to E-cadherin destruction102. Determining the binding motifs for Tns2 and Tns4 SH2 domains may help in developing inhibitors to suppress tumor cells.

HSH2D, also known as adaptor in lymphocyte of unknown function X (ALX), contains a single SH2 domain near the N-terminus, several PXXP polyproline sequences, and a potential site of tyrosine phosphorylation. It is an adaptor protein with considerable homology to another protein TsAD103. HSH2D is shown to inhibit TCR and CD28 costimulatory receptor induction of interleukin (IL-2) synthesis in the human Jurkat T leukemic cell line, suggesting it functions as a negative regulator of T cell signaling. It may also be a more important regulator of T cell function than B cell function103; 104. The

HSH2D SH2 domain is known to associates with Lck and recruits it to a complex with linker for activation of X cell (LAX) resulting in its phosphorylation. LAX can now associate with other signaling molecules such as Gads, Grb2, and PLCγ1105.

Growth factor receptor (Grb)7, Grb10, Grb14 are adaptor proteins that interact with receptor tyrosine kinases and receptor molecules. Grb7 and Grb14 share 67% amino acid identity, but their SH2 domains display differences in binding preference for receptor tyrosine kinases in whole cell extracts106. Grb10 regulates cell proliferation and apoptosis whereas Grb7 is associated with tumor-related molecules and has a direct role in cancer cell migration. This makes it a potentially therapeutic target107. Grb7 interacts strongly with erbB2 via its SH2 domain although the Grb7 binding site on erbB2 has not been determined. However, Grb14 SH2 binds to erbB2 very weakly106. On the other hand, Grb10 directly interacts with the insulin receptor (IR) and insulin-like growth factor I (IGF-IF) 108. The binding pocket for the pY+3 residue in the Grb10 SH2 is absent so it will favor binding of dimeric turn containing phosphotyrosine sequences such as the activation loop of the two beta subunits of IF and IGF-IF 108. Grb14 overexpression may lead to insulin-resistance and subsequently to type 2 diabetes109. As a result, Grb14 may be a target for anti-diabetic drugs which could disrupt its interaction with the IR.

Phosphatidylinositol kinases (PI3K) are involved in cell growth, proliferation, differentiation, motility, survival, and intracellular tracking, which are involved in cancer.

They are heterodimeric enzymes capable of phosphorylating the D-3 position of the inositol ring110. Each one is composed of the catalytic domain (p110) and a regulatory subunit p85111.

PIK3R1, PIK3R2, PIK3R3 are subunits of the PI3 regulatory subunis 1, 2, or 3, respectively. Each regulatory subunit contains an SH3 domain, a RhoGAP domain, and

89 two SH2 domains. PI3-kinases are also implicated in tumorigenesis112. Oncogenic mutations of PI3-kinase disable the ability of p85 to inhibit p110, therefore leading to cancer. Mutations in p85 have led to ovarian cancer, colon cancer, and glioblastoma111.

SH2 proteins of beta cells (SHB), SHD, and SHF are adaptor proteins that link growth factor receptors with cytosolic proteins containing enzymatic activity or transcriptional-activation domains. SHF shares 43% sequence identity with SHB and

40% sequence identity with SHD. They generate signaling complexes in response to tyrosine kinase activation. The SHB SH2 domain mediates responses in PDGF receptors, fibroblast growth factor receptor-1 (FGFR-1), TCR, and IL-2 receptors113; 114. It also binds to two ITAM motifs on the T-cell receptor ζ-chain upon CD3 stimulation of Jurkat cells or primary human T-cells114. SHB promotes apoptosis under morphogenesis while

SHF is involved in PDGF-alpha regulation of apoptosis. SHD is a physiological substrate of Abl1 and may function as an adaptor protein in the central nervous system115.

3.2. Experimental Procedures

3.2.1. Materials

Glutathione resin was purchased from Clontech Laboratories (Palo Alto, CA).

Fmoc protected L-amino acids were purchased from Advanced Chemtech (Louisville,

KY), Peptides International (Louisville, KY), or NovaBiochem (La Jolla, CA). HBTU, and HOBt, were from Peptides International. All solvents and other chemical reagents were purchased from Sigma (St. Louis, MO), Fisher Scientific (Pittsburgh, PA), or VWR

(West Chester, PA). Phenyl isothiocyanate (PITC) was purchased in 1-mL sealed ampoules from Sigma-Aldrich and a freshly opened ampoule was used in each

90 experiment. Streptavidin-alkaline phosphatase (SA-AP) conjugate (~1 mg/mL) was purchased from Prozyme (San Leandro,CA). TentaGel S NH2 resin (90 μm, 0.26 mmol/g, and ~100 pmol/bead) was purchased from Peptides International. 5-Bromo-4- chloro-3-indolyl phosphate (BCIP) disodium salt was from Sigma (St. Louis, MO).

Protein concentration was determined by the Bradford method using BSA as a standard.

3.2.2. Synthesis of the pY library

Reduced density Libraries II and III were synthesized in a similar manner as described in Chapter 2.2.2.

3.2.3. SH2 constructs

The SH2 domains were provided as GST fusion proteins by Dr. S. S.-C. Li

(University of Western Ontario). The identities of the DNA constructs were confirmed by dideoxy sequencing. The vectors were transformed into DH5α or Rosetta BL21(DE3) cells for expression and purification.

3.2.4. Protein expression and purification

The proteins were expressed and purified as described in Chapter 2.2.4. They were purified on a GST column, washed, and eluted according to the manufacturer’s recommended procedure. The proteins were concentrated down with an Amicon concentrator. The GST SH2 proteins were passed through a G-25 column or ion exchange column to remove excess glutathione. The elution buffer contained HEPES,

150 mM NaCl pH 7.5. The proteins were stored as 30% glycerol stocks or without glycerol, flash frozen in dry ice/isopropanol, and stored at -80 °C.

3.2.5. SH2 protein labeling

GST SH2 proteins were chemically labeled with NHS-biotin, NHS-chromogenic biotin, or NHS-fluorescein or Texas red. SH2 proteins (~2 mg/mL) were incubated with

1 to 2.5 equivalents of N-hydroxysuccinimidyl-biotin (NHS-biotin) or N- hydroxysuccinimidyl-chromogenic biotin (NHS-chromogenic biotin) in 1M NaHCO3 at room temperature for about 40 min. The reaction mixture was incubated with 100 µL

1M Tris pH 8.0 for 10 min to quench any unreacted NHS-biotin. The labeled protein was passed through a G-25 column to remove any excess biotin. Proteins were concentrated down and stored as 30% glycerol stocks at -80 °C.

3.2.6 pY library screening and sequence determination

The pY library was screened similarly as described in Chapter 2.2.6. Unless stated otherwise, Library II was used to screen all of the SH2 domains. Library III was used to screen SHF and PIK3R3 CSH2. The positive hit beads were sequenced as described in Chapter 2.2.7.

3.3. Results

3.3.1. Vav1 and Vav2 SH2 domains

About 100 mg of pY library was each screened against Vav1 and Vav2 SH2 domains. From the selected sequences (Table 31) it can be concluded that Vav1 SH2 selected for three peptide classes with the major peptide class selecting a hydrophobic residue at the +1 position and Pro at the +3 position. It recognized the class I consensus

XXpY(L/m)X(P/a/u), class II consensus XXpYEXP, and class III consensus

XXpY(L/M/u)N(D/e/q) (Figure 14). For Vav1 SH2, 84 class I, 17 class II, and 14 class

III sequences were obtained. Vav2 SH2 also selected for three classes of peptides (Table

32). For Vav2 SH2, 75 class I, 22 class II, and 27 class III sequences were selected. The major class preferred a hydrophobic residue at the +1 position and Pro at the +3 position.

It preferred the class I consensus XXpY(MLUe)X(P/a/u/s/d), class II consensus

XXpYEXP, and class III consensus XXpY(M/u/v/l)N(D/E) (Figure 15). The lower case letters represented less frequently selected residues and X is any amino acid. In the class

II peptide group, Glu was selected at the +1 position and Pro was selected at the +3 position. In Vav1 and Vav2 class III peptide group, Asn was selected at the +2 position similar to Grb2 and Grb7 SH2 domains. However, its +3 position selected for hydrophilic residues. There was little selectivity at the -2 and -1 positions in all peptide classes.

Table 31. Peptide sequences selected from Vav1 SH2 screening

Class I HGpYLEA HUpYLSP HUpYMVS Class III NNpYLIA MMpYLTP HGpYLNU MYpYMEQ NNpYLIA HHpYLTP HTpYLQU UGpYIND DRpYLQA PDpYLTP NQ/EpYLVU DHpYLND GRpYLVA PRpYLUP HEpYMEU GFpYLND SGpYMMA HLpYLUP VGpYUVU QYpYLNE FGpYUIA ASpYLVP EGpYVLU VYpYLNQ/E GYpYYLG NApYLVP IDpYFLV HMpYUND NPpYINI QDpYLVP NYpYMNV AGpYUNQ QGpYLEI GFpYLVP AGpYVIV HLpYVND GGpYMNI HEpYMAP YGpYLNY LGpYVNQ HFpYYGI NEpYMHP IGpYQDY GUpYMSE EDpYFLM YLpYMQP HNpYMTN FNpYLLM DQpYMQP Class II UNpYLVD EGpYITP MHpYMQP EGpYEIU YSpYUVD NNpYLAP NRpYMTP DWpYEUP DWpYEVD MFpYLEP NHpYMTP YTpYEEP DHpYLEP DVpYMTP QUpYEIP NPpYLIP I/NGpYMUP YPpYEIP NMpYLIP QLpYMUP UMpYEIP DHpYLIP QSpYMVP HHpYERP RHpYLIP DNpYMVP HFpYERP RRpYLIP NApYUEP FHpYETP TPpYLIP HIpYUEP NMpYETP EFpYLIP VGpYUHP TPpYETP PQpYLIP SGpYUHP FFpYEVP GHpYLLP NEpYUIP DYpYEVP QMpYLLP HHpYUIP HMpYEVP EQpYLQP ELpYUIP HGpYEAP RRpYLRP GFpYUTP LQpYEEP NGpYLRP DPpYUTP NFpYLRP LDpYUUP SQpYLRP SYpYUVP YDpYLRP SHpYUVP AApYLRP HEpYVTP NApYLSP YTpYYUP

Table 32. Peptide sequences selected from Vav2 SH2 screening

Class I RGpYITA EUpYMUP QGpYMSV UDpYVMN DGpYLKA TKpYMVP MRpYUVV MGpYINN QKpYLMA TGpYQVP NApYLND HLpYLUA WKpYTRP Class II NApYLND NFpYLUA GSpYTUP NEpYELA DPpYLNE HEpYLVA DSpYUAP PNpYETA GTpYMND HRpYLVA HHpYUDP NWpYEVA QDpYMNN HTpYMRA TLpYUDP FHpYEQU HUpYQNG QRpYMTA DLpYUTP HFpYEVU HRpYUND GLpYUKA PLpYUTP UMpYEUP HSpYUNQ HQpYULA GWpYUUP HHpYEDP YHpYVND NGpYULA HRpYUUP QHpYEGP NFpYEPH UHpYUQA RHpYVEP DDpYENP NLpYMRE HQpYUUA NUpYVRP HMpYENP FApYLTD HVpYUUA HGpYVTP FGpYEMP TGpYUTD YTpYVRA ASpYVTP TApYERP DGpYVTD HLpYVUA UGpYLQS MRpYEVS PQpYMVQ HEpYINF TGpYLTS THpYEUP YMpYUVD HGpYMNF ELpYMQS ARpYEEP SQpYVUD HEpYMTF SLpYMTS NUpYEEP HApYELE SGpYVQI GHpYMUS VWpYEEP DHpYIAP VVpYUFS PTpYEEP QLpYIIP PEpYUQS YQpYEIP HLpYISP HSpYUVS LRpYEVP MQpYLDP NHpYUVS TKpYEVW HTpYLEP DRpYLKU ATpYLNP TApYLQU Class III YSpYLQP HDpYLQU SRpYMEQ DFpYLSP DRpYLVU FGpYMGR DEpYLTP YQpYLVU NWpYQGW DMpYLWP ALpYMQU MGpYVGD PNpYMEP YGpYMTU NNpYMID YDpYMHP HUpYULU UQpYFLH GRpYMHP NIpYUTU NMpYMLD HGpYMIP GSpYUVU HRpYMLG HRpYMUP GDpYMLV HVpYLME

Figure 14. Sequences specificity of Vav1 SH2 domain

Figure 15. Sequences specificity of Vav2 SH2 domain

3.3.2. Rasa1 NSH2 and Rasa1 CSH2 domains

Rasa1 NSH2 was screened against 100 mg of pY library to obtain 37 partial and full sequences. 70 mg of library was screened against Rasa1 CSH2 to obtain 89 partial and full sequences. For Rasa1 N and C SH2, Pro was selected frequently at the +3 position, while small residues were mostly selected at the +1 position. The NSH2 selectivity was broader than that of CSH2. The selected sequences (Table 33) revealed that Rasa1 NSH2 selected for the consensus of XXpY(A/T/H/D/U)X(P/m/u) (Figure 16) while Rasa1 CSH2 selected for the consensus of XXpY(A/s/u/t)X(P/v) (Figure 16). The sequences for the CSH2 are summarized in Table 34.

Table 33. Peptide sequences selected from Rasa1 NSH2 domain

XSpYAKM NVpYADP HFpYEAD DEpYAGP HFpYHFD EWpYAVP XXpYLEE DUpYAIU XXpYLLQ HSpYAEV XXpYMMK IQpYAUV VApYNAG WApYDVI UHpYNEH XDpYDFM AApYSNA HHpYDEP YHpYTLH DApYDSP NEpYUWD TEpYDMV XXpYYFE (R+D,W+C)pYEHU WSpYGHP FApYRUM XXpYHGF TIpYRFY UNpYHDL TVpYKYE LEpYHAM HVpYHEM NLpYHEP YLpYHEU DNpYIEI IYpYIEP HDpYMQP YFpYNKL UYpYQEP HYpYSQP TVpYTEI TTpYTRM WDpYTEP IDpYTEP HIpYTTP AHpYTVP EApYUEM HHpYURM MHpYUUU XXpYUYU DDpYVVM

Table 34. Peptide sequences selected from Rasa1 CSH2 domain

HVpYETA N+GpYDUP AYpYRVU AEpYAFF ESpYEEP VLpYAEV HYpYEYF GGpYEVP IPpYAIV XRpYUMF NDpYFHP YDpYAIV XXpYHFI WTGQP LGpYAKV XXpYMSI VGpYHEP XXpYANV TTpYEQL IYpYHEP SPpYATV HApYLUL VKpYHGP EUpYAVV KHpYRSL EPpYHIP NNpYDFV VMpYSML IIpYHIP WDpYDLV XXpYVKL VApYHSP EEpYELV N+GpYAAP IUpYHUP KIpYGEV DYpYAEP VSpYHVP EGpYHAV AYpYAHP IGpYMUP XFpYRLV VEpYAHP TIpYSAP YYpYTQV IUpYAIP DYpYSPP NEpYTUV WVpYANP MUpYSQP HVpYUMV IFpYANP GDpYSSP GMpYSVY YIpYAQP IYpYSTP YApYTTY UGpYAQP WQpYSUP DDpYARP AYpYSUP SUpYARP TGpYSYP TPpYALH IIpYASP GPpYUIP AApYAMR HGpYASP KQpYUQP EGpYAHT GIpYATP YGpYUTP VApYAVT IIpYATP EEpYUTP AIpYIVH WNpYATP IPpYAIU XXpYIAK IVpYAUP QLpYAIU AApYMHH AUpYAVP VIpYALU XXpYMSN XXpYAVP NHpYAUU SLpYQYR TTpYU/RIP SYpYDLU WEpYDHP VGpYHEU IHpYRMH EFpYDHP YDpYHFU XHpYRTN ASpYDQP XLpYKAU UNpYDUP HGpYNQU

100

Figure 16. Sequence specificities of Rasa1 N and CSH2 domains

3.3.3. Tns2, Tns4, and HSH2D SH2 domains

105 mg of library was each screened against Tns2 and Tns4 SH2 domains.

Seventy-two sequences were obtained for Tns2 SH2. After examining the selected sequences (Table 35), Tns2 SH2 preferred the consensus XXpY(Dea)(Nu)(ILVmu)

(Figure 17). 76 class I sequences and 21 class II sequences were obtained for Tns4 SH2.

In contrast, from examining the sequences (Table 36), Tns4 SH2 selected for the class I consensus XXpY(AEdh)N(IVU) and class II consensus (FGWYI)XpY(AEHts)X(QKed)

(Figure 17).

101

100 mg of pY library was screened against 500 nM of HSH2D SH2 to obtain 144 partial and full sequences. According to the selected sequences (Table 37), there was high selectivity of Asn at the +2 position. Small residues were mostly selected at the +1 position. There was little selectivity at the +3 position. HSH2D SH2 preferred the consensus XXpY(AGHu)NX (Figure 18) similar to Bmx SH2 mentioned previously in

Chapter 2.

102

Table 35. Peptide sequences selected from Tns2 SH2 domain

HDpYAQF VVpYENI MDpYARI FTpYESI YVpYAAD WVpYARI QIpYEWI AYpYAYN ERpYANL NHpYEYI IIpYAAR MEpYARM SUpYEFL DQpYDUN TEpYARU HEpYENL FNpYEIE QSpYANV QLpYEYL MEpYEVH QEpYANV WQpYEUM WVpYHAQ YMpYAUV YGpYEYM LVpYHDR YNpYAYV MEpYEUP YYpYNVE WPpYATY IUpYEYU WNpYNUQ FIpYA(VE)(TP) MHpYEYV YSpYYGK SQpYDLI FIpYEMY SEpYSSE HWpYDNI TApYENY VLpYDNI YEpYEUY YMpYDPI WEpYGUV FDpYDIL IFpYHDI XXpYDLL HGpYHYM XXpYDNL LEpYHUU HEpYDNL WApYHUV UMpYDNL WEpYHGY YDpYDNL YNpYKUM FUpYDSL LEpYLAL WFpYDHM FVpYLRL WKpYDNM TGpYNNL LSpYDFU VEpYQAM TVpYDNU TYpYQEV NTpYDNU IEpYTUL NTpYDNU WDpYUAI TFpYDLV DEpYUFI VDpYDNV DEpYUHI DLpYDNV VIpYUTM HHpYDNV YDpYUUV UGpYDNV TKpYWEI AYpYEUF PVpYEMI 103

Table 36. Peptide sequences selected from Tns4 SH2 domain

Class I IApYAHA HHpYELI VGpYUFV DNpYAHF UNpYENI ANpYUNV KTpYAQF ILpYENI TUpYUNV XXpYARF YNpYEQI YEpYVVP LKpYAFI QRpYELL FDpYALI IHpYELL Class II UMpYANI YRpYEIM GTpYAPD IIpYAQI GNpYENM MApYAYE IEpYASI WTpYEVM FMpYAAN QGpYAML YPpYENU FYpYAHQ IKpYANL XMpYEKV XXpYANT WPpYAYP IQpYENV XXpYDUK WIpYAHU SHpYEQV NHpYEND FIpYAKU MGpYEUV GVpYEHQ YTpYANU FMpYHDI IDpYEUQ IHpYANU YIpYHRL LVpYHLG RTpYANU ALpYHEM FVpYHDK MRpYARU XFpYHNM GVpYHGS INpYAAV FApYHGU WApYIUQ YEpYANV HPpYHQU IpYMFQ GApYANV TYpYHTU WUpYQIR YHpYANY FTpYHGV FSpYSPG YHpYANY MPpYHNV INpYSMK UHpYDNI HTpYHNV YDpYTME VVpYDNI HApYHVY VDpYTTH TFpYDNI XPpYLUI XUpYUKH DGpYDKL AGpYLIU YUpYVIT KHpYDNL HYpYMYU INpYDNM FIpYRRP RGpYDNM IIpYSLF HMpYKGF TDpYDNV GApYTNL IEpYKIM SLpYDNV QUpYUNA SQpYRGU AQpYEMF XIpYUNM FApYRAY VYpYEFI PFpYUNU FUpYRTY YUpYEII LDpYUQU XXpYKEG VHpYELI YRpYUUU XXpYKAG

104

Table 37. Peptide sequences selected from HSH2D SH2 domain

LPpYANU VUpYHNM LGpYANM DDpYHNH RNpYANE NDpYING LKpYANM LGpYHNI LGpYANI SYpYKNY UDpYAUN MGpYHNI TTpYANI IApYMNU GLpYANN GKpYHNI Y+RpYANK IDpYMNQ EPpYANN TUpYHNL VDpYANL ARpYMNS TUpYANN NNpYHNP KVpYANM NApYMNY HNpYANV TDpYHNQ HApYANN TIpYNND WTpYANY QApYHNS QTpYANP XXpYNVK ESpYANY VUpYINU GIpYANQ ETpYQNU IApYUNE IApYINU IHpYANQ GFpYQNN AEpYUNE YTpYKND TTpYUNU YApYSND TMpYUNG IRpYKNM TGpYUND LQpYSNI QQpYUNK VUpYLNK DPpYUND ARpYSNS LHpYUNQ EHpYLNS FUpYUNE NSpYTNF PNpYUNS ARpYMNA GQpYUNE IGpYMNP LApYUNY VDpYMNU TEpYUNL HApYUNP HPpYDNI NNpYMNL USpYUNM IIpYANU YGpYDNK HHpYMNN PDpYUNS NFpYLNS LEpYDNK IDpYMNP VVpYUNT YFpYUNT NGpYDNP LQpYMNQ NUpYDNU EHpYHNI HQpYENH MApYQNU HYpYDTQ DTpYANA TMpYGNF TSpYSNU VYpYENM TApYANU ISpYGNI VApYSNG LYpYFNE UNpYANE UMpYGNI AHpYTNQ NGpYGNU UIpYANF IDpYGNL NTpYTNQ ISpYGGE UIpYANF LKpYGNL MUpYTNY EUpYGNF SQpYANG LPpYGNL LVpYAND QKpYGNK MSpYANH QUpYGNL HpYAUP NVpYGNM DHpYANH TIpYGNM IApYANK HFpYGNV MHpYANH LFpYGHN RLpYUNM LApYHNU QVpYANI PRpYGNN XXpYENM YYpYHSD IDpYANK UVpYGNQ FRpYFTQ VMpYHNE GEpYANL QSpYGNY FEpYANT WDpYHNE IRpYANL QGpYHNA XXpYINF TLpYHNG UGpYANM VPpYHHU AHpYKNN HNpYHNL IEpYANM MSpYHNU LGpYGNT

105

Figure 17. Sequence specificities of Tn2 and Tns4 SH2 domains

106

Figure 18. Sequence specificity of HSH2D SH2 domain

3.3.4. Grb7, Grb10, and Grb14 SH2 domains

100 mg of library was screened against Grb7 SH2 to obtain 84 full and partial sequences, whereas 145 mg of library was screened against Grb 14 SH2 to obtain 86 full and partial sequences. 130 mg of library was screened against Grb10 SH2. From the selected sequences (Table 38), Grb7 SH2 preferred the general consensus

XXpY(E/h/u)NX (Figure 19). Grb10 SH2 selected for two classes of peptide sequences

(Table 39). It selected 53 class I and 16 class II sequences. It recognized the class I consensus XXpY(E/D)X(I/U/L) and class II consensus

X(Y/E/A/w/f/m)pY(G/E/D/Y)(N/s)(T/S/G) (Figure 19). From the sequences (Table 40),

Grb14 SH2 selected a general consensus of XXpY(E/q/a/d)X(V/U/Y) (Figure 19). After examining the sequences more carefully there also appear to be a two minor classes of sequences containing the consensus XXpYEφ(I/V/u) and XXpY(D/E)X(N/e/d/q). Grb10 selected mostly for hydrophobic residues at the +3 position and had little selectivity at the

+2 position unlike Grb2 SH2. Grb7 had high selectivity of Asn at the +2 position similar

107 to Grb2 and HSH2 SH2 domains. There was little selectivity at the -1 and -2 positions in the Grb7 SH2 family.

108

Table 38. Peptide sequences selected from Grb7 SH2 screening

UMpYANA HDpYENU GApYAYQ HHpYEVU MApYANE MUpYGNY UUpYAYQ UHpYEAU UPpYANF GMpYHND IQpYEQD YGpYEYU HDpYANF HLpYHNE YFpYEID LGpYQYU HFpYANG EHpYHNG YUpYEID DKpYEQV IMpYANM VIpYHNL YQpYELD HYpYEYV LFpYANQ STpYHNL HYpYEUE HKpYAAY HPpYANU MApYHNM YApYEEE GDpYEHY MNpYANV GYpYHNQ EPpYEQQ QKpYEYY MHpYANV HQpYHNY QTpYGHE DEpYGPY ITpYANY LIpYMNE YIpYHDE LDpYGWY IPpYDNP HGpYQNE YYpYHEE IFpYQSY LGpYDNY HIpYUNA IYpYHSF VQpYSQY VKpYENF UKpYUNE ESpYHTS IIpYENF LDpYUNG YUpYIKE IIpYENH TQpYUNI XXpYMVD LTpYENI IHpYUNK YQpYSVE FYpYENK YLpYUNK YUpYUEE IKpYENK GMpYUNU QWpYUAQ HPpYENK ENpYUNV NVpYUKR LYpYENK EPpYENL Class II Class III GHpYENM LHpYAPG ESpYEQI HQpYENQ TGpYAVH XXpYHSI ISpYENT VHpYAQN INpYIYP

109

Table 39. Peptide sequences selected from Grb10 SH2 screening

Class I YYpYEEA YUpYDKV QVpYUUU LGpYEMV VFpYDGU VYpYESV LFpYDNU MPpYHVV IIpYDYU HWpYETW MFpYEIU MGpYAIY VYpYENU IUpYUEY YKpYNLU EHpYENY IDpYQTU EYpYERY KIpYEKF HPpYEWY DpYETF WKpYQDY VEpYGKF YFpYTGY HSpYASI XXpYFLI NIpYUNI IApYIQI HIpYDVI MPpYVQM EEpYEDI IHpYMNV YYpYEEI ILpYEII Class II HIpYEPI EQpYYDU DYpYEVI IYpYEIT IKpYEVI HGpYGIT HHpYEVI LWpYLNG LTpYEYI XXpYINQ IGpYMMI XXpYGNS LYpYQTI PEpYVNS EDpYTPI TApYENT LMpYAGL AMpYTNT EUpYDGL YApYDQT IVpYDGL EYpYARG MApYELL MTpYGSG LApYEVL VFpYDSN MTpYHAL AEpYGSS YApYSML DYpYETE GFpYAEM XXpYYVH GNpYATM LMpYTEM UEpYQKP 110

Table 40. Peptide sequences selected from Grb14 SH2 screening

QFpYANH YTpYHIU NIpYYQG UFpYEVP IDpYASI VKpYHAV HGpYYDI QApYEUU NDpYAML LHpYMIL NNpYYII YHpYEIV HGpYADM VSpYMUM MYpYYGL HRpYEIV QYpYALU INpYMAU EHpYYIY MNpYEUV LLpYAYU DRpYMTY GEpYANV QRpYQQI Class II Class III IPpYAVV AQpYQQI VApYEFF YYpYDRN QIpYAFY MApYQVI TGpYEII EWpYDYN HKpYDTF TRpYQLL IVpYEII EGpYEAD ESpYDUL YIpYQKU YNpYEII QRpYEME UUpYDSP GGpYQPU EHpYEII HLpYEMN IEpYDDV VIpYQRU GDpYEII IIpYEAQ MLpYDEV UDpYQAY HPpYELI NYpYDNV VEpYQAY PEpYEMI RHpYENL LLpYQKY QGpYEMI VVpYERU DApYSNI HEpYEMI HHpYERU MHpYSIP MGpYETI HRpYENY QEpYSNV HApYEVI GQpYFDI IKpYTVU YRpYEVI DYpYFSL QWpYTNV TGpYEWI NDpYFKY YKpYURF AQpYEYI MFpYGLT LWpYUSH MQpYEMM UDpYHGA EHpYUQM NEpYEUP NLpYHNA NNpYVII UFpYEVP

111

Grb10 SH2 -2 -1 1 100 80 2 60 3 % 40 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

Grb10 SH2 Class II

100 80 60 % 40 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

Figure 19. Sequence specificities of Grb7, Grb10, and Grb14 SH2 domains

112

3.3.5. PIK3R1 NSH2 and PIK3R2 CSH2 domains

Approximately 100 mg of library was screened against PIK3R1 NSH2. PIK3R1

NSH2 selected for two classes of peptides. Class I contained 123 sequences, whereas, class II contained 11 sequences. In the major class, Nle was predominantly selected at the +3 position while the minor class selected Q. However, there was little selectivity at the -1 and -2 positions. The +2 position was also not selective in each case. From the selected sequences (Table 41), PIK3R1 NSH2 selected the class I consensus

XXpY(U/e/i/v/t/m)XM and class II consensus XXpY(I/U)XQ (Figure 20).

Approximately 100 mg of library was screened against PIK3R2 CSH2. PIK3R2

CSH2 selected two classes of peptides. Class I contained 114 sequences, whereas, class II contained 7 sequences. For the main class of peptides, M was predominantly selected at the +3 position. It selected for small hydrophobic residues at the +1position similar to

PIK3R1 NSH2. From the selected sequences (Table 42), the class I consensus was

XXpY(A/U/m/e)XM, and class II consensus was XXpY(M/u)N(D/K/Q) (Figure 20). Its class II consensus was similar to Vav1 and Vav2 class III peptides.

113

Table 41. Peptide sequences selected from PIK3R1 NSH2 screening

Class I RQpYUGA MEpYEUM UGpYUIM IIpYTPU XXpYEHF MGpYEVM NQpYUKM NSpYUIU VVpYMHF DTpYEYM EUpYUKM W+TpYVKU PPpYUVF XXpYHHM DGpYUKM EUpYVLU XXpYVKF ENpYIAM XXpYULM EYpYVNU DUpYIAI SEpYILM XIpYULM NQpYVPU XXpYIAI XXpYILM XXpYULM GTpYEDV AEpYIHI XUpYIUM QDpYULM XXpYLAV GGpYUII GDpYIVM DYpYULM HFpYTHV EHpYVEI ENpYLVM XXpYUMM DDpYEFY XDpYVII UGpYMAM QHpYUMM VRpYUHY USpYVSI UGpYMEM XLpYUNM UIpYVAY XRpYAQL QGpYMHM QLpYUPM ULpYVMY XXpYAVL IGpYMIM SEpYUQM LYpYVQY GGpYEIL QSpYMMM YKpYUSM MSpYFAL GApYELL XXpYMMM XXpYUSM DEpYIIL XXpYMVM EGpYUTM Class II XXpYILL EYpYQEM QRpYUUM SIpYILE EYpYISL YGpYQFM SQpYUUM DUpYIGH IEpYTAL YEpYQIM UpYUUM XXpYUSK Y+TpYAAM SIpYSNM GEpYUUM DMpYIEQ GVpYAIM DGpYTIM VEpYUUM HQpYLLQ XLpYALM AApYTIM YGpYUUM AEpYMHQ NEpYAMM MKpYTPM FDpYUUM XXpYUNQ QEpYASM YVpYTPM SMpYUUM SGpYUPQ YTpYAUM HEpYTQM AHpYUVM NDpYWYQ VKpYEEM QApYTVM ISpYUVM XXpYEMS PTpYEFM AIpYTVM DMpYUVM VIpYMGT UPpYEIM MEpYTVM XXpYVUM XXpYEIM DYpYUAM LGpYVEP XXpYEKM IQpYUDM QApYEQU IFpYETM NUpYUEM DIpYIHU NDpYETM ITpYUEM NUpYIPU AEpYETM VNpYUIM VHpYMEU TApYEUM EApYUIM HApYMKU IIpYEUM EEpYUIM MGpYTPU

114

Table 42. Peptide sequences selected from PIK3R2 CSH2 screening

Class I Y+WpYENF QQpYAUM AVpYTPM DApYMHV QIpYMNF NPpYAVM A+FpYTQM QKpYMNV QNpYUIF ULpYAVM IGpYTRM KHpYMRV HQpYAVI PYpYAYM GYpYTUM SApYQFV DLpYDHI ARpYEHM LYpYTVM MTpYLTY YTpYEII RTpYEIM XXpYUFM IIpYSNY RApYFKI IFpYEKM QGpYUHM DDpYHMI IGpYELM HYpYUHM Class II KEpYILI NVpYEMM XXpYUMM REpYMND DWpYIUI DGpYERM MYpYUMM NUpYMAK ASpYIUI HMpYESM STpYUPM UEpYMLK AKpYLVI HSpYEUM HUpYURM PWpYUNQ FGpYULI MRpYHAM NIpYUSM AHpYMKD NGpYULI USpYHIM PpYUUM ETpYUNE F+P,D+EpYUTI NVpYHIM AMpYUUM AFpYMNG AVpYANL LRpYHKM PUpYUWM VRpYANL GHpYHKM STpYUYM QGpYEKL IHpYHLM NTpYAHU DNpYEQL MRpYHUM XKpYANU HApYMKL LIpYHUM NRpYEFU HKpYQDL AApYHVM HHpYEHU UUpYULL RRpYILM MGpYENU URpYAEM ATpYMDM ILpYLIU ESpYAEM UTpYMGM DRpYMEU XXpYAFM ATpYMGM RHpYMQU HKpYAIM IIpYMNM ITpYMQU GApYAIM XXpYMTM FGpYMRU ARpYAIM UIpYMUM EFpYTUU VEpYAKM MNpYMVM TGpYUIU ILpYANM QEpYMVM GRpYUNU DDpYANM NGpYQKM DUpYURU KGpYANM AFpYQLM LUpYUUU HDpYAQM RRpYQTM YQpYVGU ILpYARM YLpYQUM UGpYYUU HHpYARM YLpYQUM SSpYANV HHpYARM KQpYTLM QFpYHRV

115

Figure 20. Sequence specificities of PIK3R1 NSH2 and PIK3R2 CSH2 domains

116

3.3.6. PIK3R3NSH2 and PIK3R3CSH2 domains

Approximately 100 mg of pY library was screened against PIK3R3 N and C SH2 domains to obtain 133 full and partial sequences. PIK3R3NSH2 selected for the consensus XXpY(U/E/m/i)XM (Figure 21). Since PIK3R3 CSH2 exhibited possible selectivity at the +4 position according to OPAL results from the Li group (Huang), it was also screened against Library III. Out of a total of 119 partial and full class I sequences, 62 were obtained from Library III screening. A total of 38 partial and full class II sequences were obtained. 20 class II sequences were obtained from Library III screening. PIK3R3CSH2 selected a class I consensus

XXpY(V/E/T/I/m/q)X(M/I/v/u)(Q/n/t/d/f) and class II consensus

XXpY(A/M/E/I)X(H/D/E/Q)(I/V/Q) (Figure 21). The sequences are summarized in

Table 43 and Table 44. In general they both selected for hydrophobic residues and Glu at the +1 position and Nle at the +3 position.

117

Table 43. Peptide sequences selected from PIK3R3 NSH2 screening

Class I IPpYUSA IYpYUKL QFpYUSM EApYTHM DRpYAVT AIpYAVU QUpYERL GHpYUTM AYpYVAM NUpYAVN INpYUIU IUpYSRL INpYUVM UNpYVNM TTpYUPU DIpYTUL QVpYEUM EEpYVQM HYpYELU EPpYTFL ADpYEUM HHpYVQM SDpYIUU GIpYTLL QDpYEEM QDpYVVM DN/DpYIQU IRpYVML QUpYEHM VEpYVWM NGpYIVU QFpYAFM QQpYEIM YPpYYQM EUpYMEU HRpYAHM IUpYEIM DYpYIIP AMpYMKU NN pYAIM QQpYEKM GPpYIEP EEpYMPU UGpYANM LRpYEMM RDpYTPP IHpYMVU DEpYATM IYpYETM LRpYVTP YQpYTPU AEpYATM QIpYETM AMpYYYP XXpYTVU GQpYUUM QMpYEVM XXpYUPV EEpYVHU IGpYUUM IKpYEVM GGpYEQV HRpYAIF ISpYUUM MEpYIAM FTpYEYV EHpYAKF EMpYUUM IRpYILM ERpYIIV ITpYUTF QHpYUEM GNpYILM IHpYLIV QRpYGTF GTpYUFM HEpYINM EEpYMSV UEpYILF NDpYUFM XXpYITM UGpYSWV XXpYIPF XYpYUGM ETpYLHM GMpYVFV RPpYLHF DFpYUKM DUpYLKM YPpYAKY XXpYMHF QDpYUKM AHpYLTM UTpYAVY NGpYQVF LGpYUKM XXpYMAM HMpYUFY YHpYAII XXpYUKM HGpYMDM NDpYURY KIpYUAI MGpYULM UTpYMMM ESpYUUI VGpYULM P+Q/KpYMMM Class II GMpYUEI EUpYULM XXpYMMM YKpYFYD YEpYELI EVpYUMM NDpYMRM DDpYNRD HNpYIAI GDpYUNM AKpYMSM YPpYEIG HNpYIAI QRpYUPM NApYMTM GVPyLLG QRpYILI EMpYUPM XXpYMYM QLpYTEH DYpYMUI DGpYUQM NGpYSRM GIpYUSK EHpYWTI VQpYURM UVpYTAM W+EpYEQK DEpYAIL EApYURM ERpYTFM XXpYWIN FHpYUIL HKpYUSM EApYTHM EIpYMUQ

118

Table 44. Peptide sequences selected from PIK3R3 CSH2 screening

Class I DHpYEVM HTpYQEIQ QApYMDVA IUpYTNK HQpYUIA NRpYHAM MIpYQHID EGpYMNVD UUpYVLH YEpYLGA YHpYNAM YNpYQTIT GSpYQNVQ YIpYDKHV YEpYULU VApYNGM VHpYTAIQ RIpYQWVQ YSpYEMHH VYpYDNU NKpYQUM NIpYTEIM PYpYTDVI AFpYETHQ YEpYIFU VWpYRUM TQpYTNIT GGpYVNVF FDpYGFHH MEpYIMU QUpYTKM MDpYTQIR PHpYVNVS EFpYHFGQ ASpYLNU VRpYE(U/R)P GDpYVDIR GNpYYMVQ NPpYMNHN ENpYMIU IVpYYSP XXpYVMIN HIpYDFYQ FIpYSHRN VRpYNPU VNpYAEV VTpYVQIQ QApYTVGV RKpYPMU DMpYAGV HApYAPMQ Class II QGpYVTAH XXpYVEU RUpYAVV AEpYAVMQ NNpYADD SGpYVYTI VGpYVPU ERpYUPV DGpYAYMF ARpYAQQ VQpYUVI UMpYLQV NVpYDYMM USpYLVN LSpYDUI SRpYRMV QQpYENMT NEpYMGD FPpYERI GApYRRV HDpYFTMQ YHpYMAE RKpYHLI VIpYTAV NSpYHYMT DHpYMAG AYpYLRI DQpYENY INpYIDME IIpYUIE QPpYMYI XXpYIMY XXpYIIMN GGpYUNQ UGpYNTI MKpYMUY FHpYIQMH TIpYANEI DLpYRMI VTpYSFY YQpYMNMR WI/LpYETNI FGpYTUI XXpYFIAF XXpYMTMM DYpYEVTF NGpYTUI XGpYIGAN WEpYNIMH GYpYHTDV XXpYVDI XVpYIMAY YHpYQVMQ DGpYIIDT DDpYIGL QFpYENFN SIpYQWMD SVpYIMDI DTpYLML PVpYMHFQ IVpYQYMD GMpYINST DNpYYUL SQpYAFIS EQpYRIMQ SHpYMNQR YMpYAIM GLpYETIK YYpYTIMQ GSpYMTTH VTpYUUM YHpYETIQ HVpYTSMY MQpYQNEQ GEpYUIM XXpYGRIE AQpYVEMF DLpYARH UKpYUNM TLpYHDIN PEpYVHMF YEpYALR QFpYURM SVpYHSIN IGpYVIMQ DIpYAQ/GT GRpYUTM RSpYIIIS KQpYVTMT RTpYEUQ IHpYEHM MQpYISIG NIpYEPPR YGpYIVH DDpYEHM GYpYMDIW DIpYVVPA QDpYLUH EEpYEIM VEpYMMIN FDpYYNPW QTpYLDH HRpYENM TQpYMVIA TNpYANVI DNpYSKG

119

PIK3R3 NSH2 -2 -1 1 100 80 2 60 % 3 40 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

PIK3R3 CSH2 Class I -2 -1 1 100 2 80 60 3 % 40 4 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

PIK3R3 CSH2 Class II

100 80 60 % 40 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

Figure 21. Sequence specificities of PIK3R3 N and CSH2 domains

3.3.7. SHB, SHD, and SHF SH2 domains

100 mg of pY library was screened against SHB and SHD SH2 to obtain 104 and

56 full and partial sequences respectively. They mainly selected for Leu at the +3

120 position and His at the +1 position. SHB SH2 recognized the general consensus of

XXpY(H/U/a/t)(v/i/u)(L/M) while SHD SH2 selected the general consensus

XXpYH(I/V)(L/M) (Figure 22). SHF, on the other hand, selected for several peptide classes. The major class contained mostly His at the +1 position similar to SHB and

SHD SH2 domains, but also contained a wider variety of hydrophobic residues at the +3 position. SHF SH2 was screened against 190 mg of Library III due to possible selectivity at the +4 position. At the +4 position, it selected mostly for hydrophobic residues.

Seventy-six class I, 36 class II, and 26 class III full and partial sequences were obtained.

Overall, SHF SH2 selected the class I consensus

XXpY(H/R/S/D/q/t)(I/Y)(I/V/F/W)(I/V/Y) and class II consensus

XXpY(Y/f)X(Y/W/v/i)(e/d/q) (Figure 23). Their sequences are summarized in Table 45,

Table 46, and Table 47. The consensus differed a bit from the OPAL results21 with high selection for His at the +1 position for this family of SH2 domains.

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Table 45. Peptide sequences selected from SHB SH2 screening

EEpYAMU RVpYHIL HYpYEIM HUpYDYS XDpYUIF XXpYHIL DSpYGTM NFpYEAE XXpYFLF GEpYHML AYpYHUM QRpYFNH UMpYQUF EIpYHSL UUpYHUM DYpYHHD REpYVVF AApYHTL NFpYHUM UDpYWEG NEpYUQI XXpYHTL YIpYHEM URpYEWI UTpYHVL PKpYHEM WRpYHWQ XXpYFVI NYpYIQL NUpYHEM XXpYHLI XXpYITL DTpYHEM IYpYHVI GNpYIVL YYpYHEM XXpYITI/L XXpYIVL XLpYHIM NDpYAEL UApYKUL XRpYHLM AYpYAIL SHpYQVL GUpYHVM AVpYAIL NTpYQVL XXpYIVM IEpYANL NLpYRIL AUpYRIM XUpYARL GHpYTEL NApYRIM QEpYAVL UQpYTLL EYpYRLM XXpYAVL UDpYTML VFpYSVM XIpYUAL XXpYTML XXpYTUM DFpYUUL QTpYTVL UEpYTIM QTpYUUL YEpYTVL AGpYTIM EIpYULL DTpYAUM HEpYTPM XXpYUNL EApYAIM NYpYTRM MEpYUTL RVpYAIM AEpYTVM GYpYUTL QNpYAIM XXpYVUM NSpYUVL XXpYALM RXpYVIM XXpYUVL NKpYAVM XNpYVLM XXpYUVL XXpYUUM XXpYYIM XXpYDNL UApYUIM AIpYEIV UHpYEUL RRpYUIM UPpYLVV XMpYEVL XXpYUKM SSpYYQV AHpYFIL NHpYULM XXpYLVW XXpYFVL QIpYUTM AYpYHDL WApYUVM Class II YVpYHDL XXpYUVM GPpYUDE LVpYHEL DNpYEEM YYpYDME

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Table 46. Peptide sequences selected from SHD SH2 screening

XXpYAYU NFpYHDL XXpYKIL ETpYAUM GIpYHEL HSpYKTL XIpYALM NApYHIL NUpYLDM EGpYARM ETpYHIL XXpYMML PPpYAVM GIpYHIL XXpYMFM XMpYUVL QYpYHLL NLpYMSM NRpYUUM UYpYHTL RRpYMVM NNpYUIM ILpYHVL NYpYNIM HDpYUVM UUpYHVL VTpYNVM RHpYUVM RIpYHVL MRpYRMI NYpYDLL ALpYHVL XLpYRLL YRpYEIL GKpYHIM XXpYTYM PRpYEIL QQpYHIM AQpYVEF NHpYFYU AFpYHIM HMpYYUL FNpYFEL NYpYHKM UFpYYVL HRpYHAI DFpYHQM VYpYYYY UYpYHII QYpYHFN HUpYHLI NHpYHIV NFpYHVI XXpYIUM UHpYHUL XXpYIVM

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Table 47. Peptide sequences selected from SHF SH2 screening

Class I NRpYDHAI DFpYDWMY QQpYQWYY GDpYYEYD GIpYQWAI FVpYEMMQ XXpYRGYV INpYYGYI EHpYDIFY YYpYESMQ NMpYRNYY XXpYYIYM HNpYDLFI HLpYETMR IFpYSYYQ TMpYYMYG FRpYETFM GGpYSYMI FHpYYQYT FRpYHIFN XXpYMKPW Class II RQpYYVYI QGpYHIFV HYpYADVI AYpYFDAP HHpYHMFH NIpYDFVI PYpYYMAN Class III IYpYQVFR RGpYDWVF FQpYYWAR HYpYIGDQ GQpYRQFW XXpYEEVE XXpYFMFE TIpYYISR INpYRYFY HYpYEKVF RYpYYGFN RFpYAIQW NPpYRYFY HVpYGQVW IHpYYNFE FFpYDVEV AMpYTDFQ HIpYHEVA YRpYYQFH EIpYFWTV AFpYDIIY QMpYHHVW XYpYFMIQ IApYHFRF YFpYEMIW RFpYHHVE HApYYFIE HVpYHITF RYpYEYIQ QEpYIIVI IEpYYMIE XXpYIWES SPpYHIIE XRpYIIVT SHpYYMIE HIpYNRSF DQpYHQIE AYpYRFVS PDpYYTIR SNpYQWDG AFpYHQIG HIpYSFVI QQpYYVIQ MFpYQAQW HHpYHRIE VHpYSVVE XXpYFEMF XFpYRENW GYpYHSIA IFpYDHWY FNpYWYMN IIpYWFEI ETpYHYII TYpYDTWY HDpYYMMD IYpYWNTQ IGpYMDIW VIpYEMWQ HEpYFGVQ HIpYYETT FSpYMTID EEpYEWWF WEpYWVVE RYpYYGNA FEpYQIII XXpYMNWE PFpYYAVI HYpYYGTI NQpYRIIN XYpYQHWV TApYYDVY IWpYHGER DQpYRIIQ XQpYQMWV NFpYYEVD VIpYYGSV FGpYSEIW XIpYRNWI GGpYFTWQ RQpYWIHE TYpYSHIN IFpYSPWH RApYWNWD HIpYYQTR GYpYTIIQ IQpYTIWV ISpYWSWN SYpYWRNA XRpYTSII XXpYTYWS HDpYYDWG XXpYWSKY HApYTVIA IHpYEYYT PYpYYHWQ DMpYEWTV HSpYTVII QGpYMIYD XFpYYNWF XXpYFYHQ G+SXpYVIIE IFpYQEYW HIpYYSWG ANpYGYRW XRpYENI/MW GYpYQIYR DHpYFWYD XLpYDMMI HApYQVYI IDpYWHYW

124

SHB SH2

-2 -1 1 100 80 2 60 % 3 40 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

Figure 22. Sequence specificities of SHB and SHD SH2 domains

125

SHF SH2 Class I -2 -1 1 100 2 80 60 3 % 40 4 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

SHF SH2 Class II

100 80 60 % 40 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

SHF SH2 Class III

100 80 60 % 40 20 0 D E N Q H K R W F Y M L I V T U S A G P

Amino acids

Figure 23. Sequence specificity of SHF SH2 domain Note: Abu is absent in Library III used to screen this domain.

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3.4. Discussion

The specificities of SH2 domains have been previously studied using various methods mentioned in Chapter 1.2. However, with those methods individual sequences cannot be obtained. Therefore, covariance was lost. Also as a result of pool sequencing, some of the lower abundant peptides may not be detected. We developed a high throughout combinatorial approach to identify individual sequences and their covariances. Since individual sequences were obtained, they could be sorted to see if multiple peptide classes are present. These minor sequences not previously discovered with other methods, may be pY motifs of potential binding targets. Identification of new proteins may lead to a better understanding of the current roles or a discovery of new roles of SH2 domains. Seventeen SH2 domains from different protein types were individually screened and sequenced. Most of the SH2 domains had high selectivity at the +3 or +1 position with selection of hydrophobic residues at the +3 position. A couple of SH2 domains such as those from the Grb7 family, HSH2D, and the Vav family had high selectivity at the +2 position. There were differences in the general consensus of

SH2 domains between different protein families. For instance, SH2 domains from

GTPase signaling proteins preferred Pro at the +3 position, while PIK3 kinases preferred

Nle at the +3 position. As a result, it is possible that these variations in sequence consensus may depend on the type of protein the SH2 domain is in.

Most of the sequence consensus selected by the SH2 domains agreed with those identified in the literature, but some of the peptide consensus deviated from the literature results. For example, the consensus identified for the SHB family agreed more with the

127

Cantley data containing Thr, Val, and Ile at the +1 position16 than the OPAL data from the Li group containing Glu and Asp at that position. For PIK3R2 CSH2, Val was selected with low frequency at the +1 position. PIK3R1 NSH2 and PIK3R2 CSH2 mainly selected for Nle at the +3 position, while the OPAL consensus for those two domains were not as refined21. Our Grb7 SH2 consensus with high selectivity at the +2 position agreed with Cantley’s data, but not with the OPAL results. With our combinatorial approach, not only was covariance identified, multiple peptide classes selected by some SH2 domains were also identified. For example Vav1 and Vav2 SH2 domain selected for three unique classes of peptides. These minor classes of peptides may potentially be motifs of new protein targets. Some of these unique peptide sequences may be tested for binding to their corresponding SH2 domains in the future.

After validating their binding, some of the consensus of selected SH2 domains may be entered into the database to identify potential binding partners. Then, selected targets may be validated in vitro by a peptide pulldown assay or coimmunoprecipitation or in vivo by designing inhibitors based on the specificity data and observe for a change in cell activity. By differentiating the sequence specificity of SH2 domains between protein families, one could search to see if a particular group of proteins would be selected.

Once the protein groups are determined, these proteins may be characterized to further define the role of each SH2 domain in the signaling pathway. The consensus of SH2 binding motifs will help to map out the phosphotyrosine signaling network and identify altered signaling pathways in abnormal conditions such as in diseased cells. Mutations in the SH2 domains have been shown to cause diseases due to the protein overexpression or

128 loss of cell regulation in the signaling pathway. Effects of mutations on SH2 specificity for a phosphatase SHP2 will be investigated in Chapter 4.

3.5. Acknowledgments

Allison Solaru (former undergraduate researcher in Pei lab) conducted some of the screening and sequencing of PIK3R3C and PIK3R3 N SH2 domains.

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CHAPTER 4 SHP2 SH2 DISEASE-CAUSING MUTANTS

4.1. Introduction

Src homology region 2 domain-containing phosphatase-2 (SHP2), composed of two tandem Src homology 2 (SH2) domains and a phosphatase domain, is a member of the non receptor phosphotyrosine phosphatase (PTP) family. SHP2 is a positive regulator for cell proliferation, differentiation, and survival116; 117; 118. SHP2 is required for full activation of the Ras/Erk pathway by most cytokine receptors and receptor tyrosine kinases. These actions are carried out by SHP2 binding directly via its SH2 domains, to growth factor receptors, cytokine receptors, or scaffolding adaptors, such as Grb2- associated binders (Gab) proteins116; 117. In its basal state, the N-SH2 domain binds to the

PTP domain blocking the active site. Upon activation, the SH2 domain binds to phosphotyrosine peptides from a receptor or scaffolding adaptor, altering the NSH2 conformation, thus preventing its binding to the PTP domain.

SHP2, an oncogene, causes various types of childhood leukemia upon mutation and is a key downstream target of other oncogenes117. Noonan syndrome (NS) and juvenile myelomonocytic leukemia (JMML) are two conditions associated with SHP2

SH2 mutations. NS is an autosomal dominant condition that causes facial abnormalities, short stature, and cardiac defects. Some mutated sites have already been discovered. For example, T42A, a NS causing mutant, has higher binding affinity for pY peptides

130 compared to the wild type protein even though it has normal basal PTP activity116. T42A is responsible for the hydrogen bonding interactions of the phosphate group to the phosphotyrosine119. JMML is a myeloproliferative disorder characterized by overproduction of myeloid cells that infiltrate hematopoietic and non hematopoietic tissues120. Many mutations in the SHP2 gene have been studied to test their effects on structure and function121.

In this study, five SH2 mutants of SHP2 will be studied. These five mutations are functional so protein folding will not be compromised121. T42A, L43F, and T52S are located in the phosphopeptide binding cleft of the NSH2 domain, whereas E139D is located in the CSH2 phosphopeptide binding cleft. T42A and L43F are not predicted to alter the auto inhibition of the SHP2 product due to their location in the protein122. E76K is positioned near the NSH2/PTP interface essential for autoinhibition. Missense mutations in the SHP2 gene disrupt the structure and function resulting in hyperactivation of the catalytic domain. For example, mutations may disrupt the binding interface between the NSH2 domain and catalytic core necessary for the enzyme to maintain its auto-inhibited conformation. As a result, the enzyme would always be turned “on”, which leads to unregulated activity. Mutations may also disrupt the SH2 binding site, altering its sequence specificity. It is unknown whether a mutation in the SH2 binding site will alter its binding targets. As described in Chapter 1.1., protein domains recognize particular motifs corresponding to binding partners. Altered specificity may change the targets it binds to leading to a disruption of signaling pathways. The specificity of the five SHP2 SH2 mutants will be identified using our combinatorial approach as described

131 in Chapter 1.2.6. If any altered specificity is identified, the sequence consensus will be entered into a database search to identify any protein targets not previously selected by the wild type protein.

4.2. Experimental Procedures

4.2.1. Materials

All nucleotides were purchased from Integrated DNA technologies (Coralville,

IA). Antibiotics, Sephadex G-25 resin, and organic solvents were obtained from Sigma-

Aldrich (St. Louis, MO). pET22b vector and all DNA restriction enzymes were obtained from New England Biolabs (Beverly, MA). Talon resin for protein purification was purchased from Clontech (Palo Alto, CA). Reagents for peptide synthesis were from

Advanced ChemTech (Louisville, KY), Peptides International (Louisville, KY), and

Nova Biochem (La Jolla, CA). Protein concentration was determined by the Bradford method using BSA as standard unless otherwise stated.

4.2.2. Construction, expression, purification, and biotinylation of wild type SH2 domain and mutant SH2 domains

DNA sequences coding for SHP2 T42A N-SH2, SHP2 E76K N-SH2, SHP2

E139D C-SH2, and wild type SHP2 NSH2 were isolated by polymerase chain reaction

(PCR) from pcDNA3 SHP2 T42A, pcDNA3 SHP2 E76K, pcDNA3 SHP2 E139D (all provided by Benjamin Neel’s group), and SHP2 NSH2 pET28a was cloned by Dr.

Michael Sweeney29. SHP2 CSH2 was cloned and purified previously by Dr. M.

Sweeney. The following primers for wild type NSH2 and mutants were used: 5’-G

GAATTC CATATG ACA TCG CGG AGA TGG TTT CA-3’ and 5’-ATAAGAAT

132

GCGGCCGC ATCTGCACAGTTCAGAGG-3’. PCR products were digested with NdeI and NotI and ligated into pET22b-His-ybbr13 with T4 DNA ligase25. This procedure produced the wild type SHP2 NSH2 and SHP2 NSH2 mutants with a C-terminal 6-His- ybbr13 tag. The SHP2 L43F and T52S NSH2 mutants were generated by Quik-change mutagenesis according to the manufacturer’s procedure using SHP2 NSH2 pET22b-His- ybbr plasmid as the template. The following mutagenic primers, containing the mutated codon in bold text, were used:

5’-CCCTGGAGACTTCACATTTTCCGTTAGAAGAA-3’and

5’-TTCTTCTAACGGAAAATGTGAAGTCTCCAGGG-3’ for L43F; and

5’-GAAATGGAGCTGTCAGCCACATCAAGATTCAG-3’and

5’-CTGAATCTTGATGTGGCTGACAGCTCCATTTC-3’ for T52S. The identities of all constructs were confirmed by dideoxy sequencing. Protein expression, purification, and labeling were conducted in a similar manner as described in Chapters 2.2.4. and

2.2.5.

4.2.3. Library screening and sequencing

Library II was synthesized as described in chapter 2.2.2. and screened as described in chapter 2.2.6. Positive beads were sequenced by PED/MS according to the procedure described in Chapter 2.2.7.

4.2.4. Synthesis and labeling of the pY peptides

Each peptide was synthesized on ~100 mg of CLEAR amide resin (0.46 mmol/g) and labeled with NHS fluorescein according to the procedure in Chapter 2.2.8. All of the

133 peptides have a common C-terminal linker LNBK. The N-terminus was acetylated by

Ac2O.

4.2.5. Affinity measurement by fluorescence polarization

Fluorescence anisotropy measurements used to determine the binding affinity of the pY peptides against the SHP2 NSH2 mutants and wild type were performed as described in Chapter 2.2.10. Fluorescein-labeled peptide (final concentration 100 nM), 1 mM TCEP, 0.1% BSA, SH2 protein (final concentration ranging from 0 µM to 256 µM) and phosphate buffer (25 mM sodium phosphate, and 150 mM NaCl at pH 7.3) were used. The Kd was obtained from plotting a nonlinear regression fitting of the data to the following equation when the peptide concentration L is 100 nM:

2 (Amin+(Amax*Qb/Qf-Amin)*((L+[SH2]+Kd)-sqrt((L+[SH2]+Kd) -

4.3. Results

4.3.1. SHP2 NSH2 and CSH2 mutants

pY library II was screened against the proteins. They were sequenced by

PED/MS, sorted, and plotted in a histogram. In the tables, sequences from most intensely colored beads were represented by bold text, medium intensely colored beads by italized and underlined text, and least colored beads by normal text. Wild type SHP2 NSH2 and

SHP2 CSH2 were previously screened29. From the selected sequences, SHP2 NSH2 selected for four classes of peptides whereas most SH2 domains selected for a single

134 class of peptides. Class I, the major class, exhibited the consensus

(I/L/V/Nle)XpY(T/V/A)X(I/V/L/f). The other three minor classes exhibited the following consensus: class II: W(M/T)pY(y/r)(I/L)X; class III:

(I/V)XpY(L/M/T)Y(A/P/T/S/g); and class IV: (I/V/L)XpY(F/M)XP; On the other hand,

SHP2 CSH2 selected for a single class of peptide with the specificity of

(T/V/I/y)XpY(A/s/t/v)X(I/v/l).

About 100 mg of library II was screened against 500 nM of SHP2 T42A and

E76K NSH2 mutants to obtain a total of 128 and 138 full and partial sequences respectively. From the selected sequences (Table 48) the sequence specificity of T42A did not deviate from the wild type with the exception of a small switch in specificity of the +3 position from I to L. The only minor class T42A selected for was the wild type class IV peptides. From the selected sequences (Table 49), the E76K mutant specificity remained unchanged.

Approximately, 110 mg of library II was screened against 500 nM of SHP2 L43F

NSH2 to obtain 68 full and partial sequences in the major class. From the selected sequences (Table 50), the sequence specificity was similar to the wild type but with a shift in specificity from I to L at the +3 and –2 positions. It also selected for 7 wild type class IV and 38 wild type and novel class II peptides. The new class II peptides preferred the consensus (φ)(φ)pYX(φ)X where φ represents a hydrophobic residue.

About 110 mg of library II was screened against 500 nM of SHP2 T52S NSH2 to obtain a total of 199 full and partial major class sequences and 32 full and partial minor class sequences. Out of all the mutants tested, T52S is the only mutant that exhibited a

135 significant specificity switch from small hydrophobic residues to large branched ones such as Leu at the +1 position. The Ala peak was suppressed in this particular mutant whereas the Ala peak dominated in the other plots. The consensus sequence was

(L/I/V)XpY(L/U/V)X(L/i/v). Its sequences are summarized in Table 51. About 100 mg of library was screened against 500 nM of SHP2 E139D CSH2 to obtain a total of 175 full and partial sequences. Its sequence specificity did not differ much from the wild type counterpart. Its sequences are summarized in Table 52. The plots for the NSH2 mutants are summarized in Figure 24, and the plots for the CSH2 mutants are summarized in

Figure 25.

136

Table 48. Peptide sequences from SHP2 T42A NSH2 screening

Class I YMpYTIU LKpYAIL INpYURL VQpYTSV YQpYTVU VHpYAIL TTpYURL VVpYTTV IHpYTEF VVpYAIL FEpYUTL KIpYYAV IHpYAUI VVpYAKL ITpYUTL VIpYAUI IDpYALL IQpYUTL Class IV YHpYAFI IKpYALL YTpYUTL NMpYFUT VSpYAII TApYALL PVpYIQL VLpYFQT IIpYANI TMpYANL ISpYISL IMpYLQA IFpYAQI VVpYANL IHpYLQL IMpYLLP VLpYAYI VIpYANL VTpYMYL PIpYLVP VVpYUDI YRpYANL IVpYSQL IUpYMQA VQpYUII YUpYAML YFpYTHL VMpYMTA IIpYUQI LEpYAQL IUpYTML UTpYMUU IIpYUQI VQpYAQL VTpYTML ITpYMEU VUpYUQI VIpYAQL IIpYTPL ILpYMEP IQpYURI VHpYAQL VYpYTVL MLpYMMP ILpYUSI IQpYASL ITpYTYL IQpYMVP ISpYUTI IIpYAVL YIpYVEL XXpYMYP VSpYUTI IYpYAVL XXpYVIL VVpYMMS IQpYIEI VHpYAVL VTpYVNL VVpYMQT LpYIEI IIpYUAL YTpYVQL YKpYVFP ISpYMMI IHpYUUL AVpYVSL YEpYTII LRpYUUL VSpYVVL YUpYTLI TVpYUUL VSpYVVL IVpYMFD ITpYTQI YRpYUUL IVpYANM LQpYTQH VQpYVUI YUpYUUL VVpYAQM VQpYMYN INpYVDI YDpYUUL IHpYUQM IMpYHLR IRpYVEI IRpYUDL VRpYUQM VSpYVQI IQpYUHL NVpYDUM IMpYAUL ILpYUHL IEpYLTM IEpYAEL YQpYUHL AKpYVLM YEpYAEL UUpYUKL VFpYAHV IIpYAHL VTpYUKL PQpYFTV INpYAIL VMpYUQL YTpYIUV IApYAIL ITpYURL VSpYLVV

137

Table 49. Peptide sequences from SHP2 E76K NSH2 screening

Class I IEpYTRU ITpYUML IQpYFVH YMpYHIV RIpYVLU TVpYUQL LTpYMVR YMpYHYM UTpYFAR IHpYAVF VMpYUSL LTpYMVR XIpYALS IRpYGUR XXpYIFF YQpYUTL LMpYRYA XTpYQMF KUpYRPA ITpYAEI MNpYUVL MMpYHYL SVpYRNH LRpYAEI FFpYHIL QMpYTYW Class III MYpYAQI VMpYLIL TIpYIUN LNpYLYL MNpYAQI IpYNFL TVpYNFR MIpYLYR VLpYATI MYpYTEL VMpYTIS IHpYLYS IHpYUDI LHpYTTL WVpYTUY ILpYMYA IVpYUEI VHpYVUL WMpYHFG VEpYUEI VTpYVEL WMpYHFQ Class IV TMpYULI LYpYADM WMpYAHE IQpYFUU LTpYUQI ALpYUVM WFpYRIN IQpYFIU IHpYUVI GSpYEGM WIpYHIM VQpYFQU VQpYIEI AIpYEIM WMpYQIH LQpYFTU DYpYLLI FQpYGKM WFpYQLA ALpYFQG EFpYLMI LNpYMDM WIpYQLT UIpYFIP IHpYMEI QLpYSLM WIpYILN KUpYILA MQpYPRI LHpYAUV WMpYALR WEpYIEP YLpYSLI MYpYAHV WMpYULR MMpYLLP LUpYTEI SRpYGYV WMpYKLQ PMpYLVP VFpYTEI NVpYMAV WMpYNLL pYMIU IFpYTTI SQpYTEV WSpYQLF ILpYMQU IRpYTVI UMpYTRV WVpYYLA UVpYMTU IVpYVDI VIpYTVV WVpYYLE LVpYMAP WMpYYDI LSpYLVY WMpYTMS VMpYMAP MVpYAUL WTpYHMS IApYMGP LHpYAFL Class II WMpYHQK LHpYMHP LYpYATL FMpYTID WMpYHSU IIpYMTP IRpYUUL FMpYTLT WMpYFTS LLpYMTP IRpYUDL HMpYYLN WMpYYTU LHpYMNS YSpYUEL IFpYYIE WMpYQVM QLpYPUP YHpYUEL IMpYULN WIpYHYY ITpYVYP VUpYUIL IMpYNLG WIpYH WIpYYGP

138

Table 50. Peptide sequences from SHP2 L43F NSH2 screening

Class I YMpYTLU FYpYADL EMpYHVW XXpYTVU VApYAEL FTpYSHS Class IV LYpYAAI MUpYAIL FFpYELH LHpYUTP LLpYAEI ILpYANL HMpYKLT QMpYFIP LRpYAKI LYpYASL IFpYQIQ LNpYMAP LVpYANI LYpYATL IFpYQIT LVpYMUP IEpYAMI IEpYATL IIpYHID ILpYMSP LYpYATI YUpYAVL IMpYHIH ULpYMTP IVpYUAI IQpYUDL ITpYSIG LTpYVRP LLpYULI IApYUEL IFpYELN IEpYUQI YKpYUEL IMpYULG MHpYUQI IRpYUHL IMpYVLG ISpYUTI LQpYUKL IMpYELV VIpYTEI IKpYULL ITpYVNE UVpYTEI ITpYUSL IMpYRME LApYUSL LSpYVEL IMpYKYN VEpYITL INpYVSL LMpYKLT IEpYIVL IHpYVTL LMpYTMK LRpYIVL LKpYMVM QMpYYWD LVpYLEL LHpYTTM RMpYRDS LTpYLIL LYpYAUV RMpYKIN VQpYLVL LQpYUDV VMpYHLK IQpYMSL IQpYUIV VMpYITK VKpYTEL IDpYIEV VTpYNVA IHpYTLL LNpYIMV WTpYNHP IUpYTQL ISpYMUV WMpYQIP IVpYTTL LT pYMTV WMpYRIS VTpYTYL ITpYQLV WIpYTLE VKpYVUL WMpYTIV WMpYQTV VYpYVDL LHpYVAV YMpYHLE VTpYTHI LNpYVQV YMpYQLE LMpYTII HUpYQYY YMpYKLN ITpYTII XMpYHLP LYpYTLI Class II LRpYVSI EFpYYLH Class III IHpYYQI EMpYYLE MApYLYA 139

Table 51. Peptide sequences from SHP2 T52S NSH2 screening

Class I ILpYMIA MNpYIDI VQpYUHL HHpYLEL LQ pYVAL QNpYIAV QUpYIET LQpYVDA LEpYIEI MWpYUHL TUpYLEL TTpYVUL LKpYIUV MWpYIET LHpYVLA GNpYIHI VEpYUHL AQpYLHL MHpYVUL MHpYIIV LSpYIIT LHpYULU GGpYINI VKpYUHL XTpYLIL QUpYVDL LR pYIMV LKpYIVT MHpYUTU NGpYIQI YNpYUIL TApYLIL QRpYVEL VApYITV IKpYLQT IRpYUVU ITpYISI LVpYUNL TTpYLIL TH pYVEL MFpYLEV ITpYMIT HHpYILU HKpYLUI IGpYUNL LQpYLKL GApYVEL LWpYLHV IHpYMLT VKpYIVU QWpYLEI LUpYUQL MRpYLKL AQpYVIL VUpYLHV IRpYWVT LTpYIYU HRpYLLI TVpYUQL MRpYLKL TSpYVIL IQ pYLVV LKpYLUU LDpYLNI TN pYUSL VHpYLLL QH pYVML HQpYMEV Class IV ILpYLEU LHpYLTI VUpYUTL VNpYLLL MSpYVML VIpYMHV PMpYLUP LNpYLHU NGpYLVI IDpYUTL MQpYLML QHpYVQL MNpYMLV LVpYLUP LHpYLTU LRpYLVI IKpYUVL TLpYLQL GHpYVQL PV pYMQV TLpYMEP ISpYLVU LLpYMDI UNpYUVL TK pYLQL IR pYVSL VMpYTUV PLpYMQP TVpYMQU IEpYMEI LA pYUVL LVpYLSL TKpYVVL MUpYVEV II pYMTP VUpYMTU VVpYMQI GNpYUVL QTpYLTL MMpYVVL IRpYVHV IEpYMVP LApYVEU ITpYTEI YRpYUVL TTpYLTL YEpYVVL LK pYVLV VLpYMYP IRpYVVU IUpYTHI UHpYUVL VTpYLTL YHpYVVL LHpYVWV LE pYVHP LQpYUEF IRpYTKI XVpYIDL LSpYLTL LRpYVWL MNpYLVY LKpYVWP INpYUTF LRpYTPI ITpYIEL LNpYLTL VVpYUHM XXpYIDF HApYVEI AQpYIEL AHpYLTL MHpYUKM Class II LNpYLSF IYpYVEI YApYIEL QKpYLTL VMpYUNM WIpYHLD MU pYLSF VHpYVNI UNpYIHL NGpYLVL IVpYHSM QUpYULE LQpYMQF MNpYVQI GSpYIIL LK pYLVL MRpYIHM VVpYTNE IIpYRIF MKpYVVI INpYIKL LRpYLYL IKpYIHM VIpYUNG LRpYVEF LVpYADL MHpYIKL VTpYMAL VNpYIVM IIpYKYG IRpYVSF YVpYAEL AApYILL TQpYMIL INpYIWM TLpYLVG VVpYARI VRpYUUL VKpYIML LRpYMKL LVpYLEM MM pYTLH VTpYUEI TTpYUDL GG pYIQL VTpYMSL UApYMAM TWpYHIK MHpYUEI LVpYUDL IHpYISL QSpYMVL EDpYMIM IVpYHMK TQ pYUQI ISpYUEL PK pYIVL II pYTUL VKpYTFM IVpYTMK VRpYUTI QRpYUEL IKpYLUL IQpYTLL VMpYTLM LR pYVIK LTpYUTI HQpYUEL LDpYLUL VUpYTLL TKpYVLM SSpYVIK ISpYUVI QRpYUEL TTpYLDL IYpYTVL MVpYUEV VQpYILS IQ pYIAI SK pYUEL QVpYLDL ITpYTVL VRpYUQV IMpYHIT MNpYIDI MVpYUGL LVpYLDL VQpYVAL MDpYDVV VVpYHLT

140

Table 52. Peptide sequences from SHP2 E139D CSH2 screening

EMpYANA VLpYAKI NFpYAVL EGpYAFV EGpYIRG RKpYGFA YRpYALI NHpYAVL HYpY AGV GApYMKG HDpYTEA TYpYALI YEpYUUL DNpYAIV TPpYYAG THpYTFA HLpYAMI TDpYUIL VPpYANV SUpYGGH HEpYVFA ELpYARI TQpYUML TYpYAQV DQpYRQH MKpYVGA IDpYARI VQpYUQL GEpYUNV YNpYYIH ENpYAGU NKpYATI QTpYDYL PQpYUQV FYpYGNK NDpYANU TVpYATI XXpYEIL HYpYDAV RTpYIPK HGpYEGU MQpYATI MRpYGQL VQpYFMV EpYMFK RTpYHTU VIpYATI PPpYHUL TYpYILV GRpYSIK EHpYIEU VKpYATI PEpYHGL UpYILV pYAWN PDpYLFU VpYUUI DYpYIQL GEpYNIV MQpYEHN MIpYSRU NDpYUEI FDpYIYL LKpYRTV AVpYGFN ESpYMLE IKpYUEI FIpYMLL SApYSNV TUpYLAN

HGpYAHF TTpYUHI QDpYTML GFpYTQV MVpYSAN DUpYUQF HEpYUHI YEpYVVL QDpYVEV AGpYYLN

MPpYIHF HNpYUMI HEpYYNL TQpYYLV PDpYLNQ XXpYLLF VpYUMI HEpYYNL LQpYYMV UApYLTQ VFpYLLF IHpYURI GKpYYVL EGpYKVW ETpYSQQ GDpYLTF UDpYUTI HDpYL QVpYVLW IApYVIQ NTpYSNF LpYLFI TQpYAEM QUpYLTY UVpYVMQ VEpYVNF EQ pYMEI TEpYAIM YUpYLTY XXpYYIQ YNpYAUI PUpYMFI AHpYAKM XMpYMVY QSpYGAR TLpYAUI LRpYMSI SQpYALM HTpYGIP KVpYIMR YPpYAUI TApYPVI YTpYAMM TUpYIDP IHpYQHR ERpYAUI YQpYSUI QYpYARM HEpYNTP ITpYAFT AYpYADI SYpYSTI AFpYATM TDpYTTP AKpYAWT YDpYAFI HTpYTPI QTpYUQM PNpYVAP GMpYUFT LHpYAFI UApYTTI RGpYEFM NGpYVNP TNpYHWT YNpYAHI ENpYANL NNpYEVM QRpYVVP FApYSKT HDpYAHI SYpYAQL NQpYQVM XEpYWNP VGpYPYY

NEpYAII AYpYARL FMpYTRM GUpYHXX ANpYAII AQpYATL TIpYTTM PPpYXXX QRpYAKI HTpYATL UEpYTVM VGpYVUD

ISpYAKI MRpYAVL TTpYVSM IGpYVLD VTpYAKI VRpYAVL XXpYYVM WEpYUMG

141

Figure 24. Sequence specificity of SHP2 NSH2 mutants and its wild type

142

Figure 25. Sequence specificity of SHP2 E139D CSH2 mutant and its wild type

4.3.2. Binding affinities of selected peptides

To validate the screening results, seven representative peptides were individually resynthesized to test the binding affinity against the SHP2 NSH2 mutants and wild type by fluorescence anisotropy (Table 53). LVpYATI was synthesized as a control. Since L was selected by most of the mutants at the +3 position, LVpYATL was synthesized to test if it affected binding. The -2 position was essential for binding so AVpYATL was synthesized to validate this. LVpYLTL was synthesized to validate the specificity change of T52S at the +1 position. Class II and class IV peptides IMpYVLG, and

143

IIpYMTP, respectively, were also tested against all the mutant and wild type SH2 proteins for binding.

After examining the results summarized in Table 53, the E76K SH2 binding affinity for all of the tested peptides did not change, validating the screening data. Both

T42A and L43F mutants exhibited an increase in affinity compared to the wild type SH2 for all tested peptides. The dissociation constants of T52S were all similar to the wild type with the exception of binding to the pYLTL peptide, the Kd of 1.83 μM and a 6-fold difference from the wild type’s dissociation constant of 11.4 μM. This validated the specificity switch from Ala to Leu at the +1 position. As expected, the mutants and wild type bound to the class II IMpYVLG and class IV IIpYMTP more weakly than class I peptides. The CSH2 E139D mutant was also tested against LVpYATI, LVpYTEV, and

LVpYATL peptides. The binding affinity of the E139D mutant for LVpYATI only increased approximately 2-fold suggesting the little change in CSH2 binding.

144

Table 53. Dissociation constants (Kd, µM) of representative pY peptides towards the SHP2 mutants

Peptide WT NSH2 T42A L43F T52S E76K WT CSH2 E139D LVpYATI 12±3 1.6±0.2 3.1±0.2 20±2 13±3 6.9±0.9 3.3±1 LVpYTEV 9.4±1.5 1.1±0.3 6.2±1.2 9.0±2 8.1±1.9 80±13 43±6 LVpYATL 8.4±0.9 0.95±0.08 5.5±0.2 15±4 8±2 14±1 8.7±1.2 AVpYATL 63±9.0 6.4±0.6 42±0.3 86±9 97±15 ND ND LVpYLTL 11±1 1.3±0.2 5.5±0.6 1.8±0.4 11±0.5 ND ND IMpYVLG 67±13 13±1 32±4 48±6 95±10 ND ND IIpYMTP 67±16 3.4±0.5 30±3 51±8 58±5 ND ND

All peptides were acetylated at the N-terminus and contained a C-terminal linker LNBK- NH2. All peptides were labeled with NHS-fluorescein by the lysine side chain in the linker.

4.3.3. Literature search

Since the T52S mutant was the only SHP2 mutant that exhibited a switch in specificity, the T52S consensus [LIVM]XY[LC]XL was entered into the phosphosite database (http://www.phosphosite.org) to search for potentially new binding partners.

Twenty six potential substrates were identified (Table 54). Grb-associated binding (Gab) proteins 1, 2, and 3, known to bind the SHP2 NSH2 domain, were among the identified targets.

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Table 54. Human proteins predicted to have improved binding affinity for SHP2 T52S NSH2

Protein Accession Binding motif(s) Aldo-keto reductase family 1 member C1 Q04828 VRpY305LTL Aldo-keto reductase family 1 member C2 P52895 VRpY305LTL ADP-ribosylation factor-like protein 13A Q5H913 IDpY146LLL Cytokine receptor common subunit beta P32927 LEpY628LCL, QDpY882LSL Erythropoietin receptor P19235 DTpY368LVL, LKpY454LYL ERBIN, KIAA1225, LAP2 Q96RT1 IDpY1293LML Fam83B Q5T0W9 VPpY399LLL HSP90-binding immunophilin FKBP5 Q13451 MCpY327LKL flightless-1 homolog Q13045 LGpY737LEL Gab1 Q13480 VEpY627LDL Gab2 Q9UQC2 VDpY603LAL Gab3 Q8WWW8 LDpY533LAL Herc1 Q15751 LSpY2510LVL Herc2 O95714 LRpY376LTL LDB2 O43679 LNpY203LRL LOC144100 Q6IQ23 LEpY665LDL LRP1B Q9NZR2 VEpY4150LAL LRRC8C Q8TDW0 IRpY687LDL MGC45438 Q6UX73 LHpY147LKL Myosin-Id O94832 VEpY612LGL Myosin-Ig B0IlT2 VApY624LGL NRAGE Q94832 VKpY481LML PRIM1 P49642 VEpY188LSL SREBP-2 Q12772 LEpY840LKL TMTC1 Q8IUR5 MGpY312CIL 508 VRL2, VROAC TRPV4 Q9HBA0 VDpY LRL

146

4.3.4. Structural analysis of the SHP2 NSH2 domain

To determine the structural basis of the sequence specificity of T52S, the pdb file

1AYB containing the SHP2 crystal structure was downloaded from the protein databank and analyzed by PyMOL. For each mutant the amino acid residue was mutated to the desired residue and compared to the wild type crystal structure. The T52S mutation was found to be located in close proximity to the pY+1 position (Figure 26). E76K was located outside of the binding cleft so this finding validated the unchanged sequence specificity observed during screening. L43F, in contrast, appears to be in close contact with the +4 Phe of the peptide.

147

Figure 26. SHP2 NSH2 and mutant crystal structures with peptide GEpYVNIEF (in blue stick form) Top: L43F mutant structure (L43F residue in stick form) Middle: Wild type structure (T52 residue in stick form) Bottom: T52S mutant structure (T52S residue in gray stick form)

4.4. Discussion

NS and JMML are caused by SHP2 SH2 mutations. These mutations are mainly located either in the SH2 binding cleft or in the NSH2/PTP interface. Mutations in the

SH2 binding cleft may alter specificity leading to a change in its protein targets. By 148 using a combinatorial approach, we wanted to determine if mutations in the SH2 binding cleft alter specificity. Five SHP2 SH2 mutants were screened against a pY library and sequenced. To validate the screening results, selected peptides were resynthesized and were tested for binding to the mutants. Surprisingly, only T52S exhibited a specificity switch while the rest of the mutants exhibited little or no change in specificity even though some of the mutants in the binding site exhibited increased binding affinity. After examining the mutant structure using PyMOL, S52 was shown to be in close proximity to the +1 position. The substitution with a smaller residue may allow for better contact of large branched residues. After conducting a database search, some of the φXpYLXL sequences correspond to some of the Gab motifs. Gab-1, Gab-2, and Gab-3 each bind to

SHP-2 via two motifs, [V/L]XpYLXL and VDpYVXV, even though the wild type SHP2

NSH2 domain did not frequently select for the first motif29. Upon SHP2 binding to Gab protein, basal inhibition is relieved resulting in strong activation123. Increased interactions with Gab2 also lead to enhanced activation of the Erk and Akt pathways124.

Even though overall binding affinities did not increase, this specificity switch may have caused an increased recruitment of Gab protein leading to hyperactivation downstream.

While T52S exhibited altered sequence specificity, L43F and T42A did not.

However, L43F and T42A, which are located in the NSH2 binding cleft, exhibited higher peptide affinities than the wild type. T42hydrogen bonds with the phosphate of the phosphotyrosine. Substituting Ala in T42 should have led to a loss in binding affinity due to a loss of a hydrogen bond, but the pY peptide binding affinity increased significantly instead. Previously, it was discovered that an increase in enthalpy

149 contribution relative to the entropy decrease upon binding may be due to the release of strain in the pY bound structure119. On the other hand, L43F is associated with congenital heart disease but not NS119. General increased protein activity may lead to more activation of other pathways. This will enhance cell spreading and migration. This notion is also supported by previous observations that interaction of SHP-2 with Gab1 in response to EGF stimulation is enhanced by Noonan syndrome/leukemia associated SHP-

2 mutations124.

While the three mutants mentioned in the last paragraph are involved in SH2 binding, the E76K mutation disrupts autoinhibitory NSH2/PTP interactions and is basal activating119. However, it was discovered that the SHP2 E76K NSH2 mutant did not exhibit any changes in activity or specificity during this study. Since only the isolated

E76K NSH2 was studied, it is unknown whether screening with the full-length SHP2

E76K would change the binding affinity or not. Its specificity is likely not to be affected because it is located farther away from the binding cleft. E139D, located near the phosphotyrosine site of the CSH2 domain, exhibited little change in specificity with only a 2-fold change in activity. The CSH2 alone is unlikely to contribute to activation because of its minimal contact with other domains125. The NSH2 domain on the other hand is mostly involved in SHP2 regulation116. We have found some alteration in the specificity of this SH2 domain upon mutation, leading to possible increased recruitment of Gab proteins. However, it appears that most of the mutations caused an overall increase in binding affinities. There have also been diseases that are caused by mutations in other SH2 domains such as X-linked agammaglobulinemia (XLA), X-linked

150 lymphoproliferative disease (XLP), and Basal cell carcinoma (BCC) in the SH2 domains of

Btk, SH2D1A and Rasa1 respectively121. The disease-causing mutations in these SH2 domains may be studied later to see if they alter specificity. Any altered specificity in other SH2 domains may allow us to understand the mechanism of certain cancers and other diseases in more detail.

4.5. Acknowledgments

The subcloning, purification, and screenings of T42A, E76K, and E139D were conducted by Wade Duym (former Pei group member).

151

CHAPTER 5 CONCLUSION

During this study, the sequence specificity of twenty-six kinase SH2 domains and seventeen non-kinase SH2 domains were profiled. Five SHP2 SH2 mutants implicated in

Noonan’s syndrome and JMML were also examined to identify any altered sequence specificities. Using a high-throughput sequencing method developed in our laboratory, individual sequences could be obtained for each domain. The results were validated by testing the binding of selected SH2 domains to individually resynthesized peptides.

The kinase SH2 domains exhibited some overlap in their sequence specificities but with subtle differences at each position. Most of the kinase SH2 domains exhibited strong selectivity at the pY+1 and pY+3 positions. In most cases, hydrophobic residues were selected at the pY+3 position while hydrophilic residues were selected at the pY+1 position Some SH2 domains such as Fer, Fes, and Bmx SH2 domain selected pY motifs with Asn at the +2 position with little selectivity at the +3 position. Since individual sequences could be obtained using our high throughput method, covariances were identified within some of the SH2 families especially at the +3 position. For example, some SH2 domains selected more branched hydrophobic residues while another selected more small hydrophobic residues. Also, certain residues at the +3 position were present only if a particular residue was at the +1 position. We have learned that despite some sequence overlap, some SH2 domains have a narrow specificity while some bind to a

152 larger variety of binding partners (Chapter 2). To prove that our sequence specificity data could predict protein binding partners, a literature search was conducted to identify known targets of selected SH2 domains. The pY motifs of those targets closely matched to the specificity data proving it could predict new binding partners. Several minor peptide classes were also detected during screening of some of the SH2 domains. After binding validation, a few of the unique consensus sequences from Abl and Fes family

SH2 domains were entered into a protein database to search for potential binding partners. Several targets were identified. One of the protein targets was identified in literature with the identified pY motif being a potential phosphorylation site. It is unknown whether the SH2 could bind to it or not however. A pull-down assay still needs to be performed to validate those targets. Even though the specificity within the

SH2 kinase families are not very different from each other as expected, they varied in their covariances, in the minor sequence classes they bind to, and in their promiscuity.

What still needs to be determined is whether other domains such as the SH3 and kinase domains may also be involved in cross talk during signal transduction.

The specificities of 17 non-kinase SH2 domains were also profiled. Some of the results that we obtained differed from what others have found previously. First, several minor peptide classes not found previously, were identified in several SH2 domains

(Chapter 3). The non-kinase SH2 domains exhibited different specificities from each other. The binding of unique peptides still needs to be tested to see whether they can actually bind to those SH2 domains. Consensus of select SH2 proteins will be entered

153 into a protein database to identify potential binding partners. Then, these proteins will be analyzed in vitro by pull-down assay or coimmunoprecipitation.

Since it is known that mutations in the SHP2 SH2 domain may lead to aberrant binding targets resulting in diseases such as Noonan’s syndrome and JMML, those mutants were studied to see if specificity was altered (Chapter 4). Five SHP2 SH2 mutants T42A, L43F, T52S, E76K, and E139D were studied. Only the T52S exhibited altered specificity, while the other SH2 mutants T42A and L43F, located in the NSH2 binding cleft exhibited increased affinity instead. E76K, located in the SH2-PTP interface did not exhibit change in binding affinity or specificity. After entering the T52S consensus into a protein database, several potential targets, including known Gab protein, were identified. It is likely that the mutations may have either caused an increased recruitment of proteins to the SHP2 NSH2 or an increase in binding affinity to their downstream targets. These preliminary results show that one missense mutation can have profound effects on the affinity and specificity of SH2 domains. Profiling these mutant proteins may help us in determining its role in the cell and its mechanism in causing these human diseases.

Since signal transduction in the cell is complex, one needs to determine which protein interactions occur in order to understand how the signaling pathways function.

Signal transduction involves protein-protein interactions mediated by modular domains.

For example, the SH2 domain is an important modular protein that recognizes phosphotyrosine motifs. They connect receptors and downstream signaling molecules allowing signal transduction to occur within the cell. Signal transduction leads to various

154 responses such as cell differentiation, proliferation, migration, and adhesion for example.

Regulation of signal transduction is controlled by reversible phosphorylation with kinases and phosphatases acting as “on-off” switches. Deregulation on the other hand, is caused by overexpression, amplification, and mutation of components in the signal networks. It is linked to human cancer and other diseases. With the sequence specificity of the SH2 domains, new binding partners can be identified, which leads to eventual mapping of the phosphotyrosine signaling network. When wild type SH2 sequence specificities are determined, then profiling of mutant SH2 could be done to see how it affects the type of protein targets it selects. Once aberrant targets are identified, the altered signaling pathways can be mapped out. Inhibitors may be designed against target aberrant proteins in order to determine the mechanism of those diseases. Eventually drugs may be synthesized in order to suppress those mechanisms of human diseases such as cancer.

155

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