Development of a Phosphoprotein Enrichment Method to Identify and Characterize Phosphoproteins Within Leukemia Following Treatment with the PP2A Activator, FTY720

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Development of a phosphoprotein enrichment method to identify and characterize phosphoproteins within leukemia following treatment with the PP2A activator, FTY720

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Justin Charles Staubli

Graduate Program in Pathology

The Ohio State University

2012

Master's Examination Committee:

James W. Waldman, PhD, Advisor

Michael F. Freitas, PhD

Justin Charles Staubli

2012

Abstract .

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is characterized by the accumulation of CD5+ B lymphocytes in the blood and lymph organs. CLL B lymphocytes have shown a number of abnormal cellular disruptions resulting in the inhibition of several key tumor suppressors and subsequent reliance on pro-survival pathways, a term known as ‘oncogenic addiction’. This reliance on a single robust survival pathway offers a powerful new tool in cancer therapy with the development of specific molecular targets to be exploited within these diseased cells.

Given the therapeutic potential of imatinib in chronic myelogenous leukemia (CML), additional malignancies have kinases and phosphatases that are disproportionately activated or deactivated that are ripe for targeting.

Most notably, the serine/threonine phosphatase PP2A is a known tumor suppressor that has been implicated in several blood-borne malignancies including CML and CLL. Treatment with the sphingolipid derivative and S1P agonist FTY720 results in an increase in enzymatic activity of PP2A, which results in the apoptosis of CLL cells.

However, the mechanism by which FTY720 up regulates PP2A remains elusive. To fully understand the pathways affected by FTY720 in leukemia cells, an integrated, global phosphoprotein study is necessary.

Numerous methods exist for phosphoprotein investigation, including immunoaffinity and metabolic labeling. While these methods have formed the backbone of phosphoproteomic studies, unfortunately, their reliance on known cellular targets and limited scope in target identifications limits our ability to fully comprehend a phosphatase network; especially one as pervasive and intricate as PP2A. The continued development of ever-increasing sensitivity in both the hardware of mass spectrometry and the protein and post-translational modification identification software has given life to the prospect of a true ‘global phosphoproteome fingerprint’ of a phosphorylation-manipulating therapy, such as FTY720.

Mass spectrometry (MS)-based approaches have several advantages over immunoaffinity and metabolic labeling methods: MS investigation requires no prior knowledge of potential targets and can directly identify the site of phosphorylation.

Despite these advantages, MS-based phosphoproteomics techniques are time and labor intensive and often times must be tailored to individuals’ needs. Thus, the development of unique phosphoprotein enrichment methods and MS techniques are crucial. The work

I have done in this area, specifically looking at total phosphoprotein enrichment from global lysates have yielded excellent reproducibility in the global phosphoproteome fingerprint of the Burkitt lymphoma cell line Ramos, a model system for PP2A manipulation.

Utilizing a modified technique with the TALON PMAC Phosphoprotein enrichment kit from Clontech along with a modified TCA sample cleanup prior to in- solution protein digest, a highly reproducible phosphoprotein identification method has

iii been developed. With total Ramos cell lysate, technical replicates of phosphoprotein identification was as high as 93% (n=5), and biological replicates reproducibility was

88.5% (n=5). While further validation is necessary to determine the reproducibility of this method within primary leukemia cells, my work gives the first tantalizing steps toward reliable identification of a cancer ‘phosphoprotein fingerprint’ that could aid in the elucidation of phosphorylation pathways in cancer and subsequent molecular targeted therapies.

Dedication

This document is dedicated to my family, The Family, and you (all).

Acknowledgments

I would like to acknowledge the professional, intellectual and technical assistance provided by the members of the Freitas and Byrd laboratories. Your assistance was instrumental in the completion of this project.

Vita

May 2002 ...... Cambridge High School

May 2007 ...... B.S. Biology, UW – Stevens Point

June 2007 to present...... Graduate Research Associate, Department

of Pathology, The Ohio State University

Publications

1. Ghebranious N, Blank RD, Raggio CL, Staubli J, McPherson E, Ivacic L,

Rasmussen K, Jacobsen FS, Faciszewski T, Burmester JK, Pauli RM, Boachie-

Adjei O, Glurich I, Giampietro PF. A missense T (Brachyury) mutation

contributes to vertebral malformations. J Bone Miner Res. 2008 Oct;23(10):1576-

83.

2. Lapalombella R, Wang L, Ramanunni A, Yeh Y, Rafiq S, Jha S, Staubli J, Lucas

DM, Herman SEM, Johnson AJ, Lozanski A, Andritsos L, Jones J, Flynn J,

Thompson P, Algate P, Stromatt S, Jarjoura D, Mo X, Wang D, Chen C, Lozanski

G, Tridandapani S, Freitas MA, Muthusamy N, Byrd JC. Tetraspanin CD37

Directly Mediates Transduction of Survival and Apoptotic Signals. In press at

Cancer Cell. vii

Fields of Study

Major Field: Pathology

Emphasis: Cancer Immunology

viii

Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments...... vi

Vita ...... vii

Table of Contents ...... ix

List of Tables ...... xiii

List of Figures ...... xiv

CHAPTER 1 INTRODUCTION ...... 1

1.1 ONCOGENIC ADDICTION AND TARGETED MOLECULAR THERAPY .. 1

1.2 SIGNALING CASCADES AND CHRONIC LYMPHOCYTIC LEUKEMIA .. 3

1.3 THE SERINE/THREONINE PROTEIN PHOSPHATASE 2A (PP2A) ...... 6

1.4 THE PP2A-ACTIVATOR FTY720 ...... 8

1.5 CONCERTED EFFORT TO STUDY GLOBAL PHOSPHOPROTEOMICS ... 9

1.6 SUMMARY OF RESEARCH ...... 13

CHAPTER 2 PHOSPHOPROTEOMIC INDENTIFICATIONS IN RAMOS POINT TO

A POTENTIAL ROLE OF THE PP2A INHIBITOR SET IN THE MODE OF FTY720

ACTION...... 15

2.1 INTRODUCTION ...... 15

2.2 EXPERIMENTAL ...... 17

2.2.1 Materials ...... 17

2.2.2 Cell drugging and lysate preparation ...... 17

2.2.3 Phos-Tag Phosphoprotein Enrichment ...... 199

2.2.4 SDS-PAGE and Pro-Q staining ...... 19

2.2.5 Band excision and in-gel digestion ...... 20

2.2.6 Capillary ESI-HPLC-MS/MS ...... 21

2.2.7 Data analysis ...... 22

2.2.8 Western blotting and immunoprecipitation ...... 23

2.3 RESULTS AND DISCUSSION ...... 24

2.3.1 Phosphoprotein enrichment of Ramos cells by Phos-Tag Zn2+ ...... 24

2.3.2 LC-MS/MS Identification of differential phosphoproteins between FTY720

treated and untreated samples...... 25

2.3.3 Protein expression of the PP2A-inhibitor SET in Ramos and primary CLL

B cells...... 35 x

2.3.4 Potential inhibition of the Hsp90 pathway by FTY720...... 37

2.4 CONCLUSION ...... 43

UTILIZATION OF THE PHOSPHOPROTEIN ENRICHMENT KIT TALON PMAC

OFFERS A ROBUST AND REPRODUCIBLE METHOD FOR

EXPLORING THE PHOSPHOPROTEOME OF LEUKEMIA...... 45

3.1 INTRODUCTION ...... 45

3.2 EXPERIMENTAL ...... 46

3.2.1 Materials ...... 46

3.2.2 Cell Drugging and Lysate Preparation ...... 47

3.2.3 Phosphoprotein Enrichment ...... 48

3.2.4 In-Solution Digestion ...... 49

3.2.5 Capillary, ESI-HPLC-MS/MS ...... 50

3.2.6 Data analysis ...... 50

3.2.7 Western blotting ...... 51

3.3 RESULTS AND DISCUSSION ...... 52

3.3.1 Confirmation of TALON PMAC for phosphoprotein enrichment ...... 52

3.3.2 pH alterations during phosphoprotein enrichment ...... 54

3.3.3 Purification of enriched phosphoproteins by trichloroacetic acid ...... 57

3.3.4 Increasing the bead to lysate ratio for phosphoprotein enrichment...... 62

3.4 CONCLUSION ...... 66

CHAPTER 4 SUMMARY OF RESULTS ...... 69

Bibliography ...... 71

xii

List of Tables

Table 2.1 Protein list for DMSO vehicle-control-treated Ramos samples ...... 28

Table 2.2 Protein list for FTY720-treated Ramos samples ...... 31

Table 2.3 Shared protein identifications across FTY720 and DMSO treatments...... 33

Table 3.1 Summary of statistical analysis of TCA precipitated Ramos replicates ...... 67

xiii

List of Figures

Figure 1.1 PP2A Interacting Proteins Summary ...... 8

Figure 1.2 Summary of phosphoproteomic enrichment methods ...... 12

Figure 2.1 Coomassie-stained 4-15% SDS-PAGE gel for proteins following

phosphoprotein enrichment ...... 26

Figure 2.2 Identification of SET by LC-MS/MS ...... 34

Figure 2.3 SET expression in primary CLL B cells...... 36

Figure 2.4 SET expression in Ramos cells following treatment with FTY720 ...... 37

Figure 2.5 Hsp90 expression in Ramos following treatment with FTY720 ...... 40

Figure 2.6 Hsp90 expression in primary CLL B cells following treatment with

FTY720 ...... 41

Figure 2.7 Cdc37 and Akt expression in Ramos cells following treatment with

FTY720 ...... 42

Figure 2.8 Cdc37 and Akt expression in primary CLL B cells following treatment

with FTY720 ...... 43

Figure 3.1 Selective enrichment by TALON PMAC for pSTAT3 (Y705) in primary

CLL B cells following IL-21 treatment ...... 53

Figure 3.2 pH effect on phosphoprotein enrichment in Ramos cells ...... 57 xiv

Figure 3.3 Dialysis cleanup versus TCA precipitation ...... 61

Figure 3.4 TCA precipitation follows phosphoprotein enrichment in Ramos cells ...63

Figure 3.5 Increasing enrichment bead to lysate ratio for enhancing phosphoprotein

enrichment...... 64

Figure 3.6 Treatment of Ramos cells with LY294002 to determine specificity of

TALON PMAC phosphoprotein enrichment ...... 65

Figure 3.7 Venn diagram comparison of TCA precipitated Ramos replicates between

two separate LC-MS/MS runs ...... 67

Chapter 1

INTRODUCTION

1.1 ONCOGENIC ADDICTION AND TARGETED MOLECULAR THERAPY

The term ‘gene addiction’ (eventually coined ‘oncogenic addiction’) was first coined in I. Bernard Weinstein’s seminal paper, Disorders in Cell Circuitry Associated with Multistage Carcinogenesis: Exploitable Targets for Cancer Prevention and Therapy

[1]. Within the paper, Weinstein describes a multi-step process by which oncogenesis occurs within the cell, leading to a loss of homeostasis within the cell as it transitions from ‘normal’ to ‘cancerous’. However, as the cancer cell transitions to its own state of homeostasis it becomes increasingly reliant on a few specific genes for its continued existence. In several instances, this addiction relies on a single survival pathway, a cancer’s ‘Achille’s heel’ [2], if you will. And much like Achilles of Greek lore, all it takes is a single targeted arrow to bring the juggernaut down.

‘Oncogenic addiction’ is described as the initial punch in a sort of ‘pugilistic parlance’ [3], where the addiction serves as the initial punch in the process, setting the stage for the follow up knock-out punch which is a targeted molecular therapy toward a specific molecule or pathway within cancer. One of the best examples of this relationship is that of imatinib meyslate and chronic mylegoneous leukemia (CML) [4].

The identification of the Philadelphia chromosome in CML that combines the bcr and c- abl genes into the constitutively active BCR-ABL tyrosine kinase illuminated an attractive therapeutic target for this disease.

The screening of potential tyrosine kinase inhibiting compounds produced 2- phenylaminopyrimidine derivatives that occupied the ATP-binding domain of tyrosine kinases, thus preventing phosphorylation, which eventually led to the isolation of imatinib mesylate as an inhibitor of BCR-ABL [4]. The success of imatinib mesylate in

CML at inhibiting BCR-ABL with near universal response rates is legendary, and still stands to this day as one of the most successful cancer treatments in history. It has also been shown to be effective in inhibiting c-kit as well, implicating a use in GISTs [5].

Though every success story has its failures, and imatinib mesylate is no exception: there is evidence of resistance in both CML and ALL [4], where its 60-70% response rate is marred by near ubiquitous relapse within months [6]. Despite this, a plethora of tyrosine kinase inhibitors have been developed: gerfitinib and erlotinib against EGFR in non small cell lung cancer, sutent against FLT-3 in AML and sorafenib against B-Raf in melanoma and renal carcinomas [4].

This relapse could be due to imatinib mesylate not inhibiting the life-threatening addiction in ALL, or there are additional pathways that compound the affair. A well known observation is that molecular targeting of kinases works best for those kinases that have activating mutations, implying an increased signaling cascade from the kinase, and thus, increased addiction of the cancer [7]. It has been noted the underlying problem with oncogenic addiction and its targeting is in identifying the proper addiction and kinase

2 responsible for the condition [3]. A misidentification of pathway or misunderstanding of the mechanisms at work can, at best, lead to an ineffective treatment.

In a follow-up paper to his influential work, I. Bernard Weinstein notes,

“At the present time, there are no methods to fully assess the total

circuitry that controls cell proliferation, differentiation, and apoptosis in

normal or cancer cells” [2].

Weinstein does, however, indicate that advances in genomics and proteomics could offer the greatest potential for understanding the differential gene and protein profiles between cancerous and normal tissues; painting a cancer therapy tapestry to be exploited. These profiles would offer great insights into potential cancer therapeutics, an understanding of their differential functions could prove advantageous. Special attention, however, must be given to the phosphorylation signaling cascades that erupt from the kinase-addictive pathways, as the potential for tyrosine kinase inhibitors, as well as global phosphorylation manipulation has shown great promise therapeutically. Ergo, understanding of the global phosphoproteome is paramount for the development and execution of kinase inhibitory- based therapeutic strategies.

1.2 SIGNALING CASCADES AND CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic Lymphocytic Leukemia (CLL), a primarily B lymphocyte malignancy, is the most common leukemia found in adults, afflicting more than 15,000 persons a year

3 and resulting in nearly 4,400 deaths a year [16]. Depending on time of diagnosis, median survival is between 2 and 10 years [17]. There are several key diagnostic markers for

CLL. First, B lymphocytes from CLL patients test positive for CD5, CD19 and CD23

[18]. Second, the normal B lymphocyte markers CD20 and CD79b are lower compared to normal B lymphocytes [17]. Current treatments for CLL include: purine analogues (e.g., fludarabine [19] and cladribine [20], single-agent antibodies (e.g., rituximab [21] and alemtuzumab [22]), or a combination chemo-immunotherapy (e.g., fludarabine + cyclophosphamide + rituximab [23], or fludarabine + alemtuzumab [24]. Despite the effectiveness of these treatment regimes, there are several CLL patient subsets that respond poorly to these treatments. These patients can have one of a number of genetic abnormalities including: unmutated VH, 11q-, 17p- translocations [17] or abnormalities to p53 [25, 26]; although recent studies with alemtuzumab have shown promise in the treatment of these patient subgroups [27], it has serious immunological side effects.

With the success of tyrosine kinase inhibitors in chronic myelogenous leukemia, the possibility of targeting similar molecules in CLL is gaining traction, and several new avenues of drug development are currently underway. There is a need for an aggressive therapeutic that has minimal side effects which leaves the battered CLL immune system relatively intact. Much like a normal B cell, CLL B cells have an intact signaling network based around the B cell Receptor (BCR), which consists of a membrane-bound

IgM and an associated heterodimer consisting of an Ig-α and Ig-β. Engagement of the

IgM with an antigen induces phosphorylation of the cytoplasmic tails of the Ig-α and Ig-β molecules in a portion called the immunoreceptor tyrosine-based activation motif, or

4 simply ITAM. This phosphorylation is performed by the Src-kinase Lyn. The phosphorylation, in turn, results in the binding of another tyrosine kinase, Syk, which forms a complex with Lyn, Btk (another important tyrosine kinase in the BCR signaling cascade) and adaptor proteins such as BLNK. From these initial few activation steps, any number of signaling cascades can be triggered: PI3K can be activated to induce AKT signaling: PKC can activate as well, leading to MAPK signaling and NFκB [8].

CLL B cells have the BCR signaling pathway more or less intact, though with a decreased amount of surface IgM [9]. Despite this, BCR signaling is strong in CLL: Lyn is known to be constitutively active in CLL and is responsible, in part, to the survival of

CLL B cells [10], carrying the pro-survival signal through to AKT and ERK, as well as increasing levels of the anti-apoptotic protein Mcl-1 [9]. This pathway is ripe for targeting, and over the years, many agents have been developed to exploit this ubiquitously active pathway in CLL.

While dasatanib is a well known Lyn inhibitor, its efficacy and targeting in CLL

[11], especially in relapsed or refractory disease [12], remains debatable, at best. The newest inhibitor, Bafetinib, is not fairing much better [13]. Syk inhibition has shown promise both in vitro and in vivo, preventing BCR-induced AKT signaling [14], and showing a 55% partial response rate in a phase 1/2 study [9]. This response, though, is dwarfed by the tremendous response in a phase 1 trial of the Btk inhibitor PCI-32765 with an overall response rate of a staggering 64% [9]. Further downstream, the PI3K inhibitor CAL-101, which specifically targets the hematopoietic-lineage isoform of PI3K, p110δ, overcomes the protective effects of CD40L, TNFα and fibronectin on CLL B cells

[15], all well-established pro-survival factors in CLL. While not specifically ‘targeted molecular therapy’, the exciting new field of adoptive transfer of T cells expressing chimeric antigen receptors (CARs), whereby T cells are engineered to recognize tumor- specific surface molecules. A trial utilizing anti-CD19 CAR-transduced T cells is very promising, citing one complete remission in a CLL patient [9].

1.3 THE SERINE/THREONINE PROTEIN PHOSPHATASE 2A (PP2A)

PP2A is a highly ubiquitous serine and threonine phosphatase implemented in numerous cellular signaling pathways including: metabolism, transcription and translation, RNA splicing and DNA replication, development and morphogenesis, and most interesting, cell cycle progression and transformation [48]. PP2A has been found to be down regulated in numerous cancers [48-52], and has earned the unofficial title of

‘tumor suppressor’ [48, 49, 52-55] by numerous researchers. It's down regulation by the tumor-promoter Okadaic acid [56] and oncogenic-viral protein SV40 [57, 58] led to the conclusion that loss of PP2A enzymatic activity is vital for cellular transformation. PP2A is a heterotrimer consisting of a structural, scaffolding subunit (A subunit), a catalytic subunit containing PP2A’s phosphatase activity (C subunit) and a regulatory subunit (B subunit), that is believed to locate PP2A to particular tissues [59-61].

Due to its potent phosphatase activity in vivo, PP2A has a wide array of post- translational modifications for its regulation, including: phosphorylation of Tyr307 of subunit PP2AC [62], reversible carboxylation of subunit PP2AC [63], and numerous phosphorylations on the subunit PP2AB [64, 65]. Besides modifications to its own

6 structure, PP2A also has several intracellular inhibitors: I1PP2A (PHAP-I) and I2PP2A

(PHAP-II, SET, TAF-1β) [66, 67]. SET has drawn the most attention due to its evidence in cancer [68], especially leukemia [50, 69-71]. However, the exact mechanism that the two inhibitors use to inhibit PP2A remains a mystery; current belief is that PP2A inhibitors bind directly to PP2AC, hindering the phosphatase activity [72, 73]. Figure 1.1 highlights many of the known interacting proteins found to be associated with PP2A [72].

The list of direct and indirect targets of PP2A is an extensive, and implicates a number of potent tumorigenic proteins. PP2A inhibition has been shown to increase the activity of the oncogene c-Jun responsible for growth-stimulatory signals [74]. p53, a powerful tumor suppressor implicated in many cancers, is heavily regulated through its phosphorylation state [75, 76]. PP2A has been shown to dephosphorylate p53, inactivating it [77], as well as affecting the ability of MDM2 from negatively-regulating p53 [54]. Also, when PP2A is inhibited, the oncogene Rb is shown to be phosphorylated, leading to its activation [78]. In addition, PP2A regulate several key kinases that have been revealed to be involved in tumorigenesis, as well, including: ERK/MAPKs, the calmodulin-dependent kinases, PKA, PKB, PKC, IκB kinases and the CDKs [72, 79].

Also, PP2A interacts with a number of important pro-apoptotic factors: caspase-3 [80],

Bcl2 [81], and adenovirus E4orf4 protein [82]. Besides PP2A being found to be under expressed in a number of leukemias [40, 50, 83], one of the most common genetic changes inherent to B-CLL, deletion of chromosome 11q22-q23 [84], is also the location of the gene for PP2A. Ergo, the study of PP2A activation and overall function in regards

Figure 1.1 The serine/threonine protein phosphatase 2A, PP2A, is one of the most ubiquitous phosphatases within a cell. It has been implicated in dozens of pathways, and interacting with nearly as many individual pathways. Listed here are but a few of those interacting proteins. Of particular note are SV40 small t, SET, and PHAP-1; these three interacting proteins are all inhibitors of PP2A, and known cancer-inducers [72].

to CLL is of paramount importance, and restoration of its function proves an attractive avenue for therapeutic development.

1.4 THE PP2A-ACTIVATOR FTY720

FTY720, a potent S1P agonist [28-30], is a myriocin-derived compound found in the fungi Myriococcum albomyces and Isaria sinclairii [30-32] has a diverse pallet of therapeutic uses. Originally tested with great success in the treatment of renal transplant 8 immunosuppression [33-35], and multiple sclerosis [36-38], it has recently been identified as a potential treatment for myeloid and lymphoid malignancies [39-41]; in fact, during treatment of SCID mice with CLL xenograft, treatment with FTY720 proved as effective as Rituximab, a front-line treatment for B lymphocyte malignancy treatment

[39].

The reason for FTY720’s ability to cause tumor apoptosis remains elusive, though there are a number of potential mechanisms for its activity. In an NK leukemia rat model, FTY720 was shown to increase sphingosine levels and decrease Mcl-1 resulting in increased survival [42]. Its ability to inhibit sphingosine kinase 1, a known perpetrator in cancer progression [43], offers a unique avenue of drug exploitation. Adding scientific merit to this path, sphingosine kinase 1 has been noted as a major component in imatinib- induced CML apoptosis [40]. As the potential role for FTY720 in cancer begins to take shape, additional disease states are under investigation, such as breast cancer [44]. A number of additional studies also point to FTY720 interfering with nutrient transport in leukemia cells [45], as well as the autophagic-lysosomal pathway in MCL [46]. While the brush of activity of FTY720 is wide, many of these studies carry a common thread; that is, the fact that FTY720 is a potent activator of the serine/threonine phosphatase

PP2A [41] [47].

1.5 CONCERTED EFFORT TO STUDY GLOBAL PHOSPHOPROTEOMICS

Phosphorylation of proteins can alter signaling pathways in cells that are responsible for differentiation, cell proliferation, cell cycle regulation, cell

9 adhesion/movement and gene expression [85]. The phosphorylation of proteins in eukaryotes occurs on only three amino acids: serine, threonine and tyrosine. Thanks in large part to the Human Genome project, it was discovered that from these three possible sites of phosphorylation, the human genome contains over 100,000 potential sites of phosphorylation [86]. Despite their importance, the study of phosphorylated proteins has been slow and fraught with difficulty.

While it is believed that nearly one-third of proteins within a cell can be phosphorylated at any given time [87], their abundance within the cell account for a mere fraction of the global protein profile of that cell. Combine their low abundance with their transient state and susceptibility to phosphatases, pH changes and environmental factors

[88] and the study of phosphorylated proteins can become an act in futility. Thus, any study involving phosphorylated proteins must be carefully planned and executed. Until recently, a majority of phosphoprotein studies relied on immunoaffinity assays (e.g. [32P] orthophosphate metabolic-labeling, immunoblotting and immunoprecipitation).

However, only phospho-tyrosine antibodies are reliable enough to perform such immunoaffinity assays [89]. The use of phosphor-serine and phosphor-threonine antibodies in the discovery and characterization of phosphoproteins has been extremely rare [90] due to the fact that phospho-serine and threonine antibodies tend to be highly variable in their effectiveness because of the confirmation of the epitope existing at each of these phosphorylation sites [89]. And with tyrosine phosphorylations only accounting for a mere 0.05% of the phosphoproteome [30], the use of immunoaffinity assays would

10 miss a large number of phosphoproteins. A summary of common phosphoprotein techniques are reviewed in great detail from Schmidt, et al. [89] (Figure 1.2).

In recent years, a vast majority of the advances made in phosphoprotein studies has been made due to the advancement of mass spectrometers [91]. With the development of powerful mass spectrometers such as the Fourier transform-based LTQ-

Orbitrap, not only is it possible to identify phosphorylated proteins, but it is also possible to identify the specific sites of phosphorylation on the protein [92] allowing for functional studies to occur. The increased resolving power, mass accuracy and dynamic range of Fourier transform mass spectrometers offer a marked improvement over the low- resolution ion traps and mass spectrometers of old. However, despite the great strides

Figure 1.2 There are a vast array of techniques and methods currently available for the isolation and study of phosphorylated proteins. This figure highlights many of the more common methods available to researchers today. Both homemade methods and company-supplied kits have proved to be viable candidates for the study of this elusive post-translational modification [89].

achieved in mass spectrometer technology, they are not fool proof. The massive amounts of protein found in cellular lysate are still much too complex for many mass spectrometers to identify phosphoproteins or phosphopeptides accurately [86, 93]. To increase the ability of the mass spectrometer to identify phosphorylated proteins and their specific sites of phosphorylated, a dedicated enrichment process of not only phosphoproteins after cell lysis is necessary, but also the enrichment of those peptides containing phosphorylated residues (after protein digestion) is crucial for successful study of the phosphoproteome of a given cell [89, 93, 94]. One of the most widely-used enrichment process to date for phosphoproteins has been the use of immobilized metal 12 affinity chromatography (IMAC) [89, 95], which can yield a recovery of 70-90% of the phosphoproteins in a cellular lysate [96, 97]. For phosphopeptides, the use of metal oxides is essential, with Titanium dioxide being preferred for single-phosphorylated peptides [98] and Zirconium dioxide preferred for multiple phosphorylated peptides [99].

While no single enrichment or identification method is suitable for an entire phosphoproteome [100], an appropriate process can be created depending on individual investigator’s needs.

1.6 SUMMARY OF RESEARCH

The purpose of this thesis was to develop and establish a robust and efficient method for the enrichment of phosphorylated proteins in primacy CLL B cells; specifically, following manipulation of the phosphoproteome within primary CLL B cells by therapeutic agents, such as FTY720. In order to achieve this method, verification and validation was necessary on a homogenous cell population, and given the use of the

Ramos Burkitt lymphoma cell line for previous FTY720 studies [39], it was a natural candidate for validation of this method.

Chapter 2 will discuss the first kit utilized for method development, the Phos-Tag

Zn2+ Phosphoprotein Enrichment Kit, formerly available through Perkin Elmer. Through both Western blot analysis and mass spectrometry-based workflows, a paradigm was established for the kit’s effectiveness. Effectiveness not only established in the identification of phosphorylated proteins, but also in the identification of potential therapeutic targets and potential avenues of FTY720 mechanisms in the Ramos cell line.

Chapter 3 further develops phosphoprotein enrichment in the Ramos cell line utilizing the TALON PMAC Phosphoprotein Enrichment Kit from Clontech. This kit offered several advantages over the Phos-Tag technology of Chapter 2, and presented a necessary staging point for the transition to primary CLL B cells. Following Western blot validation of the TALON PMAC Phosphoprotein Enrichment Kit, mass spectrometry replicates were tested for consistency and statistical similarity. After all experiments had concluded, a technique of phosphoprotein enrichment was established through a modified method utilizing the TALON PMAC Phosphoprotein Enrichment Kit.

Chapter 2

PHOSPHOPROTEOMIC INDENTIFICATIONS IN RAMOS POINT TO A POTENTIAL ROLE OF THE PP2A INHIBITOR SET IN THE MODE OF FTY720 ACTION.

2.1 INTRODUCTION

As has been mentioned earlier, the study of phosphorylated proteins and their signaling cascades is a difficult process fraught with missed opportunities and murky results. This is due in large part to their transient nature and their low abundance within the cell. Phosphorylated proteins account for a mere fraction of the total protein load of a cell [101], and more often times than not, the phosphorylated form of a particular protein does not account for the majority of that protein’s form within the cell.

Enrichment of these proteins offers the best mechanism for their study. However, the number of techniques, kits and chemical processes to isolate these number into the dozens, with each claiming high yields of pure, unadulterated phosphoprotein goodness.

In order to understand these techniques and kits, though, you first must understand the science behind it.

A phosphorylation, at its simplest definition, is an addition of a phosphate group

(PO4) that adds a negative charge to the protein, leading to an alteration in its structure,

15 and thus, a different function is bestowed upon it. Exploiting the phosphate group’s negative charge is paramount for its enrichment; however, there exist two amino acids that also have negative charges, aspartic acid and glutamic acid, which can lead to inefficient enrichment processes. Thus, technologies must account for this.

IMAC enrichment techniques (Immobilized Metal Affinity Chromatography) are some of the most popular techniques for global enrichment of phosphoproteins and their subsequent study [101]. The simplicity of the systems allows for a robust and reproducible method to be developed and tailored for individual investigators’ needs.

At its most basic, IMAC consists of a resin-based matrix (the two most common being iminodiacetic acid and nitrilotriacetic acid [102]) that is associated with a particular metal ion. Several metal ions are currently in use for phosphoprotein enrichment, including: Fe3+ [102], Ga3+, Zr4+, and Ti4+ [103]. All have shown success in enrichment.

A variant of IMAC is a technology called Phos-Tag, which utilizes an alkoxide-bridged dinuclear Zn (II) [104] metal to enrich phosphorylated proteins. This was shown to have great success in EGF-stimulated A431 cell lysate, with an enrichment percentage of phosphoproteins greater than 90% [105], indicating a minimum loss of phosphorylated proteins. Therefore, the modified IMAC technique of Phos-Tag was utilized for preliminary studies on the mechanism of FTY720.

2.2 EXPERIMENTAL

2.2.1 Materials

In order to properly assess the reproducibility and proper enrichment of the Phos-

Tag method, all methods were optimized with the Burkitt lymphoma cell line Ramos, which is a model system for FTY720 study [39], and would make for an excellent transition to primary CLL B cells. Ramos cell line and primary CLL B cells were available through the laboratory of Dr. John Byrd. Phosphoproteins were enriched from an in-house lysis buffer through the Phos-Tag technology (Perkin Elmer Inc., MA). 4-

15% gradient SDS-PAGE gels, Coomassie Stain and Coomassie Bradford protein assays were purchased through Bio-Rad Laboratores Inc. (Hercules, CA). Nitrocellulose 3,000

MWCO filters were acquired from Millipore (Bedford, MA). Okadaic acid and 1,9- dideoxy-Forskolin were purchased from Sigma-Aldrich (St. Louis, MO). SET antibodies were purchased from Cell Signaling (Danvers, MA) and Globozymes (Carlsbad, CA).

GAPDH and Hsp90 antibodies were purchased through Santa Cruz Biotechnology (Santa

Cruz, CA). Rabbit TrueBlot with immunoprecipitation beads was available through eBioscience (San Diego, CA).

2.2.2 Cell drugging and lysate preparation

Blood was obtained from patients with immunophenotypically defined CLL as outlined by the modified 1996 National Cancer Institute criteria [106]. All of the patients had been without prior therapy for a minimum of 30 days at the time of collection and had informed consent in accordance with the Declaration of Helsinki and under a

17 protocol approved by the Institutional Review Board of The Ohio State University

(OSU). CLL B cells were isolated from freshly donated blood with Ficoll density gradient centrifugation (Ficoll-Plaque Plus, Amersham Biosciences). Enriched CLL fractions were prepared with the use of the “Rosette-Sep” kit from StemCell

Technologies according to the manufacturer’s Instructions. Both primary CLL B cells and Ramos cells were cultured in RPMI 1640 media (JRH Bioscience, Lenexa, KS), which was supplemented with 10% fetal bovine serum, 56 U/ml each of penicillin and streptomycin, and 2mM L-glutamine (Life Technologies, Carlsbad, CA) at 37°C in an

8 6 atmosphere of 5% CO2. 1x10 Ramos cells total, at 1x10 cells/ml were drugged with either 5 µM FTY720 provided by Dr. Ching-Shih Chen or an equivalent amount of

DMSO vehicle control (Thermo-Scientific, Sunnyvale, CA). Once treated, Ramos cells were incubated at 37°C in an atmosphere of 5% CO2 for an additional 4 hours, as at 4 hours PP2A enzymatic activity is at its highest [39]. Cells were then harvested, pelleted, and resuspended in ice-cold RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1%

NP-40, 1% sodium deoxycholate, 0.1% SDS) containing phosphatase inhibitors 1 and 2 and Sigma protease inhibitor (all at 1:100) (Sigma-Aldrich, St. Louis, MO), as well as 10 mM sodium fluoride and 1 mM sodium orthovanadate to ensure preservation of phosphorylations. Cellular pellets were then vortexed and placed on ice for 10 minutes and then repeated twice more to ensure total lysis. Lysates were then quantified by

Coomassie Bradford assay and stored at -80oC until further use.

2.2.3 Phos-Tag Phosphoprotein Enrichment

The enrichment process was carried out as described for the Phos-Tag enrichment protocol (Perkin Elmer Inc., MA). Briefly, approximately 2 mg of cell lysate was diluted to 0.1 mg/ml in RIPA lysis buffer. Phos-Tag Zn2+ beads are loaded onto a column, approximately 1 ml of the agarose bead slurry. The column and slurry are then washed with 5 ml of RIPA lysis buffer prior to the sample. The diluted sample is then added to the column and allowed to pass through the agarose slurry via gravity and collected as

‘nonphosphorylated protein’. The phosphoproteins, bound to the agarose beads, are eluted out with 2.5 ml of elution buffer (100 mM Na2HPO4 pH 7.0). The collected phosphoprotein fraction is then concentrated and undergoes dialysis against HPLC-grade water for 6 hours at a 1:500 ratio sample to water, with fresh water added every 2 hours.

Upon completion of dialysis, samples were placed in a SpeedVac to complete dryness and resuspended in 20 ul of HPLC water. A Bradford protein assay was then performed on the phosphoprotein elutent to determine protein recovery following Phos-Tag enrichment.

2.2.4 SDS-PAGE and Pro-Q staining

Approximately 30-50 µg of enriched phosphoproteins combined with SDS loading buffer and boiled for 5 minutes, from Ramos cells were separated by 4% - 15%

SDS-PAGE set at 90 volts for 3 hours to allow for optimal separation. Precise plus dual color protein standard ladder (Bio-Rad, Hercules, CA) is added to the gel, as is bovine casein protein, a highly phosphorylated protein used as a positive control for the Pro-Q

19 staining. Following its run, the SDS-PAGE gel was placed in 100 ml of fixing solution

(50% methanol, 10% acetic acid) and incubated at 4oC overnight to ensure complete preservation of proteins held within. Following fixing, the gel was washed three times in ultrapure water for approximately 30 minutes to remove all fixing solution. 100 ml of

Pro-Q Diamond phosphoprotein stain was used to incubate the gel, protected from light, with gentle agitation for 2 hours. Still protected from light, the gel was destained three times with 100 ml of destaining solution (20% acetonitrile, 50 mM sodium acetate, pH

4.0) for 30 minutes each. Finally, the gel washed three times with ultrapure water, 10 minutes apiece. The gel was then imagined on a Typhoon 9410 Imager (GE Healthcare,

Piscataway, NJ) by fluorescence at excitation wavelength of 523 nm. Afterward, the gel was subjected to Coomassie stain solution (25% isoproponal, 10% acetic acid, 0.25%

Coomassie brilliant blue) to stain for total protein for 1 hour. Destain solution (30% methanol, 10% acetic acid) was then added until the background was clear; typically overnight at 4oC.

2.2.5 Band excision and in-gel digestion

Following the Coomassie destaining and imaging, the SDS-PAGE gel was placed on a clear glass plate, with gel bands cut as closely as possible without extraneous gel material to aid in digestion. The gel bands were then diced and placed into a 96-well plate. Ge bands were soaked in 200 µl wash solution (50% methanol, 5% acetic acid) and allowed to soak overnight. Wash solution was then removed and dried by resuspension in 200 µl acetonitrile for 5 minutes. The gel bands were dried in a

Speedvac and 75 µl dithiothreitol (5 mg/ml in 100 mM sodium bicarbonate) was added to the gel bands and incubated at room temperature. After removal of the dithiothreitol, 75

µl iodoacetaminde (15 mg/ml in 100 mM sodium bicarbonate) was added and incubated for 30 minutes in the dark at room temperature. The iodoacetaminde was removed and the gel bands were washed with 200 µl 100 mM sodium bicarbonate, then dehydrated, then washed, then dehydrated once again, then repeated once more for a total of three wash steps and three dehydrations. After the final dehydration, gel pieces are dried in a

Speedvac for three minutes. Once dry, 30 µl of Promega sequencing-grade trypsin

(V5111) (Madison, WI) resuspended in 25 mM sodium bicarbonate was added to the gel bands and incubated at 37oC for two hours. 40 µl of extraction buffer (50% acetonitrile and 5% formic acid) was then added to the gel pieces and incubated at room temperature for 10 minutes. The solution was collected and repeated twice more. The extraction solution was then concentrated in a Speedvac to approximately 10 µl.

2.2.6 Capillary ESI-HPLC-MS/MS

In-gel digest phosphoprotein samples, following tryptic digest and reconstitution in 0.3% trifluoroacetic acid loading buffer, were then subjected to separation on a 150 cm

C18 capillary column (Michrom Biosources Inc., Auburn, CA) on an Ultimate 3000

HPLC system (Dionex, Thermo Scientific, Sunnyvale, CA) at a flow rate of 2 µl/min flow and an established gradient (5% - 50% B, 0-144 min, 50% - 75% B, 144-160 min,

75% - 90%, 160-184 min, 90% - 90% B, 184-212 min) consisting of mobile phase A

(0.1% formic acid in HPLC-grade water) and B (0.1% formic acid in acetonitrile).

Following separation, the samples would enter an LCQ-Deca XP (Finnigan, Thermo

Scientific, Sunnyvale, CA) operating in positive ion mode and data-dependent neutral loss MS3 mode. Full MS scans (m/z 300-2000) were followed by subsequent MS2 scans of the top five most abundant peptide ions utilizing normalized collision energy of 35%.

When a neutral loss of 98.0 Da, 80.0 Da, 49.0 Da, 40 Da, 32.7 Da or 26.7 Da is detected, the MS3 scan is triggered to isolate and fragment the corresponding neutral loss product ion observed in the MS2 scan.

2.2.7 Data analysis

The RAW data files from the LCQ-Deca XP are converted to the MzXML format for protein database searches with MassMatrx [107]. The MS data sets are searched against the Uniprot human protein database (VERSION DATE 2009-11-03). Trypsin was chosen as the enzyme for digestion with 2 missed cleavages. Variable post- translational modifications of phosphorylation of tyrosine, serine and threonine were allowed. The mass accuracy ranges were defined as 10 ppm for the precursor ion and 1

Da for the product ions with a peptide length of 6-35 amino acids. False discovery rates were estimated using an appended reverse decoy database as described Elias et al [108].

Protein abundance changes were determined by comparing spectral counts across treatments [109] [110]. Statistically significant differences across treatments were evaluated by use of the G-test. The resulting p-value was used to rank protein abundance difference across treatment groups.

2.2.8 Western blotting and immunoprecipitation

Cell lysates were prepared as mentioned earlier in 2.2.2 and quantified by

Coomassie Bradford protein assay. Lysates with 50 µg of total protein were separated using 10% SDS-PAGE at 90 volts for 3 hours, then transferred to 0.2-µm nitrocellulose membranes (Schleicher& Schuell, Keene, NH). The blots were probed with indicated primary antibodies at a 1:1000 ratio unless otherwise stated. Secondary antibodies with horseradish peroxidase (HRP)–conjugated goat anti-rabbit or goat anti-mouse IgG (Bio-

Rad Laboratories, Richmond, CA) were then added for detection and the chemiluminescent substrate (SuperSignal; Pierce) was added for development.

Immunoprecipitation was performed as per TrueBlot-specific method. Briefly, wash 50

µl of Anti-Rabbit IgG bead slurry and wash with kit-provided lysis buffer. To the pre- equilibrated bead slurry, add approximately 500 µg of cell lysate. Place on rocker at 4oC for 1 hour. Centrifuge at 2,500 xg for 3 minutes and transfer supernatant to a new tube.

Add 2 µg of SET antibody to the pre-cleared cell lysate and return to rocker at 4oC for an additional 1 hour. Add 50 µl of pre-equilibrated bead slurry to capture the protein- antibody complexes. Incubate overnight at 4oC. Centrifuge the tube at 2,500 xg for 30 seconds at 4°C. Remove supernatant completely and wash the beads 3-5 times with 500

μl of cold kit lysis buffer, centrifuging to pellet beads in between each wash. After the final wash, add in 50 μl of 6X SDS-PAGE sample loading Buffer to bead pellet.

Boil pellet and loading buffer for 10 minutes, then centrifuge at 10,000 xg for 5 minutes, collect supernatant carefully and load onto SDS-PAGE gel.

2.3 RESULTS AND DISCUSSION

2.3.1 Phosphoprotein enrichment of Ramos cells by Phos-Tag Zn2+

Whole proteins were extracted from Ramos cells treated with either 5 µM

FTY720 or an equivalent amount of DMSO vehicle control (1x108 cells total per treatment). An additional treatment group of Ramos cells were drugged with a combination of 5 µM Sodium fluoride and Sodium othrovanadate as a control for phosphorylation manipulation. In hindsight, proper controls for this sort of treatment would have included the PP2A activator 1,9-dideoxy-forskolin at 100 µM [111], which would mimic the properties of FTY720. The PP2A inhibitor Okadaic acid would also be used as a counter to PP2A activation at 5 nM [112]. Following an incubation time of 4 hours [39], Ramos cells were lysed, quantified by Coomassie Bradford assay and then applied to the Zn2+ column of Phos-Tag for phosphoprotein enrichment in accordance with the Phos-Tag protocol. Upon elution and subsequent clean-up, eluted phosphoprotein was quantified by Coomassie Bradford assay and approximately 50 µg of phosphoprotein-enriched protein lysate was added to a 4%-15% SDS-PAGE gradient gel for protein separation.

Direct digestion of eluted phosphoproteins would have proven problematic for

LC-MS/MS-based protein identification due to the highly robust and complicated nature of the lysate. A majority of the protein identifications garnered from such an approach would irrevocably identify highly abundant proteins such as cytoskeletal and metabolic individuals. To understand the signaling complexity of the Ramos phosphorylation

24 pathways, separation on a gradient SDS-PAGE gel was an excellent manner to allow for a simplified view of the complex proteome of the Ramos cells.

To ensure of the specificity of phosphoprotein enrichment, the SDS-PAGE gel was first subjected to Pro-Q Diamond Phosphoprotein stain. The gel unfortunately did not visualize due to the fact that the Pro-Q stain was an older batch. Instead of waiting for a fresh batch of Pro-Q to be ordered, we proceeded with Coomassie global protein stain (Figure 2.1). Despite the failure of the Pro-Q Diamond stain, the Coomassie stain was successful and between the FTY720-treated and DMSO-treated Ramos lysate lanes, a total of 12 bands (6 each from Treated and Untreated) were chosen for excision and in- gel digestion for LC-MS/MS protein identification.

2.3.2 LC-MS/MS Identification of differential phosphoproteins between FTY720 treated and untreated samples.

From the twelve bands excised from the SDS-PAGE gel, 121 proteins were identified between the Ramos cells treated with FTY720 and Ramos cells treated with the vehicle-control DMSO at the 95% confidence interval as determined by MassMatrix utilizing reverse decoys. From that list, 68 proteins were uniquely identified in the vehicle-control DMSO treatment group (Table 2.1), 45 proteins were uniquely identified in the FTY720 treatment group (Table 2.2) with only 7 proteins shared between the two treatment groups (Table 2.3). Due to the proteins from Table 2.3 existing in both treatments, a statistical comparison was made between the groups by spectral counts of each protein as described previously, and an amount of each protein was determined from

25 these counts. From there, a statistical fold change was determined. The results from this statistical analysis will be discussed in 2.3.4.

Figure 2.1 Coomassie staining of phosphoproteins enriched from Ramos lysate treated with either 5 µM FTY720 (Treated, Lanes 4 and 5), an equivalent dose of DMSO (Untreated, Lanes 6 and 7), or a combination of sodium fluoride and sodium orthovanadate to mimic inhibition of phosphatases (P. Inhibited, Lanes 8 and 9). A dual color protein ladder was utilized (DCP ladder, Lanes 1 and 10) to aid in the identification of proteins bands to be excised. Beta-casein, a highly phosphorylated protein, was utilized as a positive control for the Pro-Q diamond stain. Totally Ramos lysate was added to compare band placements to phosphoprotein enriched fractions. 12 individual bands were excised from the gel for in-gel trypsin digestion followed by LC-MS/MS protein identification.

Of particular interest in the list of proteins was a known inhibitor of PP2A;

I2PP2A (SET) [113] [73]. This was identified to be phosphorylated in the vehicle- control DMSO treatment group but was completely absent in the FTY720 treatment group’s chromatogram (Figure 2.2a), signifying a loss of SET phosphorylation following

FTY720 treatment. When a particular spectra for SET was isolated at 879.9 m/z, containing the SET-specific peptide LNEQASEEILKVEQK, that spectra was identified within the DMSO-treated sample (Figure 2.2b), but not in FTY720-treated sample.

Unfortunately, MassMatrix did not identify a phosphorylation on this particular peptide fragment.

SET has two phosphorylation sites at serine 9 and serine 24 [114], although their role in SET function remains unknown. Through phosphorylation site identification software like KinasePhos [115], one can input an amino acid sequence for a protein and the software will identify potential phosphorylation sites and the potential kinase responsible for that phosphorylation. In this case, the two phosphorylation sites on SET are phosphorylated by PKG and CKII. This would indicate that through treatment with

Table 2.1 Overview of 68 unique identified proteins from 6 excised bands from Figure 2.1 following treatment with DMSO vehicle control, phosphoprotein enrichment and subsequent in-gel digestion followed by liquid chromatography and tandem mass spectrometry.

Protein Description Protein Score Untreated

HSP 84/90 261

Beta actin 248

Heat shock 70 kDa protein 8 237

HSPCA 230 heat shock 70kDa protein 8 isoform 1 variant 218

Alpha-actin-1 198

HSP 60 194

BiP 192

SET protein (PHAP II) 122

Nuclear autoantigenic sperm protein 113

PCNA 113

Eukaryotic translation elongation factor alpha 1 variant 87

ATP synthase beta chain, mitochondrial precursor 83

Proteasome alpha 1 subunit, isoform 2 81

PSMA7 80 hnRNP K 67

Ribosomal protein S3 66

Major histocompatibility complex class I antigen-binding protein p88 62

Nucleosome assembly protein 1-like 4 60

Proteasome subunit p42 60

Tyrosine 3/tryptophan 5 -monooxygenase activation protein 58

Ras-GTPase-activating protein binding protein 1 55

TCP-1-epsilon, delta, gamma, theta, zeta, beta 54

Tumor rejection antigen 1 52 retinoblastoma binding protein 7 48 hepatoma-derived growth factor protein 1-like 48

Heat Shock 70kDa protein binding protein 45

Continued 28

Table 2.1 (continued) hnrnp A2/B1 isoform A2 45

Lupus La protein (SS-B) 42

Protein disulfide-isomerase A6 precursor 42

KCIP-1 (protein kinase C inhibitor protein 1) 41

LOC388524 40 fructosyllysine-specific binding protein/nucleolin homolog 35

Tat binding protein 1 33

IGHM 32

SFRS1 31

Heat shock 70 kDa protein 6 30

TUBB3 30

Nucleosome assembly protein 1-like 1 29 eIF4A-I 29

Nucleosome assembly protein 1-like 1 29 protein for MGC:125802 29

Calreticulin precursor 28 acidic (leucine-rich) nuclear phosphoprotein 32 family, member A 27

DnaJ homolog subfamily A member 1 26

IMPDH - II 25 acidic (leucine-rich) nuclear phosphoprotein 32 family, member E 24

MAPRE1 24 hnrnp A1 isoform a variant 24

PKCSH 23

S-adenosylmethionine synthetase gamma form 23 eukaryotic translation initiation factor 3, subunit 2 beta 23

Heterogeneous nuclear ribonucleoprotein AB 22 calcium-binding tyrosine phosphorylation-regulated protein isoform a 21

Methylosome subunit pICln 21

XPA binding protein 2 20

Continued

Table 2.1 (continued)

TFIIF-alpha 19 d(TTAGGG)n-binding protein B39=type E hnrnp 19

Complex Structure of the C-terminal Rna-binding Domain of Hnrnp D 19 eukaryotic translation initiation factor 5 18 eukaryotic translation initiation factor 4a isoform 2-like protein 17

Plasminogen 17

ATP5A1 16 hRap1 16 elongation factor 1-beta 16

PP1-gamma 15

TATA box-binding protein-associated factor 2F 15

Table 2.2 Overview of 45 unique identified proteins from 6 excised bands from Figure 2.1 following treatment with 5 µM FTY720, phosphoprotein enrichment and subsequent in-gel digestion followed by liquid chromatography and tandem mass spectrometry.

Protein Description Protein Score Treated

Treacher Collins-Franceschetti syndrome 1, isoform c 222 nucleolar and coiled-body phosphoprotein 1 (p130) 102

Nuclear ubiquitous casein and cyclin-dependent kinases substrate 93 pp52 87

KIAA0324 protein 87 structure specific recognition protein 1 72 lymphocyte-specific protein 1 isoform 2 (pp52) 60

Multifunctional protein ADE2 (PAICS) 59

YBX1 52

YB-1 52

Nopp140 (p130) 49 nucleolar and coiled-body phosphoprotein 1 49

Solute carrier family 9 39

Zinc finger protein 265 39

Glucosamine-6-phosphate deaminase 1 37

PAI-1 mRNA-binding protein 36

DEK 32 hnRNP D 30 hepatoma-derived growth factor-related protein 2 isoform 1 29

PSIP1 protein (p52, p75) 29

FUSE binding protein 1 23 hnRNP E 23 hnRNP AB 21 chromosome X open reading frame 9 20

BRG1-associated factor 60B 20 pregnancy specific beta-1-glycoprotein 11 20

Continued

Table 2.2 (continued) platelet-type 12-lipoxygenase/arachidonate 12-lipoxygenase 17

NIP30 17

WBP11 16

PPP1R2 16

OTTHUMP00000018218 15 microtubule-associated protein 4 15

Sorting nexin-4 15

2'-5'-oligoadenylate synthetase 2 14

ESR2 14

Anaphase-promoting complex subunit 4 13

Phosphatidylinositol 4-kinase type-II beta 13 complement factor H-related 3 13

Wolf-Hirschhorn syndrome candidate 1 protein isoform 3 13

ELMO2 12 adaptor-related protein complex 3 12

Gdp-Bound Rab21 Gtpase 12

ALL1 fused gene from 5q31 12

Table 2.3 Overview of 7 shared proteins identified across all 12 excised bands from Figure 2.1 following following treatment with either 5 µM FTY720 or DMSO vehicle control, phosphoprotein enrichment and subsequent in-gel digestion followed by liquid chromatography and tandem mass spectrometry.

Protein Description Protein Score Untreated Protein Score Treated

HSP 90 579 43

Nucleophosmin 152 222

NAC-alpha 105 133

Nucleolin 65 146 splicing factor, arginine/serine-rich 2 variant 33 81

ADP-sugar pyrophosphatase 16 34 immunoglobulin E variable region 15 15

Figure 2.2 From Table 2.2, the PP2A inhibitor SET was identified from several peptides by MassMatrix. According to the search engine, the SET protein was identified within excised gel bands 4 and 5 of the Untreated Ramos samples as observed in Figure 2.1. However, MassMatrix did not identify the SET protein in the excised bands from Treated Ramos samples, as seen in this chromatogram (A). One particular peptide, identified at peak 879.9 m/z was specific for SET (B). A search of that peak within the Treated Ramos sample datasets does not yield a single hit, thus cementing the fact that the SET protein was not enriched for following phosphoprotein enrichment.

FTY720, one or both of these pathways are affected by the treatment resulting in the loss of phosphorylation on SET. Ideally, either a phosphorylation-specific antibody for SET would verify the observations in the mass spectrometry protein ID. Unforunately, no such antibody exists. Phosphomimetic mutants of SET would be necessary to understand each phosphorylation site’s role in SET function as well. However, as a purely intellectual endeavor, this predictive software can aid in the direction of future experiments.

2.3.3 Protein expression of the PP2A-inhibitor SET in Ramos and primary CLL B cells.

SET has been thoroughly investigated in chronic myelogenous leukemia and has been shown to be critical for the function of FTY720 in that disease [40]. SET also has a potential role in the pathogenesis of CLL and a potential therapeutic target [116].

Besides CLL, SET was identified as being present in numerous leukemias and is known to fuse with other oncogenic instigators HRX [117] and CAN [118] in those particular leukemias.

As of yet, no data existed for SET expression levels in primary CLL B cells to the best of our knowledge; thus, it was crucial to identify the relative amounts of the two isoforms of SET in primary CLL B cells, especially in comparison to normal B cells

(Figure 2.3). Surprisingly, there was no difference between the two isoforms’ expression levels in primary CLL cells and normal B cells.

The obvious next step in the investigation of SET’s importance in the action of

FTY720 was to determine if treatment with FTY720 had an effect on SET protein

Figure 2.3 A SET Western blot in Normal, leukopack-acquired B cells were compared to CLL B cells, isolated as described previously. Ramos lysates were used as a positive control for SET, and isolated bovine β-Casein as a negative control (n=3). The SET antibody recognized two isoforms of the protein: α and β. Quantitation of gel band intensities were carried out as described previously.

expression to corroborate what had been observed in our protein list. A Western blot was performed utilizing the same Ramos lysate samples as the lysates used to generate the results of the LC-MS/MS proteomic data, Ramos cells were treated with 5 µM FTY720 or vehicle-control DMSO. As Figure 2.4 shows, there is no difference in SET protein expression observed in Ramos cells treated with FTY720 and vehicle-control DMSO.

This was not totally unexpected, however; the difference between FTY720 and vehicle-

Figure 2.4 A fraction of the Ramos cells treated as described in 2.3.1 were lysed and subsequent Western blots were performed on the individual samples; primarily, the FTY720-treated Ramos cells (Treated) and the DMSO, vehicle-treated (Untreated) samples were run (n=2).

control DMSO was at the phosphorylation level, not total protein level. This is further confirmation that the difference in SET levels as observed in Figure 2.2 was due to an actual phosphorylation state change and not an artifact of the LC-MS/MS method or a difference in total protein levels between treatment lysates.

2.3.4 Potential inhibition of the Hsp90 pathway by FTY720.

Hsp90 is an important chaperone protein within the cell; that is, it allows for the proper folding of proteins into their respective functional state. Unfortunately, this ability can allow for tumorigenesis to flourish within a cell. Known tumorigenic proteins such as HER2, EGFR, HIF1α, Raf-1, and most interesting to our phosphorylation studies,

AKT [119] are all folded by Hsp90, and most damaging, are stabilized by their interaction with the chaperone protein. Due to this role in carcinogenesis, Hsp90, in recent years, has become a focal point for targeted therapies [120].

17-DMAG was one such therapeutic in acute myeloid leukemia [121], though its further development was halted due to unfavorable side effects in a Phase I clinical trial

[122]. Retaspimycin had much more favorable outcome, with Phase I and II clinical trials in non-small cell lung carcinoma, multiple myeloma, breast cancer, prostate cancer, gastrointestinal stromal tumors, metastatic melanoma and metastatic kidney cancer, all of which are showing great promise [123].

It was both with its role in biology and therapeutic potential that Hsp90 was investigated further from the proteomic output of 2.3.2. It would be easy to dismiss the presence of Hsp90 in the dataset, however. It has long been reported that Hsp90 is one of the most prevalent proteins within a cell; cancer cells reportedly have 4 to 6 times the amount of Hsp90 as normal cells [124]. There was, however, precedence for its investigation. Utilizing in-house statistical software comparing the FTY720-treated protein list with the DMSO-treated protein list, phosphorylation enriched Hsp90 showed a statistically significant difference in abundance. The fold changes were determined from the difference in spectral counts observed for each protein across treatments.

Spectral counts have long been lauded as a straightforward approach to semi-quantitative protein expression levels in proteomic data sets [125]. Phosphorylation enriched Hsp90 was 16-fold higher in the DMSO-treated sample set than the FTY720-treated set (Table

2.3).

This finding was very interesting. It has been reported that Hsp90’s phosphorylation is directly related to its chaperoning function [123]. FTY720 treatment may alter Hsp90’s ability to chaperone tumorigenic proteins within Ramos cells, which results in cellular arrest and/or apoptosis. Western blots were performed to validate the difference in phosphorylation of Hsp90 between the two treatment groups. To enhance the detection of phosphorylation, Ramos cells were treated with 20 µM FTY720 instead of the 5 µM used previously. The point here was not to create a physiologically relevant treatment, but rather, to understand the mechanism behind FTY720 action.

Despite the apparent loss in phosphorylation, total Hsp90 levels did not differ dramatically between FTY720-treated and DMSO-treated samples (Figure 2.5). The concurrent treatment of Ramos cells with the PP2A inhibitor Okadaic Acid and the PP2A activator 1,9-dideoxy-Forskolin also did not influence Hsp90 levels in a dramatic fashion.

These data would indicate that at the total protein level, FTY720 did not alter Hsp90 levels. Also, any effect seen at the total protein level most likely did not occur because of

FTY720’s effect on PP2A, as both the PP2A inhibitor and activator studies showed no complement pattern on Hsp90 expression. Further studies with primary CLL B cells were no less conclusive, with some patients showing a mild effect on Hsp90 levels, and others showing a dramatic increase in Hsp90 levels following FTY720 treatment (Figure

2.6). This result was not surprising when taking into account the following: 1) FTY720 treatments were performed on a Burkitt lymphoma cell line and 2) the primary CLL lysates may be of poorer quality as indicated by weak GAPDH loading controls.

To understand the role in Hsp90 phosphorylation further within the Ramos cells, a Western blot was run for a co-chaperone of Hsp90, Cdc37. Cdc37 is vital for the both

Hsp90 function [126], as well as kinome function; as almost 90% of kinases require

Cdc37 for their function [127]. This offered an attractive avenue of investigation in regards to phosphoproteomic studies. Indeed, Western blot analysis indicates that at 4 hours, 20 µM FTY720 Cdc37 is decreased dramatically; by as much as 70% (Figure

2.7). The loss of phosphorylation of Hsp90, coupled with the loss of Cdc37 led to the hypothesis that AKT may become unstable as a loss of the key stability components of

Hsp90 and Cdc37, as has been reported previously [128]. Total AKT levels dropped

85% when compared to control (Figure 2.7). In spite of these observations in Ramos

Figure 2.5 Hsp90 Western blot was performed on Ramos cells as described previously. They were dosed with 20 µM FTY720, an equivalent volume of DMSO, 5 nM Okadaic acid or 100 µM 1,9-dideoxy-forskolin to mimic PP2A loss of activity or gain, respectively. Gel band intensity analysis was performed as described previously.

Figure 2.6 Hsp90 Western blot was performed on primary CLL B cells as described previously. They were dosed with 20 µM FTY720, an equivalent volume of DMSO, 5 nM Okadaic acid or 100 µM 1,9-dideoxy-forskolin to mimic PP2A loss of activity or gain, respectively. Gel band intensity analysis was performed as described previously (n=3).

cells, the results in primary CLL B cells were inconclusive (Figure 2.8), indicating little to no effect on either of these proteins within this model.

Despite the tantalizing possibility of FTY720 effecting Hsp90 stability and resulting in its loss of function, as indicated by the dramatic decreases in Cdc37 and AKT protein levels, this observation did not hold up in primary CLL B cells. The literature of

Hsp90 interplay with kinases is extensive, and points to an effective route of molecular targeted therapy in the future. However, there exist far more specific inhibitors of Hsp90

41 than FTY720 [123]. While this in an interesting observation, it is more likely a red herring finding, and thus, not the actual route for which FTY720 enacts apoptosis.

Figure 2.7 Ramos cellular lysates from Figure 2.5 were additionally probed for AKT and Cdc37 to understand the functionality of Hsp90 following FTY720 treatment. They were dosed with 20 µM FTY720, an equivalent volume of DMSO, 5 nM Okadaic acid or 100 µM 1,9-dideoxy-forskolin to mimic PP2A loss of activity or gain, respectively. Gel band intensity was performed as described previously, with values compared to DMSO (n=3).

Figure 2.8 Western blots from Figure 2.6 were additionally probed for AKT and Cdc37 to understand the functionality of Hsp90 following FTY720 treatment. They were dosed with 20 µM FTY720, an equivalent volume of DMSO, 5 nM Okadaic acid or 100 µM 1,9-dideoxy-forskolin to mimic PP2A loss of activity or gain, respectively. Gel band intensity was performed as described previously (n=3).

2.4 CONCLUSION

The understanding of the phosphoprotein signaling web within a cell is paramount for the exploitation of it in therapeutic discovery and development. The purpose of this study was to create a phosphoproteomic workflow, from lysate to proteomic analysis, which would be robust, reproducible and easily implemented and tailored to specific investigators’ needs.

We identified 121 proteins in our freshmen expedition into phosphoprotein enrichment, and from that, indicated a differential phosphoprotein profile between 45 unique FTY720-treated protein hits and 67 DMSO-treated protein hits within treated

Ramos lysates. Several key players in both cancer and specifically in PP2A activity were 43 identified within those profiles that appear to be affected by FTY720; namely, the PP2A inhibitor SET and the global protein chaperone Hsp90. Unfortunately, while Western blots verified the differential observed within the proteomic workflows for Ramos, this did not translate to primary CLL B cells. Since CLL is the disease of interest, it is necessary to translate the workflow from the successful Ramos cell proteomic studies to primary CLL B cells.

Unfortunately, the phosphoprotein enrichment kit we were utilizing, Phos-Tag

Zn2+ from Perkin-Elmer, was no longer available for further validation experiments with primary CLL B cells, no further studies with Ramos cells. This loss of product coupled with extended optimization periods and replicates fraught with contamination and irreproducible results led to the conclusion that Phos-Tag was not an appropriate system for further phosphoproteomic work. Another enrichment system would be necessary for that. And thus, the search for such a system began anew.

Chapter 3

UTILIZATION OF THE PHOSPHOPROTEIN ENRICHMENT KIT TALON PMAC OFFERS A ROBUST AND REPRODUCIBLE METHOD FOR EXPLORING THE PHOSPHOPROTEOME OF LEUKEMIA.

3.1 INTRODUCTION

Despite the successes of our initial foray into phosphoprotein enrichment utilizing

Phos-Tag, including the identification of viable FTY720 targets, our results proved either inconclusive when translated to primary CLL B cells, or our subsequent Ramos proteomic experiments were irreproducible; not matching our earliest data, either with heavy keratin contamination or incomplete data lists due to poor sample preparation, sample digestion or potential loss of sample. Our ability to adequately troubleshoot the

Phos-Tag kit was tragically cut short, though, as it was no longer available for purchase and a new method for phosphoprotein enrichment had to be found.

A paper was published in 2007 [129], Combining Protein-Based IMAC, Peptide-

Based IMAC, and MudPIT for Efficient Phosphoproteomic Analysis, from Dr. Yates’ group at The Scripps Research Institute detailing a new phosphoprotein enrichment kit utilizing similar techniques to the now deceased Phos-Tag enrichment kit from Perkin- 45

Elmer. The TALON PMAC Phosphoprotein Enrichment Kit, available through

Clontech, is based on IMAC technology, utilizing magnetic beads coupled to matrix- infused Fe3+.

This technology has a number of benefits over the Phos-Tag system originally tested. First, it requires a dramatically smaller amount of lysate to perform enrichment.

The Phos-Tag kit required nearly two milligrams of lysate for enrichment, which would prove problematic where enrichment of primary CLL B cells were concerned due to limited sample amounts. TALON requires just 200 µg of lysate, thus ensuring no sample limitations on primary CLL B cells. Second, the conjunction of the IMAC technology with magnetic beads offer a streamlined and time-efficient method over the technology of

Phos-Tag, which could suffer from poor column packing and cost twice the time of the

TALON kit. Along with its associated publication from Dr. Yates, the TALON PMAC

Phosphoprotein Enrichment Kit has the potential to fill the requirement for a robust phosphoprotein enrichment method

3.2 EXPERIMENTAL

3.2.1 Materials

In order to properly assess the reproducibility and proper enrichment of the

TALON PMAC Enrichment Kit (Clontech, Mountainview, CA), all methods were optimized with the Burkitt lymphoma cell line Ramos. Ramos cell line and primary CLL

B cells were available through the laboratory of Dr. John Byrd. Recombinant human soluble CD40L was purchased from PeproTech (Rocky Hills, NJ). TCA was obtained

46 through Sigma-Aldrich (St. Louis, MO). LY294002 was from BIOMOL Research

Laboratories (Plymouth Meeting, PA). 4-15% gradient SDS-PAGE gels, Coomassie

Stain and Coomassie Bradford protein assays were purchased through Bio-Rad

Laboratores Inc. (Hercules, CA). Nitrocellulose 3,000 MWCO filters were acquired from

Millipore (Bedford, MA). RapiGest was obtained from Waters (Milford, MA). pSTAT1

(Ser727), pSTAT1 (Tyr701), pSTAT3 (Ser727), pSTAT3 (Tyr705), total STAT1, total

STAT3, pAKT (Ser473), pGSK-3β (Ser9), total GSK-3β, pPDK1 (Ser241), and total

PDK1 antibodies were purchased from Cell Signaling (Danvers, MA), while 4G10 antibody was from Millipore (Billerica, MA). GAPDH and Actin antibodies were purchased through Santa Cruz Biotechnology (Santa Cruz, CA).

3.2.2 Cell Drugging and Lysate Preparation

Technologies according to the manufacturer’s Instructions. Both primary CLL B cells and Ramos cells were cultured in RPMI 1640 media (JRH Bioscience, Lenexa, KS),

47 which was supplemented with 10% fetal bovine serum, 56 U/ml each of penicillin and streptomycin, and 2 mM L-glutamine (Life Technologies, Carlsbad, CA) at 37°C in an atmosphere of 5% CO2. 1x108 Ramos cells, or primary CLL B cells, total, at 1x106 cells/ml were drugged with either 20 µM FTY720 provided by Dr. Ching-Shih Chen or an equivalent amount of DMSO vehicle control (Thermo-Scientific, Sunnyvale, CA).

Once treated, Ramos or primary CLL B cells were incubated at 37°C in an atmosphere of

5% CO2 for an additional 4 hours, as at 4 hours PP2A enzymatic activity is at its highest

[39]. Cells were then harvested, pelleted, aspirated dry and frozen on a bed of dry ice, then resuspended in ice-cold Extraction/Loading Buffer from the TALON PMAC

Enrichment Kit, Sigma protease inhibitor (100:1) (Sigma-Aldrich, St. Louis, MO), as well as 10 mM sodium fluoride to ensure preservation of phosphorylations. Sodium orthovanadate was not utilized because of its ability to mimic a phosphate group and affect binding of phosphoproteins with the TALON PMAC binding matrix. Cellular pellets were then vortexed and placed on ice for 10 minutes and then repeated twice more to ensure total lysis. Lysates were then quantified by A280 on a NanoDrop™ NanoDrop

1000 Spectrophotometer (Thermo-Scientific, Sunnyvale, CA) and stored at -80oC until further use.

3.2.3 Phosphoprotein Enrichment

The enrichment process was carried out as described by the TALON PMAC

Magnetic Phosphoprotein Enrichment Kit (Clontech, Mountain View, CA). Briefly, 200

µg of protein lysate was diluted to 200 µl with Extraction/Loading Buffer and was added

48 to pre-equilibrated, aspirated-dry magnetic beads. Lysate and bead mixture was then added to a rotator at 4oC to rotate for one hour. The tube was placed on a magnetic separator and the supernatant collected, which contains the nonabsorbed, unphosphorylated proteins. The beads are washed three times with 500 µl of Wash buffer. After washing, 100 µl of Elution buffer was added and then rotated at room temperature for 5 minutes. This was repeated an additional three time for a total of four elutions. The 400 µl of eluted phosphoproteins then had 480 µl (1:1.2 ratio) of 1 mM

TCA dropwise (make fresh every four weeks) added to it and placed on ice for 1 hour, then centrifuged at 4oC at 14,000 RPM for 1 hour. Liquid is aspirated off without disturbing the faint white pellet at the bottom of the tube. The pellet is washed with 500

µl ice-cold acetone, and then incubated on ice for 5 minutes. It was then spun at 14,000

RPM, 4oC, for 20 minutes. Acetone is aspirated off without disturbing the pellet at the bottom. The acetone wash was repeated an additional two times for a total of three washes. Pellet is then air-dryed and resuspended in either 2X loading buffer for Western blot analysis or 25 mM sodium bicarbonate for in-solution digestion.

3.2.4 In-Solution Digestion

To the TCA-precipitated protein pellet, 90 µl 0.5% RapiGest as per their protocol, then 3 µl Promega sequencing-grade trypsin for a 1:30 ratio was added to the protein lysate pellet. It was then incubated at 37oC with gentle shaking overnight. To the digested peptides, 27 µl 100% formic acid was added, then placed at 37oC for one hour, then finally placed at 4oC for an additional two hours. Digested sampled was centrifuged

49 at 14,000 RPM for one hour and supernatant was aspirated off. This left the pelleted, digested peptides behind, which were then resuspended in 0.3% trifluoroacetic acid.

3.2.5 Capillary, ESI-HPLC-MS/MS

In-gel digest phosphoprotein samples, following tryptic digest and reconstitution in 0.3% trifluoroacetic acid loading buffer, were then subjected to separation on a 150 cm

C18 capillary column (Michrom Biosources Inc., Auburn, CA) on an Ultimate 3000

HPLC system (Dionex, Thermo Scientific, Sunnyvale, CA) at a flow rate of 2 µL/min flow and an established gradient (5% - 50% B, 0-288 min, 50% - 75% B, 288-320 min,

75% - 90%, 320-368 min, 90% - 90% B, 368-424 min) consisting of mobile phase A

(0.1% Formic acid in HPLC-grade water) and B (0.1% Formic acid in acetonitrile).

Following separation, the samples would enter a LCQ-Deca XP (Finnigan, Thermo

3.2.6 Data analysis

The RAW data files from the LCQ-Deca XP are converted to the MzXML format for protein database searches with MassMatrx [107]. The MS data sets are searched

50 against the Uniprot human protein database (VERSION DATE 2009-11-03). Trypsin was chosen as the enzyme for digestion with 2 missed cleavages. Variable post- translational modifications of phosphorylation of tyrosine, serine and threonine were allowed. The mass accuracy ranges were defined as 10 ppm for the precursor ion and 1

Da for the product ions with a peptide length of 6-35 amino acids. False discovery rates were estimated using an appended reverse decoy database as described Elias et al [108].

3.2.7 Western blotting

Cell lysates were prepared as mentioned earlier in 2.2.2 and quantified by A280 on a NanoDrop™ NanoDrop 1000 Spectrophotometer (Thermo-Scientific, Sunnyvale,

CA). Lysates with 50 µg of total protein were separated using 10% SDS-PAGE at 90 volts for 3 hours, then transferred to 0.2-µm nitrocellulose membranes (Schleicher&

Schuell, Keene, NH). The blots were probed with indicated primary antibodies at a ratio of 1:1000 unless otherwise stated. Secondary antibodies with horseradish peroxidase

(HRP)–conjugated goat anti-rabbit or goat anti-mouse IgG (Bio-Rad Laboratories,

Richmond, CA) were then added for detection and the chemiluminescent substrate

(SuperSignal; Pierce) was added for development.

3.3 RESULTS AND DISCUSSION

3.3.1 Confirmation of TALON PMAC for phosphoprotein enrichment

Despite the literature support of the TALON PMAC Phosphoprotein Enrichment

Kit, the technique required validation of phosphoprotein enrichment. The best course of validation would be on primary CLL B cells, as that was the ultimate goal of this technique, a lesson learned at the conclusion of Chapter 2. For validation, two particular phosphoproteins were chosen: STAT1 and STAT3.

There is much literature on the phosphorylation status of both STAT1 and STAT3 in CLL, particularly on the near unanimous presence of a serine phosphorylation on both proteins at amino acid 727 [130], and with the inclusion of an IL-21 treatment [131], not only are STAT1 and STAT3 phosphorylated on their respective serine residues, but also on tyrosine residues as well (701 and 705, respectively). With both forms of eukaryotic phosphorylations present on IL-21-stimulated STAT proteins, a complete picture of the residues captured by the TALON PMAC Phosphoprotein Enrichment Kit can be established.

Primary CLL B cells were isolated from a patient as described previously and treated with 100 ng/ml of IL-21 (ZymoGenetics, Seattle, WA) for 15 minutes then lysed as per the TALON PMAC Enrichment Kit protocol, described earlier. Phosphoprotein enrichment ensued shortly afterward as per instructions and the resulting lysates were separated on a 4%-15% gradient SDS-PAGE gel. While both STAT1 and STAT3 were probed on these lysates, STAT3 gave the clearest answer (Figure 3.1), indicating the preference of the enrichment kit for phosphorylated proteins.

Figure 3.1 Primary CLL B cells were isolated from a patient as described previously and treated with 100 ng/ml of IL-21 (ZymoGenetics, Seattle, WA) and lysates were collected as per TALON PMAC Enrichment protocol, described previously. Western blots were performed and the blots were probed with phosphor-STAT3 (Tyr705), phospho-STAT3 (Ser727), total STAT3 and Actin. Two blank lanes in the resulting gel were removed from the finished blot. Their positions are indicated by a single black line.

The kit worked as described, enriching for the phosphorylated forms of STAT3, while there was no observed STAT3 in the flowthrough, which should only contain unphosphorylated proteins. The presence of phosphoproteins in the flowthrough would

53 indicate a lack of specificity of the kit, and thus, prove unfavorable for future applications. Fortunately, even on over-exposed X-ray films, there was no hint of a band at the molecular weight of pSTAT3; once again, speaking to the high degree of specificity that the TALON PMAC Enrichment Kit has for phosphoproteins.

This validation, however, is not comprehensive. Every phosphorylation affects a protein differently, folding it in shapes and contortions that are in sync with its desired function. Some of these contortions may allow for easier protein binding to the IMAC technology than others. Further Western blots will be necessary to validate the results of proteomic output, which will be covered later in this chapter.

3.3.2 pH alterations during phosphoprotein enrichment

The very nature of phosphorylations makes them a difficult entity to grasp and study. They are meant to be transient in nature; added to a protein and removed with equal ease. The slightest change in the environment can alter this nature; after all, proteins are still guided by the fundamentals of their chemical structure.

pH is one such environmental factor that must be managed with earnest when studying phosphorylations. It has been reported that the optimal pH for the binding of phosphoric acid, i.e. phosphorylated protein, is between pH 2.5 and 3 [132] [133], as phosphate becomes deprotonated in this pH range. As pH rises above 3 and approaches pH 7, there becomes a preferential binding of acidic amino acid residues (aspartic acid and glutamic acid); again, because the phosphate group becomes protonated in this pH range, the negative charge of the phosphate group cannot be exploited for IMAC

54 enrichment. Since the acidic amino acid residues are in much higher abundance than the phosphate groups attached to phosphorylated proteins, it reasons to surmise that at pH

7.4, which is the pH of the Extraction/Loading Buffer in the TALON PMAC Enrichment

Kit, that there is a preferential binding of acidic amino acid residues in this kit.

Clontech’s patent on their phosphoprotein enrichment method, US7294614, indicated that they had very specific binding of phosphoproteins when enrichment was performed with 100 mM acetic acid, which has a relative pH of 2.88 [134]. As was stated within their patent, ‘…100 mM Acetic acid, washed with 5% acetic acid had almost no non-specific binding under the current experimental conditions.’ This pH corresponds perfectly with the pH range for preferential binding of phosphorylated proteins as described earlier.

While the TALON PMAC Enrichment Kit cited its use at pH 7.4, the kit may preferentially bind acidic amino acid residues, thus taking binding sites away from phosphorylated proteins, and potentially exacerbating the loss of consistency between mass spectrometry runs. Ergo, an experiment was performed on whole Ramos lysate to determine if pH factored into the enrichment process. Ramos lysate was divided into two fractions: one fraction underwent the TALON PMAC process at the recommended pH

7.4, while the other fraction underwent the enrichment process with a pH of 2.8

Extraction/Loading Buffer, lowered by acetic acid to a comparative pH as pertained to their patent.

The resulting 4G10 Western blot, precisely looking at tyrosine phosphorylations, indicated that the TALON PMAC Enrichment Kit did not benefit from a reduction in pH

(Figure 3.2). In fact, enrichment actually suffered due to the lower pH. This was a

surprising find, as all literature to this point would indicate a lower pH would harbor a

greater amount of phosphoproteins than the neutral pH 7.4. However, our findings were

completely the opposite; the TALON PMAC Enrichment Kit actually benefited from a

neutral pH than the lower pH.

In hindsight, this observation was a logical conclusion to the experiment. While

phosphate groups may preferentially bind at a low pH, the harshness of a low pH might

result in the impaired function of the IMAC structure of the TALON PMAC magnetic

beads, thus resulting in little to no binding of phosphoproteins, as indicated by Western

blot Figure 3.2.

Figure 3.2

Figure 3.2 Ramos cell lysates underwent TALON PMAC phosphoprotein enrichment as per manufacturer’s protocol, for enrichment performed at neutral pH (7.4), save for the caveat of enrichment performed in acidic pH (2.8) conditions. Following enrichment, lysates were subjected to Western blot analysis and probing with global tyrosine phosphorylation antibody 4G10 (Millipore). 4G10 (+) was the positive control for this antibody, and the addition of Ramos input lysate to the Western blot verified proper distribution of bands in flowthrough and enriched lanes.

3.3.3 Purification of enriched phosphoproteins by trichloroacetic acid

With the conclusion that the enrichment process itself was not the source of the inconsistent nature of the proteomic output, another hypothesis was drawn up. The

Ramos replicates characterized in 3.3.2 had a weak overall signal in the spectral profile output from the LCQ-Deca-XP, which indicates a lower than calculated amount of

57 protein was loaded onto the mass spectrometer. This could result from reduced protein concentration measurements, poor sample handling or poor tryptic digestion.

The enriched phosphoproteins undergo in-solution digestion. What this entails is the addition of trypsin directly to the solution containing the proteins and allowing the enzyme to digest overnight. This ‘native digestion’ is preferable to in-gel digestion as it allows for greater coverage of the proteome, as there is no risk of protein loss in the running of a SDS-PAGE gel and subsequent excision of gel bands for digestion and analysis. Though, of course, in-gel digestion can be preferable when a particular protein is desired with a known molecular weight, as extraneous proteins and high abundance proteins can be sorted out and a protein of interest excised specifically out. In this case, breadth of protein identification was desired, so an in-solution digest was preferable.

The utilization of a detergent available from Waters, RapiGest, allows for the unfolding of proteins and subsequent enzymatic cleavage by trypsin to be significantly more efficient than a normal tryptic digestion [135]. Typically after digestion and degradation of RapiGest with a formic acid, the resulting peptide mixture, upon drying, is opaque with a slight brownish hue. However, the phosphoprotein enriched samples all had an unidentified white powder that obscured any sight of the normal opaque and brownish hue of peptides, indicating a contaminant was present.

A contaminant in the resulting sample would obscure the signal of peptides within the sample, lending to the illusion of a minimal amount of peptide added to the column, when in fact the appropriate amount was added. Typically, samples are ‘purified’ before digestion by way of dialysis, where samples are placed in micro-dialysis cassettes (Bio-

Rad Laboratories, Richmond, CA) against HPLC-grade water (VWR International,

Radnor, PA) at a 1:500 ratio for dialysis at 4oC for 6 hours, with water replaced every two hours. It is possible, though, that this step in the enrichment process is not adequate enough to fully ‘purify’ the enriched phosphoproteins for digestions and subsequent running on the mass spectrometer.

An older technique for purifying proteins away from potential contaminants is trichloroacetic acid (TCA) precipitation. While the precise mechanism of TCA precipitation remains unknown [136], there is strong evidence to support TCA binding the amine backbone of proteins, resulting in their unfolding and subsequent exposure of inner hydrophobic domains to a hydrophilic solution, resulting in proteins’ hydrophobic domains aggregating together and precipitating out of solution. This is an attractive avenue for protein cleanup, as precipitation completely bypasses any confounding variables from dialysis or incompatible buffers during the enrichment process. There are some caveats to this process, however, as certain proteins are more resistant to TCA precipitation than others, likely due to fewer hydrophobic areas than other proteins. This can be overcome with the addition of a high concentration of TCA. For all experiments hereafter, 1 M TCA was utilized for precipitation to ensure a minimal chance of TCA precipitation resistance among the enriched phosphoprotein lysate [137].

To test the hypothesis that TCA precipitation would allow for greater recovery of enriched phosphoproteins than dialysis, Ramos lysates were subjected to TALON PMAC enrichment followed by their respective cleanup protocols as described previously. The entirety of each enriched sample was then loaded onto an SDS-PAGE gel alongside its

59 respective flowthrough lysate. Figure 3.3 indicates that the two methods, while providing similar band patterns, are quite dissimilar in the amount of protein that remains following cleanup. TCA precipitation offers the greatest abundance of protein following cleanup. Dialysis, however, offers only a fraction of the intensity of bands that TCA does. This would, in part, explain the weak spectral signals from earlier proteomic outputs.

Transitioning the TCA observation to more specific target, several phosphoproteins were probed via Western blot, including the phosphorylated forms of

AKT, GSK-3β , PDK1, and BTK (Figure 3.4). A number of conclusions were drawn from these Western blots. First, the kit was specifically enriching for phosphorylated proteins, as indicated by the presence of phosphorylated proteins in the phosphoprotein enriched fraction of the TALON PMAC Enrichment kit. Second, that the phosphate groups on the proteins were not affected by TCA treatment; the epitopes remained intact.

Third, that despite the high specificity of enrichment, there remains faint traces of phosphoproteins in the flowthrough.

Figure 3.3

Figure 3.3 Following TALON PMAC phosphoprotein enrichment, enriched phosphoproteins underwent cleanup with either dialysis or TCA precipitation. Following cleanup, lysate was separated on 4-15% SDS-PAGE gradient gel (Bio-Rad) and stained with SPYRO Ruby total protein stain (Bio-Rad) and imaged as described previously. Ramos input lysate was included for verification of proper band distribution between flowthrough and enriched lanes. A total of four replicates were enriched from a single Ramos lysate, two for dialysis and two for TCA. Both sets of replicates were run on the same gradient gel.

3.3.4 Increasing the bead to lysate ratio for phosphoprotein enrichment.

This final observation of 3.3.3 was quickly remedied with an increase in the bead

to lysate ratio. Doubling the volume of beads so that the lysate to bead ratio was 1:2 all

but eliminated the phosphoprotein presence in the flowthrough (Figure 3.5). Afterward,

all subsequent phosphoprotein enrichments used the 1:2 lysate to bead ratio to maximize

the enrichment process.

As a final proof of phosphoprotein-specific enrichment from this kit with a lysate

to bead ratio of 1:2, coupled with TCA precipitation, Ramos cells were treated with 25

µM LY294002, a potent inhibitor of PI3K signaling, which would result in the

dephosphorylation of its downstream targets, including AKT and PDK1[138]. Figure

3.5 indicates that at 6 hours of treatment, AKT levels increase in the flowthrough by 60%

and decreased dramatically in the phosphoprotein-enriched fraction, indicating a loss of

phosphorylation following LY294002 treatment. Unfortunately, PDK1 did not mimic

this trend in all subsequent experiments. It remains unclear why this is. Despite this

disparity, this experiment solidifies the phosphoprotein-specific nature of enrichment for

the TALON PMAC Phosphoprotein Enrichment kit, allowing for the final steps of

validation.

Figure 3.4

Figure 3.4 Total Ramos lysates underwent TALON PMAC phosphoprotein enrichment as described previously, then purified further through TCA precipitation and subsequently separated on a 4-15% SDS-PAGE gradient gel and probed for phospho- AKT (Ser473), total AKT, phospho-GSK-3β (Ser9), total GSK-3β, phospho- PDK1(Ser241), total PDK1, phospho-BTK (Ser180) and total BTK. Ramos input lysate was included for verification of proper band distribution between flowthrough and enriched lanes.

Armored with the Western blot data, 5 global Ramos lysates, untreated, underwent TALON PMAC phosphoprotein enrichment coupled with TCA precipitation and in-solution digestion followed by ESI-HPLC-MS/MS, as well as data-dependent neutral loss MS3. Ramos replicate one, two and three were run consecutively (replicate set one), and replicates four and five were run consecutively (replicate set two), though on a different run than the first three replicates. By comparing the 95% false decoy rate protein identification list between the replicates of replicate set one, there stood 93% consensus between all three replicates for protein identification. Replicate set two was slightly higher, with 97% consensus between both replicates for protein identification.

Figure 3.5 Prior to TALON PMAC phosphoprotein enrichment, a Ramos global lysate amount was mixed with magnetic enrichment bead volume at either a 1:1 ratio (200 µg: 200 µl), 1:2 ratio (100 µg: 200 µl), or 1:4 ratio (100 µg: 400 µl). Samples were loaded onto a 4-15% SDS-PAGE gradient gel. Flowthrough samples for the 1:2 and 1:4 ratios were loaded between two adjacent gel lanes. Western blot analysis was performed with phospho-GSK-3β (Ser9) and phospho-BTK (Ser180).

Figure 3.6 Ramos cells were treated, as described previously, with 25 µM LY294002 (LY) or equivalent volume of DMSO control (Vehicle) for 6 hours, and subsequently lysed and underwent TALON PMAC phosphoprotein enrichment. Samples were then purified with TCA precipitation and separated on a 4-15% SDS-PAGE gradient gel. The Western blot was probed for phospho-AKT (Ser473), total AKT, phospho- PDK1 (Ser241), and total PDK1. Gel band intensity was measured as described previously.

While this reproducibility was impressive, the true test of this method was the comparison of the two replicate sets, as they were run on different days. When the combined lists of replicate set one were compared to replicate set two, a stunning 88.5% consistency was found between the 5 replicate sets. The results of the 5 Ramos replicates 65 are shown in Figure 3.7. The Ramos replicate protein identification amounts, spectral counts, average spectra per identified protein and statistical analysis are summarized in

Table 3.1.

The next step with these data sets would be to compare the spectral counts for each protein across all 5 replicates and observe their differences. Lundgren et. al. reported spectral count consistency between Jurkat cell replicates of 25% [125]. These data were collected with a sensitive linear triple quadrupole mass spectrometer. Their results should be considered a benchmark for all future spectral count replicate studies within the confines of this experiment. Jurkat cells are a T lymphocyte cell line, and thus, would share similar attributes with both Ramos cell lines and primary CLL B cells.

3.4 CONCLUSION

With the loss of the Phos-Tag Zn2+ phosphoprotein enrichment column, a new enrichment process was necessary. The TALON PMAC Enrichment Kit was impressive on paper, citing many advantages over the defunct Phos-Tag Zn2+ phosphoprotein enrichment column. This chapter set out to determine the effectiveness of the TALON

PMAC Enrichment Kit for global phosphoproteomic investigation. The method required

Figure 3.7 Following the results of Table 3.1, Ramos Replicates 1 thru 3 were merged into a single MassMatrix search. Ramos Replicates 4 and 5 were merged and searched separately. From the protein identification lists, 357 proteins were identified across both merged searches. Of those 357 identified proteins, 316 proteins were identified in both merged data sets. This accounts for an 88.5% consistency of protein identifications across both merged data sets.

Table 3.1 Summary of individual Ramos replicate data and statistical analysis.

67 a robust nature and a high degree of reproducibility to ensure a comprehensive understanding of the phosphoproteome.

While early results were not promising with serious reproducibility issues evident, there remained enough promise within the kit, particularly with the observed consistency between same-day Ramos treatment replicates, to dictate further analysis and interpretation. By utilizing this kit with a 1:2 lysate to bead ratio coupled with TCA precipitation, a highly reproducible method, boasting 88.5% consistency amongst protein identifications.

The enrichment and study of the phosphoproteome of primary CLL B cells was the primary goal of this thesis. The transition from the method detailed within this thesis utilizing Ramos cells to primary CLL B cells would not be difficult. The lysis procedure, phosphoprotein enrichment, TCA precipitation and in-solution digest would all translate naturally to primary CLL B cells. However, a greater population of primary CLL B cells is necessary to fully flesh out the phosphoproteome of CLL as opposed to Ramos cell lines. That will be the greatest task for the transition; procuring a large enough CLL sample population to fully understand not only its inherent phosphoproteome, but also its phosphoproteome under therapeutic duress.

Chapter 4

SUMMARY OF RESULTS

This thesis describes the basic need to understand the phosphoprotein signaling web within a cell for its exploitation in therapeutic discovery and development. That in order for this understanding to occur, a concrete, robust and reproducible phosphoprotein enrichment technique had to be developed for our particular system of leukemia: first in the Burkitt lymphoma cell line Ramos followed by chronic lymphocytic leukemia. At least, that was the basis of this study.

In Chapter 2, we identified 121 proteins in our freshmen expedition into phosphoprotein enrichment, and from that, indicated a differential phosphoprotein profile between 45 unique FTY720-treated protein hits and 67 DMSO-treated protein hits within treated Ramos lysates. The PP2A inhibitor SET and the global protein chaperone Hsp90 were identified from this initial study. Unfortunately, while Western blots verified the differential observed within the proteomic workflows for Ramos, this did not translate to primary CLL B cells.

Chapter 3 began with the loss of the Phos-Tag Zn2+ phosphoprotein enrichment column, requiring the development of a novel enrichment process. The TALON PMAC

Enrichment Kit was impressive on paper, citing many advantages over the Phos-Tag Zn2+

69 phosphoprotein enrichment column. This chapter set out to determine the effectiveness of the TALON PMAC Enrichment Kit for global phosphoproteomic investigation. While early results were not promising with serious reproducibility issues evident between

FTY720-treated and DMSO-treated groups, there remained enough promise within the kit to dictate further analysis and interpretation. By utilizing this kit with a 1:2 lysate to bead ratio coupled with TCA precipitation, a highly reproducible method, boasting 88.5% consistency amongst protein identifications, now exists for the investigation of phosphoproteomics.

While CLL was not explicitly studied in these experiments, the findings can easily translate to the study of primary CLL B cells from the detailed method described herein. Unique challenges will exist for their study, though. Unlike a cell line, which remains relatively homogeneous over studies, primary cells, particularly from CLL, will vary greatly; the heterogeneity offering its own development headaches. Care must be taken with this study, as one can easily chase false leads down deep rabbit holes without the proper controls in place. A high number of CLL patients should be studied, with replicates of each particular patient performed to fully grasp the phosphoproteome at work within this disease. Stratification of the patient population should also be taken into account, particularly in regards to prognostic factors, which can dramatically alter clinical outcomes, and thus, the treatments prescribed therein. Despite these caveats, the TALON

PMAC Phosphoprotein Enrichment Kit offers the best staging point for any phosphoproteomic study.

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