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«By ISSAM MCDOOM A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE ...»

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STRUCTURE/FUNCTION RELATIONSHIP IN THE JAK2 KINASE DOMAIN

By

ISSAM MCDOOM

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

© 2008 Issam McDoom To my family; Mom, Dad, Sof, Anu and Ro. I love you guys.

ACKNOWLEDGMENTS

First and foremost, I thank my mentor, Dr. Peter P. Sayeski. His patience and guidance have formed the foundation of my graduate training. Without these elements, I would not have succeeded.

I also thank my supervisory committee. Drs. Jeffrey Harrison, Hideko Kasahara, and David Ostrov have provided invaluable advice that has enriched my graduate work. Their impartial perspectives have highlighted the strengths and weaknesses of my work and have greatly facilitated my improvement.

I thank all of the past and present members of the Sayeski laboratory for their technical guidance and friendship. I also thank Dr. David Ostrov and Andrew Magis for their assistance with in silico modeling applications. Without their technical assistance, this work would not be possible.

I thank all of my friends for their support. Thank you for providing me with an outlet for my stress. My graduate training would not have been the same without it.

Finally, I thank my wonderful family for being an inexhaustible source of love and support. Thank you for helping me through those days that I could not see the light at the end of the tunnel. There is no way I could have completed this process without you.

TABLE OF CONTENTS

page ACKNOWLEDGMENTS

LIST OF TABLES

LIST OF FIGURES

ABSTRACT

CHAPTER

1 INTRODUCTION

Signal Transduction and the Jak/STAT Family

Janus Kinase 2

Jak2 Structure

Jak Homology (JH) Domains

Non-tyrosine residues important for Jak2 function

Tyrosine residues important for Jak2 function

Jak2-Dependent Signaling

Jak2 Activation-Ligand Dependent vs. Ligand Independent

Cell Surface Receptors

Cytokine receptor signaling

GPCR signaling

Adaptors and Regulatory Proteins

SHP phosphatase family

SH2B family

SOCS Family

Reactive oxygen species

Jak2 and ROS Pathology

Diabetes

Diabetic nephropathy

Diabetic cardiomyopathy

Atherosclerosis

Cardiac Ischemia-Reperfusion Injury

2 METHODS

In Silico Molecular Modeling of Jak2

Mass Spectrometry

Cell Lines

Cell Culture

Site-Directed Mutagenesis

Transient Cell Transfections

Immunoprecipitation

Western Blotting

Luciferase Assay

Fluorescence Microscopy

3 Y972 AND JAK2 ACTIVATION

Summary

Introduction

Results

Tyrosine 972 is a Site of Jak2 Autophosphorylation

Tyrosine 972 is Solvent Accessible

Y972 Affects Jak2 Total Tyrosine Phosphorylation and Y1007 Phosphorylation.........43 The Loss of Y972 Phosphorylation Affects Jak2 Kinase Activity, But Does Not Confer a Dominant Negative Phenotype.

Tyrosine 972 Does Not Affect Ligand-Independent STAT1 Activation.

Tyrosine 972 Phosphorylation Does Not Affect Ligand-Independent Gene Expression

Tyrosine 972 is Important for the Jak2 Response to Angiotensin II

Tyrosine 972 has Differential Effects on Growth Hormone-Dependent Jak2 Total and Y1007 Phosphorylation

Phosphorylation at Y972 Does Not Affect SH2B-β Mediated Y1007/Y1008 Phosphorylation.

The Loss of Y972 Phosphorylation Impairs Growth Hormone-Mediated STAT1 Nuclear Translocation

Discussion

4 S1120 AND JAK2 FUNCTION

Summary

Introduction

Results

Serine 1120 is Solvent Accessible

Serine 1120 is Critical for Jak2 Autophosphorylation-

The S1120A Mutant Displays a Mild Inhibitory Phenotype

The Jak2 S1120A Mutation Hinders Ligand-Independent Gene Transcription-............66 Growth Hormone-Dependent Jak2 Activation is Dependent on S1120

Serine 1120 is Critical for SH2B-β Mediated Jak2 Activation-

The S1120A Mutation Abolishes Growth Hormone-Mediated Gene Transcription......68 Discussion

5 DISCUSSION

Overview

Tyrosine 972 and Jak2 Function

Y972 and ROS Pathology

Cardiac Ischemia Reperfusion Injury

Diabetic Nephropathy

Atherosclerosis

S1120 and Jak2 Function

S1120 and Growth Hormone-Dependent Pathophysiology

Y972 vs. S1120

Reflections

Y972

S1120

LIST OF REFERENCES

BIOGRAPHICAL SKETCH

–  –  –

5-1 A comparison of S1120A-dependent and Y972F-dependent effects on several categories of Jak2 function.

–  –  –

1-1 Jak Homology (JH) domains.

3-1 Tyrosine 972 is a site of Jak2 autophosphorylation





3-2 Tyrosine 972 is solvent accessible

3-3 The effect of Y972 phosphorylation on Jak2 total and Y1007 phosphorylation...............54 3-4 Tyrosine 972 phosphorylation affects Jak2 kinase function, but not its substrate properties or Jak2 dimerization

3-5 The loss of tyrosine 972 phosphorylation does not affect Jak2-mediated STAT1 activation.

3-6 Tyrosine 972 does not affect ligand-independent gene expression.

3-7 Tyrosine 972 is critical for angiotensin II-dependent Jak2 phosphorylation

3-8 Tyrosine 972 differentially affects growth hormone-dependent Jak2 total phosphorylation and Y1007 phosphorylation

3-9 Jak2 can be activated by SH2B-β in the absence of tyrosine 972 phosphorylation..........60 3-10 Tyrosine 972 phosphorylation affects GFP-STAT1 nuclear translocation

4-1 Serine 1120 is solvent accessible

4-2 The importance of S1120 for Jak2 autophosphorylation

4-3 The Jak2 S1120A mutant displays a mild inhibitory phenotype.

4-4 Serine 1120 is important for ligand-independent gene transcription

4-5 Growth hormone-dependent Jak2 activation is eliminated by the S1120A mutation.......76 4-6 Serine 1120 is critical for SH2B-beta mediated Jak2 activation.

4-7 Serine 1120 is important for growth hormone dependent gene transcription..................78 5-1 The relative locations of Y972 and S1120 in murine Jak2

–  –  –

Chair: Peter P. Sayeski Major: Medical Sciences-Physiology and Pharmacology Janus Kinase 2 (Jak2) is one of four members of the Janus family of non-receptor tyrosine kinases. Jak2 plays a role in many physiologically relevant cellular signaling pathways and provides an essential link between the cell membrane and the nucleus. In the years since its discovery, Jak2 has been linked to a number of different disease states, including diabetes, atherosclerosis, cancer, and heart disease. These links underscore the need to fully understand Jak2 function. Like all proteins, the structure of Jak2 is intimately tied to its function. In this dissertation, we investigated two structural elements in the Jak2 kinase domain that significantly impact its function. Tyrosine 972 (Y972) is a phosphotyrosine within the Jak2 kinase domain.

We demonstrated that the loss of Y972 phosphorylation significantly affected elements of Jak2 function like autophosphorylation and ligand dependent signaling processes. However, we also determined that the loss of Y972 phosphorylation did not irreversibly affect Jak2 function. We also investigated Serine 1120 in the Jak2 kinase domain. The mutation of this residue to Alanine significantly hindered several elements of Jak2 function, including autophosphorylation, growth hormone dependent signal transduction, and Jak2 dependent gene transcription. Through the investigation of these sites, we learn more about the critical connection between Jak2 structure and its function.

–  –  –

In multicellular organisms, the ability of cells to communicate with one another is absolutely essential for the coordination of physiological processes. Most of the major human health issues in our society today have their foundations in aberrant cell signaling. For instance, diabetes mellitus is the result of either a lack of sensitivity to the biological message of insulin or the failure to produce this message in the first place. All forms of cancer, regardless of their tissue of origin, are commonly defined by dysfunctional cellular growth signaling. Finally, the progression of cardiovascular disease is inextricably linked to pathological signaling events in the vascular wall and the myocardium. These examples highlight the fact that a profound understanding of cellular signaling pathways is absolutely critical to our ability to combat the major human health issues of our time.

Discovered in the early 1990’s, the Janus family of tyrosine kinases is an indispensable component of cellular communication. This family consists of four members; Jak1, Jak2, Jak3, and Tyk2. Jak1, Jak2 and Tyk2 are ubiquitously expressed while Jak3 is mainly expressed in hematopoietic cells. Through a series of protein phosphorylation events, these kinases relay the message represented by receptor/hormone binding at the cell surface to the cell nucleus. The ultimate outcome of Janus kinase dependent signal transduction is alteration of gene transcription patterns in the nucleus. This dissertation will focus on Janus Kinase 2 (Jak2) and the structure-function relationship that governs this protein’s role in cellular communication.

Specifically, we will discuss two investigations into Jak2 structural elements. We will then discuss then discuss the relevance of these findings to several Jak2 dependent disorders.

A portion of Jak2 and Reactive Oxygen Species: A Complex Relationship is reprinted with permission from Bentham Science Publishers Ltd.

–  –  –

In 1991, shortly after the discovery of the first member of the Janus Kinase family, the Jak2 gene was cloned (1). It was identified as a member of the Janus family by virtue of its possession of both a kinase and pseudokinase domain, a trademark feature of the Jak family (1).

Initially, Jak2 was characterized as a mediator of cytokine and growth factor signaling (2). In 1993, erythropoietin (Epo) was identified as the first ligand to activate Jak2 by binding to the Epo receptor (3). Subsequent studies implicated Jak2 in the signaling pathways of growth hormone, interleukin 3, interferon gamma, prolactin, and the GP130 receptor (4-8). Thus, the early history of Jak2 only portrays it as a mediator of cytokine signaling.

As time passed, accumulating evidence subverted this narrow view of Jak2 and revealed that it is a much more versatile signaling mediator than previously imagined. In 1994, it was shown that Jak2 can be activated in response to thrombin, a ligand that binds a seventransmembrane receptor. This report was a significant development in the Jak2 field as it was a departure from the original cytokine model of Jak2 activation. It linked Jak2 activation to a whole new set of cellular processes, like G-protein coupled receptor activation, diacylglycerol/PKC signaling, and cellular changes in IP3/Ca+2 (9). In 1995, this trend was continued by discovery that angiotensin II, acting through the AT1 receptor, could activate Jak2 (10).

The relevance of Jak2 to human health grew steadily with every new discovery. Each new pathway in which Jak2 was implicated bolstered its image as a therapeutic target. For instance, the connection of Jak2 to both thrombin and angiotensin II signaling makes it an attractive target for treating atherosclerosis, a disease in which both of these pathways have been shown to play roles. One of the most monumental developments in the history of the Jak2 field was the discovery in 1998 that Jak2 could be activated by reactive oxygen species (11). This finding situated Jak2 within the field of oxidative stress signaling, an ever-growing subfield within the signal transduction literature. It also exponentially increased its pathological relevance as so many prominent human health issues have their foundations in oxidative stress. Another major event in the Jak2 field came in 2005, with the discovery of the Jak2 V617F point mutation and its relevance to myeloproliferative disorders (12). This finding cemented the position of Jak2 as a molecule that is critical to human health in many different ways.

By elucidating the links between Jak2 and human health, the history of Jak2 readily highlights the need for the continuing study of this protein. In order to further our understanding of the role of Jak2 in human pathology, we must first understand the intricacies of Jak2 function on the molecular and cellular levels. Therefore, the next two sections will be discussions of Jak2 structure and signaling. The relationship between structure and function is the dominant force that governs all Jak2 interactions. Upon this intrinsic foundation, the extrinsic elements that compose Jak2-dependent signaling combine to link the basic biochemical process of phosphorylation to the larger realm of cellular physiology and ultimately, human health as a whole.

Jak2 Structure Like all proteins, the structure of Jak2 dictates its function. In the years since Jak2 was discovered, Jak2 structure has been studied intensely in an effort to identify elements that have profound functional implications for this protein. This endeavor is necessary if we are to fully understand the role of Jak2 in human physiology and pathophysiology. Thus, the following sections are devoted to a discussion of Jak2 structure and its intimate relationship with Jak2 function. Specifically, we will discuss 1.) the organization of Jak2 primary structure into Jak Homology domains, 2.) functionally important amino acid residues (non-tyrosine), and 3.) phosphotyrosine elements of Jak2 functional regulation.



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