REGULATION OF

DBL FAMILY

GUANINE NUCLEOTIDE EXCHANGE FACTORS

By:

MEGHANA BALARAM GUPTA

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Danny Manor

Department of Pharmacology

CASE WESTERN RESERVE UNIVERSITY

January 2014 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

Meghana Balaram Gupta

candidate for the Doctor of Philosophy degree*

Dissertation Advisor: Danny Manor

Committee Chair: Amy Wilson-Delfosse

Committee Member: Bingcheng Wang

Committee Member: Vera Moiseenkova-Bell

Committee Member: Cathy Carlin

Date of Defense: December 2, 2013

* We also certify that written approval has been obtained for any proprietary material contained therein.

ii Dedication

I dedicate this thesis to my parents. Without them, I would not be the person that I am today. Their unconditional love, support, and encouragement has helped me achieve something that at times I did not think was possible.

I share the success of completing this thesis and earning my Ph.D. with my parents and my entire family. Without them, I am sure it would have been impossible.

Mom and Dad, I hope that one day, I will be as loving and supportive towards my children as you have been to me. Thank you for everything you have given me and for all that you continue to do for me. I love you both.

iii Table of Contents

CHAPTER PAGE Table of Contents ...... iv List of Tables ...... vii List of Figures ...... viii Acknowledgements ...... ix Dbl Family GEFs...... 1 Abstract ...... 1 Chapter 1 ...... 2 Literature Review ...... 2 I. Mitogenic Pathways ...... 2 II. The Ras Superfamily GTPases ...... 3 II.1 Ras Subfamily of GTPases ...... 4 II.2 Ran Subfamily of GTPases ...... 4 II.3 Rab Subfamily of GTPases ...... 4 II.4 Arf Subfamily of GTPases ...... 5 III. Rho Family GTPases: Activation and regulation of their cycle ...... 7 III.1 Rho GTPase structure: requirements for nucleotide binding, GTP hydrolysis and effector recognition ...... 7 III.2 Regulation of Rho GTPase cycling ...... 9

IV. The Dbl Family GEFs ...... 13 IV.1 The Dbl- (DH) Domain: Structure & Function ...... 14 IV.2 The Pleckstrin Homology (PH) Domain ...... 16

V. Regulation of Dbl Family GEFs ...... 18 V.1 Coupling to Tyrosine Kinases and G- Coupled Receptors ...... 18 V.2 Regulation of Dbl GEFs: Intra- and Inter- Molecular Modes of Regulation ...... 21 V.2.1 Examples of intra-molecular modes of regulation ...... 22

iv V.2.2 Release of inhibitory intra-molecular interactions ...... 22 V.2.3 Inter-molecular modes of regulation ...... 24 V.3 Other Modes of Dbl Regulation ...... 25 V.3.1 GEFs in signaling pathways ...... 25 V.3.2 Dbl GEFs mediate cross talk between pathways ...... 27

VI. GEF Dysfunction and Diseases ...... 29 VI.1 Cancers ...... 29 VI.2 Developmental/Neurological Disorders ...... 30 VI.3 Viral Infections ...... 31 Statement of Purpose ...... 32 Chapter 2 ...... 33 Plekhg4 is a novel Dbl-family GEF for Rho-family GTPases ...... 33 Capsule ...... 34 Abstract ...... 34 Introduction ...... 35 Materials and Methods ...... 37 Results ...... 40 Discussion ...... 47 Chapter 2 Figures ...... 50 Chapter 3 ...... 57 Tyrosine phosphorylation of Dbl regulates GTPase signaling ...... 57 Capsule ...... 58 Abstract ...... 58 Introduction ...... 59 Materials and Methods ...... 61 Results ...... 64 Discussion ...... 67

v Chapter 3 Figures ...... 71 Chapter 4 ...... 80 Overall Summary and Future Directions ...... 80 4.1 Overall Summary ...... 80 4.1.1 Plekhg4 ...... 80 4.1.2 Dbl...... 81 4.2 Future Directions ...... 82 4.2.1 Plekhg4 ...... 82 4.2.2 Dbl...... 82 4.2.3 510Tyr is highly conserved in other Dbl-family GEFs ...... 83 4.3 Concluding Remarks ...... 92 Literature Cited ...... 93

vi List of Tables Table 1 List depicting coupling between cell surface receptors and GEFS ...... 20

vii List of Figures Figure 1.1 Activation of cell surface receptor leads to Rho GTPase activitation ...... 3 Figure 1.2 General features of the G-Domain ...... 8 Figure 1.3 Cycling of Rho GTPases is tightly regulated ...... 10 Figure 1.4 Mechanism of GEF function ...... 11 Figure 1.5 Characteristics of Dbl family GEFs ...... 13 Figure 1.6 Crystal structure of Sos1: conserved regions of Dbl GEFs ...... 15 Figure 1.7 Representation of inter- and intra- modes of regulation ...... 21 Figure 2.1 Spatio-temporal expression patterns of Plekhg4 in murine brain ...... 50 Figure 2.2 Sequence signatures of Plekhg4 and Dbl ...... 51 Figure 2.3 Plekhg4 is a functional GEF ...... 52 Figure 2.3 (A) Plekhg4 is a functional GEF – Full gel ...... 53 Figure 2.4 Plekhg4 regulates the actin cytoskeleton ...... 54 Figure 2.5 Intracellular fate of Plekhg4 is dictated by Hsc70, Hsp90, and CHIP ...... 55 Figure 3.1 Tyrosine phosphorylation of Dbl...... 71 Figure 3.2 510Tyr is an important site of phosphorylation in Dbl ...... 72 Figure 3.3 Role of 510Tyr in Dbl’s binding to Grb2 ...... 73 Figure 3.4 Role of 510Tyr in Dbl’s association with substrate GTPases ...... 74 Figure 3.4 (A) Role of 510Tyr in Dbl’s association with substrate GTPases – Full gel .....75 Figure 3.5 Tyrosine phosphorylation of Dbl is necessary for proper activation of RhoA.76 Figure 3.5 (C) Tyrosine phosphorylation of Dbl is necessary for proper activation of RhoA – Full gel ...... 77 Figure 3.6 Role of 510Tyr phosphorylation in Dbl-induced downstream activities ...... 78 Figure 3.7 Proposed model of Dbl regulation by tyrosine phosphorylation ...... 79 Figure 4.1 510Tyr is highly conserved in Dbl family GEFs...... 84

viii Acknowledgements

There are a number of people that I would like to acknowledge and extend my gratitude, for without their guidance this thesis would not have been possible.

First and foremost, I would like to thank my advisor, Danny Manor, for his expertise, guidance, and overall commitment to my success. Danny recognized my weaknesses from the get go and continually encouraged me to do things that were out of my comfort zone. Although I definitely hated those situations, in retrospect I am grateful to him for pushing me to my limits. He has been extremely patient with me both at the bench and with writing, which has allowed me to become a more confident and independent thinker, and for that I am grateful. Over the past 5 years, with Danny’s help and encouragement, I have grown immensely as a scientist and also as an individual.

I would also like to acknowledge my labmates, past and present: Lynn Ulatowski, Varsha Thakur, Stacey Chung, and Samantha Morley. They have provided not only emotional support throughout this process but have given, without question or refrain, endless amount of scientific advice and technical help. I am sure that without them, accomplishing this thesis would not have been possible.

I would also like to thank my committee: Amy Wilson-Delfosse, Bing Cheng Wang, and Vera Moiseenkova-Bell for all their support and guidance. They continually gave me comments and suggestions that I failed to see. They helped boost my confidence and helped me find pride in my work, and by doing so helped me grow as a scientific thinker. It was obvious that my committee was vested in my overall well-being and I am extremely grateful for all their help.

Without my friends and family, I am certain that I would have lost my sanity a long time ago. I met Stephanie Balow during our interview weekend here at Case and out of the many girls that interviewed, Steph and I were the only ones to join the program. Lucky for us, we hit it off the bat. Steph, we have been through so much during graduate school, from C3MB to qualifiers and now stepping into the unknown. You are probably the only person who truly understood everything that I went through during school. Your camaraderie, weirdness, sense of humor, and randomness has kept me going.

I met my husband Nick at the beginning of graduate school. At times I definitely thought Nick was a big distraction, but the reality was that he brought balance to my life. He encouraged me to be the best that I could be, as a graduate student, but also made sure that I made time for myself and for fun. I am grateful for him, his support, and for sticking out my rollercoaster of emotions with me. ILYTIAB.

I truly believe that I have one of the best families in the world. Without their love and support I definitely would not be the person that I am today. My parents and brother have been my pillars of support throughout the years. They have been my cheerleaders and at times when I thought all hope was lost they found the words to keep me going. Words cannot express the love and gratitude I have for them.

ix REGULATION OF

DBL FAMILY

GUANINE NUCLEOTIDE EXCHANGE FACTORS

By

MEGHANA B. GUPTA

ABSTRACT

Mitogenic signals are initiated by the binding of growth factors to their receptors and culminate in a variety of cellular responses including alterations in expression, protein synthesis, cytoskeletal rearrangements, and metabolic pathways. Many mitogens exert their effects by stimulating the activity of Guanine Nucleotide Exchange Factors

(GEFs), which in turn activate small GTPases of the Rho family (Rho, Rac and Cdc42).

Perturbations in the signaling of Rho GTPases and their GEFs are often associated with several types of neurological and malignant diseases. In order to understand better the roles that GEFs play in diseased states, two main studies were conducted: (1) characterization of a novel Dbl-family GEF, Plekhg4 and (2) determination of the molecular mechanisms that regulate the activity of the proto-typical family member, Dbl.

1 CHAPTER 1: Literature Review

I. Mitogenic Pathways

Cellular communication occurs through complex signaling networks and is

necessary for the governing of basic cellular activities. Most signaling pathways are

initiated by the binding of mitogens such as growth factors to cell surface receptors,

which in turn promote the activation and progression of specific signaling pathways.

Signaling networks rely on the ability of and cellular compartments to interact,

activate, and deactivate other cellular components. Proper regulation of

pathways is essential for all aspects of cellular homeostasis, and deregulation can lead to

various diseases, such as malignant pathologies. For the most part, these mitogenic

pathways are regulated by the activity of a particular family of proteins known as the Dbl

family of guanine nucleotide exchange factors (GEFs). Members of this family activate

the Rho family of small GTPases, by catalyzing the exchange reaction from a GDP-

bound GTPase to a GTP-bound GTPase and result in the stimulation of specific

downstream effectors that control cytoskeletal architecture, , vesicular

genesis and trafficking, cell polarity, and cell cycle progression (1-5). It is therefore not

surprising that mutations in proteins of this family are associated with several types of

cancers and pathologies. Elucidating the details of the signaling cascade leading up to

the activation and regulation of Dbl-family proteins will allow us to have a better

understanding of how these pathways are disturbed in particular diseases, and as such has been a main focus of our lab.

2

II. The Ras Superfamily of GTPases

Small phosphohydrolases (GTPases) are a large class of enzymes that bind and hydrolyse GTP. Binding of growth factors to cell surface receptors promotes activation (GTP binding) of the GTPases, allowing them to stimulate downstream effectors. Subsequently, GTPases hydrolyze the bound GTP and remain in an inactive GDP-bound state until further activation occurs (Figure 1.1) (6).

Figure 1.1: Stimulation of a cell surface receptor leads to the activity of guanine nucleotide exchange factors and ultimately the activity of GTPases. Stimulation of GTPase activity leads to various cellular outcomes.

The Ras Superfamily of GTPases is a large family of highly evolutionarily conserved small GTPases and is divided into five main subfamilies based on similarities in sequence, structure, and function: Ras, Rho, Ran, Rab, and Arf (6). Ras superfamily

GTPases transmit messages from extra-cellular mitogenic stimuli to various intra-cellular signaling networks involved in gene expression, protein synthesis, cytoskeletal rearrangements, and metabolic responses. All GTPases of the Ras superfamily contain two main structural features: 1) a G-domain, which is necessary for nucleotide binding

3 and hydrolysis, and 2) a C-terminal CAAX-domain, allowing for lipid prenylation

(geranylgeranyl or farnesyl) modifications necessary for membrane attachment. Two main classes of proteins regulate the GTP binding and hydrolysis cycle of GTPases: 1) activators (guanine nucleotide exchange factors, GEFs) and 2) deactivators (GTPase activating proteins, GAPs).

II.1 Ras Subfamily of GTPases. Ras was the first identified and characterized GTPase, and as such, considered the prototypical member for small GTPases (7,8). Ras GTPases transduce signals initiating from cell surface receptors (i.e., receptor tyrosine kinases and

G-protein coupled receptors) to pathways that are involved in cell survival, proliferation, and differentiation. They do so by modulating and influencing the transcription of directly involved in cell survival, proliferation, and differentiation. All Ras superfamily

GTPases function as molecular switches and function by cycling between an inactive

GDP bound state and an active GTP bound state. These two states are mainly regulated by GEFs and GAPs. For Ras subfamily GTPases, proteins that contain a RasGEF domain function as GEFs/activators for these GTPases. Examples of such GEFs include, Sos1 and Cdc25 (9). Inactivators, or GAPs for Ras subfamily GTPases include proteins that have either a RasGAP or RapGAP domain, such as the GAPs NF1 and p120GAP (10).

II.2 Ran Subfamily of GTPases. Ran GTPases are largely known for their involvement in nucleocytoplasmic import and export through the nuclear pore complex, the assembly of the mitotic spindle, and formation of the nuclear envelope (7,8). Ran GTPases are activated by the GEFs RCC1, importin !, and RanBP10 and are deactivated by RanGAP proteins such as, RanGAP1/Rna1p (11,12).

II.3 Rab Subfamily of GTPases. The Rab GTPases are known for their involvement in

4 vesicular trafficking. They comprise the largest subfamily of GTPases as a result of gene duplications, which has led to a large expansion of this family. They regulate vesicular trafficking through the endocytic and secretory pathways (7,8). Each Rab protein is involved in a different aspect of the endocytic and secretory pathways. Activation of a

Rab GTPase allows for its association to a particular transport vesicle. This association then facilitates the binding of the transport vehicle to the proper organelle. For instance,

RabGTPases 5 and 21 mediate the initiation of receptor-mediated endocytosis, which occurs via clathrin-coated vesicles. Following internalization, sorting in the early endosome segregates molecules for return to the plasma membrane along fast (Rab4,

Rab14, Rab15) and slow (Rab11a, Rab15, Rab22a) recycling routes. Early endosomes also regulate return to the Golgi via Rab6. From late endosomes, Rab9 regulates return to the Golgi. After RabGTPases facilitate their role in sorting and trafficking, GTP is hydrolyzed and the RabGTPase is released from the membrane of its respective transport vesicle, associates with GDP and a GDP-dissociation inhibitor, remains in the cytosol and awaits further activation (13). There are four main families of GEFs that activate the Rab family of GTPases (8). These four families are classified by the presence of a: 1) Vps9 domain (14), 2) DENN domain (15), 3) TRAPP complex (16), and 4) Sec2 domain (17).

RabGEFs preferentially activate specific subtypes of Rab GTPases. For example, the

Vps9 domain-containing GEFs activate Rab5 (14) whereas DENN4 GEFs activate Rab10

(15). RabGAPS belong to a large family of TBC (Tre2/bub2/Cdc16) domain-containing proteins (17-19).

II.4 Arf Subfamily of GTPases. Arf GTPases are also involved in vesicular and membrane trafficking (7,8). These GTPases are activated by GEFs that contain a Sec7 domain (20)

5 and are inactivated by GAPs that contain an ArfGAP domain (21). Arf GTPases remain in an inactive GDP-bound state in the cytosol. Upon initiation of vesicle formation in the

Golgi, Sec-7 containing GEFs activate Arf GTPases and facilitate GTP-binding.

Activation of Arf-GTPases promotes association of Arf to Arf receptors on the Golgi membrane along with coat complexes and fatty acyl CoA, both of which are necessary for the budding of the transport vesicle. Hydrolysis of GTP inactivates Arf and promotes its spontaneous dissociation from the membrane and does not require the need for a GDP- dissociation inhibitor (22).

6 III. Rho Family GTPases: Activation and regulation of their cycle

The Rho subfamily GTPases, the focus of our lab, consists of over twenty members; the first identified and best-studied members include: Rho, Rac, and Cdc42. The Rho family is involved in signaling networks that regulate actin, cell cycle progression, gene expression, cell polarity, and hematopoiesis. Rho GTPases have two classes of activators: the Dbl-homology domain containing GEFs and those defined by a DOCK-homology domain, including and . Deactivators of Rho GTPases contain a highly conserved RhoGAP domain, as found in p50RhoGAP and BPGAP1 (7,8).

III.1: Rho GTPase structure: requirements for nucleotide binding, GTP hydrolysis, and effector recognition. Like other GTPases, two major structural features define Rho

GTPase proteins: the G-domain necessary for nucleotide binding and hydrolysis (23), and a C-terminal CAAX motif that allows for isoprenoid lipid modifications (24). These lipid modifications allow for proper localization of the GTPase in membranes (24).

The G-domain fold consists of a six-!"#$%&'&( )-!*''"( !+##,+%&'&( -.( /-helices

(Figure 1.2).

7

Wittinghofer and Vetter, Annu. Rev. Biochem. 2011. 80:943–71 (25)

Figure 1.2: General features of the G-domain. Switch regions are shown in blue and purple. "- helices are in red and !-helices are in green.

Within this domain the nucleotide-binding pocket, consisting of four to five conserved

sequence elements necessary for nucleotide binding, resides (26,27). The most important

contributions to binding are made by the interactions of the nucleotide base with the

N/TKXD motif and the P-loop (phosphate-binding loop) of the GTPase (25,28,29).

Additionally, the GTPase contains a conserved DXXG motif, which allows for nucleotide

specificity (30). The differences in GDP or GTP binding are primarily confined to two

segments referred to as the switch I and switch II domains. The conformational changes

induced by GTP binding to the GTPase are stabilized by the formation of hydrogen bonds

-'"0''%( "*'( 1-phosphate group of the GTP, and the main-chain amide groups of the

switch I and switch II domains of the GTPase. More specifically, these bonds are formed

-'"0''%( "*'( 1-phosphate group of the GTP with a highly conserved threonine in the

switch I region and a glutamine in the switch II region. Additionally, a nucleophilic water

molecule and Mg2+ ion further aid in the binding and hydrolysis of GTP to the GTPase.

Together the threonine and glutamine residues activate the nucleophilic water, which

8 promotes in-23%'(%+42',5*3234($""$46(,7("*'(1-phosphate group and thus GTP hydrolysis.

Switch from a GTP-bound state to a GDP-bound state has been described as a spring- loaded mechanism: hydrolysis of GTP leads to relaxation of the switch I and switch II domains, due to breakage of the hydrogen bonds that were formed between "*'( 1- phosphate group and the threonine and glutamine residues (31,32).

Dramatic conformational differences are observed between the GDP-bound and

GTP-bound states of the GTPase. Once GTP has been loaded onto the GTPase, the active

GTPase is able to interact with downstream effectors. Both changes in the switch I and switch II allow for proper effector recognition, but the more drastic are the ones in the switch II region. GTP binding induces a transition from a helical conformation to an ordered coil conformation, promoting reorientation of the entire "2 helix of the switch II region and generation of an effector-binding site. Directly involved in this reorientation is the aforementioned conserved DXXG motif that takes part in forming interactions with the ! and 1-phosphate groups (33).

Due to high sequence similarity (~70%), Rac1 and Cdc42 effectors contain a common sequence referred to as a CRIB (Cdc42/Rac-interactive binding) domain. The sequence identity of RhoA with Rac1/Cdc42 is only ~45% (34).

III.2: Regulation of Rho GTPase cycling. Rho GTPases function by cycling between an inactive (GDP-bound) state and an active (GTP-bound) state (see Figure 1.3). Proper cycling between these two states is crucial for proper cellular function. Studies have shown that constitutively active forms of Rho GTPases lead to cellular transformation.

Additionally, inhibition of Rho GTPase activation, through the use of dominant negative reagents, leads to inhibition in actin cytoskeletal rearrangements (35-38). As can be

9 appreciated, cycling between these two states is necessary and is highly regulated by several proteins (Figure 1.3), three of which include: Guanine Nucleotide Exchange

Factors (GEFs), GTPase Activating Proteins (GAPs), and GTP/GDP-Dissociating

Inhibitors (GDIs).

Figure 1.3: Three main classes of proteins tightly regulate the activity of small GTPases: guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and GTP/GDP dissociation inhibitors (GDIs).

10 Guanine Nucleotide Exchange Factors are the physiological activators of the

GTPases and function by catalyzing the exchange of GDP for GTP by recognizing the

GDP-bound GTPase and destabilizing the interaction between GDP and the GTPase by

displacing the Mg2+ ion necessary for nucleotide binding. Additional disruptions within

the P-loop and switch domains of the GTPase are introduced, promoting GDP

dissociation (Figure 1.4). Once GDP is displaced, the GEF strongly binds the GTPase

allowing the switch domains of the GTPase to be more flexible, promoting rapid binding

of GTP. As such, the GEF-GTPase state is short lived (8,39-41).

Vetter and Wittinghofer, Science 294, 1299 (2001) (41)

Figure 1.4: Guanine nucleotide exchange factors function by catalyzing the exchange reaction from a GDP-bound GTPase to a GTP-bound GTPase.

The intrinsic hydrolyzing activity of GTPases is very slow in comparison to the

rapid timing of the cellular activities elicited by growth factor stimulation. In order for

these mitogenic pathways to be properly regulated and turned-off, proteins that catalyze

GTP hydrolysis are necessary. GAPs function by increasing the intrinsic hydrolysis rate

11 of GTPases and are characterized by a highly conserved arginine ‘finger’ (41-43). This arginine finger inserts into the nucleotide-binding pocket of the GTPase, allowing stabilization of the GTPase switch domains by directly interacting with a highly conserved glutamine residue (discussed above). Interaction of the arginine finger with the glutamine residue promotes activation of the nucleophilic water molecule, which attacks the #-phosphate and promotes subsequent GTP hydrolysis (41,44).

RhoGDI proteins are comprised of two main domains that are necessary for Rho

GTPase interaction, an N-terminal helical bundle and a C-terminal !-sandwich domain.

The N-terminal domain interacts with the switch regions, while the C-terminal domain binds the prenylation modifications present on the C-terminal ends of Rho GTPases.

These prenylation modifications are necessary for membrane association of the GTPase.

By binding these modifications, RhoGDIs render the GTPase cytosolic (45,46). RhoGDIs are able to interact with GTP- or GDP- bound GTPases (47-50), and by doing so, it is possible that RhoGDIs maintain inactive, GDP-bound, GTPases cytosolic until activation of a cell surface receptor occurs. Additionally, it is possible they aid in the localized signal of the active, GTP-bound, GTPase (8). Although likely, evidence for regulation by

RhoGDIs is lacking. Furthermore, how RhoGDIs prevent dissociation of the nucleotide is unclear and what promotes RhoGDI release from the nucleotide bound GTPase is also unclear. However, what is certain is that by being able to interact with both states of the

GTPase, RhoGDIs halt proper cycling of the GTPase.

12 IV. The Dbl Family GEFs

Dbl family GEFs are activators of the Rho family of small GTPases (Rac, Cdc42,

Rho), and comprise over 80 family members. Proto-Dbl (from here on referred to as Dbl)

was the first identified mammalian GEF and as such considered the prototypic member of

the family (51). Dbl was initially identified as a 66 kDa oncogene product derived from a

diffuse B-cell lymphoma cDNA (51-53). It was later realized that this highly oncogenic product was produced by amino-terminal deletion of the full-length protein. Two highly conserved domains, in tandem with one another, characterize members of the Dbl family: a Dbl-homology (DH) domain and a Pleckstrin-homology (PH) domain (54) (Figure 1.5).

The DH domain is the minimal unit necessary for GEF activity by providing the major binding surface for the small Rho GTPases and catalyzes exchange of GDP (inactive

GTPase) to GTP (active GTPase). The PH domain, on the other hand, is not involved in nucleotide exchange but is thought to be crucial for proper localization of the GEF to specific cellular compartments or membranes (55-57). Once a GTPase is activated by a

GEF, it is able to bind and stimulate specific downstream effectors that control various aspects of cellular function (1-5) (see Figure 1.1).

Figure 1.5: Dbl family GEFs are characterized by a Dbl-homology (DH) domain in tandem with a Pleckstrin homology (PH) domain.

13 IV.1: The Dbl-Homology (DH) Domain: Structure & Function. GEFs function by recognizing a GDP-bound GTPase, promotes GDP dissociation, and by doing so forms a high-affinity interaction with the GTPase. Due to high levels of GTP within the cell,

GTP rapidly binds the GTPase and the interaction between the GEF and the GTPase is short lived (40) (see Figure 1.4). The minimal domain necessary for GEF function is the

Dbl-homology (DH) domain. Mutation or deletion of regions of the DH domain results in impaired GEF activity and subsequently results in an inability to catalyze the exchange reaction on downstream GTPases, this ultimately has deleterious effects on the cell (see section V). The DH domain is composed of three structurally conserved /-helical regions

(referred to as CR1–3, see Figure 1.6) separated by variable regions. It is within these regions that the necessary residues for GTPase interaction and also GTPase specificity reside. These three helices pack tightly together forming the core of the DH domain. On one face, the CR1 and CR3 contribute a region of the DH domain that is solvent-exposed, allowing for direct interaction with Rho GTPases. Additional helices pack the CR helices such that the overall secondary structure of the DH domain resembles a five-helix bundle

(58-61).

14

Stephen M. Soisson et. al., Cell, 1998. Vol. 95, 259–268 (60)

Figure 1.6: Crystal ribbon structure of Sos1 depicting overall structure of Dbl GEFs. Of note are the CR regions. CR1 is in magenta, CR2 is in peach, and CR3 is in lime green. Additional helices that pack the CR regions are in blue.

The three-dimensional X-ray crystal structure of Rho GTPases in complex with the DH–PH module of various Dbl GEFs has allowed for the speculation and understanding of how GEFs facilitate the exchange of GDP to GTP. The most important interactions for nucleotide binding occur between the phosphate groups and magnesium ion with the P-loop of the GTPase (3,41). By displacement of the magnesium ion and the generation of other structural changes, the GEF is able to promote GDP dissociation and

GTP binding. As seen in the crystal structure of Rac1 (GTPase) in complex with Tiam1

(GEF), the switch I region of Rac1 is shifted and lies in a groove between the CR1 and

CR3 of Tiam1. Interactions between CR3 of the GEF and the switch II domain of the

15 GTPase, along with hydrogen-bonding interactions formed by a highly conserved

glutamic acid residue in CR1 with main chain amide residues in the switch I domain

introduce structural changes. A side chain methyl group from the switch II region forced

into the nucleotide-binding pocket disrupts Mg2+ binding by blocking the Mg2+-binding

site. Additionally, a conserved glutamine residue from the switch II region interacts with

a lysine in the P-loop, destabilizing the interaction of the GTPase with the nucleotide.

These changes in switch II conformation are supported by interactions with a conserved

lysine from CR3 in the DH domain (61). Overall it can be appreciated that the P-loop and

Mg2+ ion of the GTPase form critical interactions with the nucleotide. GEFs, through

their DH domain, function to disrupt these interactions and promote GDP dissociation.

IV.2: The Pleckstrin-Homology (PH) Domain. Pleckstrin, the major substrate for Protein

Kinase C, is the first protein in which the PH domain was identified. In response to

thrombin stimulation, Protein Kinase C phosphorylates pleckstrin, which promotes a

signaling cascade that terminates calcium signaling in platelets (62,63). Since then, the

PH domain has been identified in various proteins involved in signal transduction.

Various studies have shown that for many Dbl family GEFs the DH–PH module is the

minimal unit necessary to promote cellular transformation in-vivo, whereas the DH

domain is sufficient for nucleotide exchange in-vitro (57,64). This supports the fact that physiologically, PH domains are necessary for proper GEF function. PH domains have multiple roles based on their ability to interact with lipids and proteins (65-67). PH

domains help in the proper association and localization of the GEF at the plasma

membrane, by binding phosphoinositides. As such, PH domains aid in the activation of

GTPases by bringing GEFs into close proximity with its cognate GTPase (4,55,56).

16 Additionally, PH domains have been shown to interact with proteins and can function as a scaffold in signaling pathways. For instance, in Dbl, the PH domain interacts with ezrin, a protein known to link the plasma membrane to the actin cytoskeleton, and is also an effector of the Rho GTPases (68). Another example of this is the GEF, Trio. The PH domain of Trio interacts with filamin to localize Trio to actin filaments and to facilitate actin-based ruffling, but has been shown to not enhance nucleotide-exchange activity of

Trio (69).

Structurally, there are varying roles for the PH domain. In some cases, the PH domain is necessary for proper binding and association to the GTPase, like in Dbs, Trio, and Dbl (70-72). However, in other cases, such as Sos1, the PH domain allosterically regulates activity of the DH domain by preventing GTPase interaction (73). Overall, depending on the cellular context, the ability of the PH domain to interact with a wide variety of molecules promotes proper localization and regulation of the GEF.

17 V. Regulation of Dbl Family GEFs

V.1: Coupling to Receptor Tyrosine Kinases and G-Protein Coupled Receptors. Rho

Family GTPases are typically activated through the activation of cell surface receptors

such as Receptor Tyrosine Kinases (RTKs) or G-Protein Coupled Receptors. In general,

activation of these receptors has wide ranging cellular effects. Ultimate activation of Rho

GTPases has effects on actin dynamics, , proliferation, and gene

expression. Receptor tyrosine kinases are cell surface receptors that are able to transduce

extracellular mitogenic signals (i.e. growth factors) to within the cell. The link between

activation of RTKs and GTPases is evident from the fact that many of the effects of RTK

stimulation lead to cellular outcomes indicative of Rho GTPase activation, such as

changes in the actin cytoskeleton. Coupling and ultimate activation of Rho GTPases by

RTK stimulation, in many cases, occurs through Dbl family GEFs. As can be appreciated

by Table 1, recruitment of Dbl family GEFs upon stimulation of an RTK allows for the

activation of Rho family GTPases.

In addition to activation by RTKs, Rho GTPases can be activated through G- protein coupled receptors (GPCRs). 8*'(/(!+-+%3"(,7("*'(912/13 family of GPCRs forms

direct interactions with RhoGEF proteins, PDZ-RhoGEF, LARG and p115-RhoGEF (74-

76), and mediates the activation of RhoA (see table below for ligand and receptor).

RhoGEF association with G12/13 /-subunits is facilitated through the RGS (regulators of

G-protein signaling) domain of the RhoGEF, which is N-terminal to the canonical DH-

:;( <,&+2'=( >9?( &,<$3%!( '%*$%4'( "*'( 3%"#3%!34( 98:$!'( $4"3@3".( ,7( *'"'#,"#3<'#34( 9/( subunits. These RhoGEFs function as signal mediators by functioning as GAPs / deactivators for the large heterotrimeric GPCRs and GEFs / activators of small Rho

18 family GTPases. 9)1(!+-+%3"!($2!,(3%"'#$4"(03"*(9AB!C(-+"("*'#'($#'(,%2.($(7'0(6%,0%( instances. For example, 9)1( !+-+%3"!( $%&( :"&D%s(3,4,5)P3 synergistically stimulate the

GEF P-Rex1 to activate Rac1, leading to lamellipodia formation and ROS production in (77,78).

19

Ligand Receptor GEF GTPase Physiological Outcome Activated

Ephrin-A5 EphA5 Vav2 ? Inhibition of migration of Schwann cells (79) Ephrin-A1 EphA2 Vav2/Vav3 Rac1 Cell migration / Vascular Assembly / Angiogenesis (80) Growth factor PDGF and Vav1/2/3 Rac1, Cytoskeletal containing EGFR RhoA, rearrangements / Cell serum Cdc42 migration (81,82) Growth factor EGFR Vav2 Rac1, Cytoskeletal containing (Rac1/RhoB), RhoA, rearrangements / Cell serum Vav3, )Pix, RhoB migration (81,83) Sos1 (Rac1), P-Rex (Rac1) Neurotrophin TrkA Ras-Grk1 Rac1 Neurite outgrowth (84) NGF, BDNF, TrkA/B P-Rex1 Rac1 Neuronal cell migration and epidermal / neurite outgrowth (85) growth factor Neurotrophin-3 TrkC Dbs Cdc42 Schwann cell migration (86) LPA 1 and LPA PDZ- RhoA Proliferation / Actin LPA2 RhoGEF, remodeling / Ca2+ LARG mobilization (74) Angiotensin II Angiotensin I LARG RhoA Actin remodeling / (AT-1) Increase in cytosolic receptor Ca2+ necessary for contraction in smooth muscle (75) Endothelin I Endothelin p115- RhoA Increase in protein Receptor RhoGEF synthesis (76)

Table 1: A short list depicting the coupling between RTKs/GPCRs and GEFs. Shown are the ligands, receptors, GTPases activated and known outcome.

20 V.2: Regulation of Dbl GEFS: Intra- and Inter- Molecular Modes of Regulation. Proper regulation of GEF activities is crucial for normal cellular function. Mounting evidence shows that Dbl GEFs, in a basal state, maintain an inhibited state by obscuring access of the GTPase to the catalytic DH domain. Upon stimulation by a growth factor or hormone, inhibition is relieved and access to the DH domain allows for controlled activation of Rho

GTPases and downstream cellular effects. Inhibitory modes of regulation are evident in the fact that many Dbl family GEFs are constitutively activated as a result of truncation of the amino terminus, suggesting that the amino terminus region of these proteins function as, direct or indirect, negative regulators of the DH-domain. Two main forms of regulation are prevalent in Dbl family GEFs: (1) intra-molecular modes of regulation and

(2) inter-molecular modes of interaction. (1) Intra-molecular modes of regulation can be considered as those in which the GEF forms direct interactions within itself to inhibit access to the DH domain. Several examples of this form of regulation involve some portion of the amino terminus directly interacting with the C-terminal end. (2) Inter- molecular interactions are those that require the involvement of other proteins in order to maintain an inhibited state. Pictorial representations of these two forms of regulation are depicted below in Figure 1.7.

Figure 1.7: Representation of intra- and inter- molecular forms of regulation, as described in the text above.

21 V.2.1: Examples of intra-molecular modes of regulation. The evidence for the regulation

of Dbl family GEFs by intra-molecular modes of inhibition is vastly mounting. Structural

evidence shows that in the GEFs, PDZ-RhoGEF, LARG, and p115, the inhibited state is

maintained by intra-molecular interactions formed by the linker region between the RGS

and DH domains and the DH domain. This interaction is disrupted by binding of the

G"12/13 to the RGS domains of these GEFs. In the case of PDZ-RhoGEF and LARG, the

PDZ domain has also been shown to mediate interaction of the RGS-RhoGEFs with

regulatory proteins to help promote the release of inhibition (87-89). A short stretch of amino acids, in ITSN1 (intersectin), located in the linker region between the N-terminal

SH3 domain and DH domain directly mediates the intra-molecular inhibition in this

GEF; however, the mechanism of release is poorly understood (90).

V.2.2: Release of inhibitory intra-molecular interactions. A common way in which intra- molecular interactions within GEFs are relieved is through phosphorylation. Upon activation of an RTK by a growth factor, RTKs can directly phosphorylate the GEF or promote the recruitment of other kinases, ultimately releasing the GEF from its inhibited state. The best-characterized example of this is seen with members of the Vav family of

GEFs. Structural determination of Vav GEFs show that they are basally inhibited via intra-molecular interactions (91-93). Specifically, in Vav1 it has been shown that the DH domain is directly, but weakly, inhibited by a helix from the adjacent acidic domain. This interaction is strengthened by contacts of the calponin homology (CH) domain with the acidic, pleckstrin homology, and DH domains (91-93). Stimulation of the receptor

(TCR) leads to stepwise relief of inhibition: two initial phosphorylation events disrupt the modulatory CH contacts, facilitating phosphorylation at the third tyrosine residue, and

22 finally stimulation of nucleotide exchange on Rho GTPases.

Similarly, Tim-family members Tim, Ngef and Wgef, remain in an inhibited state

through the formation of intra-molecular interactions. In the basal state, the catalytic

potential of Tim-related GEFs is inhibited by two main interactions: (1) a C-terminal

inhibitory helix that packs tightly against the DH domain and (2) the C-terminal SH3

domain binds an N-terminal polyproline region that further packs the inhibitory helix into

the DH domain. A poorly understood mechanism leads to the disruption of the intra-

molecular association of the SH3 domain with the polyproline region upon stimulation

with serum containing growth factors. This allows for phosphorylation by Src to occur on tyrosine residues in the C-terminal helix, which prevents reassociation of the helix with the DH domain and fully activates the exchange potential of the GEF (94).

Phosphorylation in several other Dbl family GEFs has been seen in response to

specific stimuli and has been shown to increase GEF activity; however, how these

phosphorylating events lead to the release of inhibition is still unknown. Recently, a

crystal structure of the DH-PH-PH domains FARP2 revealed the intra-molecular interactions of this GEF. Structural studies showed that the GTPase-binding site is jointly blocked by interactions formed between the last helix of the DH domain, the two PH domains, and the two inter-domain linker regions (95). The mechanism of release is not well understood, but FARP2 has been shown to be phosphorylated on tyrosine residues by Src and this may contribute to relieving the inhibited state of the GEF (96). The crystal structure of the GEF Asef has also allowed for the understanding of the inhibitory interactions regulating this GEF. The RT-loop and the C-terminal region of Asef’s SH3 domain bind intra-molecularly to the DH domain (97). Tyrosine phosphorylation of Asef

23 by Src kinase has been observed in response to EGF stimulation (98). Unfortunately, the site of tyrosine phosphorylation was not part of the solved crystal structure and it is difficult to evaluate its role in releasing the inhibition in Asef.

Tyrosine phosphorylation of other GEFs such as Tiam1 (99,100), Fgd1 (101,102), and )-Pix (103) has been shown to increase GEF activity; however, if and how this modification plays a role in the release of inhibition is not known.

It is obvious that several Dbl family GEFs have evolved mechanisms to maintain inhibition in order to prevent constitutive activation of downstream effectors. Post- translational modifications, such as tyrosine phosphorylation, seem to be a recurring theme in relieving inhibition maintained by Dbl GEFs.

V.2.3: Inter-molecular modes of regulation. Dbl GEFs can be maintained in an inhibited state via interactions with other proteins. Studies in our lab have shown that the molecular chaperones Hsc70 and Hsp90 and the U-box -protein ligase CHIP regulate Dbl by efficiently ubiquitinating and rapidly degrading the protein. This occurs by direct interaction of Hsc70, Hsp90, and CHIP with the amino terminal spectrin domain of Dbl and C-terminal PH domain. These inter-molecular interactions not only promote an inhibited state but also result in rapid degradation of the protein and low steady-state expression levels. More importantly, our studies have shown that the oncogenic form of Dbl, which lacks the spectrin domain, is unable to form interactions with the Hsc70/Hsp90/CHIP complex and thus, “escapes” regulation and maintains high expression levels within the cell. This promotes constitutive activation of downstream effectors, and ultimately cellular transformation (104,105).

In full-length Dbl the activation signal releasing the Hsc70/Hsp90/CHIP complex,

24 thereby relieving Dbl’s inhibition and promoting its activation are not well understood.

As with intra-molecular interactions we thought it possible for tyrosine phosphorylation

to function as an activating signal in proto-Dbl, thereby possibly promoting the release of

the Hsc70/Hsp90/CHIP complex, activating the GEF, and allowing it to catalyze the

exchange reaction on downstream GTPases. Understanding this potential mechanism of

regulation was one of the main focuses of this thesis.

V.3: Other Modes of Dbl GEF Regulation

V.3.1: GEFs in signaling pathways: their relocalization, and roles as scaffolds and

coordinators of signaling pathways. Other than the intra- and inter- molecular

interactions formed by Dbl family GEFs, GEFs are also regulated by their ability to

relocalize to different intracellular compartments, in response to receptor activation, and

function as scaffolds and coordinators of signaling pathways. Such changes, allow for timely activation of not only their cognate GTPases but also subsequent downstream

effectors. In many cases, GEFs remain localized to the cytoplasm, in a basal state. Upon

stimulation, GEFs relocalize to the location of its cognate GTPase, generally at the

plasma membrane. In most cases, the PH domain of the GEF mediates this relocalization,

but other domains of the GEF may also aid in association. The canonical Ras pathway is

a prime example of relocalization upon stimulation of a cell surface receptor. EGF binds

and activates the tyrosine kinase activity of the EGF receptor, promoting auto-

phosphorylation of the receptor. The adapter protein, Grb2, relocalizes and associates

with the EGFR by binding the phosphorylated residues of the receptor through its SH2

domain. This association promotes the relocalization of the Dbl-GEF, Sos1, from the

cytoplasm to the plasma membrane via EGFR and Grb2 (106). This interaction promotes

25 Sos1 exchange activity on membrane-associated Ras, and initiates a signal cascade that

culminates in transcriptional changes, proliferation, and anti-apoptotic pathways.

There are several other examples of this form of regulation. The GEF,

neuroepithelioma transforming gene-1 (Net-1) is primarily localized to the nucleus, but

upon stimulation translocates to the plasma membrane where it activates RhoA (107).

Likewise, Ect2 is localized within the nucleus during interphase but relocalizes to

microtubules and the cleavage furrow during later phases of the cell cycle. This

relocalization allows for the proper activation of Rho GTPases and for accurate cell

division to occur (108).

Not only are GEFs regulated by their relocalization but also by their ability to

directly facilitate the formation of multi-protein complexes necessary for specific

signaling of particular pathways. This is particularly important since many Dbl GEFs are

widely expressed throughout tissue types, thus allowing for specific outcomes depending

on cellular context. For example, Rac1 activation by Tiam1 is dependent on the

redistribution of Tiam1 from the cytoplasm to the plasma membrane upon stimulation of

cells with various growth factors. In one study, Tiam1 was shown to interact with

spinophilin, a scaffold protein that is present in the plasma membrane. In response to

serum stimulation, Tiam1 was recruited from the cytoplasm to the plasma membrane via

interaction with spinophilin (109). In addition to Tiam1, spinophilin also binds the Rac1

effector, p70 S6 kinase (109). With this dual ability to interact with both Tiam1 and p70

S6 kinase, spinophilin mediates the preferential activation of one Rac1 effector over

others, ultimately stimulating protein translation (109). Similarly, Tiam1 directly interacts with JNK-interacting protein-2. This interaction has been shown to occur in response to

26 serum stimulation and results in the formation of a large multi-protein complex that

promotes the activation of p38 (110). JNK-interacting protein-2 is known to function as a

scaffold. Interaction with Tiam1 to JNK-interacting protein-2 results in the activation of

Rac1, which leads to the association of MLK3 (mixed-lineage kinase-3 – a Rac effector),

MKK3 (MAPK-kinase-kinase-3) and p38 MAPK with JNK-interacting protein-2 (110).

This resulting complex promotes the specific activation of p38 and p38 effectors (110). It is obvious with these two examples involving Tiam1, different scenarios lead to different modes of regulation of the GEF and ultimately stimulation of different downstream effectors.

With the vast number of Dbl family GEFs (>80) and the number of GTPases, proper regulation of GEFs allows for specific cellular outputs. By relocalizing and taking part in signaling scaffolds in response to various stimuli, it is obvious that Dbl GEFs play crucial roles in the precise initiation and mediation of signaling pathways.

V.3.2: Dbl GEFs mediate cross talk between pathways. Although it is easier to think of signaling pathways as linear outputs, the fact of the matter is that signaling pathways are quite dynamic and involve a great deal of cross talk; this form of regulation allows for very specific cellular outputs. In many cases, communication between pathways is mediated through the function of a Dbl family GEF. Several Dbl GEFs, in addition to the canonical DH/PH module contain other domains that help mediate cross talk with other

GTPase subfamilies, such as the Ras family. Sos1 is considered both a Ras and Rho exchange factor as defined by its Ras activating domain (Cdc25) and the Rho GEF domain (DH/PH). Studies have shown that the isolated tandem DH/PH motif of Sos1 is inactive unless co-expressed with Ras. These observations suggest that Sos1 first

27 stimulates Ras activation, leading to increased PI 3-kinase activity, which then leads to the subsequent activation of Sos1’s DH/PH domain and Rac1 activation (111,112).

Tiam1, a Rac GEF, has been shown to be an immediate downstream effector of activated Ras. Studies have shown that Tiam1 contains a Ras-binding domain, which allows it to directly interact with GTP-bound Ras. Upon interaction with GTP-Ras,

Tiam1 can then activate Rac1. In this manner, Tiam1 functions as a transducer of Ras- controlled signaling pathways to outputs that are controlled by Rac1 (113).

In addition to cross talk between Ras subfamilies evidence suggest that activation of Rho GTPases can occur in a sequential manner, such that activation of one small

GTPase stimulates GTP loading of another. Dbs has been shown to preferentially bind activated Rac1, and this complex promotes the activation of RhoA (114). More specifically, studies done by Cheng et. al. show that Dbs associates with GTP-Rac1, at the plasma membrane via Dbs’s PH domain. The association is then further stabilized through phosphoinositide interactions, which may also be involved in directing the proper orientation or conformation of Dbs to activate RhoA (114).

Mounting evidence points to the importance of cross talk between and within different subfamilies of the Ras superfamily of GTPases. Coordinated regulation of nucleotide exchange factors, ensures that distinct subsets of GTPases are activated in response to a given stimulus and allows for specific biological responses to occur.

28 VI. GEF Dysfunction and Diseases

The prevalence of GEF dysfunction is becoming more and more evident in the literature. In general, mutations that result in impaired GEF activity cause improper regulation of GTPase function. Ultimately this leads to various types of diseases including: various types of cancers, developmental and neurological disorders, and viral/bacterial pathologies.

VI.1: Cancers. The rearrangement of the GEF LARG and the mixed lineage leukemia

(MLL) gene has been identified in acute myelogenous leukemias. Formation of the

MLL–LARG chimeric protein results in the loss of N-terminal sequences upstream of the

DH domain of LARG, which leads to the loss of regulation of the DH domain and constitutive GEF activity (115). A missense mutation in Tiam1’s N-terminal portion of the PH domain perturbs Tiam1 association to the membrane and promotes transformation in a poorly understood mechanism, contributing to renal cell carcinomas (116). Indirect mechanisms that promote the constitutive activation of GEFs also contribute to cancer progression. For example, a protein encoded by a tumor suppressor gene APC, regulates the GEF Asef. Mutations that lead to truncated forms of APC promote constitutive activation of Asef GEF activity and the migration of colorectal cancer cells (117-119).

Additionally, over-expression of various Dbl family GEFs have been observed.

High expression levels of Fgd1 were noted in malignancies of the breast and prostate, whereas expression levels of this GEF in matched normal tissues was negligible (120).

High expression of Tiam1 has been observed in colorectal cancers and its expression levels correlate well with disease prognosis (121-123). High expression levels of Ect2 have been reported in lung and esophageal tumors, but not in matched ‘normal’ tissues.

29 In addition to lung and esophageal cancers, Ect2 is highly expressed in various other malignancies including those of the brain, bladder, pancreas, and ovary (124). Dbl, the proto-typical family member and one of the focuses of our studies, is over-expressed in

Ewing sarcomas and tumors of neuroectodermal origin (125,126). Over-expression of these GEFs most likely leads to the constant activation of cognate GTPases and ultimately leads to malignancy; however, the exact mechanisms are unknown.

VI.2: Developmental/Neurological Disorders. Dbl-like GEFs have wide ranging roles in neuronal development and morphophology. In these types of diseases, in many cases mutations in GEFs, resulting in their loss of function lead to the loss of activation of Rho

GTPases and their downstream effectors. In the nervous system, Rho GTPases play crucial roles in the regulation of growth cone motility, axonal migration, and dendritic spine morphogenesis. As can be appreciated, mutations in GEFs that alter GTPase function can result in different forms of mental retardation and developmental abnormalities. Mutations in Fgd1 lead to X-linked skeletal dysplasia (Aarskog-Scott

Syndrome), a developmental disorder that is characterized by the malformation of skeletal structures (127). Mutations in ARHGEF10 has been linked to autosomal dominant neuropathy, as defined by slowed motor and sensory nerve conduction velocities (128). Disruption of the gene that encodes "-Pix (Cool-2) as a result of reciprocal translocation is associated with X-linked non-specific mental retardation (129). A loss of function of Alsin (Als2) is thought to be caustive for the onset of a rare form of juvenile amyotrophic lateral sclerosis. Patients with amyotrophic lateral sclerosis have mutations in Alsin that result in the premature truncation of the protein and a loss of the DH domain, and this is thought to impair proper neurite outgrowth (130).

30 Mutations and translocations affecting ARHGEF9 (collybistin) have been correlated with

epilepsy and hyperekplexia (131,132), and mutations in the gene encoding Fgd4 (frabin)

have been correlated with the onset of Charcot-Marie-Tooth disease (133).

Spinocerebellar ataxias (SCAs) are debilitating heritable neuropathies characterized by a

loss of fine motor coordination. The 16q22.1-linked autosomal dominant (ADCA) is of particular interest since mutations in Plekhg4 are highly associated with this disease. Studies have shown that various mutations in Plekhg4’s UTR significantly segregate with this particular ADCA (134-139).

VI.3: Viral Infections. Some Dbl-family GEFs facilitate the pathogenic invasion of mammalian host cells and as such can be regulated by viral proteins. T-cells infected with human T-cell-leukemia virus I (HTLV-I) show altered Vav phosphorylation, thereby most likely preventing its full activation. Since Vav is involved in T cell signaling and

Vav phosphorylation occurs upon activation of T cells, control of the activation state of

Vav by viral proteins may relate to the leukemogenic potential of HTLV-I (140). The human immunodeficiency virus type-1 (HIV-1) protein, Nef, is essential for AIDS progression. Nef directly interacts with Vav. Association with Vav mediates Rac, Cdc42, and PAK2 activation. The activation of this complex is necessary for AIDS progression

(141). Another mechanism by which HIV-1 utilizes Dbl GEFs is by inactivating p115-

RhoGEF. This inactivation in Dbl GEF activity results in the loss of RhoA function, which may aid in HIV-1 pathogenicity by perturbing T-cell migration, proliferation, and interaction with other cells (142).

31 Statement of Purpose

Dbl GEFs play vital roles in cellular function and perturbations in their function lead to the onset of various types of diseases. The overall goal of my thesis research is to understand the roles that GEFs play in diseased states. As such, two main studies were conducted: (1) characterization of an autosomal dominant spinocerebellar ataxia- associated Dbl-family GEF, Plekhg4 and (2) determination of the molecular mechanisms that regulate the activity of the proto-typical family member, Dbl.

(1) Mutations in the UTR region of the puratrophin-1 gene (Plekhg4) are associated with the neurological disorder autosomal dominant spinocerebellar ataxia

(ADCA). The molecular mechanisms underlying this pathology, prior to our studies presented here, were completely unknown. My thesis aimed to (a) examine whether

Plekhg4 is a bona fide GEF, (b) identify the mechanisms that regulate the steady-state expression levels of this protein, and (c) to understand potential mechanisms that link

ADCA to mutations in Plekhg4.

(2) Dbl is the founding member of the Dbl family GEFs for the Rho family of small GTPases Rho, Cdc42, and Rac. Studies have shown that in an unstimulated state,

Dbl remains in an inhibited state. The upstream regulatory events that regulate Dbl activity and relieve this inhibition are not well understood. My thesis aimed to identify the role that tyrosine phosphorylation plays in the activation cycle of this GEF. More specifically, I aimed to (a) identify sites of phosphorylation, and (b) understand how phosphorylation affected overall GEF activity.

32 CHAPTER 2

Plekhg4 is a novel Dbl-family guanine nucleotide exchange factor for Rho-family GTPases**

Meghana Gupta1, Elena Kamynina*2, Samantha Morley*3, Stacey Chung*3, Nora Muakkassa2, Hong Wang3, Shayna Brathwaite3, Gaurav Sharma2 and Danny Manor1, 3

Departments of Pharmacology1, and Nutrition3, School of Medicine, Case Western Reserve University, Cleveland, OH, 44106 and Cornell University2, Ithaca, New York, 14853

**Running title: Ataxia-related Plekhg4 activates Rho GTPases

*Equal contributors To whom correspondence should be addressed: Danny Manor, Department of Nutrition, Department of Pharmacology, Case Western Reserve University School of Medicine, WG-48, Cleveland, OH, 44106. Phone: 216-368-6230; Fax: 216-368-6644; E-mail: [email protected] Keywords: Dbl, guanine nucleotide exchange factor; spinocerebellar ataxia, heat shock protein 70; heat shock protein 90, CHIP E3 ligase

Published in The Journal of Biological Chemistry, 2013 May 17;288(20):14522-30.

33 CAPSULE:

Background: Plekhg4 is putative guanine nucleotide exchange factor associated with autosomal dominant spinocerebellar ataxia.

Results: Plekhg4 is regulated by the heat shock proteins and functions as a bona fide guanine nucleotide exchange factor.

Conclusion: Plekhg4 is the first RhoGEF implicated in spinocerebellar ataxia.

Significance: Aberrant GTPase signaling is a novel possible mechanism underlying autosomal dominant spinocerebellar ataxia.

ABSTRACT:

Mutations in the PLEKHG4 (puratrophin-1) gene are associated with the heritable neurological disorder autosomal dominant spinocerebellar ataxia. However, the biochemical functions of this gene product have not been described. We report here that expression of Plekhg4 in the murine brain is developmentally regulated, with pronounced expression in the newborn midbrain and brainstem that wanes with age, and maximal expression in the cerebellar Purkinje neurons in adulthood. We show that Plekhg4 is subject to ubiquitination and proteasomal degradation, and its steady-state expression levels are regulated by the chaperones Hsc70 and Hsp90 and by the ubiquitin ligase

CHIP. On the functional level, we demonstrate that Plekhg4 functions as a bona fide guanine nucleotide exchange factor (GEF) that facilitates activation of the small GTPases

Rac1, Cdc42, and RhoA. Over-expression of Plekhg4 in NIH3T3 cells induces rearrangements of the actin cytoskeleton, specifically enhanced formation of lamellopodia and fillopodia. These findings indicate that Plekhg4 is an aggregation-prone

34 member of the Dbl-family GEFs, and that regulation of GTPase signaling is critical for proper cerebellar function.

INTRODUCTION:

Guanine nucleotide exchange factors (GEFs) comprise a large family of regulatory proteins that control diverse intracellular processes such as gene expression, cytoskeletal rearrangements, intracellular trafficking and metabolism (143). Accordingly, dysregulation of GEF levels or activity have profound consequences on normal cell behavior, and lead to proliferative and developmental pathologies. For example, activating mutations in VAV1 (144), P-Rex1 (145), and Dbl (146) contribute to cell transformation and tumorigenesis, and mutations in FGD1 are associated with facio- gential dysplasia and mental retardation (147).

GEFs mediate their biological effects by facilitating the exchange of bound GDP for GTP in the nucleotide-binding pocket of small GTP-binding proteins. The activated

GTPases then stimulate specific downstream effectors that control cytoskeletal architecture, vesicular genesis and trafficking, cell polarity, and cell cycle progression

(3,148). The Dbl oncogene (51) is the prototypic member of a large family of structurally and functionally related GEFs, which activate GTPases from the Rho family and are characterized by a tandem arrangement of a Dbl homology (DH) domain and a pleckstrin homology (PH) domain (4). While the DH domain is the minimal functional unit required for nucleotide exchange (149), the PH domain is essential for proper intracellular localization and cell transformation (57). Amino-terminal to the DH/PH module are spectrin repeats that mediate association of Dbl with the molecular chaperones Hsc70 and

Hsp90, and the ubiquitin E3 ligase CHIP (105). These interactions determine the steady-

35 state expression levels of Dbl by modulating the rate of ubiquitination and proteasomal

degradation (104). It is generally believed that oncogenic mutations activate Dbl by

disrupting intra-molecular (150) and inter-molecular (104) interactions that alter GEF

activity and levels.

Multiple lines of evidence demonstrate that Dbl-like GEFs and their substrate

GTPases play important roles in development, morphogenesis, and function of the central

nervous system (151), and that they transduce signals from neuronal surface receptors

such as EphB, TrkB, NMDA and the AMPA receptor. Spinocerebellar ataxias (SCAs) are

debilitating heritable neurodegenerative disorders characterized by progressive loss of

motor coordination and balance that stem from cerebellar dysfunction (152). Of the

multiple SCA forms, the 16q22.1-linked autosomal dominant cerebellar ataxia is of

special interest. Originally, a single C-to-T substitution in the 5’ un-translated region

(5’UTR) of the PLEKHG4 gene (-16C>T) was shown to associate with the disease (134-

139). Moreover, brains of affected patients exhibited selective atrophy of cerebellar

Purkinje neurons, accompanied by cytoplasmic aggregation of the Plekhg4 protein (134).

Later studies extended this ataxia-linked genomic site to a 900 Kb region of the

PLEKHG4 promoter (139). Interestingly, ataxia-linked penta-nucleotide repeat insertions of various sizes were also observed in that locus (153). The genetic findings that link

PLEKHG4 to 16q22.1 SCA are underscored by histopathological and biochemical evidence. Specifically, cerebella samples from 16q22.1-linked SCA patients showed a significant reduction in Plekhg4 mRNA, and enhanced formation of cytoplasmic

aggregates that contain Plekhg4, G58K and spectrin (134).

36 Plekhg4’s primary sequence indicates the presence of a Sec14 domain that often mediates lipid-binding, a spectrin domain that typically mediates protein-protein interactions, and the canonical DH/PH module, which catalyzes nucleotide exchange on substrate GTPases (149). The conservation of this signature domain architecture raises the intriguing possibility that Plekhg4 functions in the cerebellum by mediating the activation of small GTPases from the Rho family. If true, this is the first case where aberrant GEF-GTPase signaling is a likely molecular culprit underlying SCA pathology.

Toward this end, we report here our initial biochemical characterization of Plekhg4 as an activator of Cdc42, Rac1, and RhoA, and the post-translational mechanisms that control its expression levels.

MATERIALS AND METHODS:

Cell Culture. NIH3T3 cells and COS7 cells were cultured in DMEM supplemented with

o 10% calf serum or 10% fetal bovine serum (Hyclone), respectively, in 5% CO2 at 37 C.

Molecular constructs. A hemaglutinin (HA)-fused Plekhg4 construct was generated by

PCR amplification of the PLEKHG4 reading frame from a commercial image clone in the pOBT vector (I.M.A.G.E. clone # 6291175; ATCC 10539799) using primers that add an in-frame amino-terminal influenza hemaglutinin (HA) tag. The resulting amplicon was ligated into a pCDNA3.1/HA/Hygro(+) vector (Invitrogen). Constructs encoding Dbl

(residues 1-925) and small GTPases were previously described (35,105,154). The pEBG-

GST-Plekhg4 construct was generated by cloning the cDNA into a pEBG-GST vector

(generous gifts of Yi Zheng, Cincinnati Children's Hospital Medical Center, and Silvio

Gutkind, National Institute of Dental and Craniofacial Research). CHIP cDNA (a generous gift from Cam Patterson, University of North Carolina) was amplified by PCR

37 and cloned into a pCDNA4.1-HisMax vector (Invitrogen) to generate the Xpress-tagged

CHIP construct. Myc-ubiquitin (in the pCW7 plasmid) was a generous gift of Ron Kopito

(Stanford University). All constructs were verified by restriction mapping and sequencing.

Reagents. MG132 (Calbiochem) was dissolved in DMSO and stored at -20º C. 17-N- allylamino-17-demethoxygeldanamycin (17-AAG; NSC 330507) was obtained from

A.G. Scientific and from the Division of Cancer Treatment and Diagnosis, National

Cancer Institute.

Antibodies. Anti-HA (clone HA.11 – both ascites and purified), anti-HA-FITC conjugates, and anti-GST antibodies were purchased from Covance Inc. Anti-Dbl antibodies (sc-89 and sc-28582) were from Santa Cruz Biochemicals. Anti-Hsc70 (SPA-

815 and SPA-820) and anti-Hsp90 (SPA-835) antibodies were obtained from StressGen

Biotechnologies. The anti-ubiquitin antibody (clone FK2) was from obtained from

Millipore.

RT-PCR. Total RNA was isolated from indicated mouse brain tissues with the

RNAqueous-4PCR kit (Ambion Inc. Austin TX, USA). RNA (500 ng) from each sample was reverse-transcribed using High-Capacity cDNA Reverse Transcription Kits (Applied

Biosystem, USA) and cDNA was amplified using primers based on the mouse brain Plekhg4 sequence. Plekhg4 primers (forward, 5’-TCC CTA AGC CTG CTG ACT

G-3’; reverse, 5’-ATG TGG GTC AGC AGG CTC-3’) were chosen to amplify a 412 bp fragment, from nucleotide 494 to 905 in the sequence (Gen Bank Accession number

EFGHHIHJIKKKL=()-actin was used as a housekeeping gene for normalizations.

38 Immunohistochemistry. Formalin-fixed paraffin-embedded brains were sagittally sectioned (20 mm), deparaffinized, and subjected to antigen retrieval step with 10 mM citrate. Sections were incubated overnight at 4$C with anti-Plekhg4 antibody (Abcam).

Plekhg4 expression was visualized using avidin-biotinylated peroxidase, and developed with 3,3’-diaminobenzidine (DAB).

Ubiquitination and chaperone association. Fourty-eight hours post transfection, indicated proteins were isolated by gluthathione-agarose precipitations or immunoprecipitations, and associated proteins were analyzed by Western blotting with the indicated antibodies.

GEF- GTPase association. GST fusion proteins of Rac1, Cdc42, and RhoA were expressed in E. coli and purified as previously described (155). cDNAs were transfected into COS7 cells using Fugene6 according to manufacturer’s protocol, and cell lysates were prepared as previously described (105). Recombinant GTPases were immobilized on glutathione agarose and washed extensively with RIPA buffer supplemented with 1%

NP-40 prior to nucleotide loading. To generate nucleotide-free GTPases, immobilized

GTPases were washed three times in 20 mM HEPES pH 7.4, 200 mM NaCl, 10 mM

EDTA, 2 mM PMSF and used immediately. To generate constitutively-active GTPases, immobilized proteins were extensively washed with 20 mM HEPES pH 7.4, 200 mM

NaCl, 10 mM EDTA, 2 mM PMSF, 100 mM GTP#S, and then with the same buffer

supplemented with 10 mM MgCl2. Immobilized GTPases prepared were incubated with

GEF-containing lysates at 4o C for 1.5 hours, washed 3 times in the respective buffer by

centrifugation, and associated GEFs were analyzed by Western blotting.

39 GTPase activation assays. To evaluate GEF activity, the fraction of GTPase that is in the

active (GTP-bound) state was determined using an established selective precipitation

approach (156,157). Briefly, COS7 cells were co-transfected with Rac1, Cdc42, or RhoA

(HA-tagged in the pKH3 vector) together with the GEF (Plekhg4 or Dbl in the

pCDNA3.1 vector). Cells were lysed in 20 mM HEPES (pH 7.4), 150 mm NaCl, 1%

Nonidet P-40, 10% glycerol, 10 mM MgCl2, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl

72+,#3&'C($%&(IH(MNO<2('$4*(2'+5'5"3%($%&($5#,"3%3%($%&(3%4+-$"'&($"(PQ(R(7,#(S(*(03"*(

!SH(MN(3mmobilized GST-fused GTPase-binding domain of mPAK3 (156) or of Rhotekin

(157). The amount of activated G-protein was visualized with anti-HA western blotting, and compared to the total GTPase levels as determined by anti-HA immunoblotting of whole-cell lysates.

Fluorescence microscopy. NIH3T3 cells were seeded on collagen-coated coverslips and transfected using PolyFect Transfection Reagent (Qiagen) according to manufacturer’s instructions. Thirty-one hours post-transfection, cells were serum-starved for 17 hours, fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with the indicated reagents. Stained slides were imaged on a Zeiss LSM 510 META confocal microscope.

For Hsp90 inhibition studies: HA-tagged Plekhg4 protein was visualized using an anti-

HA FITC conjugated antibody (Covance) and imaged on a Leica DM4100B inverted fluorescence microscope.

RESULTS:

Expression of Plekhg4 is developmentally regulated. Association between the Plekhg4 gene and spinocerebellar ataxia led us to take a deeper look into Plekhg4 expression in

40 the brain. Previously published data shows that Plekhg4 mRNA expression is high in the testis and pancreas, has mild expression in the spleen, , prostate gland, heart, placenta, lung, liver, kidney and has low expression in the ovary, small intestine, colon, peripheral-blood leukocytes, whole brain, and skeletal muscle (134). Examination of the temporal and spatial expression of Plekhg4 mRNA in the mouse brain revealed that the transcript is selectively expressed in the cerebellum, brain stem and midbrain in an age- dependent manner (Figure 1A). mRNA expression of Plekhg4 in the newborn brainstem and midbrain decreased with advancing age, leaving the cerebellum as the primary site of expression in the mature animal. To augment these results we employed immunoreactivity-based approaches aimed at detecting expression patterns of the

Plekhg4 protein. In agreement with previous reports of Plekhg4 mRNA (134), we detected high-level expression of the Plekhg4 protein in the testis (data not shown).

Additionally, we observed high expression of Plekhg4 in the cerebellum, specifically in

Purkinje neurons, as shown in Figure 1B. Interestingly, Ishikawa et al reported the existence of Plekhg4 protein aggregates in Purkinje neurons of SCA-afflicted patients

(134). Taken together, these findings strongly suggest that Plekhg4 plays important role in cerebellar function.

Domain structure of Plekhg4. Figure 2A depicts the predicted domain structures of

Plekhg4, as compared to the prototypical GEF of the Dbl family, the proto-oncogene product Dbl. Alignment of Plekhg4’s putative GEF (DH) domain with that of Dbl’s revealed 58% sequence similarity and 42% sequence identity. Importantly, high similarity is evident in residues thought to be critical for catalyzing nucleotide exchange

(Figure 2B), for binding the substrate GTPase (Figure 2C), and for dictating GTPase

41 specificity (Figure 2D;(58,59)). Taken together, the high degree of structural similarity between Plekhg4 and Dbl, suggests that, like Dbl’s, Plekhg4’s primary function is in activating the substrate GTPases RhoA, Rac1 and Cdc42.

GTPase binding and activation by Plekhg4. Despite the striking similarity to Rho-GEFs from the Dbl family, Plekhg4’s ability to stimulate nucleotide exchange on substrate

GTPases has not been reported. We therefore assessed the ability of Plekhg4 to activate the different GTPases in cultured cells, using the p21 binding domain (PBD) assay that was originally developed for Ras (158), and adapted for Cdc42, Rac and RhoA

(156,157). Specifically, we used an immobilized peptide sequence from the GTPase’s down-stream target (mPAK3 for Rac/Cdc42 and Rhotekin for RhoA) to selectively precipitate the GTP-bound form of each GTPases. As shown in Figure 3A, expression of

Plekhg4 in serum-starved cells activated Cdc42, Rac1, and RhoA, evident by the increased fraction of the GTPase that precipitates with the immobilized target PBD. To gain insight into the mechanisms of Plekhg4-stimulated nucleotide exchange, we examined the binding of Plekhg4 to its substrate GTPases. We employed an established approach in which the GEF is precipitated with immobilized GTPases in either the nucleotide-free or the GTP#S–bound forms (149). The data (Figure 3B) demonstrate that, like Dbl, Plekhg4 associated to Cdc42, RhoA and Rac1 with similar affinity. Moreover, binding was highly selective for the nucleotide-depleted form of the GTPase (i.e. affinity for the nucleotide-depleted GTPase was >100-fold tighter compared to the GTP#S–bound form), suggesting that like other GEFs, Plekhg4 stimulates substrate GTPases by catalyzing dissociation of the bound GDP (27). Taken together, our observations show

42 that Plekhg4 is a functional guanine nucleotide exchange factor for Cdc42, Rac1, and

RhoA.

Plekhg4 regulates the actin cytoskeleton. Activation of small GTPases from the Rho family stimulates specific rearrangements of the actin cytoskeleton. Specifically, GTP loading by Cdc42 enhances fillopodia formation, whereas activation of Rac1 and RhoA induces formation of lamellipodia and stress fibers, respectively (159-161). Ectopic expression of GEFs in cultured fibroblasts elicits the cytoskeletal architecture pattern typical of their substrate GTPase (35), thereby providing a convenient read-out for their in vivo signaling state. We therefore examined the effect of Plekhg4 expression on the actin cytoskeleton in cultured NIH3T3 fibroblasts. Figure 4A shows typical fluorescence micrographs of cells expressing Plekhg4 (or control vector). The data indicate that expression of Plekhg4 led to pronounced reorganization of the actin cytoskeleton.

Specifically, we observed enhanced formation of lamellipodia, fillopodia, and the beginnings of disorganized stress fiber formation (see insets in Figure 4). The subcellular expression pattern of Plekhg4 paralleled that of the actin cytoskeleton, with pronounced co-localization in areas of actin rearrangement (Figure 4). These results affirm our observation above that Plekhg4 acts as a functional GEF for Cdc42, Rac, and Rho

GTPases, and further demonstrate that at least one signaling axis of each of these

GTPases, regulating cell shape and motility, is activated by Plekhg4.

Plekhg4 associates with the chaperones Hsc70 and Hsp90, and the E3 ubiquitin ligase

CHIP. We have previously demonstrated the existence of a novel multi-protein complex that regulates Dbl signaling at the level of protein degradation. Specifically, we have shown that the spectrin domain in Dbl’s amino terminus mediates interaction between the

43 GEF, the chaperones Hsc70 and Hsp90, and the E3 ubiquitin ligase CHIP (104,105). This

multi-protein complex mediates continuous ubiquitination of Dbl and its subsequent

proteasomal degradation, such that steady-state expression levels of the protein are

maintained at very low levels (104,105). The similar domain architecture of Plekhg4 and

Dbl (Figure 2), and especially the between the two proteins’ spectrin

homology domains (28% identical, 46% similar), raise the possibility that Plekhg4’s

turnover is also subject to regulation by ubiquitination and proteasomal degradation. To

examine this possibility, we first examined whether Plekhg4 is associated with the

molecular ‘coordinators’ of protein folding and degradation, the chaperones Hsc70 and

Hsp90, and the ubiquitin ligase CHIP ((162)). We found that in growing cells Hsc70,

CHIP, and Hsp90 co-precipitated with GST-Plekhg4. These interactions were specific, as

the complex did not form in lysates expressing the control GST vector (Figure 5A).

These findings indicate the presence of a constitutive complex composed of the GEF, the

molecular chaperones Hsc70 and Hsp90, and the ubiquitin ligase CHIP.

Chaperone complex dictate the intracellular fate of Plekhg4. A delicate interplay

between the chaperones Hsp90 and Hsc70 directs the fate of client proteins toward

stabilization and refolding, or ubiquitination and proteasomal degradation, respectively

(162). To evaluate Hsp90’s role in determining the fate of Plekhg4, we examined how the

selective Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG; (163) affects Plekhg4 stability. As shown in Figure 5B, when protein synthesis was inhibited by cycloheximide, expression levels of Plekhg4 were stable over a 5 hour period (Figure 5B, upper panel). Treatment with 17-AAG caused a dramatic decline in Plekhg4 levels over time (Figure 5B middle panel), indicating that the protein is rapidly degraded. Co-

44 treatment with the proteasomal inhibitor MG132 stabilized Plekhg4 levels, thereby reversing the effect of 17-AAG (Figure 5B lower panel). These data indicate that the chaperone Hsp90 protects Plekhg4 from proteasomal degradation. In light of the established involvement of these proteins in protein ubiquitination and degradation (164), we examined the ubiquitination status of Plekhg4. When GST-Plekhg4 was precipitated from continuously growing cells, the protein was heavily modified by the endogenous ubiquitination system (Figure 5C). Similar results were obtained when we employed ectopically-expressed ubiquitin (data not shown). Moreover, Plekhg4’s ubiquitination level was markedly enhanced upon treatment with 17-AAG (Figure 5D). Finally, treatment with 17-AAG caused a dramatic change in the intracellular localization of

Plekhg4: in the absence of any treatment, the protein displayed a diffuse cytosolic expression with some localization to plasma membrane structures reminiscent of lamellopodia and actin microspikes (Figure 5E, upper panels). In the presence of 17-

AAG, Plekhg4’s localization changed to a distinct pattern characterized by dense irregular punctae (Figure 5E, red arrows). In some cells, the Plekhg4 punctae were larger, resided near the nucleus (Figure 5E, white arrows) and displayed the characteristic appearance of aggresomes (165,166). Further immunofluorescence studies in 17-AAG- treated cells revealed pronounced overlap between the intracellular localization of

Plekhg4 and that of ubiquitin (data not shown). Taken together, our data indicate that

Plekhg4 is a bona fide client protein of Hsc70, Hsp90, and CHIP. The data further show that Plekhg4 is subject to continuous ubiquitination and proteasomal degradation by these mediators of the “triage decision” (167).

45 Examination of Plekhg4’s UTR. Our interest in the Plekhg4 protein was sparked by the findings that mutations in its 5’ UTR are associated with occurrence of spinocerebellar ataxia [Ishikawa, 2005 #4892;Ohata, 2006 #6470;Ouyang, 2006 #6476;Onodera, 2006

#6471;Nozaki, 2007 #6468;Amino, 2007 #6044;]. We hypothesize that mutations in

Plekhg4’s UTR region causes an increase in Plekhg4 protein levels, and lead to aggregation of the protein. As such, we questioned whether these mutations lead to changes in predicted transcription factor recognition sites in this region. Of the several known mutations in Plekhg4, we focused on the three most prevalent SNPs (139). Two of these (positions 65,049,292 and 65,337,827 on ) are not predicted to cause any changes to transcription factor recognition. However, the most common SNP (-

16 C>T, position 65,871,434 on chromosome 16) is predicted to result in the loss of interaction with the transcription factor Yin Yang1, and does not generate a new binding site for another transcription factor. This transcription factor is widely expressed and can function as either an activator or a repressor of transcriptional activity, depending on the co-factors that it recruits. (168). For instance, studies have shown that Gon4l (Gon4-like) interacts with Yin Yang1, the transcriptional co-repressor Sin3a, and its functional partner histone deacetylase 1. Gon4l is essential for hematopoiesis and represses gene expression during hematopoietic development. In this scenario, Yin Yang1 along with these co-factors is able to mediate transcriptional repression (169). Although it is difficult to be certain for sure, due to Yin Yang1’s prevalence as a transcriptional repressor in development, it is possible that it also functions as a transcriptional repressor of Plekhg4 and helps prevent over-expression of the Plekhg4 protein. If this is the case, then loss of

Yin Yang1 association, due to mutations in Plekhg4, would likely increase Plekhg4

46 protein levels. This increase in Plekhg4 protein would overwhelm regulation by Hsc70,

Hsp90, and CHIP, thereby resulting in an increase in protein levels, and aggregation of the Plekhg4 protein. As further discussed in the “Discussion” section, aggregation of

Plekhg4 protein levels would lead to a mislocalization of the GEF, which could result in a reduction of GEF activity in the cytosol or an increase of GEF activity in the aggregate.

This would lead to impaired and altered signaling by Rho GTPases and ultimately give rise to the spinocerebellar phenotype.

DISCUSSION:

Cerebellar neurons primarily control and coordinate motor movements (170) although recent data indicate their involvement in some cognitive functions (171).

Accordingly, cerebellar pathologies manifest primarily as lack of motor coordination, generically referred to as spinocerebellar ataxias (SCAs; (172)). The vast majority of ataxias are familial, stemming from heritable alterations in distinct unrelated genes that affect diverse neurobiological processes such as growth factor signaling, synaptic transmission, lipid trafficking, and ion channel function. On the cellular level, SCAs manifest as anatomic and functional compromise in Purkinje cells, responsible for integrating cerebellar information and coordinating neural output from this region. As in many other neurodegenerative disorders, many SCAs are characterized by the formation of cytoplasmic protein aggregates, especially in Purkinje cells (173). It is generally assumed that such aggregates result from misfolding of aggregation-prone proteins, as seen in mutated versions of ataxin, beta-spectrin, atrophin or PKCg (174).

Plekhg4 was identified by molecular genetics approaches in the study of 16q22.1- linked autosomal dominant cerebellar ataxia. The protein encoded by the PLEKHG4 gene

47 (sometimes termed puratrophin-1) exhibits a diffuse cytosolic expression pattern in the

cell body of cerebellar Purkinje cells, whereas its mutated version resides in dense multi-

protein aggregates observed in atrophying Purkinje neurons (134,175). Data presented

here show that Plekhg4 is a functional activator of small GTP-binding proteins from the

Rho family i.e. Cdc42, Rac1 and RhoA. Additionally, our data show that Plekhg4 is an

aggregation-prone protein, whose steady-state levels are determined by the so-called

‘triage decision’ that determines whether the protein will be stabilized and refolded, or ubiquitinated and degraded (162). Like in other instances, the triage decision is

determined by a delicate interplay between chaperones that assist in refolding

(specifically HSP90; (176)), and those that facilitate ubiquitination and subsequently,

proteasomal degradation (namely Hsc70 / CHIP; ((177)).

The mechanisms by which insoluble aggregates of Plekhg4 cause cerebellar

dysfunction are completely unknown at present. Two alternative hypotheses can be

invoked to explain the cellular pathophysiology of Plekhg4 mutations: 1) Plekhg4

aggregation can lead to reduction in GEF activity in the cytosol or an increase in this

activity within the aggregate. Such changes will alter signaling to the substrate Rho

family GTPases, which play key roles in controlling neuronal architecture and gene

expression. Rho GTPases regulate neuronal differentiation, are involved in controlling

neuronal migration during development, and they coordinate neurite outgrowth/collapse

that define neuronal plasticity in the adult (cf. (178)). Moreover, Rho GEFs regulate

proper signaling by key neuronal hormones. For example, induction of neurite outgrowth

by NGF is mediated by the Rac-GEF Tiam1 (179) or the Rac/Rho-GEF TRIO (180), the

Rac-GEF P-Rex is essential for BDNF signaling (85), and activation of Rac and Cdc42

48 by Vav2 is required for neuron responses to the hippocampus cluster of differentiation 47

(CD47; (181). Dysregulation of Rho GTPases signaling brought about by their aggregation, is thus expected to markedly affect neuronal function. 2) Alternatively, protein aggregates can interfere with neurobiological processes directly through toxic misfolded intermediates or through indirect disruption of neurobiological processes

(182). Protein aggregates can thus lead to defects in neuronal processes such as axonal transport (183), mitochondrial function (184), and oxidative (185) or endoplasmic reticulum (186) homeostasis. Elucidation of the molecular basis underlying Plekhg4- induced ataxia awaits further experimental work.

49 CHAPTER 2 FIGURES:

Figure 2.1. Spatio-temporal expression patterns of Plekhg4 in the murine brain. A.) Top Panel: Plekhg4 mRNA levels were compared at birth (P0) and at the ages of 14 (P14) and 56 days (adult) in the indicated brain regions using real-time RT-PCR. Bottom panel: expression of actin mRNA. Shown image is representative of three independent experiments. Quantification of mRNA levels was achieved using ImageJ. B.) Left: Anti-Plekhg4 immunohistochemical staining of cerebellum section from a 2-week old mouse. Note dark DAB staining seen in Purkinje neurons. ML, molecular layer; PCL, purkinje cell layer; GL, granular layer; WM, white matter. Right: Negative control (secondary only) for DAB staining. Right: negative control stained only with secondary antibody. Scale bars =100 %m.

50

Figure 2.2. Sequence signatures of Plekhg4 and Dbl. (A). Domain structure of the two proteins was inferred using sequence prediction algorithm (Expasy). Domain size and limits were manually drawn to scale. (B-D). Sequence homology between Plekhg4 and Dbl. Shown are sequence alignments between the proteins’ DH domains (B), residues that mediate GTPase binding (C), and the residues that dictate GTPase selectivity (D). Identical and highly similar residues are shown in red and denoted by asterisks.

51

Figure 2.3. Plekhg4 is a functional GEF. (A). GTPase activation by Plekhg4. COS7 cells were transiently co-transfected with Rac1, RhoA, or Cdc42 (HA-tagged in the pKH3 vector) and the indicated GEF in the pcDNA3.1 construct. After serum deprivation for 24 hours, cell lysates were subjected to the PBD assay as described in Materials and Methods. Top panel: activated (GTP- bound) GTPase precipitated with GST-PBD, visualized after anti-HA immunoblotting. Lower panels show expression levels of the indicated proteins in cell lysates. Bait GST-PBD was visualized by Ponceau-Red staining of the blotted membranes, and fold activation was determined after computer densitometry after normalization to GEF expression. Data shown are representative of three independent experiments in which activation varied by + 15%. (B). Plekhg4-GTPase association. GST fusion proteins of Rac1, Cdc42, and RhoA were over- expressed in E. coli, purified on glutathione affinity chromatography, and their nucleotide content manipulated as described in Materials and Methods. Immobilized GTPase were incubated with lysates from HA-Plekhg4- or Dbl-expressing COS7 cells, washed, and associated GEFs were visualized by immunoblotting. GST-GTPases ‘baits’ were visualized by Ponceau-Red staining of the blotted membranes. Shown is a representative of three independent experiments.

52

Figure 2.3. (A) Plekhg4 is a functional GEF. Shown is a representative full gel depicting activation of Rac1 by Plekhg4. All westerns shown were probed with anti-HA antibody. See previous page for more details.

53

Figure 2.4: Plekhg4 regulates the actin cytoskeleton. Plekhg4 regulates the actin cytoskeleton. Cells expressing Plekhg4 (or control vector) were immunostained with HA-antibody (Plekhg4 expression) or Texas-Red conjugated Phalloidin, and visualized by fluorescence microscopy. (A): NIH3T3 fibroblasts transiently transfected with empty vector control. (B): NIH3T3 fibroblasts transiently transfected with HA-tagged Plekhg4. Insets show cytoskeletal features exhibited by Plekhg4 expression. Indicated are: stress fibers (S), lamellipodia (L) or fillopodia (F).

54

55 Figure 2.5: Intracellular fate of Plekhg4 is dictated by Hsc70, Hsp90 and CHIP. (A) Plekhg4 associates with Hsc70, Hsp90, and CHIP. GST-tagged Plekhg4 (or control vector) cDNA constructs in pEBG vector were co-transfected into COS7 cells together with cDNA encoding myc-tagged CHIP in pcDNA3.1 vector. GST-tagged proteins were then affinity precipitated with glutathione-sepharose, resolved on SDS-PAGE, and probed for interacting endogenous Hsc70, Hsp90, and ectopic myc-CHIP by immunoblotting with the corresponding antibodies. (B) COS7 cells were transiently transfected with cDNAs encoding GST-tagged Plekhg4 in pEBG vector. DMSO vehicle control (top panel), 3 µM 17-AAG (middle panel), or 3 µM 17-AAG supplemented with 25 mM proteasomal inhibitor MG132 (bottom panel) were added to cells for the indicated time prior to lysis. Cleared cell lysates were resolved on SDS-PAGE, and probed for Plekhg4 with anti-GST antibody. (C) COS7 were transfected with cDNAs encoding GST-tagged Plekhg4 or GST alone and glutathione affinity precipitations were performed. The precipitated proteins were resolved on SDS-PAGE, and probed for poly-ubiquitination by immunoblotting with the anti-ubiquitin antibodies. (D) COS7 cells transfected with cDNAs encoding GST-tagged Plekhg4 or GST alone were treated with and without 3 µM 17-AAG. Glutathione affinity precipitations were performed, precipitated proteins were resolved on SDS-PAGE, and probed for poly-ubiquitination by immunoblotting with the anti-ubiquitin antibodies. (E) HA-tagged Plekhg4 construct was transiently transfected into both COS7 and NIH3T3 cell lines. Cells were treated for 2 hrs with 3 mM 17-AAG 46 hrs post-transfection, fixed, stained with anti-HA-FITC conjugates, and visualized by immunofluorescence confocal microscopy. The arrows in the top panel show Plekhg4 localization to the cytosol and to the plasma membrane. The arrows in the bottom panel show that upon treatment with 17-AAG Plekhg4 localization drastically changes and is characterized by large punctae and strongly resembles the formation of aggresomes.

56 CHAPTER 3

Tyrosine phosphorylation of Dbl regulates GTPase signaling**

Meghana Gupta1, Xiaojun Qi2, Faping Duan3, Hong Wang2, and Danny Manor1,2,3

Departments of Pharmacology1, and Nutrition2, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, Cornell University3, Ithaca NY, 14853

**Running title: Tyrosine phosphorylation of Dbl

To whom correspondence should be addressed: D. Manor, Case Western Reserve University School of Medicine, WG-48, Cleveland – OH, 44106. Phone: 216-368-6230; Fax: 216-368-6644; E-mail: [email protected] Keywords: guanine nucleotide exchange factor (GEF); GTPases; RhoA; Cdc42; Rac1; tyrosine phosphorylation; signaling

Submitted to The Journal of Biological Chemistry as a normal article Oct 2013.

57 CAPSULE:

Background: Dbl is a guanine nucleotide exchange factor for the Rho family of small

GTPases RhoA, Cdc42, and Rac1. The upstream regulatory events that regulate Dbl activity are presently unknown.

Results: Activation of cell surface receptors leads to phosphorylation of Dbl’s 510Tyr, thus stimulating its GEF activity.

Conclusion: Dbl activation cycle is tightly regulated by the activity of tyrosine kinases and phosphatases.

Significance: Understanding signaling pathways enhances our understanding of their roles in health and disease states.

ABSTRACT:

Rho GTPases are molecular ‘switches’ that cycle between “on” (GTP-bound) and

“off” (GDP-bound) states, and regulate numerous cellular activities such as gene expression, protein synthesis, cytoskeletal rearrangements, and metabolic responses.

Dysregulation of GTPases is a key feature of many diseases, especially cancers. Guanine nucleotide exchange factors (GEFs) of the Dbl family are activated by mitogenic cell surface receptors and activate the Rho family GTPases Cdc42, Rac1, and RhoA. The molecular mechanisms that regulate GEFs from the Dbl family are poorly understood.

Our studies reveal that Dbl is phosphorylated on tyrosine residues upon stimulation by growth factors, and that this event is critical for the regulated activation of the GEF.

These findings uncover a novel layer of complexity in the physiological regulation of this protein.

58 INTRODUCTION:

The Dbl family of Guanine Nucleotide Exchange Factors (GEFs) comprises over

80 members that facilitate the activation of the small GTPases Rac1, Cdc42, and/or

RhoA. Dbl was the first identified mammalian GEF and as such is considered to be the

prototypic member of the family (51). Two highly conserved domains characterize Dbl

family GEFs: a unique Dbl-homology (DH) domain and a Pleckstrin-homology (PH)

domain. The DH domain is the minimal unit necessary for GEF activity that binds the

substrate GTPase and catalyzes the exchange of GDP for GTP in its binding pocket (54).

Following GEF-mediated activation, the bound GTP is hydrolyzed, and the GTPase

returns to its quiescent state. The PH domain is thought to mediate proper localization of

the GEF to specific sub-cellular compartments (55-57). In response to growth factor stimulation, Dbl-mediated GTPase signaling regulates numerous cellular activities such as cytoskeletal rearrangements, gene expression and vesicular trafficking, thereby promoting cell proliferation (1-5).

Since GTPases control multiple and diverse aspects of cellular physiology, proper regulation of GEF activities is crucial for normal cellular function. Indeed, mutations that cause GEF over-expression or gain-of-function result in persistent activation of downstream pathways, and lead to aberrant cell growth that manifest in developmental disorders and cancers. For example, mutations in Fgd1 cause the X-linked developmental disorder, Aarskog-Scott syndrome (127), and chromosomal rearrangements of the Bcr gene contribute to leukemogenesis (187). Mutations in Plekhg4 are associated with heritable autosomal spinocerebellar ataxia (134,175). Overexpression of P-rex1, Ect2, and Tiam1 have been noted in cancers of the breast, lung, and colon, respectively (121-

59 124,145,188), and over-expression of Dbl was reported in sarcomas and tumors of neuroectodermal origins (125,126,189,190). As our appreciation for the involvement of

Dbl family GEFs in disease grows, the need for understanding the mechanisms that regulate these proteins increases.

The upstream events that regulate Dbl’s GEF activity are poorly understood. Dbl is thought to exist in an inhibited state, which is activated by growth factor receptors; however, the molecular mechanisms that underlie the inhibited state and its release are incompletely understood. Some studies proposed an intra-molecular auto inhibited state brought about by interactions between the amino- and carboxy-termini of Dbl (150).

Other studies demonstrated that association with the molecular chaperones Hsc70 and

Hsp90 regulates Dbl activity by control its degradation in the (104,105). The mechanisms by which growth factor stimulation leads to release of Dbl’s inhibition are completely unknown.

In this study, we aimed to understand the upstream events that relieve Dbl’s auto- inhibition and stimulate its GEF activity. We show that a key feature of Dbl’s activation cycle involves reversible, growth-factor sensitive, tyrosine phosphorylation.

60 MATERIALS AND METHODS:

Cell culture: COS7 cells and HEK293T cells were cultured in DMEM supplemented

with 10% FBS (Hyclone) in 5% CO2 at 37$ C. NIH3T3 cells were similarly cultured in

DMEM supplemented with 10% Calf Serum (Hyclone).

Molecular constructs: Dbl constructs encoded the reading frame encoding the full-length

protein (residues 1-925) in the pCMV6 vector, or the pCEFL-GST vector (for focus

formation experiments). Recombinant GTPases were expressed in E. coli as glutathione-

S-transferase fusions, and purified as previously described (191). Grb2 cDNA (HA

tagged in the pCGN vector) was a generous gift from Dr. Dafna Bar-Sagi, NYU School

of Medicine. All transfections were done using PolyFect (Qiagen).

Chemicals: EGF (Sigma) was dissolved in serum-free DMEM and used at a final

concentration of 50 nM. PP2 (4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d]pyrimidine, EMD Millipore) was dissolved in DMSO and used at a final concentration of 10 µM, as was Iressa (Gefitnib, Selleck Biochemicals). Unless otherwise indicated, all treatments lasted for 30 minutes, whereas the phosphatase inhibitor activated sodium orthovanadate (Sigma) was added during the last ten minutes to a final concentration of 1 mM.

Immunoprecipitations from cell lysates: Cells were lysed in 1 ml 20 mM Hepes, pH 7.4,

1 mM EDTA, 150 mM NaCl, 1% Igepal, 20 mM NaF, 20 mM )-glycerophosphate, 1

(Covance, MMS-101R, 1:100). The lysate-antibody mixture was mixed at 4$ C for one

61 hour before the addition of 50 µl of protein A beads (Millipore) and mixing at 4$ C for

2.5 hours. Beads were washed three times with lysis buffer and spun at 3,500 rpm for 3

minutes. Precipitated beads were boiled in 2.5X Lamelli buffer and the supernatant was

resolved by 8%-12% SDS-PAGE. Tyrosine phosphorylation was visualized using anti-

phosphotyrosine antibody (4G10-platinum, Millipore, 1:1000 dilution).

Immunoprecipitations from mouse brain: Whole brain extracts from 10-days old mice were homogenized manually in 1 ml of 150 mM NaCl, 1 mM dithiothreitol, 1 mM

EDTA, 150 mM NaCl, 25 mM Tris-HCl (pH 8.0), 0.25 mM phenylmethylsulfonyl

72+,#3&'C( $%&( IH( MNO<2 each leupeptin and aprotinin. The homogenate was cleared by centrifugation at 14,000 rpm for 45 min at 4 °C, followed by immunoprecipitation with

Dbl antibody as described above.

Site-directed mutagenesis was performed according to manufacturer’s protocol

(Quikchange XL II, Agilent).

Grb2-Dbl association: HEK293T cells were co-transfected with HA-Grb2 and Dbl constructs, harvested in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-

40, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1 mM vanadate, 200

M<( 5*'%.2<'"*.2!+27,%.2( 72+,#3&'C( $%&( IH( MNO<2( '$4*( 2'+5'5"3%( $%&( $5#,"3%3%C( and centrifuged at 14,000 rpm for 20 minutes. Association was visualized by anti-HA immunoblotting of anti-Dbl immunoprecipitates.

GEF-GTPase association assays: These were designed based on the established high affinity of GEFs to their nucleotide-free form of their cognate GTPases (26,27,149).

Wild-type GTPases and their respective nucleotide-free mutants (Cdc42(T17N) or

RhoA(T19N)) were expressed in E. coli as GST fusions and purified as previously

62 described (155). The purified GTPases were immobilized on glutathione agarose and

washed extensively with cell lysis buffer supplemented with 50 µM GDP, 10 mM MgCl2

(wild-type GTPase) or 10 mM EDTA (N17/N19 GTPase). Dbl-transfected cells were

lysed in the same lysis buffer, incubated with immobilized GTPases at 4o C for 2 hours,

washed 3 times in the respective buffers, prior to SDS-PAGE and anti-Dbl

immunoblotting.

GTPase activation assays: To measure GEF activity, the fraction of endogenous GTPase

that is in the active (GTP-bound) state was determined using an established selective

precipitation approach (156-158). Briefly, HEK293T cells were transfected with Dbl

cDNA, serum-starved for 31 hours and lysed in 20 mM HEPES (pH 7.4), 150 mM NaCl,

1% Nonidet P-40, 10% glycerol, 10 mM MgCl2, 1 mM EDTA, 0.2 mM

5*'%.2<'"*.2!+27,%.2(72+,#3&'C($%&(IH(MNO<2('$4*(2'+5'5"3%($%&($5#,"3%3%=(Lysates were

spun at 14,000 rpm for 20 minutes and cleared supernatant was combined with !SH(MN(

immobilized GST-fused GTPase-binding domain of Rhotekin and rotated at 4° C for 2 h

(157). Beads were washed three times in lysis buffer and resolved on SDS-PAGE. The

amount of activated (beads-associated) GTPase was visualized with anti-RhoA

(Cytoskeleton, 1:200 dilution) immunoblotting, and compared to the total GTPase levels as determined by anti-RhoA immunoblotting of whole-cell lysates.

NF&B transcriptional activation assays: NIH3T3 cells were plated in triplicates in a 24-

well plate and co-transfected with the NF&B response element fused to the luciferase

reporter construct (pGL3B-HIV1-2+4LC( "*'( )-galactosidase-expressing pCH110

(Pharmacia), and Dbl cDNA. Thirty-six hours after transfection, cells were washed in

serum-free media and starved for 17 hours prior to harvesting cell lysates and measuring

63 luciferase $%&( )-N$2$4",!3&$!'( $4"3@3"3'!=( T+437'#$!'( 'U5#'!!3,%( 0$!( %,#<$23V'&( ",( )- galactosidase activity to account for differences in transfection efficiency.

Focus formation assays: The indicated constructs were used to transfect subconfluent

NIH3T3 cells in 10 cm plates. After 48 hours, media was replaced and cells were re-fed every other day. Approximately two weeks after transfection, cells were fixed with methanol, stained with crystal violet, and foci were manually scored.

RESULTS:

Tyrosine phosphorylation of Dbl. In a search for post-translational events that may regulate Dbl’s GEF activity, we found that anti-Dbl immunoprecipitates give a strong signal on anti-phosphotyrosine immunoblots. Importantly, the signal was only visible in the presence of the tyrosine phosphatase inhibitor sodium ortho-vanadate (Figure 1A).

The physiological relevance of this finding is evident by the observation that tyrosine phosphorylation of Dbl occurs in intact tissue where the protein is expressed, such as mouse brain (Figure 1B). Moreover, we found that when serum-deprived COS7 cells are treated with serum, Dbl phosphorylation transiently increases after 2-5 minutes, and then tapers off (Figure 1C). These observations show that Dbl is phosphorylated on tyrosine residue(s) and that this modification is a regulated event, dependent on growth factors. To begin to delineate the upstream events that regulate Dbl’s phosphorylation, we used pharmacological inhibitors of two major intracellular tyrosine kinases: Iressa (Gefitinib), which inhibits the EGF receptor tyrosine kinase, and the inhibitor of Src-family tyrosine kinases, PP2. Either treatment reduced Dbl phosphorylation significantly (70-80%), and co-treatment with both inhibitors completely abolished tyrosine phosphorylation of Dbl

64 (Figure 1D). These data indicate that phosphorylation of Dbl is mediated by mitogenic

stimulation through the activated EGF receptor, and a Src family kinase.

Site(s) of Tyrosine Phosphorylation in Dbl. We utilized a bioinformatics approach to

identify the tyrosine residue(s) that are phosphorylated in Dbl. The NetPhos2.0 algorithm

(192) identified 7 residues in Dbl that reside within tyrosine phosphorylation consensus

sequences. These putative phosphorylation sites are dispersed throughout the different

domains of the protein (217Tyr, 510Tyr, 553Tyr, 749Tyr, 780Tyr, 787Tyr, and 904Tyr see Figure

2A). We focused our attention to 510Tyr, due to its position in a high-scoring putative

phosphorylation sequence in the catalytic DH domain. Using site-directed mutagenesis,

we substituted Dbl’s 510Tyr to a phenylalanine, and observed a dramatic decrease (~80%)

in the protein’s tyrosine phosphorylation intensity (Fig. 2B). The fact that some tyrosine

phosphorylation persists in the Y510F mutant indicates that sites of tyrosine

phosphorylation other than 510Tyr exist in the protein. In attempting to identify such

residues, we found that mutation of 787Tyr did not affect the residual phosphorylation

signal observed in Dbl(Y510F) (data not shown).

Regulation of Dbl’s molecular interactions by 510Tyr phosphorylation. In order to

understand the molecular consequences of tyrosine phosphorylation in Dbl, we examined

its ability to associate with established effectors. Grb2 is an adapter molecule that

contains a Src homology 2 domain that binds phosphorylated tyrosine residues, and a Src

homology 3 domain that binds proline-rich sequences (193). Due to this dual-recognition ability, Grb2 functions as a ‘bridging’ adapter, allowing it to recruit multiple proteins to their cellular site of activation, as seen in the activation of Ras, where Grb2 recruits the

GEF, , to the tyrosine-phosphorylated EGF receptor (194,195). Since

65 Grb2 was recently reported to be associated with Dbl (196), we sought to evaluate the

role of 510Tyr phosphorylation in this interaction. As seen in Figure 3, substitution of

Dbl’s 510Tyr with a non-phosphorylateable phenylalanine greatly diminished Grb2

binding, raising the possibility that phosphorylation of 510Tyr is critical for Grb2

association.

Next, we asked whether tyrosine phosphorylation of Dbl is necessary for association of

the GEF with its substrate GTPase. Cells expressing Dbl (or its phospho-defective

mutant) were lysed and precipitated with immobilized recombinant GTPases that have

been purified in either the GDP–bound form (GST-fused wild-type GTPases), or the

nucleotide-free form (GST-RhoA(T19N) or GST-Cdc42(T17N) mutants). The rationale

for this design is based on the notion that the highest affinity of GEFs to their GTPase is

observed in the nucleotide-free form of the latter (26,27,149). As expected, we observed

a strong preference in Dbl’s association with and the nucleotide-free forms of RhoA and

Cdc42, as compared to the GDP-bound GTPases (compare lanes 1 and 2 in Figures 4A and 4B). Importantly, we observed that the affinity of the nucleotide-free GTPase to the

GEF is dramatically reduced when phosphorylation of Dbl’s 510Tyr is abolished by

mutation to phenylalanine (Figure 4A, 4B). These findings indicate that that

phosphorylation of 510Tyr is required for Dbl’s ability to bind substrate Rho GTPases.

Phosphorylation of 510Tyr is critical for Dbl’s GEF activity. To address the role of

510Tyr phosphorylation in Dbl’s activity as a GEF, we examined its ability to stimulate

GTP binding by a substrate GTPase. Since both Cdc42 and Rac1 are phosphorylated on

64Tyr ((197) and our unpublished results), whereas RhoA is not (Figure 5A), we chose to

focus on the latter GTPase. This allowed us to evaluate the effect of kinase inhibitors on

66 Dbl actions without complication arising from the inhibitors’ effect on the substrate

GTPase. We treated Dbl-transfected cells with Iressa and measured Dbl’s GEF activity by selectively precipitating GTP-bound form of RhoA ((156-158); see Materials and

Methods). We found that treatment with Iressa all but abolished Dbl’s ability to activate

RhoA (Figure 5B), indicating that EGFR-mediated tyrosine phosphorylation of Dbl is essential for its activity as a GEF. Importantly, substitution of 510Tyr to phenylalanine abolished Dbl’s GEF activity (Fig. 5C).

Next, we examined the role of 510Tyr phosphorylation on further downstream signaling events. We found that the Y510F mutant of Dbl abolished transcriptional activation of NF-&B (Figure 6A), a downstream effector of RhoA, Rac1, and Cdc42

(198-200). These results further confirm that tyrosine phosphorylation of Dbl is necessary to activate the Rho family small GTPases. Lastly, we examined the role of 510Tyr phosphorylation on Dbl’s signature biological activity: transformation of cultured murine fibroblasts (201-203). We examined the outcome of Dbl overexpression on contact inhibition by quantifying the number of three-dimensional foci formed in confluent

NIH3T3 cells transfected with Dbl cDNAs. Y510F mutation of Dbl caused a marked reduction in Dbl’s focus-formation activity (Figure 6B). Taken together our observations demonstrate that tyrosine phosphorylation of 510Tyr in Dbl plays a critical role in the protein’s activation by mitogenic signals.

DISCUSSION:

Members of the Dbl family GEFs regulate signaling pathways that emanate from their downstream targets, the Rho GTPases. As such, they control numerous intracellular processes and are critical for mediating the effects of diverse extracellular stimuli

67 (3,5,148,204,205). Underscoring the fundamental biological importance of Dbl family members is that improper regulation of their GEF activities is associated with pathological states, including cancer, developmental and neurological disorders, and viral pathogenesis (3). To-date, great progress has been made in understanding the molecular mechanisms that govern small GTPase signaling and the downstream pathways that they regulate. However, our understanding of the upstream events that control and regulate

GEF actions is very limited. Especially enigmatic are the events that couple GEFs to growth factor receptors, and the molecular mechanisms that control their activation / deactivation cycle.

A number of studies indicate that both intra-molecular and inter-molecular interactions maintain Dbl-family GEFs in an inhibited basal state that is relieved upon stimulation. Members of the Vav family of GEFs are basally autoinhibited via intra- molecular interactions between the amino-terminal calponin homology domain and the

DH domain that hinders access of Rho GTPases to the catalytic site. Stimulation of the T cell receptor leads to the phosphorylation of amino terminal tyrosines by a recruited kinase, structural relaxation of the auto-inhibited structure, and stimulation of nucleotide exchange on substrate GTPases (92,93). Similarly, autoinhibition in Ngef and Wgef, is maintained by the C-terminal SH3 domain and an N-terminal polyproline region, which is released by phosphorylation of amino-terminal tyrosines (94). In the case of PDZ-

RhoGEF, LARG, and p115, the autoinhibited state is disrupted by binding of the G"12/13 to the RGS domains of these GEFs (87-89).

Dbl was initially discovered as a potent fibroblast transforming sequence, the activity of which is stimulated upon truncation of the protein’s amino terminus

68 (2,4,51,150). Dbl has been shown to activate RhoA, Rac1, and Cdc42, and to be expressed in the central nervous system, testis, as well as in neuroectodermal cancers and

Ewing sarcomas (125,126,189,190). Upstream regulation of Dbl involves signaling concepts utilized in multiple other GEFs. The protein appears to be autoinhibited by interactions between its amino-terminal proto-oncogenic sequence, and the carboxy- terminal half of the protein (150). In addition, Dbl is regulated by interactions with the molecular chaperones Hsc70 and Hsp90 that control the rate at which the protein is degraded by the proteasome (104,105). Here, we show that Dbl is also regulated by tyrosine phosphorylation of 510Tyr. The modification is stimulated by growth factors, reversible, and essential for the transient conversion of Dbl to an active GEF. Our working hypothesis regarding the role of tyrosine phosphorylation in Dbl actions is depicted in Figure 7. According to this model, receptor stimulation leads to phosphorylation of Dbl’s 510Tyr by a yet-unidentified kinase, followed by association of

Grb2 with the phosphorylated residue, and recruitment of another kinase(s) that phosphorylates additional tyrosines. The fully phosphorylated protein is in a conformation that allows productive interactions with the substrate GTPase, and decays to the deactivated state following phosphatase-mediated dephosphorylation.

A number of important questions remain unanswered. First, the identity of the specific Src-family kinase(s) that phosphorylate Dbl is yet to be determined. Similarly, the phosphorylation site(s) that are modified in addition to 510Tyr and their impact on the protein’s activity are not presently known. Perhaps most interesting is the functional relationship that may exist between Dbl’s different modes of regulation, i.e. whether tyrosine phosphorylation affects the protein’s proteasomal degradation or its association

69 with cytoskeletal elements such as ERM proteins (68). Recently, a stable complex between Dbl and the lipid kinase PI3KC2! was observed, raising the possibility that a localized change in membranous phosphoinositides comprises an additional level of regulation (196). It is possible, and likely, that regulated phosphorylation is coupled to, or coordinated with, Dbl’s interaction with this lipid kinase or its catalytic products. These questions open the door for exciting future research avenues.

70 CHAPTER 3 FIGURES:

Figure 3.1. Tyrosine phosphorylation of Dbl. Anti-Dbl immunoprecipitates from transfected COS7 cells (A.) or whole mouse brains (B.) were imunoblotted with anti-phosphotyrosine antibodies. C.) Regulation of Dbl phosphorylation by serum. Serum-starved Dbl-transfected COS7 cells were stimulated with 10% FBS for the indicated duration, and tyrosine phosphorylation examined as in panel A. D.) Regulation of Dbl phosphorylation by EGF receptor and Src-family kinases. Dbl-transfected COS7 cells 0'#'( "#'$"'&( 03"*( WF?X( 4,%"#,2C( IH( MF( D#'!!$C(IH(MF(::SC(,#(-,"*(3%*3-3",#!(7,#(KH(<3%+"'!(5#3,#(",(2.!3!=(Y22(-2,"!($#'(#'5#'!'%"$"3@'(,7( at least three independent experiments.

71

Figure 3.2. 510Tyr is an important site of phosphorylation in Dbl. A.) Domain structure of the Dbl protein. Of Dbl’s 29 tyrosines, the top-scoring putative phosphorylation sites are indicated, as well as the immediate sequence surrounding 510Tyr. B.) Tyrosine phosphorylation of Dbl(Y510F) was examined as in Figure 3.1A. Representative of three independent experiments.

72

Figure 3.3. Role of 510Tyr in Dbl’s binding to Grb2. HEK293T cells were co-transfected with indicated constructs, and association was evaluated as described in Materials and Methods. Top Panels: presence of Grb2 and Dbl in anti-Dbl immunoprecipitates. Bottom panels: Protein expression in cell lysates. Representative of three independent experiments.

73

Figure 3.4. Role of 510Tyr in Dbl’s association with substrate GTPases. GST fusion proteins of the indicated RhoA (A.) and Cdc42 (B.) variants were over-expressed in E. coli, purified on glutathione affinity chromatography, and their nucleotide content manipulated as described in Materials and Methods. Top Panels: GEF presence in affinity precipitates. Middle panels: Dbl expression in respective cell lysates. Lower panels: GST-GTPases ‘baits’ were visualized by Ponceau-Red staining of the blotted membranes. Representative of three independent experiments.

74

Figure 3.4. Role of 510Tyr in Dbl’s association with substrate GTPases. (A) Full gel represenation of Dbl’s and Dbl Y510F’s ability to association to the RhoA GTPase. Westerns probed with an anti-Dbl antibody. See previous page for more details.

75

Figure 3.5. Tyrosine phosphorylation of Dbl is necessary for proper activation of RhoA. A.) Tyrosine phosphorylation of Cdc42, Rac1 and RhoA. COS7 cells were transiently transfected with the indicated construct and treated with orthovanadate where indicated. Tyrosine phosphorylation was examined in anti-HA immunoprecipitates. B.) Effect of tyrosine kinase inhibition on Dbl’s GEF activity. HEK293T cells were transiently transfected with the indicated constructs and serum-starved for 24 hours. Thirty minutes prior to lysis, cells were treated with 10 uM Iressa and the fraction of RhoA in the GTP-bound state was measured as described in Materials and Methods. Top panel: GTP-bound RhoA visualized by anti-RhoA immunoblotting after affinity precipitation. Lower panel: expression levels of the indicated proteins in cell lysates. C.) Role of 510Tyr phosphorylation in Dbl-stimulated nucleotide exchange. HEK293T cells were transiently transfected with indicated Dbl constructs, and RhoA activation was measured as in panel B. Each panel is representative of three independent experiments.

76

Figure 3.5. (C) Tyrosine phosphorylation of Dbl is necessary for proper activation of RhoA. Representative full gels showing endogenous RhoA activation by Dbl and Dbl mutant. RBD western was probed with an anti-Rho antibody. Lysates were either probed with an anti-Rho (GTPase expression) or anti-Dbl antibody (GEF expression). See previous page for more details.

77

Figure 3.6. Role of 510Tyr phosphorylation in Dbl-induced downstream activities. A.) Transcriptional effects. NIH3T3 cells were co-transfected with the indicated Dbl construct, an NF-&Z(#'5,#"'#(4,%!"#+4"C($%&($()-galactosidase expressing vector. NF-&B transcriptional activity was measured as described in Materials and Methods. Shown are average values of three independent transfections and statistical significance is indicated by asterix (p<0.05 compared to the vector control (*) or to Dbl (**) as determined by Student’s T-Test. B) Loss of contact inhibition. Triplicate plates of NIH3T3 cells were transfected with the indicated Dbl cDNAs and monitored for foci formation as described in Materials and Methods. Statistical significance, * p<0.05 compared to the empty vector control and ** p<0.05 compared to wildtype Dbl, as determined by a Student’s T-Test. Shown is a representative of 3 independent experiments.

78

Figure 3.7. Proposed model of Dbl regulation by tyrosine phosphorylation. In quiescent cells, both intra-molecular and inter-molecular interactions maintain Dbl in an inactive state (5). Growth factor stimulation leads to phosphorylated of 510Tyr (1), leading to the recruitment of Grb2 (2) and other kinases (3) necessary for the phosphorylation of other phosphorylation sites. Full phosphorylation relaxes Dbl’s autoinhibited structure to one that allows high-affinity binding of substrate GTPases and facilitation of nucleotide exchange (4).

79 CHAPTER 4: Summary & Future Directions

4.1 Overall Summary

Dbl-related GEFs are the physiological activators of the small Rho GTPases.

These GEFs are characterized by containing a Dbl-homology (DH) domain in tandem with a Pleckstrin-homology (PH) domain. Through their DH domain, Dbl GEFs are able to recognize and catalyze the exchange from a GDP-bound GTPase (inactive) to a GTP- bound GTPase (active). Rho GTPases, when active have broad ranging cellular effects.

As such, it is not surprising that constitutive activation of Rho GTPase function can have deleterious effects on the cell, ultimately leading to different disease pathologies. In many cases, constitutive activation of GTPase signaling stems from improper regulation of its upstream activators, GEFs. Mutations that perturb the tight regulatory mechanisms that

GEFs maintain result in the constitutive activation of Rho GTPases. In this thesis, I aimed to (1), characterize and elucidate potential regulatory mechanisms of a novel Dbl family

GEF, Plekhg4, and (2), to understand the regulatory mechanisms governing the function and activation of the proto-typical family member, Dbl.

4.1.1: Plekhg4. Mutations in Plekhg4 are associated with the heritable neurological disorder autosomal dominant spinocerebellar ataxia. Plekhg4 has been reported as a putative Dbl-family GEF; however, the biochemical functions of this protein, prior to our studies, have not been described. I sought to determine Plekhg4’s role as a GEF and to determine if it was regulated in a similar manner to Dbl. On the functional level, I demonstrate that Plekhg4 functions as a bona fide guanine nucleotide exchange factor that facilitates activation of the small GTPases Rac1, Cdc42, and RhoA. Its over- expression induces cellular rearrangements indicative of Rho GTPase activation.

80 Furthermore, my studies show that Plekhg4, like Dbl, is subject to ubiquitination and proteasomal degradation, and its steady-state expression levels are regulated by the chaperones, Hsc70 and Hsp90, and by the ubiquitin ligase CHIP. Overall, data presented here shows that Plekhg4 is an aggregation-prone protein, whose steady-state levels are determined by the so-called ‘triage decision’ that determines whether the protein will be stabilized and refolded, or ubiquitinated and degraded. A few different hypotheses can explain the onset of ADCA due to mutations present in Plekhg4: 1) Plekhg4 aggregation can lead to reduction in GEF activity in the cytosol or an increase in activity within the aggregate, or 2) protein aggregates can interfere with neurobiological processes directly through toxic misfolded intermediates or through indirect disruption of neurobiological processes. In both scenarios, improper GEF regulation leading to the misregulation of neurobiological processes, such as neurite outgrowth or axonal transport, may explain the onset of ADCA.

4.1.2: Dbl. Our understanding of the upstream events that control and regulate GEF actions is very limited. The events that couple GEFs to growth factor receptors, and the molecular mechanisms that control their activation / deactivation cycle, are not well understood. The proto-typical member, Dbl, exists in an inhibited state, which is activated in response to growth factor receptors; however, the mechanisms that relieve this inhibited state are incompletely understood. I sought to gain insight into the requirements necessary for activation of Dbl. Based on our studies I propose the following model for the activation / deactivation cycle of Dbl (see Figure 3.7). In quiescent cells, both intra-molecular and inter-molecular interactions maintain Dbl in an inactive state. Upon growth factor stimulation 510Tyr becomes phosphorylated, leading to

81 the recruitment of Grb2 and other kinases that are required for the phosphorylation of

other phosphorylation sites. Full phosphorylation relaxes Dbl’s inhibited structure,

allowing for high-affinity interaction, binding, and nucleotide exchange of substrate

GTPases.

4.2 Future Directions

4.2.1: Plekhg4. The exact mechanisms leading to the onset of autosomal dominant

spinocerebellar ataxia, due to mutations in the PLEKHG4 gene are presently unknown.

Proper neuronal function is highly regulated by Rho GEF signaling. For example, NGF

stimulation leading to neurite outgrowth is mediated by the Rac-GEF Tiam1 (179), and

the Rac-GEF P-Rex is essential for BDNF signaling (85). In order to address if mutations

in Plekhg4 have an effect on GEF signaling, it is essential to measure GEF activity in the

protein aggregates that characterize the disease, as well as non-aggregated Plekhg4.

Furthermore, measuring the ability of cells expressing either wild-type or mutant forms

of Plekhg4 to undergo growth factor-induced neurite outgrowth will reveal how ADCA- causing mutations in Plekhg4 affect this essential neuronal function. It is also possible that like Dbl, Plekhg4 is regulated in part by tyrosine phosphorylation. It will be interesting to determine how tyrosine phosphorylation of Plekhg4 affects its GEF function and the progression of ADCA (discussed in more detail below).

4.2.2: Dbl. There are a number of remaining questions left unanswered from my current studies: (1) identity of the other important sites of tyrosine phosphorylation in Dbl, (2) the sequence of events in Dbl’s activation cycle (hypothesized in Figure 3.7), and (3) the identity of other kinases that are involved in the activation of Dbl. Mutation of 510Tyr to

phenylalanine did not completely ablate tyrosine phosphorylation and elucidation of other

82 major sites through site-directed-mutagenesis based on prediction algorithms was unfruitful. Several kinases were predicted to phosphorylate Dbl (Insulin tyrosine kinase receptor, Syk, and Jak). Elucidation of the other important phosphorylating kinase(s) of

Dbl might be possible by examining the effect of specific pharmacologic inhibitors on

Dbl’s phosphorylation status and activity. Additionally, I observed that phosphorylation of 510Tyr recruits the adaptor protein, Grb2. It is tempting to hypothesize that as an adaptor protein, Grb2 recruits other kinase(s), which then phosphorylate other tyrosines in Dbl. Isolation of the Dbl-Grb2 complex, and identification of other proteins associated with this complex will provide important information regarding the identity of kinases necessary for the full phosphorylation of Dbl.

4.2.3: 510Tyr in Dbl is highly conserved in other Dbl-family GEFs. My studies from Dbl identified a previously un-described growth factor-sensitive tyrosine phosphorylation sequence (510Tyr, referred to here as TEXXYVXXL) in the catalytic DH domain of the

Dbl proto-oncogene product that regulates the protein’s activity. Importantly, I found that the TEXXYVXXL sequence is highly conserved in the catalytic DH regions of the proto- oncogenic GEFs Tiam1, Ect2, Fgd1, as well as Plekhg4 (see Figure 4.1 below).

83

Figure 4.1: Domain structure and location of the conserved tyrosine in the catalytic DH domain of the five GEFs.

These GEFs are highly disease relevant. Tiam1 (T cell lymphoma invasion and metastasis 1) is a Rac1-specific GEF that was identified as a factor responsible for lymphoma cell invasion (206). High expression of Tiam1 has been observed in colorectal cancers and its expression levels correlate well with disease prognosis (121,123). Ect2 was identified as a fibroblast transforming proto-oncogene, and high expression levels have been reported in lung and esophageal tumors, but not in matched ‘normal’ tissues

(124). In addition to lung and esophageal cancers, Ect2 is highly expressed in various other malignancies including those of the brain, bladder, pancreas, and ovary (124).

Analysis of primary non-small cell lung cancers showed that 82% of cases studied exhibited enhanced expression of Ect2 (124). Fgd1 is a Cdc42-specific GEF, mutations

84 in which cause X-linked skeletal dysplasia (Aarskog-Scott Syndrome, (207-209)). In addition, high expression levels of Fgd1 were noted in malignancies of the breast and prostate, whereas expression levels of this GEF in matched normal tissues was negligible

(120). Plekhg4 is highly associated with autosomal dominant spinocerebellar ataxia and based on our studies is an aggregation prone protein that is regulated in a very similar manner to Dbl. In all four GEFs mentioned, studies indicate that they are maintained in an inhibited state through intra- and inter- molecular interactions; however, the molecular mechanisms that underlie the regulated activation of these GEFs are poorly understood

(102,120,124,206).

Many GEFs from the Dbl family are known to be potent proto-oncogenes; however, little is known about the molecular mechanisms by which they are regulated. In light of my findings of 510Tyr phosphorylation and its role in the regulated activation cycle of Dbl, I plan to examine whether phosphorylation of the TEXXYVXXL sequence in other GEFs regulates their actions. Specifically, I will examine how the tyrosine phosphorylation state of each GEF changes upon stimulation by growth factors using

GEF-specific antibodies for immunoprecipitations and phosphotyrosine-specific antibodies for immunoblotting. I will then examine the functional consequences of

TEXXYVXXL phosphorylation in these GEFs and develop a mechanistic model that describes the physiological regulation of TEXXYVXXL-bearing GEFs. Our principal tool in these experiments will be mutant alleles of each GEF in which TEXXYVXXL phosphorylation is either abolished (Y!F) or mimicked (Y!E). I will then use these constructs to assay GEF action. For instance, I will address how mutation of this

85 conserved tyrosine in each GEF affects its ability to activate substrate GTPases and to stimulate downstream signaling pathways.

An important translational facet of the proposed studies is that each of the GEF addressed here is directly and intimately involved in the initiation and/or the progression of human cancers. Thus, I wish to evaluate the significance of the newly discovered

TEXXYVXXL phosphorylation sequence in the context of cell transformation and tumorigenesis. I will address this issue by creating cell lines that stably express wild-type, phospho-defective, and phospho-mimetic TEXXYVXXL variants of each GEF, and assess their proliferative hallmarks in vitro (i.e., motility, invasion, and cell cycle progression) and in vivo (i.e., tumor formation in xenografted nude mice). These studies will uncover the molecular mechanisms and physiological consequences of

TEXXYVXXL phosphorylation in regulating each GEF, and their involvement in proliferative disorders such as cancer.

Additionally, I will examine whether the phosphorylation status of these GEFs corresponds to carcinogenic grade of specific tumors in samples from human patients. I will generate a phospho-specific antibody raised against the TEXXYVXXL motif of each

GEF, and screen existing cell lines and primary tumor samples of various disease stages for levels of TEXXYVXXL phosphorylation. These experiments will reveal whether

TEXXYVXXL phosphorylation of each GEF correlates with the extent of cell transformation in existing cell lines, and with tumor grade in human samples.

Lastly, I would like to test the suitability of a TEXXYVXXL phospho-peptide- based reagent that will uncouple these specific GEFs from their upstream signaling cascade, thus allowing for preliminary evaluation of its utility as a potential

86 complementary therapeutic intervention approach. Briefly, peptides containing 20 amino- acid residues surrounding the consensus TEXXYVXXL sequence will be synthesized.

For each GEF, a phosphorylated version and a non-phosphorylated version will be prepared by a commercial outfit. The phosphorylated peptide (TEXXYVXXL phospho- peptide) will function as a competitive inhibitor of the wild-type GEF by sequestering interacting cellular components, for example immediate substrate GTPases. On the other hand, the non-phosphorylated peptide will function as a control to ensure that the effects I see are specific to the phosphorylated state of the GEF. Additionally, each peptide will contain an HIV-TAT sequence to facilitate cellular uptake (210). I will address how these peptides function in the functional assays that were discussed above. Specifically, I will address if introduction of the peptide can inhibit the proliferative hallmarks exhibited by these GEFs in in-vitro assays such as motility and invasion. I will also carry out the xenograft experiment described above, and deliver the peptide in lipid emulsions (211) and address if introduction of the peptide can inhibit in vivo tumor formation in xenografted nude mice. These preliminary experiments will determine the usability of this peptide sequence as an inhibitor in cells where regulatory features of specific GEFs are perturbed.

From the bench to the clinic:

Based on our studies above, the next question would be to evaluate the possible utility of targeting TEXXYVXXL phosphorylation in clinical settings. Although peptide therapeutics are becoming more and more prevalent (insulin (212), NGF (213), and glucagon-like peptide (214) are just a few examples), they are associated with several major challenges. One of the major issues encountered with peptide-based therapeutics is

87 the mode of delivery. Oral administration of a drug is the easiest and cheapest way, but is

often rendered ineffective by limited absorption and rapid catabolism in the

gastrointestinal system and the liver. To circumvent this challenge, subcutaneous or

intravenous administration of a peptide is often superior. Other modes of administration

can also be evaluated such as: inhalation, buccal, intranasal, transdermal, adeno-viral, and

nano-particle delivery systems. The efficaciousness of these modes of peptide delivery

and their usability in the clinic are currently being investigated.

It is possible that the body may mount an against the

therapeutic protein, which could ultimately even cause a harmful reaction in the patient.

However, protein-based therapies, such as with antibiotics and vaccines, are commonly

‘humanized’ to prevent immune responses from occurring.

Additionally, an issue that needs to be considered is one of specificity. In the case

of the TEXXYVXXL phospho-peptide, the synthesized peptide would need to show extreme specificity for the GEF that it is targeting as well as being tissue/cell specific.

This is necessary in order to prevent off-target effects, primarily interference with normal functioning of other Dbl family GEFs that carry this conserved sequence, leading to unforeseen complications. Luckily, the surrounding amino acid sequence varies significantly enough from one GEF to another, hopefully allowing for the generation of highly specific phospho-peptides, unique to each GEF and to the disease in which it is misregulated.

Finally, other issues that must be kept in mind are the solubility and stability of

the peptide once ingested. These two factors can be drastically affected once inside the

body. Also, peptides can carry both hydrophilic and hydrophobic characteristics, thus

88 making cellular uptake challenging. These factors can greatly hinder the possible utility of a peptide-based intervention.

Peptides, although posing several issues, have the potential of providing many benefits. As discussed above (and further discussed below), there are ways to circumvent the issues that arise when dealing with peptide-therapeutics. Additionally, one huge benefit of peptide based therapies is that for diseases in which a gene is mutated or deleted, protein-based therapies can potentially provide a therapy without the need for gene therapy, which is more complex and challenging. Considering the potential beneficial aspects that peptide-based drugs possess, one way to try and circumvent the pitfalls associated with peptides is through the generation of a small molecule compound, or peptidomimetic, that is designed to mimic the function of the peptide. They are chemically generated and modified structures of the original peptide that are designed to improve the biological activity, stability, and other issues that are associated with the original peptide.

Two major ways in which an appropriate peptidomimetic reagent can be developed are: 1) random high throughput screening of small molecule compounds to see which ones function similarly to the peptide, and 2) rational and systematic design of the peptidomimetic. The unbiased screening approach is the commonly taken approach

(215,216). However, this approach can be lengthy, expensive, and labor-intensive. The rational design is more structured, hypothesis driven, and in many cases significantly less expensive (217).

Rational design of a peptidomimetic for the TEXXYVXXL region and surrounding region could potentially result in an improvement in the biological activity,

89 stability, specificity for the GEF and disease targeted, as well as provide efficient entry into the cell as compared to the original peptide (217). The most important aspect of the rational design approach is the determination and understanding of the interaction between the peptide and its binding target. Molecular modeling and determination of the predicted spatial arrangement of the peptide ‘docking’ onto the targeted surface in the context of the entire protein will determine the appropriate chemical modifications necessary for an effective peptidomimetic reagent (217). Molecular modeling can in part be based on experimental data such as NMR spectroscopy or X-ray diffraction studies

(217). This information, since it is currently unknown for my proposed peptide and target, would be gathered though collaborations with chemists and structural biologists. It is also important to note, that in the case of TEXXYVXXL, I hypothesize that only the phosphorylated-version of the peptide will be efficacious in inhibiting perturbed GEF activity. As such, the peptidomimetic must contain the chemical modifications that mimic the phosphorylated state of the peptide. Proper choice of chemical modifications can easily mimic the phosphorylated state of the original peptide and has been proven successful in other studies (218-220). Overall, determining the structural requirements for high-affinity interaction between the peptide and the substrate will allow for a logical and systematic approach for the generation of a useful peptidomimetic reagent. These requirements will determine the location and nature of the peptidomimetic’s side chains, allowing it to function like the original phosphorylated-peptide. Once these requirements have been established, a rigid scaffold onto which the active side chains are added to must be chosen. Small scaffolds include benzene whereas an example of a larger template is a biphenyl. Both templates are rigid and aromatic, allowing for the addition of the

90 active side chains (217). Upon generation of the peptidomimetic, evaluation of the drug’s biological activity and effectiveness must be assessed. In general, rational design of a peptidomimetic can significantly improve the efficacy of a peptide-based therapy.

In summary, peptide-therapeutics come with many challenges, some of which can be eliminated through the generation of a peptidomimetic reagent. It would be interesting and exciting to investigate the possibility of a TEXXYVXXL peptidomimetic as a highly selective intervention for different types of cancers and other diseases; however, the limitations and challenges discussed above render such undertaking to be a long-term goal of this research. It is more likely that the visualization of TEXXYVXXL phosphorylation status in clinical samples can be used as a valuable screening approach.

Establishing the relationship between TEXXYVXXL phosphorylation levels of each

GEF and the respective disorder will form the basis for how TEXXYVXXL phosphorylation levels can be used in screening and diagnostic approaches. Such strategy will provide invaluable information that is necessary for the decision-making process that precedes the treatment and management of many diseases. Taken together, screening by

TEXXYVXXL phosphorylation levels may be a useful therapeutic tool allowing for more targeted therapies.

91 4.3 Concluding Remarks

As activators of the Rho family GTPases, it can be appreciated that the Dbl family

GEFs play important roles in governing proper cellular function. Understanding the regulation of this class of proteins is essential in gaining insight into how these proteins are deregulated in diseased states. As such, this thesis aimed to characterize a novel Dbl

GEF that is highly associated with spinocerebellar ataxia and also to understand the mechanisms activating the proto-typical family member, Dbl. Identification that Plekhg4 is an aggregation prone protein gives insight into potential mechanisms that lead to the onset of spinocerebellar ataxia, stemming from mutations in Plekhg4. Additionally, identification that 510Tyr is a major site of phosphorylation has allowed insight into the requirements necessary for activation of this GEF. Finally, the most interesting is the finding that 510Tyr is highly conserved in several highly disease relevant GEFs. This uncovers the possibility that several Dbl family GEFs are regulated similarly to proto-

Dbl, which may open the door to new and exciting means of screening diseases that are known to express Dbl GEFs.

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