Mitochondrial membrane binding and protein complexing of the anti-apoptotic adaptor protein GrblO.

by

Jennifer Hassard

Department of Biology

McGill University, Montreal

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of

Master's of Science

August 2001

Supervisor: Dr. David Thomas, Department of Biochemistry

© Jennifer Hassard, 2001

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0-612-78889-X

Canada ABSTRACT

GrblO is a member of the Grb7 family of adaptor proteins that also includes Grb7 and

Grb14. These three members contain multiple protein binding domains and lack enzymatic activity. Extensive two-hybrid studies have demonstrated binding of GrblO to numerous activated tyrosine kinase receptors including the insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR), as weIl as many non-receptor molecules such as MEK1, Raf-l, and Nedd4. GrblO has been implicated in IGF-I anti-apoptotic signaling regulation through interactions with Raf-l and the mitochondrial membrane.

In this report the pattern of transient Grb10 translocation following IGF-1cellular stimulation was studied. This report also demonstrates the implication of a short variable amino-terminal region of Grb10 in mitochondrial membrane association. Finally, assays were developed with the goal of identifying new GrblO binding partners. RESUME

GrblO appartient à la famille des protéines adaptatrices Grb7 qui comprend également

Grb7 et Grb14. Ces trois membres possèdent plusieurs domaines d'interaction protéine­ protéine mais n'ont pas d'activité enzymatique connue. Des études double-hybride ont montré que GrblO interagit avec des récepteurs activés par des tyrosine kinases dont le récepteur à l'insuline (IR) et le récepteur à l'IGF-I (Insulin-like Growth Factor 1) ainsi qu'avec d'autres protéines telles que MEKl, Raf-l et Nedd4. GrblO est impliquée dans la voie de signalisation anti-apoptotique régulée par IGF-I grâce à son interaction avec Raf-l et la membrane mitochondriale.

Dans ce rapport nous avons étudié la translocation de GrblO après stimulation des cellules par IGF-I. Nous avons également montré l'implication d'une courte région N-terminale variable dans l'association de GrblO avec la membrane mitochondriale. Enfin, nous avons entrepris d'identifier de nouveaux partenaires de GrblO à l'aide de différentes techniques biochimiques.

11 INDEX

Abstract & Résumé pag( i

Index iii

Acknowledgements vi

1. Introduction 1

1.1 Cellular programming for death 1

1.1.1 Morphology linked to signaling molecules 2

1.1.2 Extrinsic and intrinsic signaling pathways 3

1.1.3 Mitochondria, control centre for life or death 5

1.1.4 IGF-IR anti-apoptotic signaling 7

1.2 Orb10 and survival signaling 9

1.2.1 The Grb7family ofproteins 9

1.2.2 Grb10 binding partners 13

1.2.3 Grb10 in mitogenesis 16

1.2.4 Grb10 in anti-apoptotic signaling 18

1.2.5 Grb10 in human disease 19

1.3 Tapies of investigation in this report 21

1.3.1 Investigation ofGrb10 mitochondrial binding 21

1.3.2 The searchfor new Grb10 binding partners 22

2. Methods 24

2.1 Cloning 24

111 • 2.2 Protein preparation 25

2.3 Cell culture and transfection 26

2.4 Mitochondria isolation for immunoprecipitation & gel filtration 27

2.5 High purity mitochondria isolation for in vitro binding assays 28

2.6 In vitro binding assays 29

2.7 In vivo binding assays 30

2.8 Flag co-immunoprecipitation 30

2.9 Two-dimensional electrophoresis 31

2.9.1 1soelectric focusing 31

2.9.2 Resolution by size 33

2.9.3 Silver staining 33

2.10 Gel filtration 34

2.10.1 Mitochondrial extract separation 34

2.10.2 Column standardization 35

2.11 Western blotting 35

2.12 Fluorescence microscopy 36

3. Results 38

3.1 GrblO mitochondrial binding 38

3.1.1 Immunolocalization demonstrates a rapid Grb10 38 translocation 3.1.2 Cell counts ofimmunolocalized HeLa, a majority exhibits 39 Grb10 translocation

IV 3.1.3 In vitro assays demonstrate that mitochondrial membrane 39 binding ofGrb10 occurs via the amino-terminal and is ATP-independent

3.14 In vivo assays support the results ofin vitro assays 41

3.2 Development of assays for GrblO binding partner identification 42

3.2.1 Gelfiltration confirms ideal conditionsfor Grb10 42 complexing

3.2.2 The developed binding partner assay is not efficient enough 43 to identify binding partners

4. Discussion 45

4.1 Challenges faced in this study 45

4.2 Mitochondrial membrane binding 46

4.3 Identification of Grbl0 binding partners 50

3.4 Proposed further work 51

5. References 53

Figures included at end 1 - 12

v AC K N 0 W LED G MEN TS

l graciously thank Dr. David Thomas for supervision, guidance, and funding during the progress of this research. Thank you to Dr. André Nantel for invaluable encouragement, supervision, and expertise (and an endless supply of movie trailers) throughout this research. Dr. Nantel provided the pAN series of vectors, sorne of which were used for cellular transfections and from which many new vectors were sublconed. Dr. Anne-Pascale

Bouin is thanked for her many hours of instruction and help with co-immunoprecipitation and 2-dimensional electrophoresis (2-D) techniques. l also thank Fizah Buch for her dedication to the completion of experiments using gel filtration and her enthusiasm and energy directed towards tbis project. Fizah performed preliminary standard runs and aIl cell runs using the gel filtration system with my supervision as part of her independent research project for the McGill Department of Anatomy and Cell Biology. Anne Marcil is graciously thanked for her continued support and for my introduction to mammalian cell culture. Thank you to Daniel Dignard for sequence data and help with interpretation, along with providing good lunchtime humour. André Nantel, Anne-Pascale Bouin, Micbiei Sho,

Cunle Wu, and Marc Pelletier are aIl appreciated for lively discussions concerning research direction and for help reviewing tbis manuscript. Finally, l also thank Dr. Claude Jakob for a memorable introduction to the Eukaryotic Genetics group at the Biotechnology Research

Institute.

VI 1. INTRODUCTION

1.1 Cellular programming for death

The programmed path to cellular suicide, called apoptosis, is crucial for the development, daily maintenance, and survival of the mammalian organism. Apoptosis plays an essential role in the shaping of organs, limbs, and digits during fetal development. Using apoptotic signaling, the human body continually removes old cells to be replaced with new ones and restricts the proliferation of unwanted autoimmune lymphocytes. Apoptosis can also be used by the body to remove cells infected by a pathogen. The study of pathways that lead to apoptosis has become increasingly important in the light of the many human conditions that can arise when such signaling goes off track. Cancer, of significant importance in the field of apoptosis research, can be the result of insufficient apoptotic control. It is now demonstrated that in many circumstances the loss of proper apoptotic signaling is responsible for resistance to chemotherapeutic drugs (O'Gorman & Cotter, 2001). On the other hand, excessive apoptosis is linked with Alzheimer's Disease (AD), Huntington's

Disease (HD), and Amyotrophic Lateral Sc1erosis (ALS) which manifest as the selective and detrimentalloss of certain neuronal cell types within specific regions of the brain and peripheral nervous system (Nijhawan, et al., 2000). As our understanding of apoptotic signaling increases, so does our understanding of these related conditions, and perhaps so will our ability to better treat them.

1 1.1.1 Morphology linked to signaling molecules

Apoptosis is associated with a number of morphological characteristics. As the cell begins to shrink and lose its shape, its outer membrane eventually undergoes blebbing resulting in popcom-like shaped cells. Shrinking and budding of the nucleus is seen in conjunction with chromatin condensation and eventual DNA fragmentation. Finally, the cell fragments into apoptotic bodies that are engulfed by neighbouring cells. As opposed to necrosis, apoptosis is a tidy and specifically controlled means for a cell to die. The cellular signaling involved in apoptosis is complex and relies on a balance between pro- and anti-apoptotic signaIs.

Two groups of proteins are of great importance to apoptosis, the caspases and the Bcl-2 family members. Caspases, fittingly dubbed the "central executioners" (Hengartner, 20(0), are cysteine proteases (having a cysteine residue within the enzyme active site) which cleave after aspartic acid residues (Thomberry, et aL, 1997). Cellular protein cleavage by caspases results in many of the morphological changes mentioned above. For instance, fragmentation of nuclear DNA is known to be a result of the caspase-activated DNase

(CAD) (Nagata, 20(0). Blebbing of the cellular membrane has been attributed to caspase cleavage activation of the p21-activated kinase (PAK2) (Rudel & Bokoch, 1997). Loss of nuclear integrity and subsequent chromatin condensation is due to nuclear lamina proteolysis (Orth, et al., 1996; Rao, et al., 1996; Takahashi, et aL, 1996). Proteolytic destruction of structural proteins is the likely cause of loss of cellular integrity and structures (Thomberry & Lazebnik, 1998; Hengartner, 2000). As the pathways of apoptosis activation are tumed on, a cascade of caspase activation is initiated. This cascade results in the amplification of a small quantity of inducer signal to a small pool of activated initiator

2 caspases to a pro-apoptotic signal of activated effector caspases that is both rapid and robust (Thornberry & Lazebnik, 1998). Despite the many different initiators of apoptosis

that exist, the eventual activation of the same effector caspases explains how all types of

apoptotic stimuli result in very similar cellular morphologies. By this, studies that examine

essential apoptotic effectors have the potential for application to apoptosis on a very broad

scale.

1.1.2 Extrinsic and intrinsic signalingpathways

Two major signaling pathway types have been described for apoptosis (see Figure 1).

Extrinsic signaling initiates from extracellular ligands, such as the Fas ligand, which

activates the death-receptor pathway. Vpon Fas binding clustering of the receptor forrns a

complex that recruits procaspase-8 through the adaptor Fas-associated Death Domain

Protein (FADD). As with most caspases, caspase-8 is synthesized in the form of an inactive

zymogen. Vpon modification, typically by caspase cleavage, the pro-forrn of the caspase is

converted to an active form. It is hypothesized that the proximity of many procaspase-8

molecules at the plasma membrane induces a trans-activation by which low levels of

caspase activity present in the zymogen allow the cleavage of a few molecules. Once a few

molecules have been converted to active caspase-8 through this trans-processing, a caspase

signaling cascade begins (Juo, et al., 1998; Martin, et al., 1998; Stennicke & Salvesen,

1998). In the absence of the inhibitor c-FLIP, caspase-8 can activate procaspase-3 and Bid

by cleavage. Cleaved Bid relocates from the cytosol to the mitochondrial membrane where

3 " this Bcl-2 family member can induce mitochondrial membrane permeability and cytochrome c release. Other Bel-2 family members play important pro- and anti-apoptotic roles at the mitochondria that will be discussed shortly. Activecaspase-3 and caspase-8 induce cleavage of further caspases and cellular substrates that result in many of the morphological signs of apoptosis as described above.

Intrinsic signaling of apoptosis originates from internaI cellular signaIs such as the nuclear protein p53. DNA damage induced by ultra violet light (UV) irradiation is known to induce p53 activation of Bax. Another member of the Bel-2 family, Bax is a pro-apoptotic molecule like Bid that interacts with the mitochondria to induce membrane permeability

(Hsu, et al., 1997; Eskes, et al., 1998). Sorne Bcl-2 family members have been shown to have structural homology to bacterial toxins that form pores in membranes (Muchmore, et al., 1996; Chou, et al., 1999) and have demonstrated pore-forming activities in synthetic lipid membranes (Minn, et al., 1997; Schendel, et al., 1997). While sorne members oftms family are pro-apoptotic, such as Bid and Bax, other anti-apoptotic members counteract this effect. Anti-apoptotic effects of Bcl-2 inhibit Bax pore formation, as do the effects of

Bel-XL on Bid. Ion permeable pore formation is crucial to apoptotic induction through the release of Apoptosis Inducing Factor (AIF), cytochrome c, and eventually many other cytotoxic molecules from within the mitochondria to the cytoplasm. Cytochrome c is an important co-factor for activity of the apoptosome complex. Upon release, cytochrome c binds the apoptosis protease activating factor-l (Apaf-l) and then procaspase-9 to induce its activation. Caspase-9 can activate in turn procaspase-3, just as caspase-8 does during extrinsic signaling. Inhibitors of Apoptosis (lAPs) exist that are active inhibitors of both

4 caspase-9 and caspase-3, as well as other downstream caspases (for review, see Devereau

& Reed, 1999). The recently identified Smac/DIABLü protein, which is released from the mitochondria during apoptosis, acts as an inhibitor of the lAPs (Du, et aL, 2000; Verhagen, et aL, 2000).

While both extrinsic and intrinsic apoptotic signaling are complex pathways involving many different inductive and inhibitory players, their paths do cross at the mitochondria. It is a balance of both pro- and anti-apoptotic signaIs that converge on the mitochondria that finally decide upon the fate of the celL

1.1.3 Mitochondria, control centre for life or death

Loeffler and Kroemer (2000) propose a three-step model of apoptosis. The frrst step is premitochondrial and involves apoptosis initiation signaling from activated pathways. The decisionleffector phase is mitochondrial as it requires mitochondrial membrane permeabilization and re1ease of apoptotic effectors such as cytochrome c. Finally, the degradation phase consists ofprotease and nuclease activation that results in dramatic loss of cellular integrity. Severallines of evidence strongly support this model, the first of which cornes from the use of cell-free systems. Using many different apoptotic stimuli, these systems were shown to require the presence of mitochondria for caspases or nuclease activation (Loeffler & Kramer, 2000). Bcl-2 family members have demonstrated interactions with the mitochondrial outer membrane. While direct interaction of Bax and

5 Bal< with mitochondrial proteins induces membrane permeabilization (Marzo, et al., 1998;

Narita, et al., 1998), the binding of Bcl-2 to the same mitochondrial proteins prevents cytochrome c release(Kluck, et al., 1997; Brenner, et al., 2000).

The target sites for Bcl-2 family members on the mitochondrial membrane are components of the permeability transition pore complex (PTPC) (Brenner, et al., 2000). The voltage­ dependent anion channel (VDAC) of the outer mitochondrial membrane and the adenine­ nucleotide translocator (ANT) of the inner mitochondrial membrane are major components of the channel. Together these components play an essential role in maintaining a transmembrane potential between the mitochondrial matrix and cellular cytoplasm

(Crompton, 1999). Specifie targeting of the ANT by Bax is hypothesized to result in inner membrane permeability resulting in depolarization followed by swelling of the matrix due to solute disequilibrium and rupturing of the outer membrane (Loeffler & Kroemer, 2000).

Rupturing is explained as a result of the inner membrane having a much greater surface area than that of the outer membrane. As opposed to this concept of nonspecific channel formation for the release of cytochrome c, another proposed mechanism is the formation of a specifie pore possibly involving the VDAC and Bax (Shimizu, et al., 1999). Along with the loss of a mitochondrial transmembrane potential and release of cytochrome c, effects on cytoplasmic pH, cellular redox state and mitochondrial proton flux have been observed

(Vander Heiden & Thompson, 1999). AlI of these effects help to expl'lin the resulting cellular death.

6 1.1.4 lOF-IR anti-apoptotic signaling

The best understood pathways for the control of anti-apoptotic members of the Bcl-2 protein farnily signal via the Insulin-like Growth Factor-I Receptor (IGF-IR). IGF-IR

survival signaling plays an important role in tissue homeostasisand damage response. In addition to anti-apoptotic signaling, IGF-IR has roles in rnitogenesis, cellular differentiation, and transformation. Overexpression of the IGF-IR gene has been demonstrated in various human cancers and is believed to be a predisposing factor for

tumorgenesis (Baserga, 1999). High levels of survival signaling prevent cellular

differentiation and apoptosis, therefore preserving the cell in a totipotent and proliferative

state that is susceptible to tumorgenic mutation. Produced by the liver, insulin-like growth

factor-I (IGF-I) is the major IGF-IR ligand, is present ubiquitously and in very high

concentration in serum (O'Connor, 2000). Several examples exist to demonstrate that the

level of receptor and not the level of IGF-I is responsible for cellular survival and

transformation. For instance R-cells, which do not express IGF-IR, cannot be transformed

by the simian virus 40 (SV40) large tumour antigen (Porcu, et al., 1992). This antigen

increases IGF-I expression but lack of the receptor prevents cellular transformation (Sell, et

al., 1993). Rubini, et al. (1997) and Reiss, et al. (1998) demonstrated that different levels of

IGF-IR expression by transformation of the same R-cellline induces different responses to

the same level of IGF-I stimulation. At 3,000 receptors/cell no response occurred, but at

15,000 rececptors/cell DNA synthesis was observed. At 22,000 receptors/cell both DNA

synthesis and cell division occurred and at 30,000 receptors/cell transformation was

possible.

7 Another important aspect of IGF-IR signaling is the ability of IGF-IR overexpression to induce mitogenesis in one circumstance and to induce differentiation in another. The balance ofIGF-IR substrates determines the type of response to ligand binding. High cellular levels of insulin receptor substrate-1 (IRS-1) permit proliferation, ceIl adhesion, survival signaling, and may permit transformation. In the absence of IRS-1, high levels of

Shc protein induce cellular differentiation, which leads to eventual cell death as differentiated ceIls have a limited life span (Baserga & Morrione, 1999).

Three complementary pathways of IGF-IR signaling are known, aIl which lead to the maintenance of Bad serine phosphorylation. Bad is a pro-apoptotic member of the Bel-2 family, which can bind Bel-XL or Bcl-2 and sequester their protective roles against

apoptosis (Yang, et aL, 1995). When phosphorylated, Bad can no longer heterodimerize with other Bel-2 members and is itself sequestered by 14-3-3 protein within the cytosol

(Zha, et aL, 1996). The IRS-1 pathway involves IRS-1activation by IGF-IR followed by phosphoinositide 3-kinase (PI3-K) phospho-activation (Myers, et aL, 1994). The Akt kinase (also known as protein kinase B) is then activated and phosphorylates Bad on serine

136 (Kulik & Weber, 1998) resulting in Bad sequestration. Mitogen-activated protein kinase (MAPK) pathway activation is the second IGF-IR signaling pathway. A cascade is initiated via Shc protein activation and results in Ras, Raf, MEK-1, and ERK1 activation

(Peruzzi, et aL, 1999). ERK1 phosphorylates p90RSK which then phosphorylates Bad on serine 112 (Bonni, et aL, 1999; Shimamura, et aL, 2000) and serine 155 (Tan, et al., 2000).

The third pathway induces the mitochondrial Raf-1 pool to be activated (Peruzzi, et al.,

1999). The demonstration of mitochondrial targeted Raf-1 activitY resulting in Bad

8 phosphorylation suggests that Bad is aiso targeted by this pathway (Wang, et al., 1996). An anti-apoptotic signaling role for Raf-l has been demonstrated using a constitutively active

Raf-l mutant which protects against apoptosis and a dominant-negative mutant which increases cell death (Wang, et aL, 1996).

1.2 GrblO and survival signaling

1.2.1 The Grb7family ofproteins

In signal transduction, catalytic proteins often have a number of enzymatic substrates with a range of specificities. This places significant importance on the regulation of protein to protein binding. In 1991, Skolnik, et aL developed the cloning of receptor targets (CORT) technique that was used to clone each member of the growth-factor receptor bound protein­

7 (Grb7) family of signaling molecules. This technique used the carboxyl-terminus of the epidermal growth factor receptor (EGFR), which was tyrosine phosphorylated, to screen a cDNA expression library. Identification of Grb7 (Margolis, et al., 1992), GrblO (Ooi, et al., 1995), and Grb14 (Daly, et al., 1996) by this technique elucidated a new family of adaptor molecules. Various protein-binding domains and a lack of enzymatic activity qualify this family as a group of adaptors. Each member varies in tissue specifie expression, differential splicing, and binding specificities to different receptor tyrosine kinases and tyrosine phosphorylated proteins. Conserved sequence within this family includes an amino-terminal proline-rich region (PR domain) and Ras-associating (RA)-like

9 domain, a central pleckstrin homology (PH) domain, a carboxyl-terminal Src-homology type 2 (SH2) domain, and a BPS (between PH and SH2) domain. Amino-terminal sequence is the most variable in aH. members and is the site of most splice variation. The PR domain is highly conserved within this family but has no known function. It has been suggested that this motif may act as a common site for regulatory or effector molecule binding (Daly,

1998). Using structural homology analyses, similarity between the RA domains of Ras superfamily small GTPasesand c-Raf-l has been found with RA-like domains of the Grb7 family members and MIG10, a Caenorhabditis elegans protein involved in cellular migration (Wojcik, et al., 1999). This similarity suggests that the RA domain may be used to bind small GTPases, Ras, or other Ras superfamily members. Wojcik, et al. (1999) suggest that the presence of this domain could indicate that the Grb7 family acts as Ras­

GTP effectors and not simply as adaptor molecules. The presence of a PH domain in this family may indicate a number of different types of protein to protein and protein to

phospholipid interactions. Intracellular targeting to the plasma membrane and synergistic

control of signaling protein activation through SH3 and SH2 domains are suggested roles

for the PH domain (Pawson & Scott, 1997). Sequence analyses of PH domains has

suggested that evolutionary divergence of ligand binding and protein function has occurred

(Blomberg & Nilges, 1997), therefore supporting the concept that this domain is highly

versatile in its functions (Shaw, 1996). Phosphotyrosine-containing sequence interaction is

accomplished by the SH2 domain. For instance, extensive two-hybrid assays using various

receptor intracellular domains including Ret (Pandey, et al., 1996; Pandey, et al., 1995), the

insulin receptor (IR) (Frantz, et al., 1997), and platelet-derived growth factor ~-receptor

10 (PDOFf3-R) (Yokote, et al., 1996) have demonstrated binding with different Orb7 farnily member SH2 domains. Finally, the BPS domains ofboth OtblO and Orb14 have been implicated in IR binding (He, et al., 1998; Hemming, et a1., 2001). In conjunction with the

SH2 domain, the BPS domain may be involved in deterrnining the specificity of binding to receptors (He, et a1., 1998).

Differentiai expression of Orb7 members overlaps in sorne tissue types but is characteristic for each protein. While Orb7 is high in human pancreas, kidney, prostate, small intestine

and placenta (Frantz, et ai., 1997), as well as in mouse liver and kidney (Margolis, eta1.,

1992), OrblO expression is high in human skeletal muscle, pancreas, heart, brain (Liu &

Roth, 1995; O'Neill, et a1., 1996; Frantz, et a1., 1997), and mouse heart and kidney (Ooi, et a1., 1995). Oruppuso, et a1. (2000) have showed Orb10 in rat liver during embryogenesis but demonstrated the loss of detectable transcripts shortly after birth. Orb14 expression is highest in human testis, ovary, liver, kidney, pancreas, heart, and skeletal muscle tissue

(Daly, et ai., 1996), as weIl as in rat liver, heart, and muscle (Kasus-Jacobi, et a1., 1998).

Severallines of evidence suggest regulated roles in signal transduction for the Orb7 family

members. Both Orb7 and OrblO are tyrosine phosphorylated (Lee, et a1., 2000; Mano, et a1., 1998), while Orb7 is also threonine phosphorylated (Fiddes, et ai., 1998). AlI three

members of the Orb7 farnily are serine phosphorylated (Ooi, et a1., 1995; Daly, et ai., 1996) and have multiple receptor tyrosine kinase (RTK) targets, as was previously discussed.

Whether these three members are all regulated by the same serine/threonine kinase is yet to be deterrnined. The discovery of genetic co-segregation of the Orb7 and ErbB families may

11 shed light on the involvement of Grb7 family members in human cancer (Daly, 1998).

Stein, et al. (1994) first showed tight co-amplification of GRB7 and ERBB2 genes, both located on chromosome 17q, in human breast cancer celllines in addition to high correlation of Grb7 and ErbB2 overexpression in primary tumours. Co-amplification of these two genes has also been shown in gastric and esophageal cancer Hnes (Akiyama, et al., 1997). Correlation between GRB7 and EGFR or ERBB2 expression has been linked to extramucosal tumour invasion in esophageal cancers (Tanaka, et al., 1997). This may in part he explained by a role for Grb7 in cell mobility and metastatic invasion. Tanaka, et al.

(1998) have shown that an isoform of Grb7, which has a short hydrophobie sequence in the place of the SH2 domain, is linked to metastatic invasion in esophageal cancers and expression of a Grb7 antisense construct inhibits cellular invasion. Increased cellular mobility has also been observed in cells with overexpressed Grb7 (Han & Guan, 1999).

GRB10 and EGFR localize to the same region of human chromosome 7 (Jerome, et al.,

1997). While co-amplification of GRB10 is not yet reported, expression in sorne breast cancer Hnes has been demonstrated (Dong, et al., 1997) and EGFR is often amplified in human ovarian (Scambia, et al., 1993) and breast cancers (Pirinen, et al., 1995), as well as glioblastomas (Lihermann, et al., 1985). GRB14 and ERBB4 both localize to chromosome

2q (Baker, et al., 1996) and GRB14 has been observed overexpressed in breast and prostate cancer celllines (Daly, et al., 1996).

12 1.2.2 Grb10 binding partners

In vitro identified receptor targets of Grb10 are numerous, yet the specificity of binding in sorne cases is general to more than one Grb7 family member. In vitro demonstration of a large number of GrblO and Grb7 targets to date begs the question of how many of these targets are true in vivo targets and which are in vitro artifacts. For instance, in vitro targets determined by yeast two-hybrid assays, which re1y on expression of proteins in yeast and not mammalian cells. These targets might not bind Grb10 in vivo due to the different cellular localization or folding of these two proteins in the mammalian cell context. The

CORT cloning technique tests for in vitro interactions by screening a cDNA expression library, and not for interactions between wild type proteins. In vitro resin binding typically examines in vitro formed complexes involving GST-fused bacterially expressed Grbl0 and may therefore identify binding partners not normally found in vivo. Evidence of in vivo binding by immunoprecipitation provides a higher level of confidence than these other techniques as this technique examines in vivo formed protein complexes isolated from mammalian cells. However, the most convincing of immunoprecipitations at wild type

Grb10 expression leve1s has been shown to not he possible with the available antibodies

(Nantel, et aL, 1999). Therefore, reliance on immunoprecipitations from cells overexpressing tagged Grbl0 is the best remaining option.

Many of the demonstrated interactions between Grb10 and receptor targets require receptor stimulation by ligand, which is important for Grbl0 recruitment to the plasma membrane.

Dong, et al. (1997) originally reported large increases in Grb10 localization to the

13 membrane after insulin stimulation of Chinese hamster ovary (CHO) cells overexpressing

GrblO. More recently, Nantel, et al. (1999) demonstrated that overexpressed GrblO is mislocalized to the cytoplasm and in wild type cells is localized mainly to the mitochondria. Both serum and IGF-I stimulation resulted in recruitment of GrblO to the plasma membrane and a small amount to actin-rich membrane ruffles. These finding not only confirmed the requirement for receptor stimulation for Grbl0 recruitment but also emphasized the difficulty of drawing conclusionsfrom cells overexpressing GrblO.

Immunoprecipitation of receptor targets with endogenous level Grbl0 is therefore an important in vivo confirmation of interactions identified in vitro.

CORT cloning, two-hybrid assays, and in vitro resin binding techniques have been applied by many groups to identify various in vitro receptor targets of Grb10. These include the IR

(Liu & Roth, 1995), IGF-IR (Dey, et aL, 1996), ELK (Eph-like kinase) receptor (Stein, et al., 1996), growth hormone receptor (GHR) (Moutoussamy, et al., 1998), epidermal growth factor receptor (EGFR) (Frantz, et al., 1997; He, et al., 1998), Ret receptor (Pandey, et al.,

1995), PDGFI3-R (Frantz, et al., 1997; Wang, et al., 1999), hepatocyte growth factor receptor (HGFR), and fibroblast growth factor receptor (FGFR) (Wang, et al., 1999). Of these targets, immunoprecipitation of endogenous GrblO has not been demonstrated with

EGFR, Ret, HGFR, and FGFR, while Ret has been immunoprecipitated with overexpressed

GrblO (Pandey, et al., 1995).

Mapping of GrblO binding regions to the IR, IGF-IR, and EGFR has helped explain the varying affinities to these receptors. Laviola, et al. (1997) identified the SH2 domain of

14 GrblO using a two-hybrid screen for IGF-IR in a mouse embryo library. Similarly, two­ hybrid screens with the cytoplasmic domain of IR have demonstrated interaction with the

GrblO SH2 domain (O'Neill, et al., 1996; Frantz, et al., 1997). Characterization of the BPS domain by He, et al. (1998) demonstrated a strong interaction with the IR and EGFR. Co­ immunoprecipitations demonstrated binding of Grb10 with IR in vivo but could not do so with the IGF-IR (Laviola, et al., 1997). It was concluded that GrblO preferentially binds the

IR over the IGF-IR. This lead to the conclusion that the strong affinity of GrblO for the IR is related to interaction with both SH2 and BPS domains, while the IGF-IR and EGFR are bound with weaker affinity, as interaction is limited to only one domain (He, et al., 1998).

To elucidate the role of GrblO in signal transduction, identification of non-receptor type targets is required. In addition to oligomerization (Dong, et al., 1998), Grbl0 binding to

Tee (Mano, et al., 1998), Src (Langlais, et al., 2000), Bcr-Abl (Bai, et al., 1998), cAbl

(Frantz, et al., 1997), Jak2 (Moutoussamy, et al., 1998), pp135 (O'Neill, et al., 1996),

Nedd4 (Morrione, et al., 1999), MEK1, and Raf-l (Nantel, et al., 1998) have all been demonstrated in vitro. However, only binding with Nedd4 and Raf-l was demonstrated by immunopreeipitations of endogenous protein. Raf-l binding implicates Grbl0 in anti­ apoptotic signaling, while binding to Nedd4, a ubiquitin protein ligase (E3), suggests a role in ubiquitination. Interesting new evidence now links these two proteins in IGF-IR anti­ apoptotic signaling and demonstrates their targeting to mitochondria (Peruzzi, et al., 2001).

15 .. 1.2.3 Grb10 in mitogenesis

Inhibitory and stimulatory roles for GrblO in mitogenesis have both been argued, however the majority of reports of an inhibitory role are based on GrblO overexpression in stable

ceIllines. In 1995 Liu, et al. reported that stable GrblO overexpression in CHO-IR cells

(CHO cells overexpressing the IR) inhibited the insulin dependent phosphorylation of IRS­

1 and pp60 and the activation of PI3-K. O'Neill, et al. (1996) reported an inhibitory effect

of microinjected GrblO SH2 domain in Rat! fibroblasts on insulin and IGF-I induced

mitogenesis. He, et al. (1998) later demonstrated that this was in fact due to the BPS

domain and not the SH2 domain. Similar resultshave also been reported by Stein, et al.

(2001). Stable overexpression of Grbl0 in various mouse embryo fibroblast lines inhibited

IGF-I induced growth but not insulin induced growth (Morrione, 1997). The same

experiment also showed accumulation of cells in the Sand G2 cell cycle phases. The

dependability ofresults from stable lines, in this type of experiment, has been argued

against by O'Neill et al. (1996). They make the case that clonaI variation of selected stable

clones could result in variable results. Inhibition of GH signaling (Moutoussamy, et al.,

1998) and Tec kinase activation (Mano, et al., 1998) have also been demonstrated in cell

lines overexpressing Grb10.

A stimulatory role for GrblO in mitogenesis has been clearly demonstrated by Wang, et al.

(1999) using a regulated transient expression system of Grbl0 in normal mouse fibroblast

cells. Stimulation of DNA synthesis was observed in a Grbl0 dose-dependent manner

when signaling via the PDGF~, IGF-I, and insulin receptors was tested. The same report

16 shows that microinjection of the SH2 domain interferes with the positive stimulation via the PDGF~ and insulin receptors, as it acts as a dominant negative. Wang, et al. (1999) also tested the effects of cell-permeable GrblO domains in NIH-3T3 cells. They demonstrated that introduction of the SH2 domain also interferes with IGF-IR signaling, while introduction of the PR domain interferes only with IGF-IR and IR signaling of mitogenesis.

Transient overexpression of the entire Grb10 protein was reported to induce an increase in cellular mitogenesis based on cell counts (Wang, et al., 1999). However, other studies suggest that this may he the result of decreased cellular death (Nantel, et al., 1999).

A stimulatory role in mitogenesis now appears to be the most supported for GrblO. The dominant-negative role of microinjected SH2 domain might be explained by a sequestering effect on downstream effectors, especially considering Grb10 is an adaptor molecule with multiple protein binding capacities. This hypothesis is supported by the lack of correlation between the dominant-negative SH2 domain activity and receptor-binding activity (Wang, et al., 1999). Interestingly, receptors are the major SH2 domain binding proteins (see

Section 1.2.1) yet these results also suggest binding with downstream proteins plays a role in mitogenesis. Another explanation for variable stimulatory and inhibitory roles observed for Grb10 could be tissue-specifie effects, supported by the existence of tissue-specifie splice variants. However opposite roles have been observed in similar fibroblast celllines.

17 1.2.4 Grb10 in anti-apoptotic signaling

Grbl0 has been implicated in anti-apoptotic signaling using various lines of evidence.

Specific mutations within the SH2 domain of Grb10 are known to induce apoptosis in various transfected cell types. Rescue of these cells is possible with concomitant transfection with wild type Grbl0, suggesting that the mutant form induces apoptosis by sequestering other slgnaling proteins (Nantel, et al., 1998). Binding of GrblO to the MEKI and Raf-l kinases via the SH2 domain suggests Grb10 is involved in the mitogenic MAP kinase cascade (Nantel, et al., 1998). The RAF-MEK-ERK pathway has previously been shown to inhibit apoptosis (Gardner, et al., 1996; Xia, et al., 1995) and Raf-l has demonstrated role in anti-apoptotic signaling from the IGF-IR (Wang, et al., 1996; Peruzzi, et al., 1999). Raf-l knockout mice have recently been produced and exhibit hypocellular livers with many apoptotic cells (Hüser, et al., 2001; Mikula, et al., 2001). Binding of

GrblO to Raf-l is constitutive and has been mapped to the Raf-l amino-terminal regulatory domain (Nantel, et al., 1998). In addition, localization studies have demonstrated that

Grbl0 associates with the Raf-l pool at the mitochondria and UV treatment stimulates an increase in this association through Raf-l activation (Nantel, et al., 1999). Localization studies, using fluorescence microscopy of HeLa and Cos-7 cens, have been used to study

GrblO translocation following IGF-IR stimulation. It was shown that endogenous GrblO is rapidly translocated (within 5 minutes) from the mitochondria to the plasma membrane and then rapidly returns to the mitochondrial surface (Nantel, et al., 1999). Nante1, et al. (1999) have hypothesized that Grb10 acts as a shuttle for Raf-l between the plasma membrane and the mitochondria. This hypothesis suggests that Grbl0 translocates to the plasma

18 membrane to interact with the IGF-IR and then returns to the rnitochondria bound to activated Raf-1. The activated Raf-1 pool would then be responsible for BAD protein phosphorylation, resulting in inhibition of apoptosis.

A recent development in the research of IGF-IR signaling has shown that an interesting binding partner of GrblO, Nedd4 (Morrione, et al., 1999), also translocates from the plasma membrane to the mitochondria following IGF-I stimulation (Peruzzi, et al., 2001). Nedd4, as previously mentioned, is a ubiquitin protein ligase (E3) and therefore suggests a role for

GrblO in ubiquitination. Peruzzi, et al. (2001) have shown that Raf-1 and Nedd4 targeting to the mitochondria is dependent on serines 1280-1283 of the IGF-IR. These serines are also shown to be involved in binding 14-3-3 protein, which normally serves to sequester protein activity (Peruzzi, et al., 2001). While this group was not able to demonstrate the presence of GrblO in their cellline, it is likely that a more sensitive antibody such as the one used by Nantel, et al. (1999) could be used to do so. Peruzzi, et al. (2001) suggest that

Nedd4 targeting to the mitochondria may lead to the degradation of pro-apoptotic signals such as BAD. Nedd4 has already been identified as a target for caspases (Harvey, et al.,

1998), suggesting that it indeed has a role in anti-apoptotic signaling.

1.2.5 Grb10 in human disease

The involvement of Grb10 in a few genetic conditions has been suggested but no clear evidence exists to demonstrate this. As mentioned earlier, sorne links between Grb7 farnily

19 members with certain cancer types have been made but it is most likely that GrblO mutations wouId lead to only a predisposition to tumorgenesis. This is not unlike theories of the involvement of IGF-IR in carcinogenesis, which suggest only a predisposition to cancer (Baserga, 1999).

GrblO is a candidate gene for the Silver-Russell syndrome (SRS). This malformation syndrome is a result of retarded growth pre- and post-natal, results in cranial shape abnormalities, and asymmetry of the body (Joyce, et al., 1999). Imprinting of GRBIO has been observed in humans in a highly isoform- and tissue-specifie way. GRBIO in the brain is also the first identified gene that is imprinted oppositely in mouse and humans; paternal in humans and maternal in the mouse (Blagitko, et al., 2(00). The GrblO gene in mice is located on chromosome Il in a region that, when duplicated, induces abnormal imprinting patterns that result in aberrant pre-natal growth (Miyoshi, et al., 1998). Controversy exists over the involvement of GRBIO in SRS. Joyce, et al. (1999) and Monk, et al. (2000) demonstrated a duplicated region of chromosome 7 in a SRS patient that spans three growth related genes including GRBIO, EGFR, and IGFBP1 (insulin-like growth factor binding protein 1). Monk, et al. (2000) hypothesize that the maternaI copy duplication they observed could result in SRS from overexpression of this allele. However, Blagitko, et al.,

(2000) have completed a scan of fifty unrelated SRS patients and found no significant correlation between GRBIO alterations and the occurrence of SRS. Etiology of SRS involving GrblO is therefore not likely but may instead involve various genetic abnormalities and several genes.

20 Hirschsprung disease (HCSR) is the only other condition that has been tested for GRBIO abnormalities. Also known as congenital aganglionic megacolon, this condition has a large genetic component, affects the bowel and kidneys, and is the most common cause of congenital bowel obstruction in humans (Angrist, et al., 1998). Approximately half of known cases of HCSR are linked to mutations of the RET and related endothelins genes, leaving unidentified genes to explain the other half of cases (Attie, et al., 1995;

Chakravarti, et al., 1996; Seri, et al., 1997). GRBIO was screened as a candidate by Angrist, et al. (1998) based on known associations between Grb10 and Ret, as weIl as Grb10 expression patterns that are consistent with·a IOle in enteric nervous system development

(Pandey, et al., 1995). Using a panel of 85 patients Angrist, et al. (1998) were not able to demonstrate linkage between HCSR and GRBIO mutations.

1.3 Topics of investigation in this report

1.3.1 Investigation ofGrblO mitochondrial binding

Nantel, et al. (1999) immunolocalized GrblO using fluorescence microscopy and demonstrated translocation between the mitochondria and plasma membrane of this protein following stimulation with IGF-I. In this study, an elaboration was made on the initial immunolocalization reported. To determine the proportion of cells that exhibit IGF-I stimulated Grbl0 translocation, images ofboth HeLa and Cos-7 cells were analyzed and

21 ceIl counts were perfonned of fluorescently labeled HeLa. The proportions demonstrated exhibit a c1ear pattern of translocalized Grbl0 foIlowing IGF-I treatment.

The role of GrblO in anti-apoptotic signaling would be better understood with the knowledge of how this protein is targeted to the mitochondrial membrane foIlowing IGF-I

stimulation. Grb10 has no identified mitochondrial targeting sequence or transmembrane domain and is likely to be peripheraIly associated with the mitochondrial membrane

(Nantel, et al., 1999). It can therefore be hypothesized that an unidentified region of this

protein binds either directly or indirectly through another protein anchored. Here an in vitro

assay is used to test various Grb10 mutants for mitochondrial membrane binding and a region at the amino-tenninal outside of the PR domain is implicated. This result was

supported by a similar in vivo mitochondrial-binding assay.

1.3.2 The search for new Grb10 bindingpartners

Exhaustive use of two-hybrid assays has revealed, what is like1y to he, oruy a smaU part of

aU Grb10 binding proteins, especially considering the numerous protein binding domains of Grb10. The requirement for new techniques of protein partner identification has lead to the development of a mitochondrial-specific immunoprecipitation assay and two­ dimensional (2-D) separation technique used in this study. This analysis will be referred to

as the binding partner assay. To first establish which conditions are ideal for the study of

GrblO complexing, a gel filtration experiment was set up to resolve mitochondrial extracts

22 from HeLaS3 cells. Elution profiles and Westerns of colurnn fractions suggest that serum treated cells form sufficient Grb10 containing high molecular weight complexes at high enough concentration for use in binding partner studies. In the binding partner study, overexpression of Flag-tagged GrblO was required to produce sufficient protein for GrblO

visualization on 2-D gels. The resulting problem of non-specifie cytosolic interactions was

circumvented by the use of only mitochondrial fractions.

23 2. METHOnS

2.1 Cloning

Attempts were made to clone a series of GrblO truncation mutants from both carboxyl and amino-termini by subcloning from pre-existing vectors. A point mutant, a small deletion, and two larger carboxyl-terminal deletions were subcloned and these were applied to in vitro binding assays (see Figure 4). Only successful subcloning strategies are described here: five of the constructions attempted never yielded the desired product, as transformed

E.coli colonies never grew after repeated attempts (see Section 4.2).

The vector pAN142 encoding Maltose Binding Protein (MBP) fused Grb10 was used to subclone a Proline Rich (PR) Domain mutant that contains only the PR domain and flanking sequence. SpeI and HindIII restriction endonucleases were used to cut pAN142.

Religation of the vector resulted in a carboxyl-terminal deletion after position 211. This new Grbl0-211~ mutant encoding vector was labeled pJHll.

A similar strategy was used to produce a smaller carboxyl-terminal deletion removing all amino acids after position 413. This was accomplished using StuI and HindIII restriction endonucleases. The corresponding mutant lacks both BPS and SH2 domains along with flanking sequences. This new GrblO-413~ mutant-encoding vector was labeled pJHlO.

Nantel, et al. (1998) previously described the GrblO-RL point mutant Arg-~B5-Leu which induces apoptosis as it prevents all interactions of the SH2 domain and prevents the recruitment of Grbl0 to the plasma membrane from the mitochondria. A vector encoding

24 GrblO-RL fused to the Flag epitope, pAN200, was cleaved using EcoRi and XbaI. This insert was ligated to EcoRI/XbaI cleaved pMAL-c2 vector (New England Biolabs) to produce a MBP fused RL mutant vector, labeled pJH12.

The GrblOÔPR mutant, which has a deletion of the Proline Rich domain only, was generated as a MBP fusion. The Flag-tagged ôPR mutant, which lacks amino acids 135 to

144, was obtained by cleaving the pAN207 vector with EcoRi and XbaI and ligating to the same pMAL-c2 fragment as used to make pJH12. This final vector was labeled pJH13.

AH newly constructed vectors were confirmed by restriction mapping, DNA sequencing of ligation junctions, and correct expressed protein size.

2.2 Protein preparation

AH confirmed vectors described above were purified by miniprep and transformed into the

UT5600 strain of Escherichia coli, a strain that 1acks certain protease activities and therefore favours protein expression. Strains were then grown in 1.5 L of Rich Broth with

0.2% glucose and 100Jlg/mi ampicillin from overnight cultures. After the culture reached

an Optical Density at 600 nm (OD600) of about 0.5, the expression of GrblO mutant protein expression was induced by the addition of O.3mM IPTG. After 2 hours of inductive growth, cells were cold centrifuged at 4000g for 20 minutes and resuspended in 50ml bicarbonate

0.7ul/ml~­ column buffer (20mM NaHC03 at pH 8.0, 20ûmM NaCl, 1mM EDTA, and mercaptoethanol to prevent disulfide bond formation upon lysis). The lysate was then

25 sonicated at full power and continuous setting for four 30 seconds. After centrifuging at

9000g for 30 minutes at 4oC, the supernatant was then diluted five fold and incubated with amylose resin (New England Biolabs) at 4°C on a rotary shaker for between 1 and 4 hours

(MBP-LacZ required only 1 hour while GrblO mutants required between 3 and 4 hours for optimal binding). MBP extracts were purified by loading of the incubated extract onto a column, rinsing with Il volumes of column buffer, and eluting with 20ml column buffer containing lOmM maltose. Eluates were tested for protein levels using the Bio-Rad Protein

Assay and loading on SDS-PAGE gels. Coomassie staining allowed deterrnination of relative concentrations of each sample.

2.3 Cell culture and transfection

Cos-l, HeLa, and HeLaS3 cells were initially grown from liquid nitrogen stored stocks in

Dulbecco's Modified Eagle's Medium (DMEM) + 10% Fetal Bovine Serum (FBS) at 37°C in 6% atmospheric COz' Upon reaching 80% confluency, HeLaS3 cells were scrapped using a plastic cell scrapper and put into suspension. To prevent cadherin adhesion calcium-free media (DM-) was used and the cells were shaken continuously. To serum starve any of the above cell types, cells were resuspended in media-lacking FBS. IGF-I treatment consists of 24 hour starvation and resuspension of cells in the appropriate media containing 0.1 % Bovine Serum Albumin (BSA) and lOOng/ml IGF-1 warmed to 37°C.

BSA is added to insure efficient delivery of IGF-I to cells by sequestering IGF-I binding proteins and preventing non-specifie binding to the plastic dishes used. For UV treatment, 26 • cells were starved for 24 hours and then were irradiated with 100 J/m2 UV at 254 nm. To allow sufficient time for apoptotic induction, cells were incubated for 4 hours in DMEM lacking serum.

Transfection of Cos-1 cells was done using Lipofectamine Reagent (Life Technologies,

Inc.) according to the manufacturer's instructions. Cells were grown to about 40% confluency, incubated with 15Jlg/ml Lipofectamine for 5 hours, and harvested 48 hours

1ater. The control vector pcDNA3 (Invitrogen), from which pAN185, pAN200, and pAN207 are aH based, was used as an empty vector control in all transfection experiments.

2.4 Mitochondria isolation for immunoprecipitation & gel filtration

Isolation of mitochondria for immunoprecipitation and gel filtration experiments was done by cellular fractionation using differential centrifugation. Cells were harvested using a cell scraper, if attached, and then were centrifuged in a cold centrifuge at 1000rpm for 5 minutes. Subsequent steps were carried out at 4°C. Cell pellets were rinsed twice with

Phosphate Buffered Saline (PBS) and pelleted again. The pellet was then suspended in

1.5ml MS buffer (21OmM mannitol, 70mM sucrose, 5mM Tris pH7.5, 1mM EDTA at pH7.5) and lysed using a Polytron at setting 6.5 for 10 seconds. Nuclei and unbroken cells were removed by centrifuging three times at 1300g for 5 minutes, keeping the supernatant each time. The mitochondria were then pelleted at 17,000g for 15 minutes yielding the medium speed pellet (MP). The pellet was then washed once with 500~1 MS with a

27 Protease Inhibitor pellet (Roche Molecular Biochemicals), 200 !-lM sodium vanadate and

0.1 % Triton-X 100. The MP was then resuspended in 500!-l1 of the same buffer and rotated for 1 hour on a LabQuake shaker to lyse mitochondrial membranes. Lysis for gel filtration was performed instead in Column Buffer (5% sucrose, 20 mM HEPES/NaOH, 0.1 %

CHAPS, 5mM dithiothreitol (DTT), pH 7.0) with Protease Inhibitor, 200 !-lM sodium vanadate and 0.1 % Triton-X 100. Lysate was then centrifuged at 17,000g for 10 minutes to remove membranes and the pellet discarded. This supernatant fraction contains the mitochondrial fraction, which was either loaded directly onto a gel filtration column or incubated with various resins for immunoprecipitation. Nantel, et al. (1999) have previously shown this method to he highly efficient at isolating purified mitochondrial fractions lacking contamination from other subcellular compartments.

2.5 High purity mitochondria isolation for in vitro binding assays

In vitro binding assays require highly purified mitochondria to he certain that MBP-fused proteins are binding strictly to mitochondria and not contaminants from other cellular fraction. To accomplish this, a discontinuous sucrose gradient was applied. Cultured

HeLaS3 cells were grown until 50 million cells could be harvested. Initial preparation of a

MP fraction was done as described above (see Section 2.4) and again all steps were performed at 4°C. The MP was then resuspended in 2ml MS buffer and 1ml was layered over 6ml MS lM sucrose on top of 6ml MS 1.5Msucrose in ultracentrifuge tubes. Tubes were then ultracentrifuged at 28,000 rpm for 25 minutes using a SWi28.1 rotor. Removal of

28 the mitochondriallayer at lI1.SM interface was perforrned using a needle that was cleaned and wiped thoroughly after each removal. Two-fold dilution of the mitochondriallayer was done slowly, adding one drop of MS at a time while vortexing gently, to prevent mitochondrial damage from rapid tonicity change. Purified mitochondria were then centrifuged at 17, OOOg for lS minutes, washed with 1rnl MS, recentrifuged, and resuspended in SOOI-tl MS. Bio-Rad Protein Assay was then used to assay for total protein concentration, samples were aliquoted and snap frozen in liquid nitrogen. These samples

were then stored at -SO°C until required.

2.6 In vitro binding assays

Based on the deterrnined relative concentrations of produced MBP-fused proteins (see

Section2.2) relative loading volumes were decided upon for each, where MBP-LacZ was

the most concentrated and 1jtl of it was loaded. The loading volume of each was added to

MS with 1% BSA to give 200ul of total volume. After centrifuging at 27,000g for S

minutes at 4°C to pellet out contaminants, 2jt1 of each sample was added to 0 or 2Sjtl of

purified mitochondria (see Section 2.S). The volume was then completed to 200jtl with

MS with 1% BSA and incubated at room temperature or 4°C for 1 hour. From each tube

lOjtl were saved as Loading Control (Le) and 70jtl of IX SDS Sample Buffer (SOrnM

TrisHCI pH 6.S, 2% SDS, 0.1 % bromophenol blue, 10% glycerol) was added. Samples

were then centrifuged at 17,000g for S minutes at 4oC, lOjtl of supematant was removed from each as the Unbound Fraction Control (S), and 70jtl of IX SDS Sample Buffer (SB) 29 was added. Pellets were washed three times by gently vortexing with fresh MS and pelleted each time centrifuging at 17,OOOg. Each pellet was then resuspended in 80jû IX SB as the

Bound Fraction (P) and aIl samples were loaded on 10% SOS-PAGE gels for anti-MBP

Western blotting.

2.7 In vivo binding assays

Cos-1 cells were transfected with pcONA3 and various mutant GrblO encoding vectors

(see Section 2.3). After harvesting, mitochondrial extracts were then prepared by differential centrifugation (see Section 2.4). Extracts were then loaded on SOS-PAGE gels and Western blotted for Grbla (see Section 2.11) to demonstrate the binding of Grb10 mutants in vivo. Western blotting for cytochrome c was used to control for the amount of mitochondria loaded.

2.8 Flag co-immunoprecipitation

For each experiment, eight large 125cm2 petri dishes of Cos-1 cells at 95% confluency were transfected with pAN185 (overexpressed GrblO-Flag) or pcONA3 (empty vector) (see

Section 2.3). Based on the estimated protein concentration of mitochondrial extracts prepared by differentia1 centrifugation from these transfectants, dilutions were performed to make protein leve1s approximately equal. M2 affinity gel Flag resin slurry (Sigma) was

30 washed and resuspended in MS buffer, maintaining a concentration of 1.Smg/ml. Resin slurry was added to each mitochondrial extract in al:1 ratio by volume and incubated on a rotary shaker at 4°C for 18 hours. This 1: 1 ratio was determined as optimal by testing a range of ratios by GrblO Western blot (not shown) that allowed for a high leve1 of GrblO recovery without excessive resin use. Loading of each sample in a 1ml syringe packed with desiliconized glass wool allowed washing by centrifugation at SOOg for 2-3 minutes with a minimum of 10m! MS with 1% Triton-X 100. This wash buffer was se1ected as optimal for

Flag-tagged protein recovery after testing several common wash buffers. Samples were then re-e1uted three times with 200pl of isoelectric focusing (lEP) buffer (9M urea, 4%

CHAPS, SOmM DTT, lOmM spermine pHIl) by centrifuging. When Western analysis of samples was required without two-dimensional separation, washed resin was instead boiled in lOOpl 3X SB.

2.9 Two-dimensional electrophoresis

2.9.1 Isoelectricfocusing

The following technique was adapted from Rabilloud, et al. (1994, #1). Eluted immunoprecipitations were prepared for isoelectric focusing by adding SpI of IPG buffer

(Pharmacia) to give a final concentration of 2% and 2.5}t1 of either orange G (500}tg/ml) or bromophenol blue (200}tg/ml) dye. Samples were centrifuged for a 2-3 seconds and the volume completed to 2S5pl ± 5pl with IEF buffer. Immobiline DryStrips (Pharmacia) with

31 a length of 13cm and pH range of 3 to 10 were rehydrated overnight in a rehydration chamber (Pharmacia) with the prepared samples and immersed in oil. DryStrips were then

loaded in a MultiPhor unit (Pharmacia) on an e1ectrophoresis plate using a supplied plastic tray to assist in proper alignment between electrodes. Moistened strips of Whatman paper

(3M) were used to create a contact between the DryStrips and assembled electrodes.

Mineral oil was used to immerse the strips, to ensure efficient temperature distribution, and

a thermostatic circulator was set to 18°C. Maintenance of a sufficiently cool temperature

was important to prevent overheating due to the high voltages applied, but cooling below

18°C was not desired as IEF buffer can crystallize at lower temperatures. The following

voltage increases were programmed into a programmable EPS 3501 XL Power Supply

(Pharmacia):

step voltage duration (minutes)

1 0-250 20

2 250 25

3 250-500 20

4 500 25

5 500-3500 120

6 3500 210

Upon removal from the MultiPhor, DryStrips were equilibrated in equilibration buffer A

(30% glycerol, 6M urea, 2.5% SDS, O.lM bis-TrisHCI, 65JlM DTT) and equilibration

32 buffer B (30% glycerol, 6M urea, 2.5% SOS, O.lM bis-TrisHCI, 0.2M iodoacetamide) for

10 minutes each. OryStrips were either stored in equilibration buffer B at -20°C for a maximum of two weeks or immediately loaded on SOS-PAGE gels.

2.9.2 Resolution by size

Following IEF, OryStrips were loaded on 16cm x 18cm 12% SOS-PAGE gels using a 1% low melting point agarose bed made from SOS-PAGE gel stacking buffer (0.5M Tris, 0.4%

SOS, pH6.8). Gels were run for 18 hours at 50V and then either transferred to polyvinyldifluoride (PVDF) membranes for Western blotting (see Section 2.11) or silver stained (see below).

2.9.3 Silver staining

Staining of 2-D gels was accomplished using a silver nitrate with tetrationate protocol adapted from Rabilloud, et al. (1994, #2). AlI solutions were prepared with Milli-Q filtered water and abso1ute ethanol. First, gels were fixed in a 30% ethanol, 10% acetic acid solution for 1 hour and a 30% ethanol, 5% acetic acid solution overnight. Gels were washed for 10 minutes in 20% ethanol, for 10 minutes in 10% ethanol, and twice for 10 minutes in water. Gel were then sensitized by a 15 second wash in lJlM sodium thiosulfate and washed twice in water for 15 seconds. Gels were stained for 30 minutes in a 12JlM

33 silver nitrate solution and washed again with water for 15 seconds. To develop the gels a

250JlM potassium carbonate, SOnM sodium thiosulfate, 0.01 % formaldehyde solution was applied for about 15 minutes. Development was stopped with a 330JlM Tris, 2% acetic acid solution applied for 30 minutes. Gels were then scanned using Scantatic (Epson) and manipulated using Photoshop (Adobe). Gels were also dried for permanent storage.

2.10 Gel filtration

2.10.1 Mitochondrial extract separation

A 16mm x 60cm high-resolution column (Pharmacia) was packed with Sephacryl S-300

(Pharmacia) according to manufacturer's instructions and equilibrated with Column Buffer

(see Section 2.4). Experiments were performed using 25 million HeLaS3 cells which were either serum treated, serum starved, IGF-I treated, or UV treated (see Section 2.3).

Mitochondrial extract, prepared by differential centrifugation (see Section 204) and resuspended in 200JlI Column Buffer, was loaded on the column. The initial 15ml were discarded as the column void volume. Elution of proteins was performed at a flow rate of

OArnI/minute and 2ml fractions were collected at 4°C. Odd-numbered fractions were concentrated to 100~.tl using Vivaspin 2 (Vivascience) concentrator columns with a 10 kiloDalton (kD) cut off. Concentrated fractions were then analyzed by Bio-Rad Protein assay to plot a total protein profile and by Western blot analysis to determine the presence of Grb10 and associated proteins. A control sample of non-separated total mitochondrial extract was also analyzed by Western blot as a positive control (not shown).

34 2.10.2 Column standardization

To calibrate the column, standards were run between 12.3 and 150kD. Rat IgG (l50kD),

BSA (66kD), ovalbumin (45kD), and cytochrome c (12.3kD) were aIl run through the column by the same method as mitochondrial fractions. Detection of each was done by

Bio-Rad protein assay and by Western blot for aIl but ovalbumin. Cytochrome c was also

detected using the OD660 •

2.11 Western blotting

Blotting ofproteins was done by resolving on 8.5% SDS-PAGE gels for anti-GrblO and anti-Raf-l Westerns, 10% gels for anti-MBP, anti-IgG, and anti-BSA, and 15% gels for anti-cytochrome c. Samples were diluted with 3X SDS Sample Buffer (150mM TrisHCl pH 6.8, 6% SDS, 0.3% bromophenol blue, 30% glycerol). Electrotransfer to Immobilon-P

PVDP membranes (Millipore) according to manufacturer's instructions was foIlowed by overnight incubation with 1:1000 of anti-GrblO (Nantel, 1998), anti-Raf-l (Santa Cruz), anti-BSA (Sigma) or anti-cytochrome c (PharMingen) antibodies, or 1:20,000 anti-MBP

(New England Biolabs). IgG Westerns were incubated directly with anti-mouse secondary antibody. Incubation with the appropriate secondary antibody (anti-rabbit IgG or anti­ mouse IgG) conjugated to horseradish peroxidase (HRP) at a 1:2000 dilution was performed for 1 hour. Blots were developed using the chemiluminescent Lumi-Light

Western Blotting substrate (Roche Molecular Biochemicals) and exposed on Biomax emulsion film (Kodak).

35 2.12 Fluorescence microscopy

An eight well Permanox slide (Nunc) was seeded with 7,SOO HeLa or Cos-7 cells per well in DMEM with 10% FBS. After an ovemight incubation, cells were then starved for 24 hours and then IGF-I treated for 0,2, S, or 20 minutes (see Section 2.3). Fixing was performed by treating each well with 400Jll fresh 4% paraformaldehyde for 10 minutes.

Each well was then rinsed twice with PBS, cells were permeabilized with 400Jll 0.2%

Triton-X 100 for 2 minutes, and rinsed again with PBS. Cells were then blocked for 30 minutes with PBS containing 1% FBS. Incubation for 1 hour with both primary antibodies,

1:10 of polyclonal anti-GrblO (Nantel, 1998) and 1:S0 of monoclonal anti-cytochrome C

B4abm (Molecular Probes), in lS0JlI PBS with 1% FBS was performed. Washing with

PBS was repeated three times for 10 minutes each and secondary antibodies Lissamine

Rhodamine goat anti-rabbit IgG (Jackson ImmunoResearch) and Fluorescein

Isothiocyanate (FITC)-conjugated AffiniPure F(ab')z fragment donkey anti-mouse IgG

(Jackson ImmunoResearch) (both diluted 1:S0) and DAPI stain (Sigma) (diluted 1:SOO0) for the visualization of nuclei were applied in lS0JlI PBS with 1% FBS for 30 minutes to 1 hour. Cells were then washed three times for 10 minutes each with PBS and a glass slide was mounted using ProLong anti-fade reagent (Molecular Probes). After 20 minutes of drying, slides were visualized by oil immersion with a Leitz Aristoplan microscope coupled to a Princeton Instrument CCD camera. DAPI staining allowed for rapid scanning of the slide, and cytochrome c staining helped to confirm mitochondrial structures. Images were manipulated using Eclipse (Empix Imaging Inc.) and Photoshop (Adobe) software.

36 Approximate1y 100 HeLa cells at each time point were scored for plasma membrane localizationn of GrblO.

37 3. RESULTS

3.1 Grb10 mitochondrial binding

3.1.11mmunolocalization demonstrates a rapid GrblO translocation

Immunofluorescence microscopy was performed on both HeLa and Cos-7 cells to demonstrate the transient translocation of GrblO from the mitochondria to the plasma membrane. Mfinity purified GrblO antibody was used to label endogenous GrblO and cytochrome c antibody was used as a mitochondrial marker to confirm co-Iocalization.

Fixed Cos-7 cells were digitally photographed as morphology of these provided good readable images. HeLa cells exhibit lower endogenous Grb10 levels and higher background making them more difficult to photograph clearly (not shown). Figure 2 demonstrates typicallocalization patterns for Cos-7 following treatment with 100ng/ml IGF-I. While starved cells exhibit almost no plasma membrane staining of GrblO, cells treated with IGF­

1 for 2 minutes have a fair amount of staining visible at the membrane. Cells treated for 2 minutes maintain a high level of mitochondrial staining, as is seen in starved cells. Cells treated for 5 minutes. exhibit strong plasma membrane signais, which are almost entirely depleted at 20 minutes. At this final sampling time, Grb10 has returned to mitochondrial localization.

38 3.1.2 Cell counts ofimmunolocalized HeLa, a majority exhibits Grb10 translocation

Immunofluorescence microscopy of HeLa cells demonstrates a distinct pattern of Grb10 translocation that was scored by counting of cells. Scoring of the number of cells exhibiting plasma membrane localization against those that do not allowed for the plotting of cel1 count percentages in Figure 3. Less than 2% of starved HeLa exhibit membrane

10caIization of GrblO, which increases to 40% at the 2 minute treatment time point. By 5 minutes of IGF-I treatment 55% of cells show membrane localization, but this is reduced to

12% by 20 minutes. This demonstrates that more than haIf of the cell population responds to IGF-I stimulation by translocation of GrblO after 5 minutes of treatment. However, this response is somewhat heterogeneous as a large proportion of cells do not exhibit plasma membrane localization of GrblO at the peak time of 5 minutes. This is possibly due to variances in IRS-l and Shc levels within the cells (see Section 1.1.4).

3.1.3 In vitro assays demonstrate that mitochondrial membrane binding ofGrb10 occurs via the amino-terminal and is ATP-independent

In vitro binding assays were used to examine the ability of bacterially expressed, MBP­ fused GrblO mutants to bind the memb'rane of purified mitochondria. The four MBP-fused mutant proteins described in Figure 4, in addition to LacZ and a full-length form of GrblO, were affinity purified. Proteins were then incubated with mitochondria from HeLaS3 cells that were purified using a discontinuous sucrose gradient.

39 Many controls were used in this assay. Load controls demonstrate that approximately the same quantity of protein is used for each sample. Unbound fraction controls demonstrate the amount of protein remaining in the supernatant after pelleting of mitochondria. Lack of binding of the MBP-LacZ construct controls against adverse binding that may occur from the MBP tag. Pelleting of incubated proteins in the absence of mitochondria controls against protein aggregation. Incubation of mitochondria alone demonstrates that the anti­

MBP antibody used does not cross-react with mitochondrial proteins.

Positive binding of GrblO-211~and of GrblO~PRimplicates amino-terminal sequence outside of the PR domain in mitochondrial membrane binding. These two mutants have only amino acids 1 through 211, minus a deletion between position 135 and 144, in common.This is supported by the positive binding of GrblO-413~to the mitochondrial membrane, as this mutant lacks onlythe BPS and SH2 domains. The GrblO-RL mutant, which contains an arginine to leucine point mutation in the SH2 domain, serves as a positive control. This mutant was previously described in vivo to lack Grb10 binding to kinases and receptors, likely preventing GrblO translocation, (Nantel, et al., 1998) and was therefore expected to bind to the mitochondrial membrane. Degradation explains the presence of numerous bands for each sample. This experiment was also performed at 4°C to test the energy requirements of GrblO to mitochondria binding. As all mutant proteins are found in the mitochondrial pellets at this incubation temperature, it is assumed that mitochondrial membrane binding is ATP-independent. As the band intensities in the mitochondrial pellets for mutants of Grb10 are weaker than for fUlllength Grb10, other explanations for energy requirements to bind mitochondria cannot be exc1uded. While the

40 lack of an effective negative control cannot allow unspecific binding of GrblO to the mitochondrial membrane to be ruled out, Nantel, et al. (1999) have previously shown that mild hypotonie lysis and alkali treatment both result in a disruption of this interaction. In addition, the use of BSA containing buffer reduces the possibility of unspecific interactions.

3.14 In vivo assays support the results ofin vitro assays

Cos-l cells transfected with either GrblO-Flag or one of two Flag-tagged mutants, Grb10­

RL and GrblO~PR, were used to test GrblO mitochondrial membrane binding in vivo.

GrblO Western blots of cell and mitochondrial extracts (Figure 6) demonstrate that aH three proteins are present in the mitochondrial fraction. Transfection with the empty vector pcDNA3 serves as a negative control and demonstrates that only a low level of endogenous

Grb10 is visible in mitochondria-rich fractions. Cytochrome c Western blots (also in Figure

6) were used to demonstrate approximately equalloading of each sample. In vivo assays therefore partially confirm the results of in vitro assays as both the GrblO-RL and

GrbIO~R mutants were demonstrated to bind the mitochondrial membrane. The high intensity of these two mutants, compared to the wild type form, is in part a result of increased cell survival following transfection.

41 3.2 Development of assays for GrblO binding partner identification

3.2.1 Gelfiltration confirms ideal conditions for Grb10 complexing

The Sephacryl S-300 size exclusion gel filtration column used in this study was first calibrated using four molecular weight standards. Rat IgG (l50kD), BSA (66kD), ovalbumin (45kD), and cytochrome c (l2.3kD) were eluted through the column individually and eluted fractions were assayed for protein content. Unexpectedly, the generated protein profiles (see Figure 7) revealed an early elution for BSA and ovalbumin, both before rat IgG. It is hypothesized that this is a result of protein agglutination due to the high sucrose content of the column buffer used. Elution of IgG and cytochrome c were as expected, with cytochrome c eluting much later. The IgG elution peak at 70ml provides a useful guideline for the size determination of complexes eluting close to this volume. This

is especially true as the protein complex of interest in this experiment, Grb10 with Raf-l, is

approximated at 150kD in size.

Following growth in suspension, mitochondrial extracts were prepared from HeLaS3 cells.

This cell type was selected for gel filtration studies because HeLaS3 can be grown to high

concentrations and therefore yield high quantities of endogenous proteins. Different growth

conditions permitted the study of Grbl0 in various states of complexing. Mitochondrial

extracts were loaded on the column and eluted fractions were collected. Protein profiles

were generated for each treatment (see Figure 8) and reveal that serum, IGF-I and UV

treated cells all exhibit peaks at smaller elution volumes than serum starved ceUs.

Therefore, conditions that favour the activation of Raf-l (Nantel, et al., 1999) also favour

42 the formation of higher molecular weight complexes found in mitochondrial extracts.

GrblO and Raf-l Westerns were performed (see Figure 9) and demonstrate the presence of these two proteins in higher molecular weight complexes in serum treated cens than in starved cens. In addition, Westerns of serum treated cens also show that the strongest

GrblO and Raf-l bands are present in the same elution fractions at approximately 42ml, which corresponds to about 150kD. These runs were repeated and produced Westerns that are identical to those shown. Westerns of UV and IGF-I treated cens also show GrblO eluting in earlier fractions than in starved cens. These results therefore suggest that Grb10 and Raf-l associate under conditions that favour Raf-l activation. Due to the higher protein yield, serum treatment is therefore the best condition for the study Grb10 complexing in mitochondrial fractions.

3.2.2 The developed binding partner assay is not efficient enough to identify binding

partners

A Grb10 binding partner assay was developed using transfected Cos-l cens. Cos-l cens were used in this experiment as they yield high levels of overexpressed GrblO fonowing transfection. Expression of Grb10 fused to Flag was used based on previous work demonstrating no deleterious effect of this tag (Nantel, et al., 1998). Transfection of cens with GrblO-Flag encoding vector (pAN185), mitochondrial fractionation, Flag co­ immunoprecipitation with an anti-Flag conjugated resin, and SDS-PAGE or 2-D separation was performed. Comparison was made against empty vector (pcDNA3) transfected cells.

43 Westerns of SDS-PAGE gels (see Figure 10) demonstrate the presence of both GrblO and

Raf-1 in total and mitochondrial extracts. Note that several forms of Grb10 are present, either due to splicing variation or phosphorylation. The Raf-1 Western demonstrates that this protein is clearly co-immunoprecipitated by Grb10-Flag.

Figure Il shows the Grb10 Western blots of 2-D electrophoresis of Flag immunoprecipitates. Grb10-Flag exhibits a distinct spotting pattern that shows several forms of this construct, which are likely due to the presence of four consecutive aspartic acid residues within the Flag-tag that can affect the migration of GrblO-Flag during lEP.

Attempts to demonstrate the presence of Raf-1 by Western blotting of 2-D gels failed, likely due to an insufficiently low concentration following co-immunoprecipitation to be detected. Raf-1 has several identified phosphorylation sites (Morrison, et al., 1993) and therefore may focus to severallow concentration spots on 2-D gels.

To visualize all co-immunoprecipitated proteins resolved by 2-D gels, silver staining was performed (see Figure 12). Spots appearing on both control and GrblO-Flag gels indicate non-specifie immunoprecipitated proteins. The large number of these spots prevents the identification of Grb10-Flag. A number of spots were selected from the Grb10-Flag gel that did not appear on the control gel. These spots are candidates for Grb10 associating proteins.

44 4. DISCUSSION

4.1 Challenges faced in this study

Molecular studies of Grb10 are challenging due to severallimitations. Grb10 truncation mutants are difficult to clone, as seen in this study. This may explain why progress in characterizing the function of this protein has been slow. The use of point mutants and small deletions has therefore become very important to studies of this kind. A second problem with this protein is the difficulty obtaining new results from yeast two-hybrid assays. While non-receptor type binding partners of GrblO have been identified using this and other techniques (see Section 1.2.2), only a few have been demonstrated by immunoprecipitation of endogenous protein. It is highly likely that the numerous domains of Grb10 are involved in binding to more effector molecules than already known. For this reason, studies that approach the identification of binding partners of GrblOin new ways

are very important. The use of a proteomic approach in this study helps to attack the problem ofpartner identification in a new and potentially powerful way. A third problem posed by the study of GrblO is its mislocalization upon overexpression. Nantel, et al.

(1999) demonstrated that overexpression of GrblOin HeLa and Cos-1 cells results in an

abnormally high level of GrblO in the cytoplasm. This fact explains why previous localization studies incorrectly identified Grb10 as a mainly cytopHismic protein and

failed to notice a pool present at the mitochondria (Dong, et al., 1997; Frantz, et al.,

1997). It has also been recently shown that approximately 10% of cellular GrblO is

present within the nucleus (A. Nantel, personal communication) although the function of

45 this nuc1ear pool is currently unknown. The development of an inducible promoter system for lower levels of Grb10 expression has been accomplished (A. Nantel, personal communication) but was not ideal for use in this study because maximized expression leve1s were important for the optimization of 2-D separation of immunoprecipitated cellular fractions. Strong overexpression using the SV40 promoter was used but cell extracts were fractionated to enrich for mitochondrial content. By this technique, co­ immunoprecipitation of Grb10 binding partners is limited to those found in mitochondrial fractions, which is the cellular location of interest in this study. It is acknowledged that the use of overexpressed GrblO in this study may have an effect on signaling pathways within the cell but any candidate binding partners identified by this technique would be tested for Grb10 in vivo immunoprecipitation using an antibody against a tagged form of the candidate. This wou1d ensure that the protein identified in fact binds GrblO in vivo.

4.2 Mitochondrial membrane binding

IGF-I stimulated GrblO translocation was previously demonstrated by Nantel, et aL

(1998). This study elaborates on their results by investigating the pattern and extent of translocation. Immunofluorescence microscopy shows the pattern of translocation, demonstrating a rapid re-Iocalization of Grb10 from the mitochondria to the plasma membrane and back. In Cos-7 cells, this translocation is completed within 20 minutes. If this translocation indicates the recruitment of Grb10 to the activated IGF-IR, as was suggested by Nantel, et al. (1999), then the pattern observed implies that a very rapid cellular survival response exists. Counts of cells with translocated GrblO reveal a

46 heterogeneous response to survival signaling. The extent of GrblO translocation was limited to approximately 55% of cells counted. It is known that the IGF-IR signaIs in more than one way, depending on intracellular levels ofIRS-1 and Shc (see Section

1.1.4). It is therefore hypothesized that the variable responses to IGF-IR observed by microscopy are a result of different IRS-l and Shc levels in each cell.

Assays used implicate the Grbl0 arnino-terminal region, excluding the PR domain, in mitochondrial membrane binding. GrblO, when expressed in cells in vivo, is differentially spliced to give many variants and the amino-terminal region is the most variable (see www.cbr.nrc.ca/thomaslab/grb7.html for a review of GrblO splice variants). The differential expression and varying cellular functions of GrblO have already been partially examined (Blagitko, et al., 2000) but the extent of this variation is not well understood. It can be hypothesized that the identified mitochondrial membrane­

associating region may be involved in variations in Grb10 function based on isoform differences.

How GrblO associates with the rnitochondria is still unknown. Nantel, et al. (1999)

suggest that peripheral binding is more probable than insertion into the mitochondrial membrane, because overexpression mislocalizes GrblOto the cytoplasm and because use

of Raf-l as a protein anchor is stoichiometrically not possible. In addition, lack of ATP requirements for binding favours the theory of peripheral association with the

mitochondrial membrane over involvement of active insertion of GrblO into the

membrane. The PH domain was initially a strong candidate for membrane association, as

PH domains have previously been described as lipid interacting sequences (Shaw, 1996)

47 but this now appears unlikely. In in vitro binding assays, the association of Grb10 variants with the mitochondrial membrane through oligomerization with wild type Grb10 is excluded due to the positive binding of the GrblO-21l~ mutant. Oligomerization of

GrblO has previously been determined to occur through the SH2, BPS, and PH domains

(Dong, et al., 1998), but the GrblO-211~ mutant lacks aIl three of these domains and is still found in the mitochondrial pellet. It is possible that Grb10 interacts directly with the mitochondrial membrane by an unknown mechanism, as there are no known proteins that associate with the implicàted GrblO amino-terminal region. Further studies to determine the mechanism of Grbl0 association with the mitochondrial membrane and to identify other mitochondrial proteins associating with Grb10 are important to determine how

Grbl0 binds the mitochondrial membrane. A recent candidate for mitochondrial membrane targeting of Grb10 is the E3 ubiquitin ligase, Nedd4. A translocation of Nedd4 with Raf-l from the plasma membrane to the mitochondria has been demonstrated following IGF-I stimulation (Peruzzi, et al., 2001). As Nedd4 has been demonstrated to bind GrblO in vivo (Morrione, et al., 1999), this protein could be involved in targeting of

Grbl0 from the plasma membrane to the mitochondria following IGF-I treatment.

However, Peruzzi, et al. (2001) studied Nedd4 translocation after 35 hours of IGF-I treatment, which is not easily compared to the Grb10 translocation described after 5 minutes of treatment. In addition, binding of GrblOto Nedd4 has 1;>een demonstrated to involve a region containing the SH2 and BPS domains (Morrione, et al., 1999), which are both truncated from the GrblO-413~ and Grbl0-211~ mutants found to bind mitochondrial membranes in this study. It is therefore unlikely that Nedd4 is involved in

48

fil GrblO mîtochondrial membrane association but might instead play a role in anti­ apoptotic targeting of BAD protein for degradation.

A mode! for GrblO signaling proposed by Nantel, et al. (1999) suggests that GrblO plays an important role in anti-apoptotic signaling from the IGF-IR. By this model, the activation of the IGF-IR by IGF-I signaling results in the recruitment of GrblO to the plasma membrane from the mitochondrial membrane. GrblO then binds MEKI and Raf-l and retums to the mitochondrial membrane. From the results in this study it can be further hypothesized that this translocation is rapid (completed by 20 minutes after the application of IGF-I) but only occurs in a subset of the cellular population (likely due to variances in IRS-l and Shc levels). To extend this hypothesis, GrblO-Raf-1 complexes found at the mitochondrial membrane may serve to recruit Bc1-2, which then phosphorylates Bad protein also found on the mitochondrial membrane. As previously discussed, the phosphorylation of Bad results in sequestering by 14-3-3 protein and the inhibition of apoptosis (see Section 1.1.4). The amino-termînal region of GrblO is implicated in mitochondrial membrane binding in this study. Considering there are no known proteins that bind to this region, it is also hypothesized that the binding of Grb10 to the mitochondrial membrane occurs in an ATP-independent manner through a direct interaction or an unidentified binding partner. Two important experiments follow, in addition to GrblO binding partner identification, to test this hypothesis. The production of amino-terminal deletions would be useful to confirm that this region is responsible for mitochondrial membrane targeting by applying them to the in vitro and in vivo binding assays used in this study. Ifthis is the case, the same mutants could also be used to

49 examine the effect of loss of mitochondrial membrane binding of Grb10 on cellular signaling using transfection studies. Unfortunately, in this study and aIl others published on Grb10 to date, no amino-tenninal deletions have been produced. This may be due to some toxic effect on E. coli during cloning, which is currently not understood but may in part be due to protein sequestering and interference with the transcription mechanism. A second important experiment would test in vitro Grb10 binding to liposomes containing lipids found in mitochondria. This may allow the determination that Grb10 binds directly to the mitochondrial membrane without the use of a protein anchor.

4.3 Identificationof GrblO binding partners

Grb10 has previously been shown to associate with the mitochondrial Raf-1 pool (Nantel, et al., 1999). Gel filtration studies support this by demonstrating the presence ofboth proteins in the same eluted fractions, which correspond to the predicted molecular weight of the GrblO-Raf-1 complex. The binding of each GrblO and Raf-1 with other proteins yielding complexes of this same molecular weight cannot be excluded but is unlikely.

The presence of higher molecular weight complexes in cells treated with serum, IGF-I or

UV to induce IGF-IR signaling suggests that activation of Raf-1 favours binding with

Grb10. As serum treatment of cells yields high molecular weight complexes and high concentrations of overall protein, this condition is the best for identifying Grb10 binding partners. For instance, UV treated cells also yield high molecular weight complexes but eventual induction of apoptosis results in too much cell death to yield sufficiently high protein concentrations.

50 Attempts to visualize co-immunoprecipitated Raf-l from serum treated cells overexpressing GrblO-Flag by 2-D electrophoresis unfortunately failed. The Flag-binding resin used was only efficient enough to co-immunoprecipitate Raf-l to levels detectable on SOS-PAGE. The lack of ability to visualize GrblO-Flag on silver stained gels, likely due to insufficient concentrations of this overexpressed protein, prevented the identification of Grbl0 binding partner candidates. The requirement of large amounts of protein for 2-D electrophoresis makes this a difficult technique to perform. The application of this technique using higher protein concentrations may lead to success. It would also be useful to test the silver staining efficiency of bacterially expressed Grb10­

Flag on 2-D migrations, as it is known that sorne proteins do not stain well by this method. However, the use of a different immunoprecipitation system may be more useful due to the large number of non-specifie immunoprecipitates seen in silver stains. These non-specifie proteins indicate a lack of efficient immunoprecipitation and the selection of a new system with higher antibody specificity, such as mye tagging, is suggested. Given the ability to reproduce candidate spots identified in Figure 12 or new candidate spots for

Grb10 binding partners, spots could be eut and sequenced by mass spectrometry for identification and tested for the ability to immunoprecipitate Grbl0 at endogenous levels.

3.4 Proposed further work

In addition to the use of a new immunoprecipitation system for the identification of

Grb10 binding partners, other types of experiments would be interesting to examine the effects of produced Grb10 variants on apoptotic and mitogenic signaling. Conflicting

51 results exist conceming the function of GrblO. For instance, both stimulatory and inhibitory roles have been proposed for GrblO in mitogenesis (see Section 1.2.3). As most groups studying Grb10 rely on a single splice variant, of which there are at least seven, conflicting results are not surprising. To further examine the role of the amino­ terminal region of GrblO, the function of GrblO isoforms, and to clarify sorne of the existing conflicts in results, research that involves comparisons of splice variants in the same system is necessary. Cloning of human isoforms from multiple cDNA libraries, cellular transfection, and in vivo binding assays may provide new information conceming the mitochondrial membrane binding of these isoforms. Assays for apoptosis and mitogenesis of the same transfected cells should also provide usefuI information on

Grb10 function.

The very recent production of GrblO knockout mice has been accomplished (c. Stewart, personal communication) and provides an exciting opportunity to study the effects of lacking Grb10 or expressing Grb10 mutants on a null background. Expression of the mutants used in the in vitro binding assays in knockout cells would permit the examination of in vivo mitochondrial membrane binding and the effects of these mutants on apoptosis and mitogenesis, without interference from wild type signaling.

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65 i) extrinsie Iii) antl-apoptotlc slgnaling Fas ligand slgnaling

FADO proeaspase-8 /'\. She IRS-1 BI. , :.fl!h--ll 0 1 1. easpase-8 c::::::> ---...., l 1 ~~aved Ras'" PI3-K ! a J- -+0 l o - o Raf·1 Akt proeu;u.~ J1 '"r·~ ! MEK1

?/othercaspases ~ ERK1 Apaf-1 tr1 ,IAPs~ easpase-9V' \ Smae/DIABLO l . 1. p90 RSK APopt:some cytoehrome c Bel.XL Grb10 AIF Raf-1 14-3-3

B~B".2 :...------.Ph::!!.Jted ii) intrinsie t BAD signaling p53 .."A DNAdamage Figure 1. Pro- and anti-apoptotic signaling pathways. Pro-apoptotic signaling occurs through two major pathways. Extrinsic signaling (i) originates from Fas ligand binding to the Fas receptor and initiates the death receptor pathway. A caspase signaling cascade initiates relocalization of Bid to the mitochondria. Intrinsic signaling (ii) is initiated by DNA damage and activated p53 leads to the targeting of Bax to the rnitochondria. Both extrinsic and intrinsic pathways induce the release of cytochrome c, AIF, and Smac/DIABLü from the mitochondria. A weIl known anti-apoptotic signaling pathway (iii) occurs via the IGF-IR. Shc and IRS-1 can both be activated by IGF-I stimulated receptor binding. Multiple downstream effectors result in the phosphorylation of BAD, which is sequestered by 14-3-3 protein. GrblO and Raf-1 are bound at the mitochondrial membrane and may modulate the phosphorylation and activation of Bad. Starved IGF-1, 2 min IGF-1, 5 min IGF-1, 20 min

Figure 2. Immunolocalization of endogenous GrblO in IGF-I stimulated Cos­ 7 cells. Cells were starved or treated with lOOng/ml IGF-l for either 2,5, or 20 minutes. Localization was performed using an affinity-purified anti-GrblO antibody. Images represent typicallocalization patterns for each sampling tîme. Mitochondria appear as a mesh-like pattern throughout the cytoplasm and surrounding the nucleus. High background from nuclei results in sorne overexposure of this region (especially at 5 minutes).

I.no IGF-1.IGF-1, 2 minDIGF-1, 5 min .IGF-1, 20 mini GO

~ ...... 40 -1------al ë ::1 8 30 -/------­ !!J. fl iU 20 -/------..o 10 -/------

o -t------.l---- membrane localization

Figure 3. Percent ofIGF-l stimulated HeLa cells with membrane localized GrblO. Cells were starved and treated with lOüng/ml IGF-l for either 0,2,5, or 20 minutes (n=lOO). Localization was performed using an affinity-purified anti­ GrblO antibody. Cells exhibiting membrane localized GrblO were scored against those that did not. PR RA PH BPS SH2

413 Grb10-4134

211 Grb10-2114

Grb10-RL

Grb104PR v

Figure 4. Structure of GrblO and bacterially expressed mutants. Pive domains have been described in GrblO; the amino-terminal proline-rich region (PR), the Ras-associating (RA)-like domain, the central pleckstrin homology (PH) domain, the carboxyl-terminal Src-homology type 2 (SH2) domain, and the BPS (between PH and SH2) domain. Indicated amino acid ranges are for the longest human splice variant hGrblŒ;, which is the fuIllength GrblO used in aIl experiments. A 49 amino acid overlap exists between the RA and PH domains. The bacterially expressed mutants used in in vitro binding assays are represented here; A) GrblO-413~lacks the BPS and SH2 domains, B) GrblO-211~ contains only the PR and flanking sequence, C) GrblO-RL contains an arginine to leucine point mutation in the SH2 domain, and D) GrblO~PR lacks the PR domain. o o i) RT :!: -'Ë E u 0 U 0 ..JU)a.c ..JU)a.c

MBP-LaeZ

MBP-Grb10

MBP-Grb10-4136

MBP-Grb10-2116

MBP-Grb10-RL

MBP-Grb10-6PR

mita anly

Figure 5. GrblO binds the mitochondrial membrane in vitro. Mitochondrial membrane binding of bacterially expressed MBP fused to fulliength GrblO and LacZ (as a control) were compared to four mutants: GrblO-413~, GrblO­ 211~, GrblO-RL, and GrblO~PR. Mitochondria were incubated with these proteins in isotonie buffer with 1% BSA (to prevent aggregation), pelleted, washed, and lysed. Anti-MBP Western blots were prepared with a load control (Le) showing the total protein used in the assay, an unbound fraction control (S) showing the amount of protein remaining in the supernatant, and a Bound Fraction (P) showing the amount of protein in the mitochondrial pellet. To control against protein aggregation all sampIes were pelleted after incubating in buffer without mitochondria (no mito), To control against mitochondrial cross-reactivity with the anti-MBP antibody mitochondria were pelleted after incubating alone (mito only). The same assay was incubated at room temperature (RT) (i) and 4°e (ii), each for 1 hour. Sand P band intensities are as intense as and not equal to the Le fraction intensities likely due to the difficulty of pipeting small volumes. Total Mito extract extract CG RL PR CG RL PR

Grb10

cyt c

Figure 6. Transfected GrblO binds mitochondria in vivo. Cos-l cells were transfected with one offour vectors encoding overexpressed Flag­ tagged fusions of either GrblO (G), GrblO-RL (RL), Grb108PR (PR), or the empty vector pcDNA3 (C) as a control. Cells were then grown over a period of48 hours, harvested, and subjected to mitochondrial extraction by differential centrifugation. Total cell extracts and mitochondrial extracts were tested for GrblO and cytochrome c (as a control for the amount of protein loaded) by Western blots. Cytosolic fractions (not shown here) rendered no cytochrome c or GrblO signal. -~~-----'~i--'---'------~------'-r------' 1.4 ! : --- B5A :: - ovalbumin 1.2 : i ---IQG : ,.CytC 1 1 ~ 1l '1 .3 1 1l '1 "o 1 1 1l '1 ~ 1 1 0.8 1 1 Il / 1 /l ' 1 1 1 80.6" / 1 1 1 1 1 ~" l '\ li 0.4 / t \._ 1 1 0.2 ) r, /---", ",,/ ...... --./ ,_ ...... - ...... "'-'" \ ..../ o ----- ..." ._~ .....,...--'--<' o 20 40 60 80 100 120 140 160 elUllon volume (mil

Figure 7. Elution of gel filtration column standards. Proteins standards were resolved using a Sephacryl S-3ÜO gel filtration column and assayed for total protein concentration to estimate the molecular weight range for each fraction. Unexpectedly, ovalbumin (45kD) and BSA (66kD) both eluted before the 70ml peak of rat IgG (l50kD), possibly due to agglutination. Cytochrome c (12.3kD) peaked very late, at 134ml, as expected.

0.7,------,

--- serum 06 -starved ······IGF-I _··-W '" 0.5 ~ a 0.4 ! E ~ 03 u ~" i(., \ 02 ... ,.~--. Q. 1 :! \ './

0.1 j ....:/ \ .. _--~,.,_/~\ /- l '-_.-." ...... -....~- .....~~-. --~/I • • ...... -'_._._._...... ~ O+-"----"""'---,=::::.::.:.-"--r---~---~---~--~ o 20 40 60 80 tOO 120 olutlon yolwno (ml)

Figure 8. Protein profiles of column runs. Eluted proteins from the cell runs shown in figure 6 were assayed for total protein concentration. IGF-l and UV treated cells have comparable profiles. Serum, UV, and IGF-I treated cells aIl exhibit major peaks at approximately the same high molecular weight fraction (approximately 44mI). elution volume (ml)

2 6 10 14 18 22 26 30 34 38 42 46 50 54 58 62 66 70 74

Grb10 starved

Raf-1

Grb10 serum treated Raf-1

uv Grb10

IGF-1 Grb10

Figure 9. GrblO and Raf-l co-elute from a gel filtration column. HeLaS3 cells were harvested and mitochondria were extracted by differential centrifugation. Extract was then run through a Sephacryl S-3OO gel filtration system that eluted endogenous Grb10 and Raf-1 in many of the same fractions, as seen by Western blot. Cells were either serum starved, serum treated, UV treated (100 J/m2), or IGF-I treated (loong/ml). Raf-1 was not detectable from UV and IGF-I treated cells, as total protein levels were low. Earlier elution (smaller volume) is indicative of higher molecular weight complexes. T M ip CG CG CG Grb10

Raf-1

Figure 10. Raf-l is co-immunoprecipitated by GrblO-Flag. A GrblO Western blot of extracts from GrblO-Flag overexpressing Cos-7 shows that GrblO is present in total (T), mitochondrial (M) and immunoprecipitated (ip) extracts. A Raf-l Western blot ofthe same extracts demonstrates that Raf-l is immunoprecipitated in GrblO-Flag (G) transfected cells but not in cells transfected with the empty vector (C). i) control

83-

kD 62-

48-

ii) Grb10-Flag

83-

kD 62-

48-

3 4 5 6 7 8 9 10 pH

Figure 11. Immunoprecipitated GrblO is resolved by 2-D electrophoresis. Grb10 Westerns of co-immunoprecipitated Cos-7 mitochondrial extracts resolved by 2-D electrophoresis. Cells transfected with the pAN185 vector (H) demonstrate the presence of GrblO-Aag, which is absent in cells transfected with empty vector (i). Arrow indicates GrblO-Aag. i) control

kD

ii) Grb10-Flag 175-

83-

62-

48-

kD

33-

25-

16-

pH

Figure 12. Silver staining of 2-D separations reveals a high number of immunoprecipitated species. Sïlver staining of co-immunoprecipitations of mitochondrial extracts from pcDNA3 (i) and pAN185 (ii) transfeeted eells resolved by 2-D eleetrophoresis shows a high number of non-specifie proteins are immunoprecipitated (spots present in both). Arrows indieate spots found in gels from pAN185 transfeeted cells but not in gels from peDNA3 transfeeted cells.