RHO G PROTEINS AND FILAMIN C IN INSULIN ACTION IN MUSCLE CELLS
By
Alexandra Koshkina
A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology University of Toronto
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Alexandra Koshkina Master of Science
Graduate Department of Physiology University of Toronto
2008 Abstract
Insulin-induced GLUT4 translocation in muscle cells involves the activation of
Akt and Racl-mediated actin remodelling, both downstream of PI3K. Our study was aimed at establishing the nature of the relationship between these two signalling arms.
The first aim was to test the existence of crosstalk between Akt and Racl, in an acute fashion. Our results confirm that Akt does not regulate Racl activation or actin remodelling. Interestingly, we observed an Akt-dependent activation of Cdc42 in response to insulin. The second aim was to analyze the function of a cytoskeletal protein,
FLNc, as a "molecular bridge" between the Akt and Racl signalling cascades. The results of this thesis establish that FLNc is an in vivo substrate of Akt. However, we failed to see any functional significance of FLNc in insulin action. Our present study offers further support to the theory of bifurcation of PI3K signals into Akt and Racl-dependent actin remodelling.
ii Acknowledgements
First of all, I would like to thank my MSc supervisor and my mentor Dr. Amira Klip.
Without her constant believe, support, kindness and guidance I would not be here today.
She accepted me into her laboratory without extensive biochemistry and cell biology experience and gave me the opportunity to learn many new and exciting aspects of science. I would also like to thank the members of my graduate committee, Dr. Denise
Belsham and Dr. Tony Lam. I want to thank them for always challenging me, for their scientific advice and for their kind support throughout this journey. Of course none of this could be accomplished without the help and constant support of the Klip lab members past and present. I want to thank our Research Associate, Dr. Phil Bilan for his technical support and advice in the lab. I want to offer a special thanks to Dr. Victor
Samokhvalov, Dr. liana Talior and Dr. Varinder Randhawa for their friendship and valuable scientific discussions over Starbucks coffee. I would also like to thank Dr.
Costin Antonescu, Dr. Wenyan Niu, Dr. Hilal Zaid, Dr. Shuhei Ishikura, Dr. Jonathan
Schertzer, Dr. Lellean JeBailey, Amanda Tung, Dr. Farah Thong, Kevin Foley, Tim Chu,
Dr. Constantine Samaan, Ariel Contreras and Aida Lieva Gonzalez for their help and for making lab work, fun. Lastly, I want to thank my family (Irene, Vadim, Maria, Lydia,
Mikhael and Paul) for always being t)iere for me and for encouraging me every step of the way. Finally, I want to thank my husband, who patiently listened to me talk about all that "weird science stuff for many years and whose advice, love and support I could not do without.
Hi The work presented in this M. Sc. Thesis was performed from 2006-2008 in Programme in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada, under the supervision of Dr. Amira Klip. Financial stipend was provided by Dr. Amira Klip and
Restracomp (Research Institute, Hospital for Sick Children).
iv Table of Content
Abstract ii Preface iii Acknowledgements iv Table of Content v List of Figures vii Chapter 1: Introduction 1 1.1 Normal Physiology and Regulation of Glucose Homeostasis 2 1.1.1 Glucose Transporter Isoform 4 3 1.2 Insulin Resistance and Type II diabetes mellitus 4 1.3 Insulin Signalling 5 1.4 Insulin-Stimulated Actin Remodelling 7 1.4.1 Mechanism of actin remodelling 9 1.4.2 Actin binding proteins (ABPs) 10 1.5 Rho family of G proteins 10 1.5.1 Racl, Cdc42, Rho A, TC10 12 1.5.2 WASP/WAVE family 13 1.5.3 PAK (1-3) and ROCK 13 1.5.4 Rho G proteins in insulin-stimulated GLUT4 translocation 14 1.5.4.1 Actin remodelling and Rho G proteins in GLUT4 traffic in 3T3L1 adipocytes 14 1.5.4.1.a. TC10 14 1.5.4.1. b.Cdc42 15 1.5.4.1.C Racl 16 1.5.4.2 Rho family of small G proteins in insulin-stimulated actin remodelling in muscle cells 17 1.5.4.2 a. TC10 17 1.5.4.2 b. Racl 17 1.6 FLN, a GLUM interacting protein 21 1.6.1 Filamin Structure and Tissue Expression 21 1.6.2 Filamin function in health and disease 23 1.6.2.1 Filamin in actin remodelling 24 1.6.2.2 Filamin acts as a scaffold 26 1.6.2.3 Filamins and Insulin Signalling 28 1.7 Rationale and Hypothesis 31 Chapter 2: Materials and Methods 34 2.1 Tissue culture 35 2.2 Cell treatments 35 2.3 siRNA transfection 36 2.4 Preparation of whole celllysates 36 2.5 Immunoblotting 37 2.6 Antibodies 38 2.7 Immunodetection of GLUT4myc at cell surface 38 2.8 GST-protein preparation and GTPase assay 39
v 2.9 Fluorescence Microscopy 40 2.9.1 Confocal Microscopy 41 2.10 Statistical Analysis 41 Chapter 3: Crosstalk between Akt and Rho GTPase signalling 42 3.1 Summary 43 3.2 Results 44 3.2.1 Effect of Aktl/2 inhibition on Racl-mediated actin remodelling in response to insulin in L6 cells 44 3.2.2 Activation state of Cdc42 in L6GLUT4myc cells 49 3.2.3 Effect of Aktl and Akt2 inhibition on Cdc42 activation 51 3.3 Discussion 53 3.3.1 Insulin signals bifurcate downstream of PI3K into two arms, Akt-dependent signalling and Racl-mediated actin remodelling 53 3.3.2 Inhibition of Akt phosphorylation did not inhibit insulin-induced GLUT4 translocation 55 3.3.3 Cdc42 is activated in response to insulin in an Akt-dependent manner 57 3.4 Future Directions 61 3.4.1 Independence of Rac and Akt signalling 61 3.4.2 Temporal and spatial activation of Rac and Cdc42 61 3.4.3 Cdc42 contribution to the insulin-mediated GLUT4 translocation 62 Chapter 4: FLNc in insulin signalling 63 4.1 Summary 64 4.2 Results 65 4.2.1 Filamin C in L6 cells 65 4.2.2 Filamin C phosphorylation 66 4.2.3 Filamin C function in insulin signalling 71 4.3 Discussion 79 4.3.1 Filamin C is phosphorylated by Akt in vivo 79 4.3.2 Filamin C in insulin-mediated GLUT4 translocation 80 4.4 Future directions 82 4.4.1 Expression of FLNc dominant-interfering mutant 82 4.4.2 Expression of FLNc phosphorylation mutants 82 Chapter 5: Conclusion 84 Chapter 6: References 84
vi List of Figures
Figure 1.1: Insulin Signalling Pathway in Muscle Cells 7
Figure 1.2: Insulin-Induced Actin Remodelling in L6 Myoblasts 9
Figure 1.3: Regulation of Rho G Proteins 11
Figure 1.4: Signalling Downstream of the Rho G Proteins 13
Figure 1.5: Insulin-Induced Racl Activation in L6 Myoblasts 18
Figure 1.6: Filamin Structure 22
Figure 1.7: Filamin is a Multifuncional Protein 25
Figure 1.8: Objectives of this thesis 33
Figure 3.1: Akti 1/2 inhibits insulin-induced Akt phosphorylation 44
Figure 3.2: Akti 1/2 does not affect insulin-induced actin remodelling 46
Figure 3.3: Akti 1/2 does not affect insulin-induced Racl activation 48
Figure 3.4: Akti 1/2 does not inhibit insulin-induced GLUT4 translocation 49
Figure 3.5: Cdc42 is activated in response to insulin in L6 myoblasts 50
Figure 3.6: Cdc42 activation in response to insulin is inhibited by Aktil/2 52
Figure 4.1: FLNc is expressed in L6 myoblasts and myotubes 65
Figure 4.2: FLNc is phosphorylated in response to insulin, but not in response to other stimuli that induce GLUT4 translocation 67
Figure 4.3: Phosphorylated FLNc localizes to the insulin-induced membrane ruffles with GLUT4 and F-actin 68
Figure 4.4: FLNc phosphorylation is inhibited by PI3K inhibitors LY294002 and wortmannin 69
Figure 4.5: FLNc phosphorylation is inhibited by Akti 1/2 70
Figure 4.6: FLNc expression is decreased using siRNA 71
vii Figure 4.7: FLNc siRNA does not affect insulin-induced Akt, ERK 1/2 or JNK phosphorylation 73
Figure 4.8: FLNc siRNA does not affect insulin-induced Racl activation 75
Figure 4.9: FLNc knockdown does not affect insulin-induced actin remodelling 76
Figure 4.10: FLNc knockdown does not affect insulin-stimulated GLUT4 translocation 78
viii List of Abbreviations
ABPs Actin binding proteins ANOVA Analysis of variance AS 160 Akt substrat of 160 kDa ATP Adenosine triphosphate AMP Adenosine monophosphate AMPK 5'-AMP-activated protein kinase Akti 1/2 Akt isoform 1 and 2 inhibitor Arp2/3 Actin-related protein 2/3 complex BSA Bovine serum albumin Ca2+ Calcium ion CA Constituitively active cDNA Complementary DNA CRIB Cdc42/Rac-interactive binding domain DMSO Dimethyl sulfoxide DN Dominant negative DNP 2,4,-Dinitrophenol ECL Enhanced chemoluminescence EDTA Ethelyenediamine tetraacetic acid FBS Fetal bovine serum FLNa Filamin A FLNc Filamin C GAP GTPase activating protein GEF Guanine nucleotide exchange factor GDI GTPase dissosciation inhibitor GDP Guanosine diphosphate GTP Guanosine triphosphate GLUTs Glucose Transporters GUJT4myc wye-tagged glucose transporter-4 HRP Horseradish peroxidise IgG Immunoglobulin IRS Insulin receptor substrate K+ Potassium ion kDa KiloDalton uM Micromole/litre mg Milligram min Minute mL Millilitre Na+ Sodium ion NaF Sodium fluoride Na3V04 Sodium orthovanadate nM Nanomole/Litre NP-40 Nonidet P-40 OPD O-phenylenediamine dihydrochloride PAK p-21 activated kinase
IX PBS Phosphate-buffered saline PDK Phosphoinositide-dependent kinase PFA Paraformaldehyde PI3K Phosphoinositide 3-kinase PIP2 Phosphatidylinositol (4,5) bisphosphate PIP3 Phosphatidylinositol (3,4,5) trisphosphate PKC Protein kinase C PM Plasma membrane PMSF Phenylmethylsulfonyl fluoride PVDF Polyvinylidene fluoride ROCK Rho kinase SDS-PAGE Sodium dodecylsulfate-polyacrylamide gel electrophoresis siRNA Small interfering RNA SH2 Src homology 2 T2DM Type 2 diabetes mellitus TGN trans-Golgi network WASP Wiskott Aldrich syndrome protein WAVE WASP family verproline homologous WT Wild type
x Chapter 1: Introduction
1 2
1.1 Normal Physiology and Regulation of Glucose Homeostasis
The maintenance of homeostasis depends on the strict regulation of many parameters within the body. Glucose, a primary energy source for the brain, is one of the parameters. Glucose homeostasis requires the co-operativity of multiple organs in the body, including the pancreas, liver, brain, adipose and importantly skeletal muscle. A failure or a dysfunction in one or more of these key organs will inevitably lead to a dysregulation of glucose homeostasis, which soon develops into hyperglycemia, insulin resistance and potentially type 2 diabetes mellitus with its variety of side effects.
As a meal is ingested, the absorption of carbohydrate from the intestine leads to an increase in the overall blood glucose concentration. Glucose enters into the pancreatic
(3 cells by facilitative diffusion via a glucose transporter isoform 2 (GLUT2). Once inside, glucose is metabolized into pyruvate, which is shuttled into the mitochondrion for further metabolism (171). As a result, the overall ATP to ADP ratio within the cell increases, leading to the closure of ATP-sensitive K+ channels and subsequent 3 cell depolarization.
Voltage-gated Ca2+ channels open following depolarization to allow for the exocytosis of insulin-granules (171).
An increase in plasma insulin levels leads to a number of physiological events. In the liver, insulin inhibits glucose production by inhibiting both glycongenolysis and gluconeogenesis. Glycogenosis is in part inhibited by the activation of glycogen synthase that promotes glycogen synthesis from glucose. Inhibition of gluconeogenesis is accomplished by the suppression of key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphotase (7). Other than affecting the liver, insulin is also important for the central control of food intake and glucose production 3
(137). A blockade of insulin action in the hypothalamus of normal rats causes hepatic insulin resistance, increased hepatic gluconeogenesis, and increased food intake—giving support to the importance of central insulin action in the control of glucose homeostasis
(116,117).
Two other insulin sensitive tissues are skeletal muscle and adipose tissue. Insulin leads to a variety of physiological and biochemical events in these tissues, most notably an increase in glucose uptake. Furthermore, skeletal muscles are responsible for approximately 80 % of the bodies' total glucose disposal after a meal (32). The insulin- stimulated increase in glucose flux is accomplished by an increase in the cell surface expression of glucose transporter isoform 4 (GLUT4) (95, 103).
1.1.1 Glucose Transporter Isoform 4
GLUT4 is a member of the SLC2 facilitative sugar transporter family (80, 173).
Of the 13 members, GLUT4 is the insulin-responsive transporter that is expressed predominantly in the muscle and adipose tissues (152). In the basal state, GLUT4 is mostly cytosolic; insulin stimulation enhances plasma membrane (PM) GLUT4 and subsequently glucose uptake into cells (70). The steady-state distribution of GLUT4 in the basal state is maintained by fast endocytosis and slow exocytosis. Insulin shifts the steady-state distribution of GLUT4 favouring the PM by largely elevating the transporter exocytic rate, a process known as GLUT4 translocation. GLUT4 resides in multiple internal compartments that include the trans-Golgi network, the endocytic recycling compartment (ERC), and a compartment termed the 'GLUT4 specialized compartment' 4
(SC) or 'GLUT4 storage vesicle' (GSV), which is segregated from the ERC and the TGN
(99,175).
1.2 Insulin Resistance and Type II diabetes mellitus
All of the above-described biological systems must work in concert with each other in order to maintain glucose homeostasis and/or health. In recent years, there has been a significant increase in obesity, insulin resistance and type 2 diabetes mellitus
(T2DM) throughout the world. In the US alone, there are approximately 2 to 4 million new diabetics every year (96). In Canada, there are around 60,000 new diabetic cases yearly with costs estimated at 9 billion dollars per year (Health Canada). T2DM can be characterized by a P cell dysfunction, which manifests itself as impaired insulin secretion, along with insulin resistance seen as increased hepatic gluconeogenesis and a defect in insulin-regulated plasma glucose clearance by the skeletal muscles (31). Insulin resistance precedes frank T2DM, a condition where P cells are capable of compensating for insulin resistance initially by elevating the plasma insulin levels (31).
T2DM is a disease that results from a combination of genetic and environmental factors. The genetic predisposition has primarily been demonstrated by the study of identical twins that had an almost 100% concordance rate for T2DM (6). In terms of environmental factors, many studies have shown a strong association of lack of exercise, high calorie diet and obesity with the development of T2DM (96). Extensive scientific effort has focused on determining the underlying cause(s) of this pathology. However, this disease is a multi-organ dysfunction, making it very difficult to resolve. Nevertheless, 5 different components of the insulin signalling pathway have been implicated for their involvement in insulin resistance.
1.3 Insulin Signalling
Insulin signals can be divided into two broad categories, mitogenic and metabolic.
Mitogenic signals are primarily responsible for regulation of gene transcription. Insulin also functions to increase cell metabolism, and in muscle and adipose tissues to increase glucose uptake. Insulin signals through its receptor (IR), a member of a transmembrane tyrosine kinase receptor family, by inducing a conformational change and IR autophosphorylation (121). Mice lacking the IR gene die shortly after birth from severe ketoacidosis (16). Tyrosine phosphorylated IR recruits downstream signalling molecules that contain a phosphotyrosine binding domain (PTB) and Src homology 2 (SH2) domain, such as insulin receptor substrate (IRS) 1-4 and protein She (4, 152) (Figure 1). The mitogenic effects of insulin are transduced by engaging the Ras-Raf-MEK-MAPK signal cascade (4). In contrast, its metabolic effects are signalled predominantly through the well characterized IR-IRS-PI3K (phosphatidylinositol-3-kinase), and leads to a variety of cellular events (152).
In skeletal muscles, IRS-1 is primarily required for the translocation of insulin-responsive
GLUT4 to the cell surface, thereby increasing glucose flux into muscles (69) (Figure 1.1).
Indeed, IRS-1 knock-out mice exhibit peripheral insulin resistance as well as impaired glucose tolerance (146). Once tyrosine-phosphorylated, IRS binds the SH2 domain of
Class la PI3K. The predominant substrate of the activated PI3K is phosphatidylinositol-
4,5-bisphosphate (PI(4,5)P2), the phosphorylation of which leads to the production and 6
subsequent transient rise in phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) levels
(126,152). Numerous studies using pharmacological inhibitors of PI3K (wortmannin or
LY294002) or PI3K mutants (mutant delta p85 subunit), have highlighted the necessity of PI3K activation for insulin-stimulated translocation of GLUT4 to the PM (26, 87, 90,
147). Downstream of PI3K lies the activation of two serine/threonine kinases, Akt and atypical protein kinase C (aPKC) 7JC,, both of which were shown to be necessary for the gain in surface GLUT4 (47,152,168). Akt isoforms 1 and 2 are activated in response to insulin in muscles and adipose tissues (170). The importance of Akt activation for insulin-stimulated glucose uptake and GLUT4 translocation was established with the use of the constitutively-active (CA) and phosphorylation-deficient mutants of Aktl in L6 myoblasts, siRNA for Aktl and Akt2 in 3T3L1 adipocytes, and more recently a specific allosteric inhibitor of Aktl and Akt2 (60, 88, 168). Mice deficient in Akt2 showed signs of hepatic and skeletal muscle insulin resistance, further emphasizing its physiological importance in the insulin signalling cascade (25). Recently, there has been a significant focus on substrates downstream of Akt activation, specifically on the role of Akt
Substrate of 160 kD (AS 160) in GLUT4 translocation. AS 160 is thought to function as a
Rab-GTPase activating protein (GAP). Akt phosphorylation of AS 160 inhibits its GAP
activity, thus allowing for the activation of its downstream Rab GTPases (73). The
signals above, in concert with insulin-stimulated actin remodelling (see below) are necessary for the metabolic effect of insulin in muscle and adipose tissues, especially the
gain in surface GLUT4 and elevated glucose flux. Mice with a tissue-specific knock
down of GLUT4 in either muscle or fat, exhibited severe insulin resistance and impaired 7 glucose tolerance, emphasizing the importance of this glucose transporter in the regulation of glucose homeostasis (1,178).
f-attm
Figure 1.1: Insulin Signalling Pathway in Muscle Cells. Insulin leads to the activation of metabolic and mitogenic pathway in muscle cells. The metabolic pathway is mainly downstream of the activated PI3K, which leads to the activation of atypical PKC, Akt and Racl-dependent actin remodelling. The combination of these signals leads to the mobilization of GLUT4 from the intracellular compartments to the PM.
1.4 Insulin-Stimulated Actin Remodelling
There is considerable evidence indicating that another important event takes place
downstream of PI3K in muscle and adipose tissues, namely the remodelling of actin
filaments. This process requires the dynamic changing of the actin cytoskeleton, which 8 can be visualized as an actin mesh-like structure beneath the PM in muscle cells. Insulin-
induced actin remodelling in muscle creates membrane ruffles or lamellipodia (Figure
1.2), normally within 3 minutes of insulin stimulation (89). The importance of this phenomena for insulin-stimulated GLUT4 gain has been demonstrated by using pharmacological actin depolymerizing (latrunculin B, cytochalasin D) and stabilizing
(jasplakinolide, swinholide A) agents that prevent actin remodelling, and in turn inhibit insulin-stimulated GLUT4 translocation (89,153,157). In 3T3L1 adipocytes, actin remodelling is also apparent, with intense changes in polymerization both at the cell cortex and within the perinuclear regions of these cells (84, 168).
The exact function of the remodelled actin remains to be elucidated, but at least two scenarios have been suggested. Firstly, remodelled actin might serve as a scaffold that collects signalling molecules to allow for proper signal transduction. This theory is
supported by evidence of co-localization of signalling molecules such as the IRS, PI3K
subunits, Akt with the remodelled actin after insulin treatment in L6 cells (89,151,153).
Secondly, there is evidence that the remodelled actin may serve as tracks for motor proteins that pull the GLUT4 vesicles towards the PM (42). These two functions are not
mutually exclusive, and moreover, it is clear that the induction of insulin resistance in
muscle cell reduces insulin-induced actin remodelling, further emphasizing its
physiological and cellular importance to GLUT4 traffic (76, 153). 9
Figure 1.2: Insulin-induced actin remodelling in L6 myoblasts. Representative image of actin remodelling detected using immunoflourescence in L6 myoblasts. Cells were serum-deprived for 3 hours and then treated with or without 100 nM insulin for 10 min. The samples were fixed and permeabilized as described in Materials and Methods. Actin filaments are stained with rhodamine-phalloidin.
1.4.1 Mechanism of actin remodelling
Actin exists in two states: as monomers (G-actin) or as polymerized filamentous actin (F-actin). Filamentous actin, along with a large variety of the actin binding proteins, constitutes the actin cytoskeleton, which plays many vital roles within a cell (142). The actin cytoskeleton is required for cell motility, cell division, vesicular traffic, transmembrane signalling events as well as cell-cell and cell-substrate interactions (136).
Actin molecules can bind ATP in their monomeric state, and this ATP is hydrolyzed to ADP as monomers polymerize (136). Actin filaments are polarized, such that the rate of polymerization on one (i.e., plus/barbed) end is much more thermodynamically favourable compared to its other (i.e., minus/pointed) end. The rates of filament polymerization and depolymerization, the amount of actin branching, filament stabilization and severing are all organized events, and are directly controlled by a variety 10
of actin binding proteins (ABP). Many of the ABPs are controlled by intracellular factors
like calcium (Ca2+) and phosphoinositides (75).
1.4.2 Actin binding proteins (ABPs)
Several ABPs modulate different stages of the actin polymerization process. The
availability of actin monomers for polymerization is tightly regulated by the first
identified monomer ABP, profilin. In the presence of PI(3,4)P2, profilin releases actin monomers allowing for their addition to existing actin filaments or for de novo actin polymerization (136). In addition to G-actin availability, F-actin polymerization is
controlled by actin capping proteins that bind to the filaments' barbed-ends, thus preventing further polymerization. Gelsolin is an actin severing and capping protein,
which bends and severs actin filaments, afterwards capping them in a Ca2+-dependent
manner (8, 105).
Actin filament crosslinking is another very important process that controls its
organization. Actin filaments can be gathered into bundles, this is accomplished by the
binding of the small globular ABPs (e.g., a-actinins). Filaments can be also gathered into
orthogonal networks and this is accomplished by larger ABPs (e.g., filamins) (54).
1.5 Rho family of G proteins
Small G proteins of the Rho family play a pivotal role in reorganization of the
actin cytoskeleton in response to different stimuli. Rho proteins belong to the Ras
superfamily of small GTPases. The Rho family consists of 22 members in mammals (74).
Similar to other Ras GTPases, these proteins act as molecular switches going from the 11
"ON" or GTP-bound state to the "OFF" or GDP-bound state. Structurally, Rho GTPases contain two segments that are commonly referred to as Switch 1 and Switch 2 domains
(64). Switch 1 and 2 undergo a conformational change after binding a GTP molecule, providing a platform for interaction with downstream effectors (37). Rho GTPases also contain five G domains that are responsible for nucleotide (GDP/GTP) binding and a C- terminal CAAX motif that undergoes posttranslational modifications, most significant of which is isoprenylation of the conserved cysteine residue (64,124). The isoprenylated
CAAX motif allows for Rho GTPases to be targeted and inserted into the PM (33).
Figure 1.3: Regulation of Rho G Proteins
The activity of Rho GTPases is controlled by three types of proteins: GAPs,
Guanine Nucleotide Exchange Factors (GEFs) and Guanine Nucleotide Dissociation
Inhibitors (GDIs) (Figure 1.3). GAPs bind the GTP-bound Rho GTPase and increase its
intrinsic rate of GTP hydrolysis, promoting the GDP-bound inactive state of the protein
(94). GEFs trigger the release of the GDP molecule from the inactive GTPase allowing a
GTP molecule to bind, hence promoting GTPase activation (122). GDIs bind the
prenylated CAAX motif of some Rho family proteins, preventing their interaction with 12 membranes and instead sequestering them in the cytosol. GDIs can also function by preventing GTPase binding to their downstream effectors (33, 108).
1.5.1 Rad, Cdc42, Rho A, TC10
The best-characterized Rho family proteins are Racl, Cdc42 and Rho A.
Although all three are involved in the organization of the actin cytoskeleton, they play different roles in this phenomenon. Racl, a ubiquitously expressed isoform, is mostly present at the PM, where its activation induces lamellipodia formation or membrane ruffling (45). Lamellipodia can be defined as the leading edge of a motile cell; however, once the edge detaches from the substrate reaching vertically, it is referred to as membrane ruffling (2, 93). Furthermore, cells can form circular dorsal ruffles that rise vertically from the dorsal surface of the cell and are observed after treatment with growth factors (eg. platelet-derived growth factor (PDGF)) (93, 107). Racl gene deletion studies,
support the requirement of this GTPase in lamellipodia, membrane ruffling and circular
dorsal ruffle formation (93, 162). Cdc42, also partly localized at the PM, induces
formation of fillopodia that resemble finger-like projections of the PM filled with parallel bundles of actin filaments (93). However, expression of constitutively-active (CA) Cdc42 mutants induced lamellipodia formation, although smaller in size than seen with Rac
expression (115). Rho A GTPase, found both at the PM and within the cytosol, induces
stress fibre formation (45).
Rho GTPases function by regulating the activity of downstream proteins that
directly affect actin remodelling (Figure 1.4). Several of these key downstream effectors
include the Wiskott-Aldrich syndrome protein (WASP) and WASP family verproline- 13 homologous (WAVE) family, p-21 activated kinases (PAKs) and Rho kinases (ROCK).
These proteins transduce the Rho GTPase signals facilitating the remodelling of actin.
Lamellipodia Filopodla Stress fibers
\^yRac/Cdc42 \_y Rho A
\ / \ WASP/WAVE Pak ROCK DRFs \ / LIMK Gelsolin Actin polymerization * Actin I (Parallel fibers) \Capping 1v Arp2/3Compl \ Cofilin—P \j i Actin ^Severing Actin Polymerization (Branched fibers) Actin Remodelling
Figure 1.4: Signalling Downstream of the Rho G Proteins
1.5.2 WASP/WAVE family
The activation of WASP/WAVE proteins by Rho GTPases leads to the activation of the Actin-related protein 2/3 (Arp2/3) complex. This 7 protein complex binds to the side of the actin filament and induces actin branching (132). WASP-dependent Arp 2/3 activation can also direct de novo actin nucleation, otherwise kinetically unfavourable
(109).
1.5.3 PAK (1-3) and ROCK 14
Racl and Cdc42 can activate p-21 activated kinases (PAK) 1-3 by interacting with the Cdc42/Rac-interactive binding (CRIB) domain on PAK proteins (177). This results in the activation of LIM kinase (LIMK), and subsequent inactivation of cofilin, the latter being an actin severing protein (39). RhoA can also activate LIMK, via stimulation of another serine/threonine kinase, namely ROCK (101,144).
The proper remodelling of the actin network first requires the severing of existing actin filaments, followed by a burst of actin polymerization. This is facilitated by the dynamic regulation of the actin severing and polymerizing proteins highlighted above.
1.5.4 Rho G proteins in insulin-stimulated GLUT4 translocation
Insulin-stimulated GLUT4 translocation requires dynamic changes in the actin cytoskeleton that occur downstream of Rho family G proteins in both muscle cells and adipocytes. The specific Rho GTPase facilitating this process, however, is cell-type
specific: Racl and TC10 modulate insulin-responsive actin remodelling in muscle and adipose cells, respectively (76, 85, 89).
1.5.4.1 Actin remodelling and Rho G proteins in GLUT4 traffic in 3T3L1 adipocytes l.SJ.l.a. TC10
The less well-characterized Rho GTPase TC10 elicits general cellular effects on the actin cytoskeleton that can be most closely related to those of Cdc42 (Neudauer,
1998). A few years ago, TC10 was shown to be involved in insulin-stimulated GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, presumably, at least in part, as a 15 regulator of actin dynamics (24, 85). In fact, TC10 is required for insulin-induced actin remodelling in 3T3-L1 adipocytes (24, 84, 85). Moreover, in a semi in vitro system,
TC10 participated in creating comet tails on GLUT4 vesicles (86). In contrast to Racl, which depends on PI3K input, TC10 is activated downstream of the PI3K-independent
Cap/Cbl signalling cascade in response to insulin (24).
1.5.4.1.a.l Downstream of TC10
TC10 has high sequence homology to Cdc42 and Racl and can activate many of the same downstream effectors (83). While the search for the signalling pathway downstream of TCI 0 is still underway, a recent study implicated a member of the WASP family of Rho GTPase effectors, N-WASP, in insulin-stimulated GLUT4 translocation.
Using a yeast two-hybrid screen, TC10 was shown to interact with N-WASP as well as with all three PAK isoforms (113). Over-expression of a dominant-negative (DN) N-
WASP attenuated insulin-stimulated actin polymerization, reduced insulin-induced
GLUT4 traffic as well as interfered with actin polymerization induced by CA TC10 expression, making it a strong candidate as a downstream effector of TC10 (78, 85, 86).
1.5.4.1. b. Cdc42
The role of Cdc42 in insulin-stimulated GLUT4 traffic in 3T3-L1 adipocytes is more controversial. There is conflicting evidence regarding its activation and/or its role in actin remodelling by insulin. In contrast to CA TC10, expression of CA Cdc42 in 3T3-L1 adipocytes had no effect on the structure of cortical actin (85). Furthermore, treatment of
3T3-L1 adipocytes with wild type (WT) or DN Cdc42 mutants had no effect on insulin- 16
stimulated glucose uptake or GLUT4 translocation (24). This same study demonstrated that Cdc42 does not get GTP loaded by insulin stimulation. Contrastingly, a more recent study by Usui et al. showed using siRNA to Cdc42 or microinjection of anti-Cdc42 antibody a significant decrease in insulin-responsive GLUT4 translocation in 3T3-L1 adipocytes (159). This study placed Cdc42 downstream of Gaq/n in the signalling cascade, and upstream of PI3K and PKCX, input (159). More research remains to be done to fully elucidate the involvement ofCdc42 in this process.
1.5.4.I.e. Racl
Racl participation in GLUT4 traffic and actin remodelling in 3T3-L1 cells is also not fully understood. In an early study, transient expression of CA or DN Racl mutants were without effect on glucose uptake; however, DN Racl expression did perturb actin remodelling by insulin. Moreover, stable expression of CA Racl did not rescue wortmannin-inhibited GLUT4 translocation (102). A caveat of that study is that while actin remodelling was measured in the fibroblast stage, when the mutants were clearly expressed, glucose uptake and surface GLUT4 were measured upon differentiation of the cells into adipocytes, when the expression of DN Racl was not confirmed. On the other hand, JeBailey et al demonstrated activation (i.e. GTP-loading) of endogenous Racl in
3T3-L1 adipocytes by 5 minutes of insulin treatment (76). This activation was PI3In dependent (76). Further work remains to be done to determine the exact contribution of
Racl to this pathway in adipose cells. 17
1.5.4.2 Rho family of small G proteins in insulin-stimulated actin remodelling in muscle cells
1.5.4.2 a. TC10
L6 muscle cells express significantly lower levels of TC10 compared to 3T3-L1 adipocytes (76). However, as with adipocytes, TC10 transfected into L6 myoblasts was also GTP loaded within minutes of insulin stimulation. Yet transfection of WT, CA or
DN mutants of TCI 0 into the L6 cells did not attenuate insulin-stimulated actin remodelling or GLUT4 translocation, despite causing alterations in the basal actin morphology seen only in cells expressing CA TC10 (76). Taken together, this evidence offers further support to the notion that Rho GTPases function in a cell-specific manner to elicit the cell surface gain in GLUT4 in response to insulin (explored below).
1.5.4.2 b. Racl
Racl activation in response to insulin is observed as early as 1 minute of treatment in L6 muscle cells (73) (Figure 1.5).
Insulin Treatment (min) 0 1 Rac-GTP ""^7:™""
Total Rac l^^-iggB^
Figure 1.5: Insulin-induced Racl activation in L6 muscle cells, adapted from Ishikura etal (73). L6 myoblasts were exposed to 100 nM insulin for 1 min. GTP-bound Racl was precipitated using glutathione-S-transferase fusion protein of the p-21 kinase 18
CRIB (Cdc42/Rac interactive binding) domain conjugated to glutathione beads. The insulin-activated, GTP-bound Racl and the total Racl from cell lysates were detected by immunoblotting.
This is necessary for the insulin-dependent net gain of surface GLUT4, as expression of
DN Racl mutant or siRNA-directed inhibition of Racl expression in L6 myotubes abolished the increase of PM GLUT4 following insulin stimulation without alteration in basal levels (77, 89). In addition, DN Racl and siRacl prevented the formation of membrane ruffles by insulin, without changing the morphology of actin stress fibres in the basal or insulin-stimulated states (77, 89). These results emphasize the importance of functioning Racl in transducing the insulin stimulus into actin remodelling and subsequent GLUT4 translocation in muscle cells. Cellular transfection with WT or DN
RhoA was not able to prevent the formation of membrane ruffles in response to insulin, further reinforcing the specificity of the results seen with Racl mutants (89).
1.5.4.2 b.l. Signalling Upstream of Racl in L6 muscle cells
Having established that insulin induces Racl activation in L6 cells, it became imperative to uncover the upstream signalling cascade that leads to this phenomenon.
Inhibition of PI3K using wortmannin abolishes Racl activation and actin remodelling in response to insulin (76). Importantly, inhibition of Aktl using a DN mutant, decreased insulin-induced GLUT4 gain at the PM, but had no effect on actin remodelling (168).
This highlighted a potential bifurcation of the signalling pathway downstream of the
PI3K, where Akt and Racl activation represent two arms leading to insulin-mediated
GLUT4 translocation in parallel. To further bolster this point, Racl siRNA, did not 19 impinge on insulin's ability to phosphorylate Akt in L6 myotubes, while inhibiting
insulin-induced actin remodelling and GLUT4 translocation (77). However, this potential
bifurcation is so far established using long-term inhibition of either signalling arm, and further verification is required using acutely interfering strategies. This thesis explores
one approach to rapidly inhibit the Akt signalling arm.
Further support for the bifucation model comes from studies in cells rendered
insulin-resistant, which showed that, these two signalling arms have slightly different
susceptibility to the cellular insults (77). JeBailey et al used two in vitro models of insulin resistance, C2-ceramide (C2) and glucose oxidase (GO), to examine their effect on
insulin-stimulated GLUT4 translocation in L6 muscle cells (77). C2 is a fatty acid
derivative that has been shown to accumulate in muscles of insulin-resistant humans (3).
GO, on the other hand, generates extracellular peroxide that diffuses into cells to induce
oxidative stress, which leads to insulin resistance (14, 68). This study found GTP-loading
of Racl to be more susceptible to insulin resistance than Akt phosphorylation, as higher
concentrations of both GO and C2 were needed to impair the latter (77). The result of
insulin resistance on actin remodelling in L6 cells correlates with impaired actin
remodelling seen in the skeletal muscles of insulin resistant Zucker fatty rats (106).
1.5.4.2.b.2. Downstream effectors of Racl in L6 muscle cells
While Racl participation in GLUT4 traffic is clear, its downstream effectors still
remain largely unidentified, but PAK and LIMK are two targets in other cells, especially
during cell migration. Interestingly, there is evidence that PAK and LIMK are both
activated in response to insulin, but this has not been linked to metabolic outcomes of the 20
hormone. LIMK is involved in insulin-induced membrane ruffle formation in KB cells, a human carcinoma cell line, since the transfection of kinase-inactive LIMK suppressed membrane ruffling in response to insulin (174). The physiological significance of insulin action in these cells is unknown. More relevant to this thesis, PAK was shown to be
activated in response to insulin in L6 muscle cells in a PI3K-dependent manner (156).
Furthermore, cellular insulin resistance induced by C2 and GO decreased the insulin- induced activation of PAK 1, confirming its place in the insulin signalling cascade (77).
Although, PAK1 and LIMK get activated by insulin, their actual modulatory roles in
GLUT4 traffic are unknown.
Following another line of evidence, PKC£ has been implicated to signal downstream of Racl and to contribute to the insulin-mediated actin remodelling in muscle cells. Liu et al. showed PKC£ colocalizing with the remodelled actin mesh in L6 cells in response to insulin, in a PI3K-dependent manner (98). As well, they observed that
PKC^ overexpression induced membrane ruffle formation, a small increase in surface
GLUT4 levels, and increased glucose uptake in the absence of insulin stimulation.
Expression of CA Racl led to a colocalization of PKC£ with the remodelled actin that was wortmannin-insensitive, but inhibited by PKC^ pseudosubstrate (98). The results of these experiments can potentially place PKC£ downstream of Racl in the insulin- mediated actin remodelling pathway. Nevertheless, the search for more proteins in the
insulin-signalling cascade leading to GLUT4 translocation is still ongoing, with a special
focus on actin remodelling. 21
1.6 FLN, a GLUT4 interacting protein
One of the studies conducted in our laboratory is aimed at uncovering new proteins that participate in and regulate insulin-stimulated GLUT4 translocation by interacting with the GLUT4 transporter. This study utilized a stable isotope labelling by amino acids (SILAC) technique followed by immunoprecipation of the myc-tagged
GLUT4 (GLUT4myc) and mass spectrometry analysis to identify GLUT4 interacting proteins and the effect of insulin on these interactions (52). This technique identified 603 proteins that interact with GL\JT4myc (52). One of these was the cytoskeletal protein,
Filamin C (FLNc), whose association with GLUT4 was slightly increased in response to insulin (52).
1.6.1 Filamin Structure and Tissue Expression
FLN is a -280 kDa actin-binding and crosslinking protein that consists of 24 repeated sequences of-100 residues each (Figure 1.6). These repeats form antiparallel p- sheet domains, which overlap to create rod-like structures (61). FLN also contains an actin binding domain (ABD) composed of two calponin homology domains on its amino terminus and a dimerization domain on its carboxy terminus (161). Recently, a study conducted by Washington et ah, demonstrated that it is particularly the ABD of FLN that directs its cellular localization (169). Specifically, FLN's ABD alone showed the same localization as the full length protein, and it was distinct from the localization of the ABD of a-actinin (another actin crosslinking protein) (169). FLNs also have two hinge regions, positioned between repeats 15-16 and 23-24, which give the protein its flexibility and potentially structural adaptability (130, 143). FLNs form tail-tail homodimers that can 22 crosslink two actin fibres to make an orthogonal structure, and are identified as the most potent actin filament crosslinking protein (13, 143).
N CH l Dimerization !b. 3 \i 3 Domain \ • JO" i \~L- '5-.\16 18 20 22 \ - '< . •• 17 i 19/ll 2S/-J^ r *" TS* ~f TT- •* Til
Actin Binding , ,* 1« 18 20 22 . ^*C Domain ,a >,'•• 17 19 2l 23 » - 9 , .-11, •* ,' —» . iir T / • 6 H/nge Regions
Figure 1.6: Filamin Structure: Filamin contains 24 immunoglobulin-like repeats with two hinge regions, an amino-terminal actin binding domain and a dimerization domain on the carboxy terminus.
In mammals, three FLN genes help code for the three filamin isoforms: FLN a, b, and c. In addition to these three isoforms, there are a number of different splice variants.
The three FLN isoforms show up to 70 % sequence homology, with the exception of the two hinge regions and an 81 amino acid insert in repeat 20 of FLNc. FLNa and FLNb are ubiquitously expressed, although their levels of expression may differ depending on cell type (161). FLNc expression is largely restricted to skeletal and cardiac muscles (150).
FLNs were found to localize to actin stress fibres, cortical actin networks as well as in membrane ruffles of migrating cells (161). The C-terminal region of FLNa was also
found to localize to the nucleus, where it translocates together with the androgen receptor
(100,123). In skeletal muscle cells, FLNc was found to localize along the Z-lines, myotendinous junctions and with the cortical actin beneath the PM (58, 59, 127, 150). 23
1.6.2 Filamin function in health and disease
FLNs are crucial for normal morphogenesis and embryogenesis (49). Mutations in
FLNs leads to abnormal development of brain, bones, cardiovascular system and skeletal muscles (49). FLNa loss-of-function mutation leads to periventricular heterotopia, in which a portion of neurons fail to migrate to the cerebral cortex from the lateral ventricular zone (53, 81). Recently, four more diseases have been identified with mutations localized to FLNa: otopalatodigital syndrome types 1 and 2, frontometaphyseal
dysplasia, and Melnik-Needles syndrome (133). Mutations in the FLNb gene give rise to
a class of diseases that manifest as abnormal vertebral segmentation, joint formation and
skeletogenesis (91). With FLNc expression restricted to striated muscles, mutations in this protein give rise to muscular dystrophies and have recently been connected to a novel
form of myofibrillar myopathy (28,164).
In addition to their ability to cross-link actin, FLNs play an important role in
relaying cellular signals that contribute to changes in the actin cytoskeleton. They are
known to interact with over 20 different proteins, and this number continues to grow as
new binding partners are uncovered (129). As potential scaffolds for different signalling
molecules, in this role FLNs are involved in the regulation of transcription, membrane
receptor localization and stabilization, and cellular protein traffic (129) (Figure 1.7). 24
IRhoG proteins § Insulin Signalling - m
i - •' r ' ) \ ).* Actin Remodelling Pyrin, CFTR,FCyRI J' \ > Signalling scaffold _ > CD At r* V,- #CD28# ;• *..
»• *..- ' " y
- *• •• X, X . ,_. 1 Actin Crosslinking
Figure 1.7: Filamin is a Multifunctional Protein. FLN functions as an actin crosslinker and participates in actin remodelling. It can interact with many proteins involved in a variety of cellular processes. The position of these interactions has been mapped in a number of cases with most protein interacting with the C-terminal section of FLN. By binding to other proteins FLN can function as a signalling scaffold and take part in the insulin signalling cascade.
1.6.2.1 Filamin in actin remodelling
As mentioned previously, actin remodelling regulates multiple cellular processes.
An increasing body of evidence points to FLNs as key mediators of actin remodelling in response to a variety of stimuli. FLNa constitutively (GTP-independently) binds to the
Rho GTPases, Racl, Cdc42 and RhoA, which can regulate actin structures (104). FLNa is also able to interact with Ras-related small GTPase Ral A in a GTP-dependent manner and induce filopodia formation in Swiss 3T3 fibroblasts (119). Furthermore, RalA was not able to induce filopodia in the absence of FLNa in the M2 cell line (a cell line derived 25
from human malignant melanoma that does not express FLNa) (119). Interestingly, RalA
was recently implicated in insulin-stimulated GLUT4 traffic in 3T3L1 adipocytes, where
it is suggested to interact with the motor protein Myolc and promote GLUT4 translocation to the PM (22).
The ability of FLNa to transduce cell signals into actin remodelling was further
established by evidence of its interaction with a Racl and RhoG GEF named Trio (10).
Expression of one of the domains of Trio, Trio GEFD1—responsible for Rho GTPase
activation, is able to induce characteristic dorsal membrane ruffling (11). Utilizing the
M2 cells, Bellanger et al. demonstrated the necessity of FLNa and FLNa—Trio
interaction for actin remodelling and membrane ruffling seen with Trio GEFD1
expression. Expression of FLNa in A7 cells (M2 cell line stably transfected with FLNa
cDNA), rescued the effect of Trio GEFD1 on cytoskeleton remodelling (10).
Recently, Ohta et al. identified a novel Rho GTPase GAP, FilGAP, with specific
Racl activity (118). FLNa was shown to bind FilGAP and target it to the epidermal
growth factor (EGF)-induced membrane ruffles. The interaction of FilGAP and FLNa
and their proper cellular localization was able reduce the cellular levels of GTP-bound
Racl and reduce lamellae formation (118).
In addition, a study by Vadlamudi et al. demonstrated that FLNa is essential for the actin assembly mediated by Pakl activation (160). Using activators of Pakl
(heregulin, serum and sphinoglipids), known to induce actin remodelling and membrane
ruffle formation, this group demonstrated that Pakl interacts and phosphorylates FLNa
on a Ser2152 residue (160). Furthermore, expression of FLNa with Ala2152Ser mutation
or using the M2 cell line prevented Pakl-induced ruffle formation. Finally, Pakl 26 activators failed to induce cofilin phosphorylation in the absence of FLNa normally seen in the A7 cells (160). Taken together, these results indicated that FLNs can interact with small Gproteins, as well as their upstream regulators and downstream effectors; thereby integrating the cellular signals to allow the remodelling of the actin cytoskeleton.
1.6.2.2 Filamin acts as a scaffold
One of the key functions of FLN is its ability to act as a scaffold in a variety of signalling events and to direct the cellular traffic and membrane expression of a variety of receptors. One of the proteins whose intracellular traffic depends on FLNa is furin, a membrane-associated serine endoprotease. FLNa directly interacts with furin in vivo, tethering it to the cell surface, and thus regulating its rate of internalization (97). FLNa is also necessary for the sorting of furin from the early endosomes to the trans-Golgi network (97).
Similar to furin, FLNa also controls the membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) (149). FLNa interacts with CFTR on the membrane and tethers it to the underlying actin networks. Importantly, a disease causing mutation in CFTR decreased its interaction with FLNa, resulting in the internalization and degradation of the receptor (149).
Recently, FLNa was found to play a role in the accumulation of lipid rafts at the T cell immunological synapse (148). FLNa interacts with CD28 (a co-stimulatory molecule) in activated T cells and is recruited into the immunological synapse along with various lipid raft markers (148). Knockdown of FLNa expression by siRNA significantly diminished the accumulation of lipid rafts in the activated T cells. Furthermore, part of 27 the CD28 function is to promote Cdc42-dependent actin remodelling (135). Expression of mutant CD28 that lacks the FLNa-interacting region or siRNA-mediated knockdown of
FLNa expression prevented the activation of Cdc42 in response to T cell activation (148).
These results suggest that CD28 uses FLNa as a scaffold to integrate signalling pathways that lead to actin remodelling and lipid raft accumulation at the T cell immunological synapse.
FLNa has an additional scaffolding role in immune cells. Beekman et al. uncovered a novel role for FLNa, the control of FcyRI (Class I IgG receptor) surface expression and lysosomal degradation (9). FcyRI surface expression was nearly undetectable in the M2, FLNa-deficient, cell line compared to the A7, FLNa-positive cell line. Additionally, M2 cells had decreased cytosolic levels of FcyRI, suggesting lower protein expression (9). The authors showed that in the absence of FLNa, FcyRI is preferentially recruited into the lysosomal compartment, where it is degraded (9).
Yet another role of FLNa is in actin-dependent HIV receptor clustering and HIV infection (79). FLNa organizes F-actin rearrangements required for the envelope glycoprotein, gpl20, to mediate HIV receptor clustering on the PM. The binding of gpl20 activates RhoA GTPase, subsequently leading to cofilin phosphorylation and increased actin polymerization. FLNa directly interacts with CD4 (HIV receptor) and
CXCR4 (HIV co-receptor), connecting these receptors to the actin cytoskeleton (79).
Perturbing FLNa binding to either prevents RhoA activation, actin polymerization and hence viral infection of HIV (79).
The calcitonin receptor (CTR) also interacts with FLNa. Under normal conditions,
CTR undergoes tonic endocytosis and subsequent recycling back to the cell surface that 28 are both increased in the presence of calcitonin (138). CTR expression in the M2 cell line significantly reduced PM CTR expression and increased its degradation, without changes in its synthesis (138). FLNa was necessary for the recycling of CTR back to the PM, whereby loss of FLNa enhanced CTR degradation. Calcitonin treatment also decreased the amount of FLNa cleaved by its endogenous protease, calpain, suggesting that this regulation allows for the enhanced recycling of FLNa-CTR in response to calcitonin
(138).
Taken together, evidence from a variety of studies establishes the role ofFLNs as scaffolding proteins, acting as integrators of cellular signalling cascades, protein traffic and protein (receptor) membrane expression.
1.6.2.3 Filamins and Insulin Signalling
A few studies have identified FLNs as mediators of the insulin-signalling cascade, although its overall role remains for the most part unknown. First, SHIP-2, a 5'- phosphatase and negative regulator of insulin signalling, colocalizes together with FLNc in membrane ruffles in response to EGF treatment (38). SHIP-2 and FLNc also colocalized at the sarcolemma on analysis of skeletal muscle sections (38). EGF treatment of M2 cells failed to induce SHIP-2 translocation from the cytosol to the transiently appearing membrane ruffles, an effect rescued by stably expressing FLNc.
That observation suggested a requirement for an interaction between these proteins for the EGF-mediated translocation of SHIP-2 to membrane ruffles. This interaction also proved necessary for the SHIP-2-mediated decrease in PIP3 levels and P-actin accumulation in the membrane ruffles (38). FLNc has also been shown to interact with 29
LL5p, a PIP3 binding protein (125). Although the exact cellular function of LL5|3 remains to be defined, it offers further evidence for FLNc involvement in the pathway downstream of PI3K.
Interestingly, FLNc was also found to be an in vitro substrate of Akt at residue
Ser2213 (111). This FLNc residue was phosphorylated in response to insulin and EGF in
C2C12 mouse myoblasts, with a similar time course of activation as Akt. Moreover, the use of wortmannin fully prevented FLNc phosphorylation in response to EGF and insulin in C2C12 cells, while the inhibitors of the MAPK cascade and the mTOR pathway had no effect (111). Finally, insulin-responsive FLNc phosphorylation was also impaired in the cardiac muscle of 3'-phosphoinositide-dependent kinase 1 (PDK1) knockout mouse
(111). This offered further support for positioning FLNc downstream of Akt in the insulin-signalling pathway. Further, FLNc phosphorylation is specific to insulin, since it is not detectable in response to exercise in human skeletal muscles (34). In contrast, exercise induced the phosphorylation of the FLNa isoform in human skeletal muscles as detected by the phospho-Akt Substrate (PAS) antibody (34). However, it has been proposed that the PAS antibody can also detect phosphorylation by AMPK. The latter point still remains to be verified in the case of FLNa.
Like FLNc, FLNa has also been implicated in the insulin signalling pathway. He et al. observed that tyrosine phosphorylation of She downstream of insulin was markedly reduced in the FLNa expressing A7 cells, compared to M2 cells that lack FLNa (65). Akt phosphorylation was unaffected, while ERK phosphorylation levels were markedly reduced in FLNa expressing cells in response to insulin or IGF-1 treatment (65). FLNa constitutively interacts with the IR, and expression of a truncated FLNa that is unable to 30
interact with the IR significantly increased ERK phosphorylation without affecting Akt
(65). Thymidine incorporation, a measure of DNA synthesis, was enhanced in the insulin-treated M2 cells. Moreover, translocation of She, ERK and phospho-ERK was impaired in A7 cells.
In summary, FLNs participate in the insulin pathway at many different levels
(highlighted above). However, despite this extensive body of evidence implicating FLNs downstream of insulin signalling, their exact physiological function remains elusive, and was the subject of examination in this thesis. 31
1.7 Rationale and Hypothesis
Insulin-stimulated GLUT4 translocation requires activation of both the signalling pathway downstream of Akt phosphorylation as well as Racl-mediated actin remodelling.
Furthermore, evidence suggests that these pathways bifurcate downstream of the PI3K in
L6 muscle cells (77, 168). Indeed, chronic inhibition of either arm prevents the insulin-
responsive gain in surface GLUT4. However, there is no knowledge about the effect of
acute inhibition of Akt phosphorylation on Racl activation and actin remodelling in response to insulin. A newly described allosteric inhibitor of Akt isoforms 1 and 2 (Akti
1/2), can provide the means to study the effect of acute inhibition of Akt on Racl
activation and fully determine if crosstalk exists between these two pathways (60, 176).
Furthermore, there could be additional input into the insulin-induced GLUT4 translocation and actin remodelling pathways in muscle cells from other Rho G proteins.
Cdc42 is implicated in the GLUT4 translocation pathway in 3T3L1 adipocytes (159).
Activated Cdc42 has been shown to function upstream of Racl in EGF-stimulated
membrane protrusions in MTLn3 carcinoma cells (40). Moreover, lamellipodia formed
by Racl activation were shown to nucleate from Cdc42 formed filopodia in NIH3T3
fibroblasts (62). Taken together, Cdc42 is a candidate to have a functional role in insulin-
mediated GLUT4 translocation.
A lot of questions remain unanswered about the insulin signalling cascade that
leads to the translocation of GLUT4 to the PM. It is known that PI3K signals direct both
actin remodelling and Akt phosphorylation; however the point of intersection of these
two pathways has not been determined. FLNc may be the protein that lies at the
intersection of the two signals. FLNc is highly expressed in skeletal muscles and may be 32 affected by Akt signalling since it is an in vitro target of Akt phosphorylation. FLNc crosslinks actin fibres, hence it may position the signalling molecules in the area of actin remodelling. FLNa, a close homolog of FLNc, binds to Rho GTPases and their downstream molecules, and directs membrane ruffle formation. In addition, FLNs have been identified as scaffolding proteins in a number of signalling pathways. Taken together, FLNc is a candidate scaffold protein that coordinates insulin signalling and actin remodelling in the pathway that leads to GLUT4 translocation.
Hence the purpose of this study is twofold. Firstly, the purpose is to analyze the effect of acute inhibition of Aktl/2 on insulin-stimulated actin remodelling and Racl activation, to confirm the independence of these two signalling arms. As well as analyze the activation of Cdc42 in response to insulin and the potential input of Akt on this Rho
GTPase. Secondly, the purpose is to examine the contribution of FLNc to the insulin pathway in muscle cells and its potential role as a scaffold for the signalling molecules involved.
The hypothesis of this study is that insulin signalling downstream of PI3K bifuricates into an arm leading to actin remodelling and an arm leading to Akt activation; that Cdc42 is activated in response to insulin; and that FLNc is necessary for insulin- stimulated GLUT4 translocation and actin remodelling, uniting the two signalling arms. 33
Figure 1.8: Objectives of this thesis. This thesis has two objectives. The first objective is to analyze the crosstalk between Akt and Racl signalling downstream of the PI3K in response to insulin, as well as to analyze the activation of Cdc42 in response to insulin in L6 cells. The second objective is to analyze the importance of FLNc in the process of insulin-induced GLUT4 translocation and its role in integrating Racl and Akt signals. Chapter 2: Materials and Methods
34 35
2.1 Tissue culture
All experiments were performed in L6 rat muscle cell line that stably expresses
GLUT4myc. GUJT4myc cDNA (from Rattus norvegicus) was constructed by inserting the human c-myc epitope (AEEQKLISEEDLIK) into the first ectodomain of GLUT4 and subcloned into the pCXN2 vector (82), stably transfected in L6 cells and characterized
(51,158,167). L6GLUT4myc cells (L6 muscle cells) were maintained as monolayer myoblasts in medium of aMEM (Wisent Inc, Montreal, QC) supplemented with 10%
FBS (v/v; Wisent Inc. Montreal, QC) and 1% antibiotic/antimycotics (v/v; Wisent Inc.
Montreal, QC) in an atmosphere of 95% O2 and 5% CO2. The myoblasts were subcultured every 48 h by trypsinization (0.05% trypsin; Wisent Inc. Montreal, QC).
Myotubes were grown in aMEM (Wisent Inc, Montreal, QC) supplemented with 2% FBS
(v/v) and 1% (v/v) antibiotic/antimycotic. On average fully differentiated myotubes were formed 6-8 days after seeding. The cells are seeded at a density of- 5x10 cells/mL and
2xl04 cells/mL for studies in myoblasts and myotubes, respectively.
For immunoflourescence experiments cells were seeded onto 18 mm diameter glass coverslips in 12-well tissue culture plates and used 2-8 days later. For cell surface
GLUT4myc detection experiments, cells were seeded at a density of- 2xl05 cells/mL in
24-well plates. For total cell lysate or GST-CRIB pull-down experiments, cells were
seeded on 6-well or 10cm plates.
2.2 Cell treatments
For all experiments, prior to acute treatment with stimulus, myoblasts or myotubes were incubated in serum-free aMEM for 3-5h. For insulin treatment, 100 nM insulin (human insulin (humulin); Eli Lily Canada Inc, Toronto, ON) was administered for indicated time. Membrane depolarization was achieved with high K isotonic solution
+ consisting of 20 mM HEPES (pH 7.4), 2.5 mM MgS04,5 mM p-D glucose, 120 mM K gluconate and 25 mM NaCl, for 20 min (Sigma, St Louis, MO). 2,4-dinitrophenol (DNP;
Sigma St Louis, MO) 0.5 mM in aMEM occurred for 20 min. PI3K inhibition was achieved by pre-incubating the cells with 100 nM Wortmannin (in DMSO) or 25 uM
LY294002 (in DMSO) for 20 min prior to insulin treatment (Biomol; Plymouth Meeting,
PA). Akt inhibition was achieved by pre-incubating the cells with 10 uM isozyme selective Akt inhibitor (Akti 1/2) in DMSO (Calbiochem, San Diego, CA).
2.3 siRNA transfection
All knockdown experiments were performed in L6GLUT4myc myotubes using siRNA to FLNc (200 nM of oligonucleotides) using the calcium-phosphate-based transfection reagent (CellPhect Transfection Kit; Amersham Biosciences, Baie d'Urfe,
QC) as per manufacturer's instructions. The sequence for FLNc was GAC GGU ACC
UGC AAA GUC A (29). The sequence for non-related siRNA control (NRCon SeqX;
Qiagen Inc, Mississauga, ON) is AUU CUA UCA CUA GCG UGA C. Cells were transfected on day 5-6 after seeding, and experiments were carried out 48 h post- transfection.
2.4 Preparation of whole cell lysates
Myoblasts or myotubes seeded on 6 well plates were left untreated (basal) or treated as indicated. Cells were washed twice with ice-cold phosphate-based saline (PBS, 37
154 mM NaCl, 5.6 mM Na2HP04, 1.1 mM KH2P04). Total cell lysates were made with made with 2 x Laemmli sample buffer containing phosphotase inhibitors (1 mM Na3V04,
100 nM okadaic acid) and protease inhibitor cocktail (ImM benzamidine, 10 uM E-64,1 uM leupeptine, 200 uM phenylmethylsulfonyl fluoride (PMSF); Sigma, St Louis, MO), all at 4°C. Cell lysates were then passed through a 27-gauge needle, centrifuged for 20 min at 13,000 rpm and heated for 5 min at 95° C.
2.5 Immunoblotting
Fifteen-twenty (xg of proteins were resolved by 7.5-10% SDS-PAGE and electrotransferred onto polyvinylidene fluoride (PVDF; Bio-Rad Laboratories, Hercules,
CA) membranes for 2h at 100V. The membranes were blocked in Tris-buffered solution
(500 mM Tris-base, 150 mM NaCl pH 7.5, 0.5% Tween-20 and 0.5% NP-40) with 3% bovine serum albumin. Membranes were then immunoblotted overnight at 4° with primary antibody as specified in the text. Membranes were then washed for 1 h before applying horseradish peroxidise (HRP)-conjugated secondary antibodies for 1 h at room temperature followed by 1 h wash. Immuno-labelled protein bands were visualized using an enhanced chemoluminescence (ECL; PerkinElmer Life Sciences Inc, Boston, MA).
The immunoblots were quantified with ImageJ software (National Institutes of Health,
Bethesda, MD). 38
2.6 Antibodies
For measuring protein expression in cell lysates resolved by SDS-PAGE and in immunofluorescence experiments, we used primary antibodies for FLNc (1:1000 for immunoblotting and 1:100 for immunofluorescence; Dr. James Hastie, Division of Signal
Transduction Therapy, University of Dundee, Scotland), a-actinin-1 (1:30,000; Sigma-
Aldrich, St. Louis, MO), Racl (1:1000, Upstate Biotechnology, Billerica, MA) and
Cdc42 (1:1000; Cell Signaling, Beverly, MA). For measuring protein phosphorylation, membranes were immunoblotted with anti-phospho FLNc (Ser2213) (1:1000 or 1:100 for immunofluorescence; Dr. James Hastie, Division of Signal Transduction Therapy,
University of Dundee, Scotland), anti-phospho-Akt (Ser473), anti-phospho-JNK
(Thrl83/Tyrl85) and anti-phospho-ERKl/2 (Thr202/Tyr204) (all 1:1000, Cell Signaling
Technology, Beverly, MA). The secondary antibodies used were: HRP-conjugated rabbit- anti-sheep, sheep-anti-mouse and goat-anti-rabbit IgG (Jackson ImmunoResearch, West
Grove, PA) as well as anti-mouse IgM (Sigma-Aldrich, St. Louis, MO).
2.7 Immunodetection of GLUT4myc at cell surface
The amount of GLUT4myc at the surface of intact cells was measured by an antibody-coupled colorimetric assay, as was described previously (167). All solutions were made in PBS (pH 7.4) containing 0.49 mM MgCl2 and 0.68 mM CaCl2. In brief, following incubation with inhibitors and stimuli at 37°C, cells were washed three times with ice-cold PBS, blocked with 5% goat serum (v/v) for 10 min, and incubated primary polyclonal anti-myc antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA). for 1 h, all 4°C. Cells were then washed 5 times with PBS, fixed with 4% paraformaldehyde 39
(PFA; Canemco, St. Laurent, QC) for 10 min, and quenched with 0.1 M of glycine
(Bio Shop Canada Inc, Burlington, ON) for 10 min, all at 4°C. The cells were then washed with PBS and incubated with secondary antibody (HRP-conjugated goat anti-rabbit IgG in 1:1000 dilution) for 1 h. Following the incubation with secondary antibodies, the cells were washed 5 times with PBS and all the liquid was aspirated from the wells. Briefly, 1 mL of 0.05M phosphate-citrate buffer (pH 5.0) containing 0.4 mg/mL o- phenylenediamine dihydrochloride (OPD) reagent and 12xl0"3% hydrogen peroxide (v/v;
Sigma, St Louis, MO) was added for up to 20 min at room temperature. The reaction was stopped by 0.25 mL of 3M HC1. The amount of GLUT4myc on the cell surface was measured by optical absorbance of the supernatant at 492 nm by an optical plate reader
(Molecular Devices, Sunnyvale, CA). Background colour was determined by performing the entire experiment without adding primary antibody. The value for the background
samples was subtracted from each experimental condition.
2.8 GST-protein preparation and GTPase assay
To examine the activation of Racl or Cdc42 with insulin, cell lysates were
subjected to pull-down experiments with a GST-fusion protein of the CRIB domain of
PAK conjugated to glutathione beads that bind GTP-bound Racl or Cdc42 (12, 35). Cells
in 10 cm plates were treated with inhibitors and stimuli. Cells were washed twice with
ice-cold PBS and lysed with GTPase buffer containing 150 mM NaCl, lOmM MgCl2, 5 mM HEPES pH 7.5, 2% glycerol, 1% Igepal, 1 mM NaV04, lmM EDTA, 20 ug/ml protease inhibitor cocktail (BD Biosciences, Oakville, ON) and 1 mM PMSF. Lysates
were centrifuged at 12,000g for 1 min at 4°C, 20 uL were removed for total lysate analysis, and the rest was incubated with GST-CRIB conjugated glutathione Sepharose beads. After 30 min rotation at 4°C, the samples were centrifuged briefly at 12,000 g, washed three times with lysis buffer, eluted with 2xLaemmli sample buffer, and heated
for 5 min at 100°C. Pulled down proteins were resolved by 10% SDS-PAGE gel,
followed by immunoblotting using anti-Rac and anti-Cdc42. The negative and positive
controls were obtained by treating non-stimulated cell lysates with excess of either GDPP or GTPyS, respectively, the nonhydrolysable analogs.
2.9 Fluorescence Microscopy
Cells were grown on 18mm diameter glass coverslips and left untreated or treated with siRNA or inhibitors as described in the text. Cells were serum deprived for 4 h and
treated with or without 100 nM insulin for 10 min at 37°C. Cells were fixed with 3%
paraformaldehyde (v/v) in PBS for 10 min at 4°C, quenched for 10 min with 0.1M
glycine. Cells were briefly permeabilized using 0.1% Triton X-100 (v/v) (Bio-Rad,
Hercules, CA) in PBS for 3 min, and blocked for 15 min with 5% milk (v/v) in PBS.
Cells were incubated either with polyclonal sheep anti-FLNc, anti-phospho-FLNc or anti-
myc (T.200) primary antibodies for 1 h at room temperature, followed by extensive
washing with PBS. Cells were then incubated with rhodamine-coupled phalloidin and
secondary antibody conjugated to the fluorophore Alexa 488 and Cy3 for 1 h at room
temperature, protected from light (1:1000; Molecular Probes, Invitrogen, Carlsbad, CA).
Cells were then washed with PBS five times and mounted with Daiko for imaging
analysis (DakoCytomation, Carpinteria, CA). 41
2.9.1 Confocal Microscopy
Images were obtained by laser confocal microscopy (Zeiss LSM 510). For acquisition of actin images the focal plane was chosen to best the actin structures in basal and insulin-stimulated states. To analyze the actin morphologies, the whole cell was scanned along the z-axis by taking optical slices of 1 um width a single composite image was generated using LSM5 Image software (Carl Zeiss, Thornwood, NY).
2.10 Statistical Analysis
All the data points within each set of experiments were normalized to the control basal value from one of the experimental repeats (randomly picked). In a few indicated experiments the data points from each experimental repeat were normalized to their respective control basal to negate some of the external variability. Statistical analyses were performed using GraphPad Prism version 5.0 for Windows (GraphPad Software,
San Diego, CA) Student's paired t-test was employed when two groups were compared.
One-way ANOVA with Tukey post-hoc test was used to examine differences in cell treatments and stimuli. Data are presented as means ± SE. Chapter 3: Crosstalk between Akt and Rho GTPase signalling 43
3.1 Summary
In muscle and adipose tissue the main effect of insulin is seen as an increase in
GLUT4 translocation from the intracellular compartments to the PM. This phenomenon
depends on a number of signalling events, most importantly the activation of Akt and
Racl-dependent actin remodelling, both positioned downstream of PI3K. Previous
studies from our laboratory using chronic inactivation of either Rac or Akt signalling,
alluded to a signalling bifurcation model, wherein Racl-dependent actin remodelling and
Akt phosphorylation are mutually independent signalling cascades. The present study
offers further support to this model, since the acute inhibition of Aktl and Akt2 had no
effect on insulin-induced Racl activation or actin remodelling. In contrast to this, Cdc42
activation in response to insulin, presented in this study, is downstream of Akt and is
significantly inhibited by Akti 1/2. The exact function of Akt-dependent Cdc42 activation
in L6 cells remains to be examined. Nonetheless, this newly described signalling phenomenon, points to the increasing complexity of the insulin signalling cascade
downstream of the PI3K and suggests a potential input of Akt into actin, microtubule or
aPKC function. 44
3.2 Results
3.2.1 Effect of Akt1/2 inhibition on Rad-mediated actin remodelling in response to insulin in L6 cells
Insulin-induced actin remodelling is crucial for GLUT4 translocation in both L6 muscle cells and 3T3L1 adipocytes (84,157). The involvement of activated Racl in insulin-induced actin remodelling in L6 cells has been well established with DN Racl mutants and siRNA-mediated Racl knockdown (77, 89). Racl knockdown, although preventing GLUT4 movement to the PM in response to insulin, did not affect insulin- induced Akt phosphorylation (77). Conversely, chronic inhibition of Akt phosphorylation prevented insulin-dependent GLUT4 movement, while having no visible effect on actin- remodelling (168). To fully establish the interdependence of these two pathways, Akt phosphorylation was inhibited using a newly described allosteric inhibitor of Akt isoforms 1 and 2 (Akti 1/2) (176).
Acute treatment of L6GLUT4myc with Akti 1/2 significantly reduced insulin- induced phosphorylation of Akt on the serine 473 residue (0.6 ± 0.3, p < 0.001) compared to the Akt phosphorylation in response to insulin in vehicle-treated cells (4.9 ± 0.6, p <
0.001) (Figure 3.1).
A)
DMSO Akti 1/2 Insulin - + - + 45
B)
C^DMSO ^ Akti 1/2 5- < « l%2 4- 1= J5 o 3-
2- Norm a
osphor y elativ e t 1- 0. ^ 0- Basal Insulin
Figure 3.1: Akti 1/2 inhibits insulin-induced Akt phosphorylation. A) Akt phosphorylation on Ser473 in response to 100 nM insulin treatement for 10 min in DMSO (vehicle) or Akti 1/2 treated cells. Cells were serum-deprived for 3 hours. Cells were then pretreated with lOuM vehicle or inhibitor for 1 hr prior to insulin stimulation. Total cell lysates were resolved by SDS-7.5% PAGE and immunoblotted with anti- phospho Akt and anti-Cdc42 antibodies. Cdc42 is a loading control. B) Quantification of inhibition of Akt phosphorylation by Akti 1/2; Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. The values were normalized to the vehicle-treated basal within each experimental repeat. The quantified values represent the means ± SE, #p < 0.01 vs basal control, *p < 0.01 vs insulin-treated control, n = 3.
Since this inhibitor exhibited near complete inhibition of insulin-induced Akt phosphorylation, it provided a great tool for studying the effect of acute Akt inhibition on actin remodelling and the Racl pathway. Interestingly, Akti 1/2 had no effect on actin remodelling seen as membrane ruffle formation in the insulin-treated cells (Figure 3.2A).
Membrane ruffles are formed on the dorsal surface of the cells and are best seen after 10 min of insulin treatment. Actin remodelling was quantified by counting the number of cells with visible dorsal membrane ruffles in the insulin-treated conditions (Figure 3.2B).
There was no significant difference in the number of cells containing membrane ruffles 46 following Akti 1/2 treatment (0.89 ± 0.04, p = 0.94) compared to the vehicle-treated controls (0.88 ± 0.08, p = 0.94).
A)
DMSO AfcUl/2 insulin
pFUNc
Actln 47
B)
Q
© C 2 100-, .a I i 4E) E 75- .c .t: w 50- — 3 © "- u «^ 25- o *-c» rc e a> DM SO Akti 1/2 a.
Figure 3.2: Akti 1/2 does not affect insulin-induced actin remodelling. A) Representative image of actin remodelling detected using immunoflourescence in L6 myoblasts. Cells were serum-deprived for 3 hours. Cells were then pretreated with 10uM vehicle (DMSO) or inhibitor for 1 hr prior to being treated with or without 100 nM insulin for 10 min. The samples were fixed and permeabilized as described in Materials and Methods. Actin filaments are stained with rhodamine-phalloidin, phosphorylated FLNc is stained with anti-phospho FLNc antibodies followed with fluorophore- conjugated secondary antibodies (Alexa 488). Phosphorylated FLNc levels serve as a control for Akt inihibition. B) 1.5X magnification of representative images of vehicle and Akti 1/2 pretreated cells after insulin stimulation. The arrows indicate the cells with visible peripheral membrane ruffles. C) Quantified percent of cells that contained 48 peripheral membrane ruffles after insulin-treatment in vehicle or inhibitor-treated conditions. Shown are representative cells for each treatment, n = 3.
Also, cells treated with Akti 1/2 maintained a significant activation of Racl following insulin treatment (3.3 ± 0.6, p < 0.01) compared to Akti 1/2 cells in the basal conditions (1.3 ± 0.3, p < 0.01). There was no significant difference in insulin-induced
Racl activation in Akti 1/2 (3.3 ± 0.6, p = 0.23) and vehicle-treated cells (4.1 ± 0.4, p =
0.23) (Figure 3.3).
A)
DMSO Akti 1/2 in vitro Control Insulin GDP GTP Rac-GTP
RacTCL
B)
o I DMSO a X I Akti 1/2 1 I 5 30- — ffi © 2.5- 2.0- 1.5- 75 ^ "« 1.0- § I 0.5- o *~ 0.0 Basal Insulin
Figure 3.3: Akti 1/2 does not affect insulin-dependent Racl activation. A) Endogenous activated Racl was detected using Racl pull down assay described in Materials and Methods. Cells were serum-deprived for 3 hours, then pretreated with lOuM vehicle (DMSO) or inhibitor for 1 hr prior to being treated with or without 100 nM insulin for 10 min. Pulled down psroteins were resolved on SDS-10% PAGE and immunoblotted with anti-Racl antibodies. Total cell Racl was used as a loading control. In vitro controls with nonhydrolysable GDP and GTP are shown as indicated (see Materials and Methods). B) Quantification of Racl activation after Akti 1/2 treatment. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. The values were also normalized to the DMSO-treated basal from one of the repeats of the experiment. The quantified values represent the means ± SE, *p < 0.01 vs corresponding basal, n = 6.
Surprisingly, Akti 1/2 did not cause a reduction in insulin-induced GLUT4 translocation, while causing a slight increase, although not statistically significant, in the basal PM GLUT4 levels (Figure 3.4). This was an unexpected result as the same
concentration of the Akt inhibitor partially inhibited insulin-induced GLUT4
translocation in 3T3L1 adipocytes (60).
3n DMSO •* « Akti 1/2 H (0 ~) n _l .a o v>4- 1 ac e iv e la t ur f OT (r e OJL-I I I I 1^^ Basal Insulin
Figure 3.4: Akti 1/2 does not inhibit insulin-induced GLUT4 translocation. L6 myoblasts after 3 hours of serum starvation were pretreated with lOuM vehicle (DMSO) or inhibitor for 1 hr prior to insulin stimulation. Cells were treated with 100 nM insulin for 30 min. Values were normalized to DMSO-treated basal within each experimental repeat. Cell surface GLUT4 was measured and expressed as means ± SE, *p < 0.05 vs corresponding basal, n = 4.
3.2.2 Activation state of Cdc42 in L6GLUT4myc cells
A considerable amount of crosstalk has been observed amongst Rho G proteins
(17). For instance, Cdc42 can induce Racl activation acting via PAK and PAK-
interacting exchange protein (PIX), which can function as a Cdc42 or Rac GEF (48).
Cdc42 has been observed to function upstream of Rac in EGF-stimulated membrane 50 protrusions in MTLn3 carcinoma cells (40), and RhoA can regulate the coupling of Rac versus Cdc42 to the actin remodelling machinery (41). Taking into account the extent of
Rho G protein crosstalk, as well as the documented role of Cdc42 in GLUT4 traffic in
3T3L1 adipocytes (159), it became crucial to examine the activation state of Cdc42 in L6 muscle cells.
To examine the activation state of Cdc42, L6GLUT4myc myoblasts were treated with 100 nM insulin for 10 min. This time of stimulation was chosen as a starting point for the examination of Cdc42 activation, since Racl activation is maximal at 10 min, hence if Cdc42 acts upstream of Racl it would most likely remain in the activated state at this time. Compared to untreated (basal) cells which showed a low level of Cdc42 loading with GTP (0.9 ± 0.2, p < 0.05), a significant increase in Cdc42 activation was observed in response to insulin (3.0 ± 0.6, p < 0.05) (Figure 3.5).
A)
insulin I m vitro control Cdc42-GTP GDP GTP
Cdc42TCL
B)
4n Basal Insulin 5 Figure 3.5: Cdc42 is activated in response to insulin in L6 myoblasts. A) Endogenous activated Cdc42 was detected using Cdc42 pull-down assay described in Materials and Methods. Cells were serum-deprived for 3 hours, and then they were treated with or without 100 nM insulin for 10 min. Pulled down proteins were resolved on SDS-10% PAGE and immunoblotted with anti-Cdc42 antibodies. Total cell Cdc42 was used as a loading control. In vitro controls with nonhydrolysable GDP and GTP are shown as indicated (see Materials and Methods) B) Quantification of Cdc42 activation. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. Valued were normalized to one of the basals. The quantified values represent the means ± SE, *p < 0.05 vs basal, n = 4. 3.2.3 Effect of Akt1 and Akt2 inhibition on Cdc42 activation Having established that Cdc42 is activated in response to insulin in muscle cells, it became of interest to elucidate the position of Cdc42 in the insulin signalling cascade. Since our main goal was to examine the interaction of Akt with the Rho GTPase- dependent actin remodelling pathway, we used Akti 1/2 to determine the dependence of Cdc42 activation on Akt phosphorylation. Surprisingly, insulin-dependent Cdc42 activation was significantly inhibited by Akti 1/2 (0.8 ± 0.2, p < 0.05) when compared to the DMSO-treated cells (2.6 ± 0.8, p < 0.05) (Figure 3.6). This result places Cdc42 activation in response to insulin downstream of Akt in muscle cells. A) DMSO Akti 1/2 Insulin + + Cdc42-GTP B) 52 3 DMSO HI- Akti 1/2 Hi": Z ° -^ 0.5- 0.0- Basal Insulin Figure 3.6: Cdc42 activation in response to insulin is inhibited by Akti 1/2. A) Endogenous activated Cdc42 was detected using Cdc42 pull-down assay described in Materials and Methods. Cells were serum-deprived for 3 hours, and then pretreated with lOuM vehicle (DMSO) or inhibitor for 1 hr prior to insulin stimulation. Cells were treated with or without 100 nM insulin for 10 min. Pulled down proteins were resolved on SDS-10% PAGE and immunoblotted with anti-Cdc42 antibodies. Total cell Cdc42 was used as a loading control. B) Quantification of Cdc42 activation after Akti 1/2 treatment. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. The values were also normalized to the DMSO-treated basal within each experimental repeat. The quantified values represent the means ± SE, *p < 0.05 vs insulin-treated control, n = 3. 53 3.3 Discussion 3.3.1 Insulin signals bifurcate downstream of PI3K into two arms, Akt-dependent signalling and Rac1-mediated actin remodelling Evidence from previous studies has alluded to a possible split in the insulin signalling cascade, into an Akt-dependent arm and an arm leading to actin remodelling. This theory was initially suggested when the overexpression of the DN Akt mutant in L6 cells did not alter the remodelling of actin in response to insulin (168). Our observations in the present study offer further support to this signalling bifurcation model. Specifically, acute inhibition of Akt 1 and Akt2 did not perturb insulin-mediated Racl activation or actin remodelling. However, the lack of Akti 1/2 effect on actin remodelling was determined based on the visual "all or nothing" presence of membrane ruffles in insulin- stimulated L6 myoblasts. No other method was used to examine the remodelled actin and membrane ruffles qualitatively. Hence if a small difference in actin remodelling and membrane ruffle structure is present it may not be accounted for with this experimental approach. The independence of actin cytoskeleton remodelling from Akt phosphorylation can also imply that Akt phosphorylation lies downstream of actin remodelling, and this is the basis for a second theory. Although siRNA knockdown of Racl did not inhibit Akt phosphorylation in response to insulin, protein knockdown with siRNA is achieved over time, which could potentially allow for some compensatory mechanisms to come into play. Therefore, it is essential to employ acute inhibition of Racl to fully prove the signalling bifurcation theory (see Future Directions). Indeed, a number of studies suggest that activated Rho GTPases, specifically Racl and Cdc42, can lie upstream of both actin remodelling and Akt in T lymphocytes in 54 response to T-cell antigen receptor engagement, in Rati fibroblasts in response to PDGF, and in HEK293 cells in response to PDGF treatment (19, 57, 66). In COS-1 and NIH3T3 cells, expression of CA mutant of Racl induced, while the DN Racl mutant inhibited, Akt phosphorylation on both serine 473 and threonine 308 (66). Furthermore, expression of DN Rac partially inhibited PDGF-induced Akt phosphorylation in Rati fibroblasts and HEK293 cells (19, 66). Interestingly, coexpression of a DN Akt mutant suppressed the CA Rac-induced cell motility (without affecting actin remodelling), further reinforcing the position of Akt downstream of Racl (66). In addition, overexpression of Dbl, a Rho GTPase GEF, induced Akt phosphorylation in NIH3T3 and HEK293 cells, which again places Akt downstream of the Rho GTPases (110). Overexpression of CA Akt in 3T3L1 adipocytes treated with latrunculin B (LB) (an actin depolymerising agent) induced GLUT4 translocation to the same level as in response to insulin in cells with intact actin (46). On the other hand, the disruption of actin remodelling by LB decreased insulin- mediated activation of PI3K and Akt phosphorylation in 3T3L1 adipocytes (46). However, results from our lab suggest that actin depolymerization per se does not inhibit insulin-induced PI3K activation in L6 muscle cells or 3T3L1 adipocytes, but rather reduces the relocation of active PI3K to the GLUT4 containing compartment (157,166). The theory of Akt activation being downstream of actin remodelling proposes that the remodelled actin functions to organize the insulin signalling complexes. This theory is supported by the fact that PI3K and its product PI(3,4,5)P3 are segregated into the remodelled actin mesh in L6 myotubes, which then allows for the recruitment and activation of Akt and subsequent GLUT4 translocation (126). The results from the present study that demonstrate a lack of input from the Akt signalling arm into Racl- 55 dependent actin remodelling in response to insulin, can fit with both of the above presented models. The independence of actin remodelling and Racl activation from Akt phosphorylation can imply that the two signalling arms are entirely independent or that Racl-induced actin remodelling lies upstream of insulin-induced Akt phosphorylation. Therefore, a study examining the acute inhibition of Rac activation will be able to fully weed out the interaction and dependence of the Akt and actin remodelling arms of insulin signalling pathway. 3.3.2 Inhibition of Akt phosphorylation did not inhibit insulin-induced GLUT4 translocation There has been increasing debate regarding the contribution of Akt signalling to the event of GLUT4 translocation in response to insulin treatment. This debate comes from the seemingly conflicting evidence among a number of studies. One line of evidence suggests that Akt activation is the key event downstream of the IR, responsible for insulin-mediated GLUT4 translocation. This concept is supported by a number of studies that utilized membrane-targeted C A Akt mutants, which on their own were able to mimic the effect of insulin on GLUT4 translocation in both L6 muscle cells and 3T3L1 adipocytes (46, 63,168). In addition, a recent study by Ng et al. utilized a method that allowed for an acute activation of Akt2 in 3T3L1 cells (114). They demonstrated that the rapid induction of Akt2 was able to mimic the effect of insulin on GLUT4 translocation. However, glucose uptake in these cells was only half of that observed with insulin treatment, suggesting the importance of other signalling components in the insulin cascade (114). The other line of evidence places doubt on the primary role of Akt in insulin-dependent GLUT4 translocation. First of all, siRNA-mediated knockdown of 56 Akt2 in 3T3L1 adipocytes led to only a 40% inhibition of insulin-dependent GLUT4 translocation to the PM, while siRNA-mediated knockdown of Aktl had no effect on this process (60). In this same study, Akti 1/2 treatment of 3T3L1 adipocytes led to a 65% and 30% inhibition of GLUT4 translocation after InM and 170 nM insulin treatment (60). Furthermore, transfection of L6 muscle cells with the DN Akt mutant required a very significant amount of overexpression of the construct to observe a mere 50% inhibition in insulin-induced GLUT4 translocation (168). Surprisingly, in the present study Akti 1/2 failed to have any effect on insulin-stimulated GLUT4 translocation. It is not however clear if both Akt isoforms 1 and 2 have been inhibited in the present study, although Akti 1/2 has been demonstrated to do so at the same concentration as seen here (176). Aktl is the primary isoform activated in response to insulin in rat skeletal muscles with some contribution from Akt2 (165). However, Akt3 is also activated in response to insulin in L6 myotubes and would not be inhibited at the Akti 1/2 concentrations used in the present study (165). It is yet unknown which Akt isoform contributes to insulin-induced GLUT4 traffic in L6 cells. Hence, it is currently difficult to speculate on reasons for the lack of effect of Akti 1/2 on GLUT4 traffic. One possible explanation is that a very small amount of phosphorylated Akt seen in Figure 3.1, which remains after Akti 1/2 treatment, is sufficient to fulfill its role in the GLUT4 translocation pathway. This theory is in line with a recently published study that emphasizes the lack of linearity between Akt phosphorylation and GLUT4 translocation in 3T3L1 adipocytes (67). Specifically, insulin was able to stimulate maximal GLUT4 translocation at concentrations that induced phosphorylation of only 5% of the total Akt (67). Therefore, a small increase in Akt phosphorylation is sufficient to induce a maximal GLUT4 response. Furthermore, the 57 inhibition of Akt phosphorylation might not translate into the inhibition of Akt kinase activity; however this issue is addressed in Chapter 4. There is a growing body of evidence that an alternative pathway exists downstream of PI3K and functions in parallel with Akt via aPKCCfk. Results from our laboratory have failed to concretely support the importance of this pathway (126). PKCA, failed to localize with PIP3 in the newly formed membrane ruffles in L6 myotubes in response to insulin unlike Aktl. However, aPKC kinase activity significantly increased following insulin treatment and was wortmannin-sensitive, as determined by an in vitro kinase assay (5, 140, 141). Furthermore, aPKC£ pseudosubstrate, which functions as an inhibitor, inhibited insulin-dependent GLUT4 translocation and glucose uptake in L6 cells (5). The involvement of aPKC was further established by expressing CA and DN PKCA, mutants in 3T3L1 adipocytes, where CA PKCA, induced an insulin-like effect on GLUT4 and DN PKCX inhibited insulin-induced GLUT4 movement to the PM (71). Overall, it remains possible that aPKCs work in parallel with Akt, potentially alleviating the effect of Akt inhibition on insulin-mediated GLUT4 translocation. 3.3.3 Cdc42 is activated in response to insulin in an Akt-dependent manner Our results demonstrate that Cdc42 is activated in response to insulin in L6 cells. This is a novel finding and opens a new signalling arm to explore. Even more intriguing is that this activation is Akt-dependent as was demonstrated using the Akti 1/2, which inhibited insulin-dependent Cdc42 activation. The activation of Cdc42 or its position downstream of Akt does not necessarily imply that Cdc42 is important in the context of GLUT4 translocation. This possibility is something we intend to examine in the future. 58 However, theoretically, there are a few potential functions of activated Cdc42 in the insulin signalling cascade. Cdc42 activation downstream of Akt has been observed in THP-1 cells in response to anti-inflammatory lipoxin A4 (131). This study suggested that Akt-activated Cdc42 plays a role in the regulation of the actin cytoskeleton in response to lipoxin A4 in the process of phagocytosis (131). However, from our studies it seems unlikely that Cdc42 directly participates in insulin-induced actin remodelling, since this event is Akt- independent. Interestingly, Cdc42 has been shown to control the localization of the Rac- dependent membrane protrusions in primary rat embryonic fibroblasts (REF) (18). In the process of cell migration, REFs form actin-based protrusions along the leading edge of the cell. After the expression of DN Cdc42 or the WASP-CRIB domain (it functions as a dominant-interfering mutant), while still maintaining the ability to form actin-rich membrane protrusions, membrane ruffles were no longer localized to the leading edge of the cell and could be found throughout the cell periphery (18). In the same study, DN Rac mutants inhibited both the localized membrane protrusions at the REF's leading edge and the delocalized protrusions when coexpressed with WASP-CRIB (18). Hence, this leaves the possibility that although Cdc42 inhibition (via the inhibition of Akt) did not affect the overall ability of L6 muscle cells to form membrane ruffles, it may have affected their cellular localization and function. A recent study has also implicated Cdc42 in the activation of an actin severing protein, cofilin (56). This is a very novel concept, since the role of Rac/Cdc42 in cofilin inactivation via LIMK has been well established (39). Cofilin phosphorylation level, hence the level of inactive protein, was markedly increased in Cdc42 knockout neurons compared to wild-type (39). It appeared that cofilin-specific 59 phosphatases were inhibited in the cortical extracts of Cdc42 knockout mice, thus explaining the increase in phosphorylated cofilin levels (39). Cofilin is activated in response to insulin in L6 muscle cells and the siRNA-mediated knockdown of this protein reduced insulin-mediated GLUT4 translocation (unpublished observation by Patel and Klip). The newly acquired result of Cdc42 activation after insulin treatment and the potential ability of Cdc42 to activate cofilin provides a potential model for this pathway. Interestingly, in addition to actin remodelling, Cdc42 is also implicated in microtubule dynamics and interaction/activation of aPKC X/C, via the Par complex. There is a growing body of evidence implicating microtubules in insulin-stimulated GLUT4 traffic. A number of studies have demonstrated the importance of intact microtubules for insulin-induced GLUT4 translocation (23, 50, 120). Furthermore, insulin stimulates a PI3K-aPKCX,-dependent KIF3 motor protein binding to microtubules, and the microinjection of anti-KIF3 antibodies prevents GLUT4 exocytosis (71). Cdc42 is implicated in controlling microtubule dynamics and determining the orientation of the microtubule-organizing center (18,44). One of the ways in which Cdc2 regulates microtubules is by activating PAK65, a member of the p21 -activated kinase family, which phosphorylates and inactivates the microtubule destabilizing protein, stathmin (15, 30). Interestingly, PAK65 is phosphorylated in response to insulin in L6 myotubes and this phosphorylation is PI3K- dependent, potentially placing Cdc42 upstream of PAK65 in this signalling cascade (156). Cdc42 can also affect microtubule dynamics via its interaction with the Par complex (18,44). The polarity Par complex consists of Par-3, Par-6 and aPKC, which function in various cell polarization events (145). There is evidence that activated Cdc42 binds to the Par complex via Par-6, changing its conformation and leading to aPKCs activation (18, 55). aPKC can then phosphorylate downstream glycogen synthase kinase 3p (GSK3(3), which participates in regulating microtubule dynamics (43). Interestingly, the expression of CA Cdc42 can change the localization of aPCKX, from nuclear to cytoplasmic in NIH3T3 cells, offering another possible point of crosstalk between these two proteins (27). Overall, there are many possible roles for activated Cdc42 in insulin-mediated GLUT4 translocation. The determination of whether it is regulating the actin or microtubule cytoskeleton or regulating signalling of aPKCs will be the focus of our future work. 61 3.4 Future Directions 3.4.1 Independence of Rac and Akt signalling The results from this study indicate that Akt is not upstream of insulin-dependent actin remodelling or insulin-dependent Racl activation. However, the question remains whether an acute inhibition or activation of Racl will affect Akt signalling as well as other insulin-mediated events. One possible method to test this involves using a Tat- GTPase fusion protein (163). Tat is a 36 amino acid fusion protein that can be efficiently absorbed into all cell types in a receptor-less fashion with the results seen within 15-60 min (139). Using Tat-GTPase fusion protein will allow us to use DN and CA mutants in an acute fashion. This can be a great strategy to examine the function of Racl in L6 cells without any chance of compensation from other proteins or pathways. 3.4.2 Temporal and spatial activation of Rac and Cdc42 So far it has been determined that Racl and Cdc42 get activated in L6 cells (76). The time course of Racl activation has been partially determined by performing the Rac- activation assay at different intervals of insulin treatment (73, 76). Racl activation was seen as early as one minute after insulin treatment (73) (Figure 1.5). However, there is no information about the localization of the activated Rho GTPases in muscle cells after insulin stimulation. Furthermore, since both the localization and activation of Rho GTPases is highly dynamic, it is necessary to examine activated Rac and Cdc42 in live cells to have a full grasp of their regulation and function. A way to examine the localization of activated Racl in living cells is to use a bimolecular FRET (fluorescence resonance energy transfer)-based probe (128). This probe works by expressing a GFP- 62 Rac fusion protein and microinjecting into cells a PAK-CRIB domain that is labelled with an acceptor dye (Alexa-546) (92). Since CRIB-PAK binds selectively to GTP-bound Rac, FRET occurs only when the two proteins bind, thus resulting in decreased GFP emission and increased Alexa-FRET emission (92). Another method exists for the detection of activated endogenous Cdc42 in live cells, as described by Nalbant et al. (112). It involves expressing a domain of WASP that is covalently labelled with a dye and binds the GTP-bound Cdc42. The binding of endogenous activated Cdc42 to the labelled WASP protein increases the fluorescent intensity of the dye allowing for easy detection of activated Cdc42 in live cells (112). The use of these two probes will allow us to map the time course of activation and the localization of these Rho G proteins in the presence of insulin in L6 cells. 3.4.3 Cdc42 contribution to the insulin-mediated GLUT4 translocation Much remains unknown about the function of Cdc42 in the insulin signalling cascade that leads to GLUT4 translocation. A way to address the function of this protein is to use a set of Cdc42 mutants: WT, DN and CA. Since L6 cells do not have a very high efficiency of transfection with cDNA, this method will only allow us to perform single- cell assays, so insulin-induced GLUT4 translocation will have to be determined from the detection of cell surface myc epitope by fluorescence confocal microscopy. We will also be able to examine the role of Cdc42 in insulin-mediated actin remodelling by using rhodamine-phalloidin staining of the F-actin followed by confocal microscopy. Expressing these mutants in L6 cells will allow us to examine the potential contribution of functional Cdc42 to the insulin-dependent processes in muscle cells. Chapter 4: FLNc in insulin signalling 63 64 4.1 Summary As stated in the Background section, FLNc is a muscle-specific actin binding protein that has an ability to cross link actin into orthogonal networks. FLN has been shown to interact with a number of different signalling molecules, as well as be involved in a variety of signalling cascades, actin remodelling and protein traffic. It has been identified as an in vitro Akt substrate, it localizes to the remodelled actin structures upon growth factor treatment and a study from our lab identified it as a protein that interacts with GLUT4 in response to insulin. All of this evidence suggested a possible role for FLNc in insulin-induced GLUT4 traffic, potentially functioning as a signalling scaffold. In the present study we confirmed that FLNc is highly expressed in L6 cells and gets phosphorylated following insulin treatment. Furthermore, we demonstrate that FLNc is an in vivo Akt substrate, since its phosphorylation is inhibited by a specific inhibitor of Akt. Unfortunately, we were unable to elucidate the exact function of FLNc phosphorylation, except that it is unlikely important for actin remodelling. siRNA- mediated protein knockdown was used to examine FLNc function in the insulin signalling cascade and its involvement in GLUT4 translocation. Our results suggest that FLNc is not needed for insulin-induced GLUT4 translocation, actin remodelling and Racl activation. It is possible that FLNc functions only as a structural protein at the muscle Z-disk and the sarcolemma. However, it will be necessary to use another experimental approach, such as dominant-interfering FLNc mutant, to fully establish FLNc function in insulin-signalling. 65 4.2 Results 4.2.1 FilaminCin L6 cells FLNc, an actin crosslinking protein, was identified as an in vitro target of Akt phosphorylation and was coimmunoprecipitated with GLUT4myc in muscle cells ((111); unpublished observation). This experimental evidence, as well as the characteristic role of FLN as a signalling integrator, renders FLNc as a potential scaffolding protein in the insulin signalling cascade that leads to GLUT4 exocytosis. The initial step of the study was to confirm the expression of FLNc in the undifferentiated myoblasts and differentiated myotubes. FLNc expression was determined by resolving equal amount of cell lysates (determined as (xg of protein) by SDS-PAGE followed by immunoblotting with FLNc-specific antibodies. Furthermore, FLNc expression in each sample was normalized to the expression of lactate dehydrogenase (LDH), which is not affected by cell differentiation. L6 cells express significant levels of FLNc at both stages of developments (Figure 4.1a). However, FLNc expression is upregulated in L6 myotubes (1.4 ± 0.2, p < 0.01) compared to myoblasts (1.0 ± 0.2, p < 0.01) (Figure 4.1b). The upregulation of FLNc expression in L6 myotubes is consistent with its functional role in muscle cell differentiation and the maintenance of the elongated myotube structure (29). A) MB (WT FLNc - 280 kDa LDH I w^"**** -~ 140 kDa B) c •—oi CO re s CO a 2 UXi (0 _l o o _zi u. a> •a > N 9 T5 ^ •E_ o z Figure 4.1: FLNc is expressed in L6 myoblasts and myotubes. A) Expression of FLNc in L6 myoblasts (MB) and myotubes (MT). Total cell lysates were resolved by SDS- 7.5% PAGE and immunoblotted with anti-FLNc and anti-lactate dehydrogenase (LDH) antibodies. LDH served as a loading control. B) Quantification of FLNc expression in L6 cells. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. Values were also normalized to one MB value. The quantified values represent the means ± SE, *p < 0.01 vs MB, n = 3. 4.2.2 Filamin C phosphorylation Murray et al. identified the residue of FLNc that gets phosphorylated by Akt in vitro as a serine 2213. Moreover, they demonstrated that this same residue gets phosphorylated in C2C12 myoblasts in response to insulin (111). To confirm FLNc phosphorylation in vivo in L6 muscle cells, cells were treated with and without 100 nM insulin. Cells were lysed and the total cell lysates were resolved by SDS-PAGE and followed by immunblotting with anti-phospho FLNc antibodies, which detect a phosphorylation on the serine 2213 residue. In vivo FLNc phosphorylation increased 3.6 ± 1.1 (p < 0.05) fold compared to control (untreated) cells (Figure 4.2). There are other stimuli that are known to induce GLUT4 translocation in muscle cells, namely 2,4- dinitrophenol (DNP) mitochondrial uncoupler and high K+ solution that depolarizes the 67 PM. Unlike insulin, these two stimuli use alternative signalling pathways and do not require actin remodelling (134,172). Interestingly, neither DNP nor high K+ treatments increased FLNc phosphorylation above basal levels, confirming that FLNc phosphorylation is specifically due to insulin treatment (Figure 4.2). A) K+ DNP insulins' Insulin 10' Stimuli + - + - + - + pFLNc ACFN1 B) •a 0) 10 +•*m £• ra o £1 I 1 H 2 firm B K+ B DNP B INS Figure 4.2: FLNc is phosphorylated in response to insulin, but not in response to other stimuli that induce GLUT4 translocation. Cells were serum-deprived for 3 hours. Cells were then either left untreated or were treated with 100 nM insulin for 10 min, or 0.5 mM DNP for 20 min, or 120 mM K+ gluconate for 20 min. Total cell lysates were resolved by SDS-7.5% PAGE and immunoblotted with anti-phospho FLNc and anti-a- actinin-l(ACTNl) antibodies. ACTN1 is a loading control. B) Quantification of FLNc phosphorylation by different stimuli. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. The values were also normalized to the respective basal within each treatment. The quantified values represent the means ± SE, *p < 0.05 vs corresponding basal, n = 3. Having established that FLNc is phosphorylated in response to insulin, it was interesting to examine the localization of the phosphorylated protein in L6 myoblasts 68 with respect to F-actin and GLUT4. Permeabilized L6 myoblasts were stained for phosphorylated FLNc, F-actin and GLUT4myc as described in Material and Methods. This was followed by treatment with fiuourophore-conjugate secondary antibodies and confocal microscopy. As can be seen in Figure 4.3, insulin induced the formation of membrane ruffles at the cell periphery, within which there was a clear colocalization of GLUT4myc, phosphorylated FLNc and F-actin. The colocalization is visualized as white in the merge image, thus suggesting a close proximity of these proteins within the resolution of fluorescence microscopy (Figure 4.3). pFLNc Actin GLUT4myc Merge Figure 4.3: Phosphorylated FLNc localizes to the insulin-induced membrane ruffles with GLUT4 and F-actin. Representative image of L6 myoblasts in the basal and insulin-stimulated conditions. Cells were serum-deprived for 3 hours and then treated with or without 100 nM insulin for 10 min. The samples were fixed and permeabilized as described in Materials and Methods. Actin filaments are stained with rhodamine- phalloidin, phosphorylated FLNc is stained with anti-phospho FLNc antibodies and GLUT4myc is stained with anti-myc antibodies followed with fluorophore-conjugated secondary antibodies (Alexa 488 and Cy5 for pFLNc and GLUT4myc, respectively). Shown are representative cells for each treatment, n - 3. 69 In the next step of the study we were interested in determining the position of FLNc in the insulin signalling cascade in vivo. Firstly, we wanted to confirm the position of FLNc downstream of PI3K in L6 muscle cells. We used two pharmacological inhibitors of PI3K, wortmannin and LY294002. The concentrations of LY294002 and wortmannin used in this study inhibit only the class IA of PI3K, of which the main product is PIP3 (20, 36, 72). PI3K inhibitors led to an almost complete inhibition of insulin-dependent FLNc phosphorylation (Figure 4.4). The inhibition of insulin-induced FLNc phosphorylation was more efficient with wortmannin than with LY294002. However, this experiment needs to be repeated and quantified to determine if there was a significant difference between the two PI3K inhibitors. Nevertheless, FLNc phosphorylation is clearly affected by the inhibition of class IA PI3K. UNT LY294002 Wortmannin Insulin -+- + - + Figure 4.4: FLNc phosphorylation is inhibited by PI3K inhibitors LY294002 and wortmannin. Cells were serum-deprived for 3 hours. Cells were either left untreated or were pretreated for 20 min with 100 nM and 25 uM of wortmannin and LY294002, respectively (20, 36, 72). Cells were then either left untreated or were treated with 100 nM insulin for 10 min. Total cell lysates were resolved by SDS-7.5% PAGE and immunoblotted with anti-phospho FLNc and ACTN1 antibodies. ACTN1 is a loading control, n = 2. Secondly, it was important to verify whether FLNc is an Akt substrate in vivo. For this purpose, we used Akti 1/2, a specific allosteric inhibitor of Akt 1 and Akt 2 previously described by Zhao et al. and tested in the present study (Figure 3.1) (176). 70 Akti 1/2 led to a near complete inhibition of insulin-stimulated FLNc phosphorylation (1.9 ± 0.3, p < 0.05) normalized to ACTN1 expression, compared to cells treated with insulin alone (5.0 ± 0.6, p < 0.05) (Figure 4.5). A) Akti-1/2 Akti-1/2 UT DMSO [iuM] [IOUMJ Insulin + + - + - + pFLNc • -_ ---^-r-T-^ -^-=; ~~r ..^. • .^.J ACTN1 ^^ f%57$v£ M* .,^ £?&*• --*r.+ij B) IUNT I DMSO lAkti 1/2 Basal Insulin Figure 4.5: FLNc phosphorylation is inhibited by Akti 1/2. A) FLNc phosphorylation in response to 100 nM insulin treatement for 10 min in untreated (UNT), DMSO (vehicle) or Akti 1/2 (1 or 10 uM) treated cells. Cells were serum-deprived for 3 hours and then pretreated with or without the above-listed inhibitor, this was followed by incubation with or without 100 nM insulin for 10 min. Total cell lysates were resolved by SDS-7.5% PAGE and immunoblotted with anti-phospho FLNc and ACTN1 antibodies. ACNT1 is a loading control B) Quantification of inhibition of FLNc phosphorylation by Akti 1/2. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to ACTN1. The values were also normalized to the one of the vehicle-treated basal values. The quantified values represent the means ± SE, #p < 0.05 vs corresponding basal, *p < 0.05 vs insulin-treated control, n = 3. 71 Interestingly, inhibition of FLNc phosphorylation (via the inhibition of Akti 1/2) did not prevent insulin-induced actin remodelling in L6 myoblasts (Figure 3.2). This result suggests that the phosphorylation of FLNc in response to insulin is not required for actin remodelling. 4.2.3 Filamin C function in insulin signalling Having established that FLNc is expressed in L6 cells and is strongly phosphorylated by Akt in response to insulin, the next aim of the study was to examine the function of FLNc in this signalling cascade. For this purpose, the expression of FLNc was decreased using siRNA-mediated protein knockdown. The siRNA sequence was acquired from the study by Dalkilic et al. that examined the structural consequences of FLNc deficiency in muscle cells (29). Since FLNc is an abundant structural protein, it was difficult to achieve a high degree of protein knockdown. However, using the differentiated L6 myotubes and siRNA at 200 nM concentration, FLNc expression level was decreased to 45 ± 10 % (p < 0.05) of that seen in cells treated with a non-related (NR) siRNA sequence (Figure 4.6). A) NR FLNc Insulin + + 72 B) 1.00-1 *,OJH I~0.25H 0.00- NR FLNc Figure 4.6: FLNc expression is decreased using siRNA. A) Myotubes were transfected with 200 nM siRNA for FLNc or a non-related (NR) sequence for 48 hr or left untreated (UNT). Cells were serum-deprived for 3 hours. Cells were then treated treated with 100 nM insulin for 10 min. Total cell lysates were resolved by SDS-7.5% PAGE and immunoblotted with anti-phospho FLNc and ACTN1 antibodies. ACTN1 is a loading control B) Quantification of FLNc knockdown. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. The values for insulin-treated samples were also normalized to the NR siRNA-treated cells within each experimental repeat. The quantified values represent the means ± SE, *p < 0.05 vs NR insulin-treated, n = 4. We next examined the effect of such partial knockdown of FLNc on selected markers of insulin signalling cascades, specifically Akt, ERK1/2 and JNK. As described above, Akt is important in transducing the metabolic effects of insulin, specifically GLUT4 translocation in muscle and adipose tissue (152). On the other hand, ERK1/2 and JNK are both members of the MAP kinase family. They are activated in response to insulin, downstream of Ras GTPase and are responsible for gene transcription regulation and cell proliferation (154). Interestingly, insulin-induced Ras activation was abolished after disruption of the actin cytoskeleton, suggesting that intact actin and possibly actin remodelling are necessary for the proper signalling to occur (155). Hence it was interesting to examine whether or not the knockdown of cytoskeletal protein, FLNc, will 73 have an effect on the transduction of either signals. Suprisingly, FLNc siRNA did not affect insulin-induced activation of Akt, ERK 1/2 or JNK as compared to cells treated with NR siRNA (Figure 4.7). A) NR FLNc Insulin + . + MW pFLNc *-. ' "-.. 280 ACTO1 __ 100 pAkt 65 pJNK m m 50 . 45 pERK m • B) 6- NR CC 5-\ < _ i FLN TJ "O o -a Q •u •o 0) 20n INR fr £ T IFLNC £ 3 g * 10- N 0) — .£ E 15 s JD o- Basal Insulin D) •o 12.5n te d ra re NR £C 10.0H FLNc fr +-> X .oe c a X 8* 7.5H •c z P? "•-» •- Z 5.0H •o o N 0> 2.5H ^(0 > I t=l or m 0.0- , rel a z ~' Basal Insulin Figure 4.7: FLNc siRNA does not affect insulin-induced Akt, ERK 1/2 or JNK phosphorylation. A) Myotubes were transfected with 200 nM siRNA for FLNc or a non- related (NR) sequence for 48 hr. Cells were serum-deprived for 3 hours. Cells were then treated treated with or without 100 nM insulin. Total cell lysates were resolved by SDS- 7.5% PAGE and immunoblotted with anti-phospho FLNc, anti-phospho ERK1/2, anti- phospho-Akt (serine 473), anti-phospho-JNK and ACTN1 antibodies. ACTN1 is a loading control B) Quantification of Akt phosphorylation. C) Quantification of ERK1/2 phosphorylation D) Quanification of JNK phosphorylation. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. All the values were normalized to the NR siRNA-treated cells (basal). The quantified values represent the means ± SE, *p < 0.05 vs corresponding basal, n = 3. Our original hypothesis was that FLNc might function as a signalling scaffold, bridging Akt-dependent signalling and Racl-dependent actin remodelling. To test this 75 hypothesis, it was crucial to examine the effect of FLNc siRNA on Racl activation and Racl-mediated actin remodelling. Racl activation was examined by pulling down GTP- bound Rac (refer to Materials and Methods). Cells treated with siRNA to FLNc maintained a significant increase in insulin-induced Racl activation (1.5 ± 0.1, p < 0.05) compared to the FLNc siRNA-treated cells in the basal conditions (0.3 ± 0.1, p < 0.05). Although there was a slight decrease in insulin-induced Racl activation with FLNc siRNA treatment (1.5 ± 0.1) compared to NR siRNA (2.2 ± 0.4), this did not reach statistical significance (Figure 4.8). A) NR FLNc Insulin - + - + pFLNc Rac-GTP — — RacTCL ——* .*- B) 3n # INR 1,1. x I FLNc Basal Insulin 76 Figure 4.8: FLNc siRNA does not affect insulin-induced Racl activation. A) Endogenous activated Racl was detected using Racl pull down assay described in Materials and Methods. Myotubes were transfected with 200 nM siRNA for FLNc or a non-related (NR) sequence for 48 hrs. Cells were serum-deprived for 3 hours, and then treated with or without 100 nM insulin for 10 min. Pulled down proteins were resolved on SDS-10% PAGE and immunoblotted with anti-Racl antibodies. Total cell Racl was used as a loading control. B) Quantification of Racl activation after FLNc knockdown. Immunoblots were scanned within the linear range, quantified by Image J software and normalized to the loading control. The values were also normalized to one of the NR siRNA basal. The quantified values represent the means ± SE, *p < 0.05 vs corresponding basal, p < 0.01 vs corresponding basal, n = 4. Consistent with this, insulin-induced actin remodelling was unaffected by FLNc siRNA treatement (Figure 4.9). Actin remodelling was evaluated in permeabilized myotubes stained with rhodamine-phalloidin followed by confocal microscopy, and was seen as dorsal membrane ruffle formation. A) NR siRNA FLNc siRNA Insulin - + - + 77 B) (0 •£ 40- ^ 3 fl) l- 30- o Ifc- 20- 4-o1 10- oc o ft- a> u-« a. Figure 4.9: FLNc knockdown does not affect insulin-induced actin remodelling. A) Representative image of actin remodelling detected using immunoflourescence in L6 myotubes. Myotubes were transfected with 200 nM siRNA for FLNc or a non-related (NR) sequence for 48 hrs. Cells were serum-deprived for 3 hours and treated with or without 100 nM insulin for 10 min. The samples were fixed and permeabilized as described in Materials and Methods. Actin filaments are stained with rhodamine- phalloidin, total FLNc is stained with anti- FLNc antibodies followed with fluorophore- conjugated secondary antibodies (Alexa 488). Arrows point to peripheral membrane ruffles on cells. B) Quantified number of cells with positive ruffles after insulin-treatment in NR and FLNc siRNA treated cells. Shown are representative cells for each treatment, n = 3 The last aim of our study was to determine whether or not FLNc plays a role in insulin-induced GLUT4 translocation. Since the L6 cells used in this study stably overexpress GLUT4 with a myc epitope inserted in the first extracellular loop, this allows for an easy detection of the myc in unpermeabilized cells. In this case, myc is representative of the number of transporters fused with the PM (167). FLNc siRNA did not affect insulin-responsive GLUT4 translocation to the PM (2.3 ± 0.5, ns) as compared to cells after NR siRNA treatment (2.9 ± 0.3, ns) (Figure 4.10). 78 3.5-i NR :=• 3.0- ;* re FLN 3 re " X. _J -Q O o 20- u ® 1.5^ (0 .2 •g 2 1.0- W £ 0.5- 0.0- Basal Insulin Figure 4.10: FLNc knockdown does not affect insulin-stimulated GLUT4 translocation. L6 myotubes were transfected with 200 nM siRNA for FLNc or a non- related (NR) sequence for 48 hrs. After 3 hours of serum starvation cells were treated with or without 100 nM insulin for 30 min. Values were normalized to the NR-basal within each experimental repeat. Cell surface GLUT4 was measured and expressed as means ± SE, #p < 0.001, *p < 0.05, n = 5. 79 4.3 Discussion 4.3.1 Filamin C is phosphorylated by Akt in vivo Murray et ah, identified FLNc as an in vitro Akt substrate that is phosphorylated on the serine 2213 (Ser2213) residue (111). Moreover, they demonstrated that FLNc was phosphorylated on this site in response to insulin and growth factor stimulation in a PI3K-dependent manner in C2C12 myoblasts (111). The present study confirms these observations in L6 muscle cells. Furthermore, we clearly identify FLNc as an in vivo substrate of Akt, since FLNc phosphorylation in response to insulin is nearly completely inhibited after Akti 1/2 treatment. This result is further supported by the fact that FLNc remains unphosphorylated after DNP or high-K+ treatments, which induce GLUT4 translocation in an Akt-independent manner (151,172). K+-induced membrane depolarization and DNP-dependent mitochondrial uncoupling are cellular models for the study of exercise-induced GLUT4 translocation (172). Intriguingly, a recently published study by Desmukh et al. identified FLNa as a protein that gets phosphorylated in response to exercise in human skeletal muscles, detected by anti-phospho-Akt substrate antibodies, while FLNc remained unphosphorylated (34). This observation is again consistent with the results seen in the present study. High K+ or DNP-induced GLUT4 translocation does not require an intact actin cytoskeleton, which is necessary for insulin- induced GLUT4 traffic, suggesting a potential function of FLNc phosphorylation (172). However, our results indicate that FLNc phosphorylation is likely not required for insulin-induced actin remodelling, since the Akt inhibitor prevents FLNc phosphorylation, but not dorsal membrane ruffle formation in L6 myoblasts. It is important to note that insulin-induced Akt activation has many different roles in muscle cells beyond its 80 requirement for the GLUT4 translocation pathway. Other roles of Akt include: glycogen synthesis, cell growth, cell cycle and apoptosis regulation. Hence it is possible to speculate that Akt-dependent FLNc phosphorylation can be involved in one of these cellular pathways. On the other hand, this study shows that phosphorylated FLNc localized specifically to the peripheral membrane ruffles following insulin treatment in L6 myoblasts. This observation suggests that although Akt-dependent FLNc phosphorylation does not on its own direct actin remodelling and membrane ruffle formation, it might allow FLNc to localize to these structures in response to insulin. However, at this point the exact purpose of FLNc phosphorylation remains unresolved and will be the focus of future studies. 4.3.2 Filamin C in insulin-mediated GLUT4 translocation To assay the function of FLNc in the insulin signalling cascade in L6 muscle cells, FLNc expression was decreased using FLNc-specific siRNA. FLNc expression was decreased by approximately 55% in the differentiated L6 myotubes after 48 hours of transfection. However, we were unable to concretely ascertain the function of FLNc in the insulin pathway. Our results suggest that a reduction in FLNc expression does not alter insulin-induced actin remodelling, Racl activation or GLUT4 translocation. In addition, the limited FLNc knockdown attained, does not affect insulin-dependent phosphorylation of Akt, ERK1/2 and JNK kinases. The ability of actin to remodel in the absence of FLNc was the most surprising result given FLNc function as an actin cross- linker and its localization to the remodelled actin structures in L6 cells. 81 There are a few concerns with the results presented above that need to be addressed in the future. Firstly, FLNc is highly abundant in skeletal muscle cells, thus the remaining ~ 45% of the protein could be sufficient to fulfill its cellular functions, consequently not manifesting in any phenotype. Secondly, a ubiquitously expressed and highly homologous isoform of FLNc, FLNa, could hypothetically compensate for the loss of FLNc by taking over some of its native functions. However, it is also possible that FLNc functions as a structural protein with little scaffolding and signalling functions. Dalkilic et ah, demonstrated that C2C12 muscle cells deficient of FLNc, form multinucleated "myoballs" upon differentiation rather than myotubes (29). In addition, mutations in FLNc were shown to be causative for a form of myofibrillar myopathy, which is characterized by myofibrillar disintegration and cytoplasmic accumulation of Z- disk proteins (164). An alternative, more effective approach to silence FLNc will be necessary to confirm and elaborate on the results presented in this study. 82 4.4 Future directions 4.4.1 Expression of FLNc dominant-interfering mutant In the presented study, many of the experiments designed to investigate FLNc function, relied on the use of siRNA-mediated protein knockdown. However, as addressed in the discussion, the percentage of the remaining protein was high and the siRNA-treated cells did not exhibit any phenotype. Therefore, before any conclusions can be made about the function of FLNc in the insulin cascade in L6 cells, an alternative strategy needs to be used to support the above described results. A useful approach is to overexpress a segment of the wild-type FLNc protein in L6 cells. Since the expressed construct is not a full length protein, it will act as a dominant-interfering mutant, not permitting the endogenous FLNc to function properly. Using this approach, we will be able to analyze the effect of disruption of FLNc signalling on actin remodelling and GLUT4 translocation to the PM using immunofluorescent single-cell assays. 4.4.2 Expression of FLNc phosphorylation mutants A part of our study was aimed at analyzing the functional significance of the Akt- dependent FLNc phosphorylation in muscle cells. So far, we established that FLNc phosphorylation likely does not play a role in insulin-induced actin remodelling. To further examine the functional role of this phosphorylation, we intend to overexpress FLNc phosphorylation mutants in L6 cells. The phosphorylation mutants are segments of FLNc that contain the Ser2213 residue with either an alanine, to make it unphosphorylatable, or glutamate, to make it pseudo-phosphorylated, mutations. Using 83 these two mutants will hopefully shed some light on the function of FLNc phosphorylation in the insulin signalling cascade in L6 cells. Chapter 5: Conclusion 84 85 The insulin signalling cascade that directs GLUT4 translocation to the PM is of great complexity. It involves diverse signalling proteins and the cytoskeletal machinery, all of which are required for the exocytosis of vesicles containing this glucose transporter (152). Actin remodelling, a required step in GLUT4 translocation in both muscle and adipose cells, is affected by insulin resistance-causing conditions with higher sensitivity than are other insulin signalling counterparts (21, 77, 153) This emphasizes the physiological importance of studying actin dynamics as part of insulin signalling. A great deal of effort has been aimed at examining the crosstalk between the insulin-induced actin remodelling and the signalling molecules downstream of the PI3K, specifically Akt. There is increasing evidence suggesting that the two signalling pathways are mutually independent and that PI3K bifurcates into separate arms downstream (77, 168). From the results of this thesis using a specific inhibitor of Aktl and Akt2, we can for the first time confirm that Akt does not play a regulatory role in insulin-induced Racl-dependent actin remodelling. A second new finding of this thesis is that insulin leads to a rapid activation ofCdc42 in muscle cells. Moreover, although acute inhibition of Akt did not disrupt Racl activation or actin remodelling, it inhibited the activation of Cdc42. Although, the function of Cdc42 activation by insulin in L6 cells is unknown, its dependence on Akt in the insulin signalling pathway makes it an important research topic for the future. Intriguingly, our results cast doubt on the necessity of phosphorylated Akt for the increase in GLUT4 gain at the PM in response to insulin treatment. Acute inhibition of Akt did not inhibit insulin-stimulated GLUT4 translocation, a result that although is surprising can be corroborated by the lack of linearity between Akt phosphorylation and 86 GLUT4 translocation in general. Specifically, a very small amount of phosphorylated Akt is sufficient to induce a maximal GLUT4 response in 3T3L1 adipocytes following insulin treatment (67). The second part of this study was focused on examining the function of a cytoskeletal protein FLNc in the insulin signalling cascade in L6 muscle cells. FLN is a fascinating protein, implicated in many signalling pathways, often playing a role of a signal integrator (143). FLNc characteristics suggested its prospective role as a signalling scaffold in the insulin-induced GLUT4 translocation pathway in muscle cells, integrating actin dynamics and Akt signalling. In this thesis, were able to define FLNc as an in vivo Akt substrate, which gets phosphorylated in response to insulin. However, the insulin-dependent phosphorylation of FLNc does not play a role in actin remodelling, since this was qualitatively unaffected by the inhibition of FLNc phosphorylation. As well, we were unable to functionally connect FLNc to the insulin-induced GLUT4 translocation pathway. siRNA-mediated FLNc knockdown was likely insufficient to yield any functional effects on actin remodelling, Racl activation and GLUT4 translocation. Other experimental techniques will be necessary to fully elucidate the function of FLNc in the insulin signalling cascade. All the data acquired in this study points to the independence of Akt signalling and Racl-mediated actin remodelling with the absence of crosstalk or a signalling scaffold. Future experiments with acute Racl inhibition/activation as well as alternative methods of impairing FLNc function will be necessary to fully establish this signalling bifurcation. Chapter 6: References 87 1. 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