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Ionomycin-induced activation of LFA-1 is independent of calpain-mediated cleavage of talin Dreolini, Lisa Faye 2005

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IONOMYCIN-INDUCED ACTIVATION OF LFA-1 IS INDEPENDENT OF CALPAIN-MEDIATED CLEAVAGE OF TALIN by LISA FAYE DREOLINI B.Sc, University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Genetics THE UNIVERSITY OF BRITISH COLUMBIA February 2005 © Lisa Faye Dreolini, 2005 ABSTRACT Integrins are heterodimeric surface receptors that play a critical role in many cellular processes due to their ability to mediate the interactions between cells as well as between cells and the extracellular matrix. Integrin function can be regulated by conformational changes in their extracellular domains that increase their affinity for their ligand. Inside-out signalling is thought to initiate conformational changes through the binding of cytoplasmic proteins such as talin to integrin cytoplasmic domains. Calpains, calcium-dependent proteases, cleave talin upon activation, generating a free talin head domain that exhibits higher affinity for integrins than the intact talin protein. Therefore, the activation of calpains by receptor-induced calcium fluxes may lead to talin cleavage and could be an important step in the activation of integrins via inside-out signalling. In this study, the effects of the calcium ionophore ionomycin on the activation of LFA-1 in murine T cells were tested. Ionomycin treatment induced LFA-1-mediated adhesion in the helper T cell hybridoma line T28, in in vitro generated cytotoxic T cells, and in primary resting splenic T cells. The calpain inhibitors calpeptin and PD 150606 abrogated LFA-1 activation by ionomycin, implicating calpains in this process. However, calpeptin also inhibited LFA-1 activation by PMA, which did not induce calcium influx, suggesting that calpeptin is not entirely specific to calpains. Additionally, both calpain inhibitors induced the rapid apoptosis of cells treated with ionomycin, indicating that their effects on adhesion are non-specific. In contrast, two other calpain inhibitors, namely A L L N and calpain inhibitor III, did not impair LFA-1 activation by ionomycin. Western blotting did not detect cleavage of talin following ionomycin treatment of T cells, or cleavage of a-spectrin, another well-established substrate of calpains. These ii findings suggest that ionomycin-induced activation of LFA-1 is independent of the calpain-mediated cleavage of talin. iii TABLE OF CONTENTS TITLE PAGE i ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES v LIST OF ABBREVIATIONS vi ACKNOWLEDGMENTS x CHAPTER 1 Introduction 1 1.1 LFA-1 1 1.1.1 LFA-1 Structure and Function... 1 1.1.2 Regulation of LFA-1 Activation 6 1.2 Talin 10 1.2.1 The importance of talin for integrin function 10 1.2.2 Talin structural features 12 1.2.3 Regulation of talin interaction with integrins 16 1.3 Thesis objectives 20 CHAPTER 2 Materials and methods : 23 2.1 Tissue Culture 23 2.2 Antibodies 26 2.3 Production and purification of sICAM-1 28 2.4 Cell adhesion assay 29 2.5 Quantitation of intracellular free calcium 30 2.6 Detecting talin cleavage by Western blotting 30 2.7 Talin co-immunoprecipitation with LFA-1 34 2.8 Calpain cleavage assay 35 2.9 Spectrin cleavage assay 35 2.10 Apoptosis Assay 36 2.11 Rap activation Assay 36 2.12 Akt phosphorylation assay 38 2.13 Analysis of talin distribution by confocal microscopy 38 CHAPTER 3 40 3.1 The role of calpain activation and talin cleavage in ionomycin stimulation of murine T cells 40 3.2 Potential mediators of ionomycin-induced activation of LFA-1 61 CHAPTER 4 Discussion 68 APPENDIX '. 77 REFERENCES .': 81 iv LIST OF FIGURES Page Fig 1.1 Structural model of LFA-1 4 Fig. 1.2 Structural features of talin 12 Fig. 1.3 Hypothesis of the mechanism of activation by LFA-1 by ionomycin 22 Fig. 2.1 Purity of splenic resting T cell seperation and in vitro CTL culture 25 Fig. 3.1 Ionomycin treatment of T cells activates LFA-1 41-42 Fig. 3.2 Ionomycin induces intracellular calcium flux 43 Fig. 3.3 Murine talin is cleaved by both Calpain I and Calpain II 45 Fig. 3.4 Talin is not cleaved following ionomycin stimulation 46-47 Fig. 3.5 Talin head fragment does not co-immunoprecipitate with LFA-1 50 Fig. 3.6 Calpeptin inhibits both ionomycin and PM A induced LFA-1 53-54 activation Fig. 3.7 Not all calpain inhibitors prevent ionomycin-induced 55 activation of LFA-1 Fig. 3.8 Calpain cleavage in murine cells could not be detected 57 Fig. 3.9 Spectrin is not cleaved in response to ionomycin stimulation 58 in T28 cells Fig. 3.10 Calpeptin and PD 150606 induce apoptosis in a 60 dose-dependent manner Fig. 3.11 Rapl is not activated by ionomycin stimulation in T28 cells 62 Fig. 3.12 PMA activation of LFA-1 is through a PKC-dependent pathway 63 Fig. 3.13 PI3K inhibitors prevent ionomycin but not PMA induced LFA-1 65-66 activation in T28 cells Fig. 3.14 Phosphorylation of Akt is unchanged by ionomycin stimulation 67 Suppl. Fig 1 Talin consistently localizes to the contact site with sICAM but 79-80 not a-LFA-coated beads vi LIST OF ABBREVIATIONS 7-AAD 7-amino-actinomycin D APC antigen presenting cell BCA bicinchoninic acid BSA bovine serum albumin CTL cytotoxic T lymphocyte DAG diacylglycerol DMEM Dulbecco's modified Eagle medium ECL enhanced chemiluminescence EGF epidermal growth factor ERM ezrin-radixin-moesin FACS fluorescence activated cell sorter FAK focal adhesion kinase FCS fetal calf serum PERM band four-point-one/ezrin/radixin/moesin FITC fluorescein isothiocyanate FRET fluorescence resonance energy transfer GST glutathione-s-transferase HBSS Hank's balanced salt solution HRP horseradish peroxidase ICAM intercellular adhesion molecule Ig Immunoglobulin IL Interleukin JAM junctional adhesion molecule kb kilobase kDa kilodalton LAD leukocyte adhesion deficiency LFA-1 leukocyte function-associated antigen-1 mAb monoclonal antibody Mac-1 macrophage antigen-1 MIDAS metal ion-dependent adhesion site NMR nuclear magnetic resonance p70S 6 K ribosomal p70S6 kinase PBS phosphate buffered saline PE phycoerythrin PI3K phosphatidylinositol-3-OH kinase PI phosphatidylinositide PIP2 phosphatidylinositol 4,5-bisphosphate PIPKly phosphatidylinositol phosphate 5-kinase Type ly PKB protein kinase B PKC protein kinase C PMA phorbol 12-myristate 13-acetate PSI plexins, semaphorins, integrins PTB phosphotyrosine binding PTP-1B phosphotyrosine phosphatase-IB PVDF polyvinylidene fluoride RBD Rap binding domain rpm rotations per minute SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis TCR T cell receptor VLA-4 very late antigen-4 i x ACKNOWLEDGEMENTS There are many people I would like to thank for their support and encouragement throughout the research and writing stages of this thesis. First, I would like to thank my supervisor, Dr. Fumio Takei, for his excellent teaching, patience, and stimulating discussions. I am also indebted to my supervisory committee, Dr. Michael Gold and Dr. Pauline Johnson for their suggestions and critical review of my thesis. Dr. Motoi Maeda and Dr. Reza Marwali provided excellent technical assistance. The girls of the Takei laboratory (Linnea Veinotte, Nooshin Tabatabaei-Zavareh, Dr. Erica Wilson, Emily Mace, and Evette Haddad) made every day in the lab enjoyable, and I am grateful for their friendship and support. Numerous family members and friends have also provided encouragement during the tough times, and essential diversions from my studies. Finally, I would like to express my deepest gratitude for my partner Jonathan Duncan. His unwavering love and support have made this thesis possible. CHAPTER 1 INTRODUCTION 1.1 LFA -1 Integrins are well known for their ability to facilitate adhesion between cells and between cells and the extracellular matrix (Bazzoni and Hemler 1998). Leukocyte function-associated antigen-1 (LFA-1) is an integrin that is involved in the cell-cell interactions of a number of cell types of the immune system. The absence or impairment of LFA-1 results in severe immune impairment in human leading to early mortality, highlighting the importance of LFA-1 in immune function (Hogg, Smith et al. 2004). Although LFA-1 has been extensively studied, the exact mechanisms underlying LFA-1 regulation remain elusive. Recent studies have suggested that the cytoskeletal-associated protein talin may play a key role in the activation of integrins (Calderwood 2004). Therefore, I initiated my research project to investigate the role of talin in activation of LFA-1 on T cells. 1.1.1 LFA -1 Structure and Function Integrins contain two non-covalently associated, type I transmembrane glycoprotein subunits termed the a and P subunits. The extracellular domains are relatively large, while the cytoplasmic domains are short (Shimaoka, Takagi et al. 2002). LFA-1 is a member of the leukocyte-specific p2 family of integrins, and is composed of the common p2 (CD 18; 95 kDa) chain and a unique a L (CD1 la; 150 kDa) chain (Sanchez-Madrid, Simon et al. 1983). There are four P2 integrins described to date: CD1 la/CD 18 (LFA-1, aLp2), CD1 lb/CD 18 (Mac-1, aMp2), CD1 lc/CD18 (axP2), and CD1 ld/CD18 (aDp2). LFA-1 is expressed on all leukocytes including T cells (Kishimoto, Larson et al. 1989), while Mac-1, axP2, and O.DP2 are expressed primarily on cells of the myeloid lineage (Sanchez-Madrid, Nagy et al. 1983; Miller, Schwarting et al. 1986; 1 Van der Vieren, Le Trong et al. 1995). The ligands for LFA-1 are ICAM (intercellular adhesion molecule)-1 (Marlin and Springer 1987), ICAM-2 (Staunton, Dustin et al. 1989), ICAM-3 (de Fougerolles and Springer 1992; Fawcett, Holness et al. 1992), ICAM-4/Landsteiner-Wiener blood group antigen (Bailly, Tontti et al. 1995), ICAM-5/telencephalin (Tian, Yoshihara et al. 1997) and the recently identified JAM (junctional adhesion molecule)-A (Ostermann, Weber et al. 2002). ICAM-1 is the most extensively studied of the LFA-1 ligands, and is commonly used in assays to detect LFA-1 activation. It is expressed on a wide variety of cell types including endothelial cells, epithelial cells, fibroblasts, macrophages, and lymphocytes, and its level of expression is upregulated upon inflammatory stimulation (Dustin, Rothlein et al. 1986). The N-terminal region of integrin a subunits contains seven segments of approximately 60 amino acids each that have weak homology to one another and fold into a 7-bladed P-propeller domain (Springer 1997; Xiong, Stehle et al. 2001) (Figure 1.1). The p-propeller of LFA-1 contains a domain of approximately 200 amino acids known as the inserted (I) domain or von Willebrand factor A domain (Larson, Corbi et al. 1989). This domain is inserted between sheets 2 and 3 of the P propeller domain (Springer 1997), and is the ligand-binding site for integrins that contain I domains (Diamond and Springer 1993; Michishita, Viderh et al. 1993; Randi and Hogg 1994). The I domain exists in three conformations of low, intermediate, and high affinity, and these conformations are dependent on the position of the seventh a helix (Shimaoka, Xiao et al. 2003). This controls the conformational state of the magnesium-containing metal ion-dependent adhesion site (MIDAS) motif that is important for ligand binding (Lee, Rieu et al. 1995; Shimaoka, Xiao et al. 2003). The position of the a helix is controlled by interactions with an I-like domain in the p subunit, and this interaction in proposed to mediate a pull-spring mechanism where intersubunit interactions result in the downward movement of the seventh a helix during ligand binding or integrin activation (Yang, Shimaoka et al. 2004). This movement coordinates the active conformation of the MIDAS motif (Lee, Bankston et al. 1995; Shimaoka, Xiao et al. 2003; Yang, Shimaoka et al. 2004). The region C-terminal to the P-propeller consists of three p-sandwich domains, designated the thigh, calf-1 and calf-2 domains, and make up the stalk region of the a subunit (Xiong, Stehle et al. 2001). The N-terminus of the P chain of integrins contains a region of 50 amino acid residues that has been named the PSI (plexins, semaphorins, integrins) domain for its homology to membrane proteins including plexins, semaphorins and the c-Met receptor (Bork, Doerks et al. 1999). C-terminal to the PSI domain is the I-like and hybrid domains. The I-like domain shares homology with the a-chain I-domain and contains a MIDAS motif (Lee, Rieu et al. 1995). It is inserted in the hybrid domain and assumes the nucleotide-binding fold found in the a I-domain (Xiong, Stehle et al. 2001). The I-like domain appears to directly bind ligand in integrins that lack I domains in their a subunit, and to indirectly regulate ligand binding by integrins that contain I domains (Takagi and Springer 2002). C-terminal to the hybrid domain is four tandem cysteine-rich repeats that each assume the structure of a type I EGF (epidermal growth factor) fold, followed by the P-tail domain, which make up the stalk of the P subunit (Xiong, Stehle et al. 2001). Integrin a and P subunits fold to form an extracellular globular headpiece that is connected to the membrane by two stalks (Carrell, Fitzgerald et al. 1985). The globular headpiece is formed by the P-propeller domain of the a subunit and the I-like and hybrid domains of the p subunit (Xiong, Stehle et al. 2001). The ligand binding site of integrins is formed at the interface where the a subunit P-propeller domain and p subunit I-like domains make extensive contacts in the headpiece (Xiong, Stehle et al. 2002; Takagi, Strokovich et al. 3 integrin stalks are composed of a series of globular domains in the a and P subunits that incorporate a highly flexible genu/knee (Xiong, Stehle et al. 2001; Takagi, Petre et al. 2002). The complete crystal structure of an integrin containing an I domain has not been reported, so the exact position of the I domain in LFA-1 is not known. Cytoplasmic domains Dustin ML Nat. Immunol. 2004 5(4): 363-372 Figure 1.1: Structural model of LFA-1 The ligand binding site of LFA-1 is formed by a magnesium ion bound by the MIDAS motif within the I domain. The blue cylinder of the I domain is the C-terrninal a-helix, whose position controls ligand-binding affinity through interactions with the I-like domain of the P-subunit through a "bell-rope/pull spring" mechanism. The angle between the hybrid domain and the I-like domain of the p subunit may control its ligand binding status and therefore the overall ligand binding affinity of LFA-1. 4 LFA-1 is critical to a number of steps in immune surveillance and the mounting of an immune response by T cells, and was originally identified by monoclonal antibodies that block cytotoxic T cell (CTL)-mediated killing (Davignon, Martz et al. 1981). Insights into LFA-1 function have been gained by the study of LFA-1 deficient mice, as well as through the use of monoclonal antibodies that block LFA-1 function. LFA-1 is involved in the firm adherence of lymphocytes to endothelial cells, which facilitates transmigration to peripheral lymph nodes, bone marrow, and splenic white pulp. Deletion of LFA-1 does not completely abolish lymphocyte homing, due to compensation by a4 integrins including VLA-4 (very late antigen-4) (Andrew, Spellberg et al. 1998; Warnock, Askari et al. 1998; Berlin-Rufenach, Otto et al. 1999; Lehmann, Jablonski-Westrich et al. 2003; Lo, Lu et al. 2003). In the initial mounting of the immune response, LFA-1 is important for the activation of naive T cells by antigen-presenting cells (APCs) (Shier, Otulakowski et al. 1996; Bachmann, McKall-Faienza et al. 1997; Abraham, Griffith et al. 1999; Shier, Ngo et al. 1999; Abraham and Miller 2001; Kandula and Abraham 2004). The requirement of LFA-1 for full activation is mediated both by LFA-1-dependent adhesion to the APC, and by co-stimulatory signals provided by LFA-1 ligation (Kuhlman, Moy et al. 1991; van Seventer, Newman et al. 1991; Abraham and Miller 2001). The binding of CTLs to target cells is also mediated by LFA-1, and is required for target cell killing (Davignon, Martz et al. 1981; Davignon, Martz et al. 1981; Kaufmann, Golstein et al. 1982; Martz, Davignon et al. 1982; Sanchez-Madrid, Krensky et al. 1982; Krensky, Sanchez-Madrid et al. 1983; Shier, Ngo et al. 1999). The importance of LFA-1 in immune function is evident in humans with leukocyte adhesion deficiency (LAD) type I, which is a rare heritable disease, characterized by recurrent 5 bacterial infections and impaired wound healing. LAD type I patients have either defective (32 expression levels (Kishimoto, Hollander et al. 1987), or express non-functional P2 integrins (Kuijpers, Van Lier et al. 1997; Hogg, Stewart et al. 1999; Harris, Shigeoka et al. 2001; McDowall, Inwald et al. 2003). Loss of functional P2 integrins results in the absence of migration of neutrophils and monocytes to sites of inflammation. Defects in lymphocyte function have not been reported, possibly due to compensation by VLA-4 (Wehrle-Haller and Imhof 2003). Therefore, LFA-1 is essential for immune responses in humans, and plays a key role in the homing, activation and function of lymphocytes in mouse. 1.1.2 Regulation of LFA-1 Activation Circulating T cells are continually exposed to ICAM-1 and ICAM-2 on the vasculature (Hogg, Smith et al. 2004). However, LFA-1 expressed on resting lymphocytes is in an inactive state, ensuring that the cells do not bind inappropriately to their ligands and can freely circulate throughout the body. However, LFA-1 can be rapidly converted to an active state to allow for an efficient response of the cells to activation signals. Ligation of cell-surface receptors, such as the T cell receptor (TCR) or chemokine receptors, initiates intracellular signaling cascades that increase LFA-1-mediated adhesion. This is termed inside-out activation. Engagement of activated LFA-1 with ligand initiates outside-in signaling, and can influence a number of cellular responses including lymphocyte activation (Pardi, Bender et al. 1989; Van Seventer, Shimizu et al. 1990). The activation of LFA-1 by inside-out signaling is proposed to be regulated by two major mechanisms: changes in conformation, resulting in an increased affinity for ligand (Carman and Springer 2003), and clustering of molecules on the cell surface (van Kooyk and Figdor 2000). Both of these processes are components of the avidity regulation of integrins. 6 1.1.2.1 Control of LFA -1 clustering Avidity is a broad term that refers to the overall strength of adhesiveness, and encompasses a number of processes that influence interactions with ligand. It is governed by the affinity of an integrin for its ligand (discussed below), two-dimensional kinetic rates, receptor lateral mobility, receptor clustering, membrane deformability, and the effects of force (Dustin, Bivona et al. 2004). Discussions of the avidity regulation of integrins commonly focus solely on the process of integrin clustering on the cell surface. The clustering of LFA-1 is thought to be controlled by interactions with the actin cytoskeleton. Single-molecule tracking studies demonstrated that LFA-1 on resting cells is immobile on the cell surface. Treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA), which induces LFA-1-mediated adhesion, results in the increased mobility of LFA-1 molecules. Treatment with cytochalasin D, which disrupts the actin cytoskeleton, increases the mobility of LFA-1 and activates LFA-1-mediated adhesion in a B cell line (Kucik, Dustin et al. 1996). Similarly, treatment of resting peripheral blood lymphocytes with cytochalasin D increases LFA-1 -mediated adhesion to ICAM-1 and induces clustering of LFA-1 on the surface (Lub, van Kooyk et al. 1997). These observations have led to the model where non-activated LFA-1 is restrained by the cytoskeleton. Activation releases LFA-1 from the cytoskeleton, which leads to increased mobility on the cell surface and subsequent clustering (Hogg, Henderson et al. 2002). LFA-1 clustering has been detected following stimulation with a number of agonists that increase LFA-1 adhesiveness, including phorbol esters, calcium mobilizers, CD3 cross-linking, and chemokines (Lub, van Kooyk et al. 1997; Stewart, McDowall et al. 1998; Constantin, Majeed et al. 2000). These observations have led to the belief that clustering is an integral component of LFA-1 activation by a number of stimuli. However, a recent study demonstrates 7 that stimuli that activate LFA-1-mediated adhesion do not activate clustering in the absence of ligand (Kim, Carman et al. 2004). Therefore, LFA-1 clustering may stabilize integrin-ligand interactions after the integrins bind to multivalent ligands, as opposed to functioning in the initial activation of LFA-1 adhesiveness. 1.1.2.2 Affinity regulation Affinity regulation has been defined as changes in the monomeric affinity for ligand that are coupled to alterations in integrin conformation (Carman and Springer 2003). The idea that conformational changes in integrins leads to increased affinity for ligand is supported by the observation that many monoclonal antibodies bind specifically to the activated forms of integrins (Bazzoni and Hemler 1998). Global structural rearrangements in the extracellular domains of integrins have been directly visualized by electron microscopy (Takagi, Petre et al. 2002; Takagi, Strokovich et al. 2003). Integrins exist in at least three conformational states: integrins with low affinity for ligand exist in a bent conformer, with the ligand-binding headpiece folded over the legs due to a bend at the knees. Binding of manganese, which activates integrins, results in a switchblade-like opening of the integrin to an extended conformation. This conformer has a closed headpiece with the stalks close together, and is thought to represent intermediate-affinity integrin. Ligand binding produces an extended conformation with an open headpiece, with the stalks separated due to an outward swing of the (3 subunit. The crystal structure of the complete extracellular domain of avP3 confirms the low affinity and extended, closed headpiece conformations (Xiong, Stehle et al. 2001) that were observed by electron microscopy. These global conformational changes are coupled to inter and intra-domain rearrangements in the head region that stabilizes the high affinity state of the ligand 8 binding site (Shimaoka and Springer 2003; Xiao, Takagi et al. 2004; Yang, Shimaoka et al. 2004). The outward swing of the P subunit provides a potential mechanism for the transmission of signals to the cytoplasm during outside-in signaling (Takagi, Petre et al. 2002; Xiao, Takagi et al. 2004). Although the cytoplasmic domains of integrins have not been resolved by electron microscopy or crystallographic studies, the separation of the cytoplasmic tails of LFA-1 following ligand binding has been detected by FRET (Kim, Carman et al. 2003). Therefore, the outward swing of the p subunit is likely to induce the separation of the transmembrane and cytoplasmic domains of integrins, mediating outside-in signaling. How signals from inside the cell are transmitted across the membrane to activate integrin function during inside-out activation is incompletely understood, however it is thought to be mediated by the separation of integrin cytoplasmic tails. The cytoplasmic domains of integrins control their affinity state (O'Toole, Katagiri et al. 1994). Mutations that cause association of the cytoplasmic domains maintain integrins in an inactive state, while mutations that prevent their association activates integrins (Hughes, Diaz-Gonzalez et al. 1996; Lu, Takagi et al. 2001; Takagi, Erickson et al. 2001). Integrin a and P cytoplasmic tails associate through multiple electrostatic and hydrophobic contacts in their membrane proximal regions (Vinogradova, Velyvis et al. 2002). These contacts are thought to form a clasp in maintaining the receptor in a low-affinity state. Dissociation of the clasp results in the spatial separation of the a and P tails, initiating conformational changes in the extracellular domains of integrins that increase affinity for ligand (Dustin, Bivona et al. 2004). This unclasping hypothesis is supported by NMR and FRET studies that have demonstrated a disruption of the interactions between a and P tails under conditions that activate integrin 9 function (Vinogradova, Velyvis et al. 2002; Kim, Carman et al. 2003; Vinogradova, Vaynberg et al. 2004). Inside-out activation of integrins has been proposed to be mediated by the binding of proteins to the cytoplasmic tails of integrins, disrupting their association and increasing affinity for ligand (Dustin, Bivona et al. 2004). Rapl is an important signaling effector for multiple stimuli that activate pi , P2 and P3 adhesion (Bos, de Rooij et al. 2001), and controls LFA-1 activation through its recently identified effector RapL(Katagiri, Maeda et al. 2003). RapL interacts with the OIL subunit of LFA-1, and through this interaction modulates the distribution of LFA-1 on the cell surface and increases the affinity of LFA-1 for ligand. Association is dependent on lysine residues (1097 and 1099) in the a subunit that are proximal to the conserved GFFKR motif. This motif is known to stabilize a and P-subunit heterodimer formation of integrins (Hughes, Diaz-Gonzalez et al. 1996). Therefore, RapL is a candidate protein for. mediating integrin cytoplasmic tail unclasping, however this has not been directly tested. Talin is another protein that may mediate inside-out signaling of integrins by inducing the separation of cytoplasmic tails. NMR and FRET studies have directly demonstrated that talin fragments disrupt the membrane proximal interactions between the a and p subunits of multiple integrins (Vinogradova, Velyvis et al. 2002; Kim, Carman et al. 2003; Ulmer, Calderwood et al. 2003; Vinogradova, Vaynberg et al. 2004). However, the physiological relevance of this interaction is still under investigation. 1.2 Talin 1.2.1 The importance of talin for integrin function Talin is an abundant and widely expressed cytoskeletal protein, and talin binding to 10 integrin cytoplasmic tails has been proposed to act as the final common step in integrin activation (Critchley 2000; Tadokoro, Shattil et al. 2003). In Caenorhabditis elegans and DrosophUa, talin knockout embryos display a phenotype similar to that seen with integrin null embryos (Brown, Gregory et al. 2002; Cram, Clark et al. 2003), indicating that talin is required for normal integrin function in vivo. Targeted disruption of the murine talin gene is embryonic lethal, with a failure of cell migration during gastrulation (Monkley, Zhou et al. 2000), and talin_/" undifferentiated embryonic stem cells have defective focal adhesion assembly (Priddle, Hemmings et al. 1998). Therefore, the loss of talin affects the function of a number of integrins, suggesting that talin may represent a conserved mechanism for integrin activation. The analysis of talin function has been complicated by the recent identification of a second talin gene (TLN2) in mammals. The predicted human talin2 protein (2532 residues) is almost identical in size to the talin 1 protein (2541 residues) and the amino acid sequences are strikingly similar (74% identity, 86% similarity) (Monkley, Pritchard et al. 2001), suggesting that the two proteins may have identical functions. However, the expression patterns of the two genes are different. In the adult mouse TLN1 gives rise to an ~8-kb mRNA that is detected in all tissues, with high expression in the heart, liver, kidney, lung and spleen. TLN2 gives rise to transcripts of varying sizes (3.9-10.0 kb) with the highest levels in the heart and testis, lower levels in the lung and liver, and virtually undetectable expression in the spleen (Monkley, Pritchard et al. 2001). The two talin genes may be differentially expressed depending on cell type, and the TLN2 isoform expressed may not share the same functional characteristics as talin 1. The defects in embryogenesis seen with the deletion of TLN1 indicates that TLN2 cannot compensate for the loss of talinl in mesoderm migration (Monkley, Zhou et al. 2000). 11 Additional studies with antibodies that are specific to each talin protein will elucidate the contribution of each to integrin function in various cell types. 1.2.2 Talin structural features talin head ~30 kDa * • « -talin head integrin binding site (186-435) talk tail/rod ~220 kDa Calpain cleavage site (433-434) C-terminal integrin binding site (1984-2113) Santa Cruz anti-talin head Ab (H-300) 1-300 -Sigma mAb (Sd4) 4S2-636 v Santa Cruz anti-talin tail Ab (C-20) C-terminus (exact residues unknown) Figure 1.2: Structural features of talin The talin head and talin tail/rod domains within the full-length talin protein (2541 amino acids) are shown. The FERM domain (86-400) is within the head domain, and contains the integrin binding site that mediates integrin activation. The site of calpain cleavage, which separates the head domain from the rod domain, is indicated. Binding sites for a number of molecules are shown. Actin binding sites are indicated by grey boxes (102-497, 951-1327, 2269-2541). Vinculin binding sites are shown with hatched boxes (607-636, 852-876, 1944-1969). The focal adhesion kinase (FAK) (225-357) and phosphatidylinositol 5-kinase Type ly (PIPKy) (150-480) binding sites in the head domain are indicated by black and hatched ovals respectively. The regions of talin recognized by the antibodies used in this study are shown in italics. (Hemmings, Rees et al. 1996; Critchley 2000; Ling, Doughman et al. 2002; Critchley 2004) 12 1.2.2.1 Integrin binding by talin and the role of talin in integrin activation Talin contains a number of structural features that may explain its importance for integrin function (Figure 1.2). It consists of an N-terminal -50 kDa globular head domain and an -220 kDa C-terminal rod domain (Rees, Ades et al. 1990). The talin head domain contains a predicted FERM domain (band four-point-one/ezrin/radixin/moesin homology domain) (Rees, Ades et al. 1990) that mediates interactions of ezrin-radixin-moesin (ERM) proteins with the cytoplasmic tails of transmembrane proteins such as integrins (Bretscher, Edwards et al. 2002). As predicted, talin head fragments interact with the cytoplasmic tails of pi (Pfaff, Liu et al. 1998; Calderwood, Zent et al. 1999), p2 (Kim, Carman et al. 2003), and P3 (Calderwood, Zent et al. 1999; Patil, Jedsadayanmata et al. 1999; Calderwood, Yan et al. 2002; Tadokoro, Shattil et al. 2003) integrins. Talin also contains an integrin binding site within the C-terminus of the rod domain (Calderwood, Zent et al. 1999; Xing, Jedsadayanmata et al. 2001; Yan, Calderwood et al. 2001; Calderwood, Yan et al. 2002; Tremuth, Kreis et al. 2004). However this site exhibits a much lower affinity for integrin cytoplasmic tails than the head domain (Yan, Calderwood et al. 2001). The major integrin binding site in talin is within a phosphotyrosine binding (PTB)-like F3 subdomain within the FERM domain (Calderwood, Yan et al. 2002). PTB domains often recognize proteins containing P turns formed by NPXY motifs, and integrin P tails contain conserved NPXY motifs. Mutation of the tyrosine residue within the first (membrane proximal) NPXY motif in pi or P3 integrins disrupts binding of talin head fragments (Pfaff, Liu et al. 1998; Calderwood, Zent et al. 1999; Calderwood, Yan et al. 2002; Garcia-Alvarez, de Pereda et al. 2003; Tadokoro, Shattil et al. 2003). Crystallographic analysis demonstrates that the NPXY motif of P3 (744-747) forms a reverse turn, with the tyrosine residue pointing into a hydrophobic 13 pocket within the talin F3 domain (Garcia-Alvarez, de Pereda et al. 2003). Residues upstream also make important contacts with the talin F3 structure, including a highly conserved tryptophan residue (739) that is required for talin binding to P3 tails (Garcia-Alvarez, de Pereda et al. 2003; Tadokoro, Shattil et al. 2003). PTB domains were initially characterized as domains that bind to phosphotyrosines in NPXY motifs. However there are now a number of examples of phosphorylation-independent interactions (Schlessinger and Lemmon 2003). Tyrosine phosphorylation is not required for talin to bind to P tails because unphosphorylated P tail fragments bind talin (Calderwood, Zent et al. 1999; Calderwood, Yan et al. 2002). Additionally, talin binding to pi integrins is disrupted by tyrosine phosphorylation of NPXY motifs (Tapley, Horwitz et al. 1989). The pocket in PTB domains that recognize phosphotyrosines is strongly basic, whereas the tyrosine-binding pocket of the talin F3 domain is uncharged and hydrophobic (Garcia-Alvarez, de Pereda et al. 2003). This indicates that the tyrosine binding is mediated by hydrophobic interactions, and may explain how talin can bind to P2 integrins, which possess phenylanine residues in place of tyrosine in their NPXY motifs. Overexpression of talin head fragments containing the FERM domain activates P2 (LFA-1) (Kim, Carman et al. 2003) and P3 (allbp3) (Calderwood, Zent et al. 1999; Calderwood, Yan et al. 2002; Vinogradova, Velyvis et al. 2002; Tadokoro, Shattil et al. 2003) integrins. Overexpression of a talin fragment containing the C-terminal integrin binding site does not increase P3 activation (Tremuth, Kreis et al. 2004), localizing the integrin activating function of talin to the N-terminal head domain. Activation of integrins by talin head is mediated by affinity modulation (Tadokoro, Shattil et al. 2003). Talin head fragments disrupt the association of a and P cytoplasmic tails, which as previously discussed may initiate conformational changes in the 14 extracellular domains of integrins that increase affinity for ligand (Vinogradova, Velyvis et al. 2002; Kim, Carman et al. 2003; Ulmer, Calderwood et al. 2003; Vinogradova, Vaynberg et al. 2004). The separation of integrin cytoplasmic tails may be caused by conformational changes initiated by the PTB-like interaction between talin and the C-terminus of integrin p tails (Calderwood 2004). This is supported by the finding that talin head cannot activate integrins with mutations in the NPXY motif (Calderwood, Yan et al. 2002; Tadokoro, Shattil et al. 2003). NMR and biochemical analyses have demonstrated that talin also binds to a second site in P tails, in a membrane proximal region (Patil, Jedsadayanmata et al. 1999; Vinogradova, Velyvis et al. 2002; Tadokoro, Shattil et al. 2003; Ulmer, Calderwood et al. 2003; Vinogradova, Vaynberg et al. 2004), that overlaps with the region involved in interactions with a cytoplasmic domains (Vinogradova, Velyvis et al. 2002). Therefore, talin interactions with multiple regions in p cytoplasmic tails may act in concert to mediate the full separation of the a and P tails. 1.2.2.2 Actin and vinculin binding Talin contains multiple actin binding sites in both the head and rod domains (Hemmings, Rees et al. 1996) By virtue of its ability to bind to both integrin cytoplasmic domains and actin, talin might provide the link between integrins and the actin cytoskeleton (Critchley 2004). Talin also contains multiple binding sites for vinculin throughout its length (Hemmings, Rees et al. 1996). Vinculin contains a binding site for actin (Menkel, Kroemker et al. 1994), and the vinculin tail region inserts into acidic phospholipids vesicles, suggesting that it can interact with the plasma membrane (Johnson, Niggli et al. 1998). Therefore, vinculin may function to cross-15 link and therefore stabilize interactions between talin and the actin cytoskeleton, as well as talin and the plasma membrane (Critchley 2000; Critchley 2004). 1.2.3 Regulation of talin interaction with integrins In ERM proteins, the FERM domain is a globular structure that can be completely masked by interactions with the extended C-terminal tail (Pearson, Reczek et al. 2000). In this "dormant" form, the C-terminal actin and N-terminal membrane protein binding sites are unable to bind ligand (Bretscher, Edwards et al. 2002). The talin FERM domain may also be similarly masked, with talin existing in a dormant state until activated. Although these intramolecular interactions have not been directly detected for talin, talin exists in an equilibrium between monomers and dimers, with the monomeric form taking a globular shape (Molony, McCaslin et al. 1987). In the dimeric form, talin forms antiparallel homodimers (Goldmann, Bremer et al. 1994; Isenberg and Goldmann 1998) which has also been detected with ERM proteins (Bretscher, Edwards et al. 2002). This suggests that the tertiary structure of talin resembles that adopted by ERM proteins, and that FERM masking may occur in talin as well. This concept is further supported by the finding that the cleavage of talin by calpains in vitro separates the head domain from the rod and increases the affinity of talin for P3 cytoplasmic tails (Yan, Calderwood et al. 2001). Therefore, the interaction of talin with integrin cytoplasmic tails may be regulated by controlling the unmasking of the talin head domain. There are two models that have been proposed to explain how the talin head is unmasked so that it can mediate binding to integrin cytoplasmic tails. In the first model, the full length talin protein is regulated by conformational changes, in a similar manner as other ERM proteins. In the second model, calpain-mediated proteolysis of the native talin protein separates the talin 16 head from the talin rod domain, and the masking of the talin head is therefore relieved by the physical separation of the two domains. 1.2.3.1 Regulation by PIP2 and phosphorylation Two pathways contribute to ERM protein unmasking and activation: phosphorylation of a C-terminal threonine residue in the tail, and interactions with phosphatidylinositides such as phosphatidylinositol 4,5 bisphosphate (PIP2) (Bretscher, Edwards et al. 2002). Threonine phosphorylation weakens the FERM/tail interaction (Matsui, Maeda et al. 1998), and in the presence of phospholipids, unmasks the membrane protein and F-actin binding sites of ERM proteins (Simons, Pietromonaco et al. 1998). Phosphoinositides including PIP2 are required to unmask the F-actin binding site of phosphorylated moesin in vitro (Nakamura, Huang et al. 1999), indicating that both events are required for full ERM protein activation. PIP2 binding to talin enhances the interaction with pi cytoplasmic tails in vitro, and PIP2 is required for proper localization of talin in focal adhesions (Martel, Racaud-Sultan et al. 2001). This suggests that talin may be regulated in a manner analogous to other ERM proteins. Interestingly, talin also binds PIPKly (Ling, Doughman et al. 2002; Barsukov, Prescot et al. 2003), an enzyme responsible for PIP2 synthesis (Anderson, Boronenkov et al. 1999). The binding of PIPKy to talin is adhesion-dependent, and is stimulated by the phosphorylation of PIPKly by FAK. Binding of PIPKly to talin activates its kinase activity in vitro (Di Paolo, Pellegrini et al. 2002). PIPKly interaction facilitates the membrane targeting of talin, and PIPKly activity is crucial for talin recruitment into focal adhesions (Ling, Doughman et al. 2002). 17 1.2.3.2 Regulation of talin by calpain-mediated cleavage 1.2.3.2.1 Calpain activation There are two ubiquitous calpain isozymes, u-calpain (Calpain I) and m-calpain (Calpain II), which are active at micromolar and millimolar calcium (Ca2+) concentrations, respectively. Exposure to calcium at concentrations of 5-50 uM (u-calpain) and 200-1000 uM (m-calpain) activates both calpains in vitro (Reverter, Sorimachi et al. 2001). Calpain I and II are composed of a distinct large catalytic (80 kDa) subunit, and a common small regulatory (30 kDa) subunit. Crystallographic studies have shown that binding of two Ca 2 + ions within the proteolytic core of the large subunit induces conformational changes that align the active site of u-calpain. The conformation of the active site is also regulated by constraints imposed by the three-dimensional structure of the calpain molecule; calcium-dependent conformational changes in regions in both the large and small subunits have been proposed to alleviate these constraints (Moldoveanu, Hosfield et al. 2002). Calpains were initially thought to be activated by increases in intracellular calcium, however the calcium concentrations required to activate calpains in vitro are not generally attainable under physiological conditions (Glading, Lauffenburger et al. 2002). Consequently, several mechanisms have been proposed to lower the calcium requirement of calpains. Binding of phospholipids such as phosphatidylinositol (PI) or PIP2 decreases the Ca 2 + requirement for calpain activation in vitro. Calpains autolyse in the presence of calcium, and the shortened 2_|_ protein requires a lower Ca concentration for activation than the intact one. Multiple activator proteins have been identified for both u- and m-calpain that decrease the concentration of Ca required for activation in vitro. Finally, phosphorylation of both calpains has been detected, and 18 is another potential activation mechanism for lowering the threshold of activation. Most of these studies have been performed in vitro, so further investigation is needed to determine if they are relevant in vivo. Besides these mechanisms, calpain activity is also regulated by the endogenous inhibitor calpastatin, which binds to and inactivates calpains through each of its four repetitive inhibitory domains. The release of calpain from calpastatin correlates with increased calpain activity, although this is not sufficient for activation (Glading, Lauffenburger et al. 2002; Goll, Thompson et al. 2003). 1.2.3.2.2 Calpains and integrin function Although the large subunits of each calpain isozyme are unique, the substrate specifities of u- and m-calpain are very similar if not identical (Croall and DeMartino 1991). Both calpains can cleave a number of proteins involved in cytoskeletal regulation, including talin, ezrin, vinculin, spectrin, filamin, and a-actinin, among many others. As a result, calpain has been proposed to be an important regulator of cell spreading and migration (Glading, Lauffenburger et al. 2002; Goll, Thompson et al. 2003). Recently, attention has focused on the possibility that calpain may be involved in the initial activation events of integrins as well. Calpain I and II cleave talin in vitro and in vivo (cell culture) to yield two pieces, a ~47-kDa head and a ~200-kDa tail fragments (Oda, Druker et al. 1993; Inomata, Hayashi et al. 1996; Calderwood, Zent et al. 1999; Hayashi, Suzuki et al. 1999; Kulkarni, Saido et al. 1999; Yan, Calderwood et al. 2001; Liu and Schnellmann 2003; Franco, Perrin et al. 2004). As discussed above, the separation of the talin head domain from the rest of the polypeptide by calpain cleavage increases the affinity for integrins, and overexpression of 19 talin head fragments activates integrins. This suggests that the talin head fragment generated by calpain cleavage may be an important regulator of integrin activation. Treatment of T cells with the Ca mobilizers, ionomycin, 2,5-di-t-butylhydroquinone and thapsigargin activates LFA-1-mediated cell adhesion. Furthermore, activation of LFA-1 by thapsigargin-induced calcium fluxing is prevented by pre-treatment with the calpain inhibitor calpeptin, suggesting that activation of calpains may be responsible for LFA-1 activation. Although it was suggested that activated calpains might cleave talin, this was not directly tested (Stewart, McDowall et al. 1998). Calcium fluxing accompanies physiological stimulation of T cells through the TCR or by chemokine receptors (Wolff, Hong et al. 1993; Sanchez-Madrid and del Pozo 1999). Therefore, this finding supports the idea that calpain-mediated generation of talin head may represent a physiological mechanism of talin regulation in T cells. 1.3 Thesis objectives As discussed above, there is an abundance of data that implicates talin as a key regulator of integrin activation. However, how talin is regulated is not understood. Two models have been proposed: conformational changes induced by phosphorylation and PIP2 binding, and talin head liberation by calpain-mediated cleavage. The finding that calpain inhibitors prevent LFA-1 activation by calcium mobilizers suggests that the latter model may be relevant in T cell adhesion induced by calcium fluxing. The objective of this thesis was to test the following hypothesis (Figure 1.3): ionomycin-induced increases in intracellular calcium activate calpain proteolytic activity in T cells. Activated calpains cleave talin, resulting in the generation of the talin head fragment. This fragment binds to P2 cytoplasmic tails, activating conformational changes in LFA-1 that increase the affinity for its ligand, ICAM-1. To test this hypothesis, I 20 evaluated whether talin cleavage occurs after ionomycin stimulation in T cells, generating talin head fragments that interact with the LFA-1 (32 subunit. The effects of calpain inhibitors on ionomycin-induced LFA-1 activation were also tested, and the proteolytic activity of calpains was assessed by a-spectrin cleavage assay. Ionomycin-induced increases in intracellular calcium activated LFA-1-mediated adhesion in the T28 cell line, in resting splenic T cells, and in in vitro generated CTLs. However, the results presented in this thesis suggest that ionomycin-induced activation of LFA-1 is independent of the calpain-mediated cleavage of talin. 21 Step 1 Step 2 Ionomycin Figure 1.3: Hypothesis of the mechanism of activation of LFA -1 by ionomycin Step 1: Ionomycin treatment of T cells induces increases in intracellular calcium concentrations that activate the calcium-dependent protease calpain. Step 2: Activated calpains cleave full-length talin, separating the talin head domain from the talin tail domain. Step 3: The talin head fragment binds to the cytoplasmic tail of the P chain of LFA-1. This separates the cytoplasmic domains, initiating conformational changes in LFA-1 that increase its affinity for ICAM-1. 22 CHAPTER 2 MATERIALS AND METHODS 2.1 Tissue Culture 2.1.1 Mice C57BL/6 (B6) mice were obtained from Jackson Laboratories (Bar Harbor, ME) and HY TCR transgenic Rag T1' C57BL/10 (Rag-HY) mice were obtained from Taconic Farms (Tarrytown, NY). These mice were bred in the Joint Animal Facility of the BC Cancer Research Centre (BCCRC) according to animal welfare guidelines 2.1.2 Cell lines The murine T cell hybridoma line T28 has been described (Dang, Michalek et al. 1990), and was a gift from Dr. K.L. Rock (Dept. Path, U. Mass. Medical School). The human prostate cell line LNCaP was obtained from Dr. M . Sadar (BCCRC, Vancouver, BC, Canada). Murine fibroblast L cells were from American Type Culture Collection (ATCC, Rockville, MD). T28 and L cell lines were maintained in Dulbecco's modified Eagle medium (DMEM) plus 5% fetal calf serum (FCS), penicillin (100 U/ml)/streptomycin (0.1 mg/ml), and L-glutamine (200 uM; Stem Cell Technologies, Vancouver, BC, Canada). LNCaP was propagated in phenol red-free RPMI1640 (RPMI) medium (Gibco/Invitrogen, Carlsbad, CA) with the additions described above. 2.1.3 Primary murine T cell isolation Splenic resting T cells were isolated by the Spin Sep murine T cell enrichment kit (Stem Cell Technologies). A single cell suspension was prepared from the spleens of two to six month old male B6 mice using a 70 um nylon cell strainer (Falcon). This splenocyte mixture was processed according to manufacturer's protocol to isolate T cells, which resulted in > 92% purity (Figure 23 2.1 A) as assessed by fluorescence-activated cell sorter (FACS) analysis using fluorescein isothiocyanate (FITC)-conjugated anti-CD3s monoclonal antibody (mAb; from BD Pharmingen, Mississauga, ON). 2.1.4 Generation of CTLs To isolate dendritic cells, a single cell suspension was prepared from the spleens of two to four month old male B6 mice. Red blood cells were lysed with 0.8% ammonium chloride in 0.1 mM EDTA, and Fc receptors were blocked with 2.4G2 hybridoma supernatant (ATCC, supernatant produced in house) for 15 minutes at 4°C. Dendritic cells were labelled with 0.6 pg/ml phycoerythrin (PE)-conjugated CD1 lc antibody (BD Pharmingen) for 15 minutes at 4°C, followed by separation using the Easy Sep PE selection kit (StemCell Technologies) according to the manufacturer's instructions. A single cell suspension was prepared from the spleens and mesenteric lymph nodes of six to eight week old female Rag-HY mice. Red blood cells were lysed as above, and lymphocytes were mixed with irradiated (30 Gy) dendritic cells at a 20:1 ratio. Mixed cells were seeded at 3-4 x 106 cells/ml in a 12 well culture plate (2.5 mis per well) and grown in the presence of 20 U/ml IL-2 (Peprotech Canada, Ottawa, ON)-supplemented RPMI medium plus 10 % FCS, 2mM L-glutamine, 5 x 10"5 M P-mercaptoethanol, and penicillin/streptomycin for six days. By day 6, the culture contained 93 % T cells, as assessed by FACS using anti-CD3s-FITC staining (Figure 2.IB). On day six, CTLs were centrifuged, then resuspended in medium lacking IL-2 and grown for a further 36-48 hours before harvesting for experiments. 24 Figure 2.1: Purity of splenic resting T cell separation and in vitro C T L culture (A) Splenic resting T cells obtained by Spin Sep were analyzed for purity by flow cytometry. Grey shaded histogram indicates staining with isotype control Ab, and open histogram indicates staining with CD3s-FITC. Results shown are representative of two independent trials. (B) CTL populations were analyzed for purity by flow cytometry. Grey shaded histogram indicates staining with isotype control Ab, and open histogram indicates staining with CD3s-FITC. 25 2.2 Antibodies 2.2.1 Commercially available antibodies 2.2.1.1 Primary Antibodies Antibody name Company Species raised in Monoclonal or Polyclonal ? Dilution for Western blotting Anti-talin (8d4) Anti-talin tail Sigma (St Louis, MO) Mouse . Monoclonal •10 ug/ml Anti-talin (C-20) Santa Cruz Biotechnology (Santa Cruz, CA) Goat Polyclonal Anti-talin (H-300) Anti-talin head Santa Cruz Rabbit Polyclonal 1:200 Anti-u-calpain (CL4Calpain 1) Cedarlane Rabbit Polyclonal 1 pg/ml Anti-m-calpain (CL3Calpain2) Cedarlane Rabbit Polyclonal 1 ug/ml Anti-a-spectrin (non erythroid) (MAB 1622) Chemicon (Temecula, CA) Mouse Monoclonal 1:1000 Anti-Rap 1 (sc-65) Santa Cruz Rabbit Polyclonal 1:200 Anti-phospho Akt (Ser 473) Cell Signaling Technology (Beverly, MA) Rabbit Polyclonal 1:1000 Anti-GAPDH (6C5) Research Diagnostics, Inc (Flanders, NJ) Mouse Monoclonal 1: 15 000 26 2.2.1.2 Secondary Antibodies Antibody name Catalogue number Company Conjugated to Dilution used Anti-rat IgG 112-036-062 Jackson ImmunoResearch Laboratories, Inc (West Grove, PA) Horseradish peroxidase (HRP) 1:5000 Anti-mouse IgG 715-036-150 Jackson HRP 1: 10 000 Anti-rabbit IgG 711-035-152 Jackson HRP 1:5000 Anti-mouse IgG A-11017 Molecular Probes (Eugene, OR) Alexa-488 5 pg/ml (Confocal Microscopy) 2.2.2 Monoclonal antibody purification from hybridoma supernatants The YN1/1.7.4 (YN1) rat mAb recognizing murine ICAM-1 was generated in our laboratory (Takei 1985; Horley, Carpenito et al. 1989), and was purified previously in our lab by Jose Rey-Ladino. The rat hybridomas TIB 213 (anti-LFA-1) and TIB 218 (anti-CD 18) were from ATCC. They were grown in DMEM + 10% FCS (plus additions described in 2.1.1) for 10-13 days to overgrowth. Cultures were centrifuged at 2300 rpm for 30 minutes, followed by filtration through a 0.22 pm bottle top filter (Falcon) to remove cellular debris. TIB 213 mAb was purified by running the cleared supernatant through a Hi Trap Protein G column (Amersham Biosciences, Piscatway, NJ) at a flow rate of 1 ml/minute at 4°C. The column was then washed with 20 ml 20 mM sodium phosphate buffer (pH 7.0), and the bound protein was eluted with 0.1 M citric acid (pH 2.7) in four 1 ml fractions. Fractions were neutralized with 1 M sodium carbonate, and then diluted once with 0.2 M sodium bicarbonate (pH 8.0). The fractions were run alongside bovine serum albumin (BSA) standards on SDS-PAGE using the Mini-Protean 3 27 electrophoresis system (Bio-Rad, Hercules, CA) according to manufacturer's instructions. Coomassie staining of the gels allowed for quantitation of the purified antibody. 2.2.3 Preparation of monoclonal antibody-coupled beads YN1 was coupled to beads for use in affinity purification of soluble ICAM-1 (2.3), while TIB 213 was coupled for use in LFA-1 immunoprecipitations (2.7). The YN1 immunoaffinity column was made by mixing 2 ml of Affigel-10 Active Ester Agarose beads (Bio-Rad) with 4 mg of purified YN1 mAb in 0.1 M NaHC0 3 overnight at 4°C. TIB 213-coated beads were prepared by mixing 4 mg of purified mAb with 3 ml beads overnight at 4°C. The beads were then washed with 100 mM Tris (pH 8.0) to block residual coupling sites, and stored in 10 mM Tris (pH 7.5), 0.15 M NaCl, 0.2% azide at 4°C. 2.3 Production and purification of sICAM-1 The soluble ICAM-1 producer line 2706C was previously generated in our laboratory (Welder, Lee et al. 1993). 2706C cells were seeded into DMEM + 5% FCS (plus additions described in 2.1.1), and grown for 10 days. The culture supernatant was harvested by centrifugation at 2300 rpm for 30 minutes, followed by filtration through 0.22 um bottle top filter to remove cellular debris. sICAM-1 was purified from culture supernatants by affinity chromatography using YN1-coupled beads (2.2.3). One litre of cleared supernatant was loaded onto the YN1 column (2 ml bed volume) at a flow rate of approximately 50 ml/hour. The column was then washed with 100 ml of 10 mM Tris (pH 7.5), 0.15 M NaCl at a flow rate of 60 ml/hour and sICAM-1 was eluted with 0.1 M glycine (pH 2.8), 0.15 M NaCl at a flow rate of 60 ml/hour. Nineteen 1 ml fractions were collected into tubes, and neutralized with 1.5 M Tris buffer. The collected fractions were monitored by SDS-PAGE and Coomassie staining. Fractions with the highest concentrations of 28 sICAM-1 were pooled, and the concentration was determined by SDS-PAGE alongside BSA standards, followed by Coomassie staining. 2.4 Cell adhesion assay LFA-1-mediated cell adhesion to immobilized ICAM-1 was performed using a modification of the method described by Welder et al. (Welder, Lee et al. 1993). Nunc Maxisorp 96-well plates were coated at room temperature for 60-90 minutes with 10 ug/ml sICAM-1 diluted in 0.1 M sodium bicarbonate buffer (pH 8). The wells were washed three times with phosphate buffered saline (PBS) and blocked with 0.5 mg/ml heat inactivated BSA in PBS for 30 minutes at room temperature. Cells were resuspended at 1 x 106 cells/ml in Hank's balanced salt solution (HBSS), and stained with 1 ug/ml Calcein-AM (Molecular Probes) for 15 minutes at 37°C. Cells were washed twice with HBSS + 2% FCS and resuspended to 8 x 105 cells/ml in HBSS + 2% FCS. The cell suspension (500 pi per condition) was aliquoted into microfuge tubes, and stimulated with the indicated concentrations of ionomycin (Calbiochem), or 50 pg/ml PMA (Sigma) for 10 minutes at 37°C. For specificity control, 1.5 pg anti-LFA-1 mAb (TIB213, 2.2.2) was also added to the appropriate tube. Following stimulation, cells were gently resuspended and 100 pi of cell suspension was dispensed to each sICAM-coated well in triplicate. Cells were incubated for 25 minutes at 37°C and non-adherent cells were washed away with five washes of 100 pi HBSS + 2% FCS pre-warmed to 37°C per well. The fluorescence intensities of the cells before and after the washes were measured by CytoFluor2300 (Millipore, Medford, MA). The percentages of cell adhesion were determined by the ratio of the postwash over prewash fluorescence values after subtracting the background fluorescence values. 29 2.4.1 Inhibitor assays: To assess the effects of inhibitors on LFA-1-dependent adhesion, inhibitors were added at the indicated concentrations to cell suspensions and incubated at 30 minutes at 37°C prior to the addition of ionomycin or PMA. Primary cells were incubated with calpeptin for 15 to 30 minutes prior to ionomycin stimulation. The inhibitors used were the p/m-calpain inhibitors calpeptin, PD 150606, ALLN, or Calpain inhibitor III, the PKC inhibitor Bisindolymaleimide I (BIM I), and the PI3K inhibitors LY294002 (all from Calbiochem) and wortmannin (Sigma). The % inhibition was calculated by the equation: % inhibition = % cell adhesion (- inhibitor) -% cell adhesion (+ inhibitor) X 100% % cell adhesion (-inhibitor) 2.5 Quantitation of intracellular free calcium: T28 or resting T cells were washed once with Tyrode's buffer (10 mM HEPES pH 7.4, 130 mM NaCl, 5 mM KCI, 1.4 mM CaCl 2, 1 mM MgCl 2 , 5.6 mM glucose, 0.1 % BSA), then resuspended at 1 x 106 cells/ml in Tyrode's buffer containing 2 uM FURA-2/AM (Molecular Probes). Cells were incubated at room temperature for 45 minutes in the dark, allowing FURA-2/AM to accumulate in the cytoplasm of the cell. Cells were then washed twice to remove extracellular FURA-2/AM and resuspended to 2 x 106 cells/ml in Tyrode's buffer. One ml of cell suspension was placed into a quartz cuvette and stimulated with ionomycin or PMA. Cytoplasmic Ca 2 + influx was measured in real-time by monitoring fluorescence intensity at 510 nm (after excitation of the sample at 340 and 380 nm) using an MC200 spectrophotometer/ monochromator from SLM-Aminco (Urbana, IL) controlled by the 8100 V3.0 software program. 30 2.6 Detecting talin cleavage by Western blotting 2.6.1 Western blotting SDS-PAGE gels were run using the Mini-Protean 3 system and transferred to polyvinylidene fluoride (PVDF; Pall, Pensacola, FL) membranes at 100 volts.for 2 hours (unless otherwise indicated) in a Hoefer TE 22 (Amersham Biosciences) transfer apparatus according to the manufacturer's instructions. Proteins on blots were visualized by specific antibodies and the appropriate HRP-conjugated anti-IgG secondary antibody, followed by chemiluminescence using an ECL system (Amersham) according to the manufacturer's protocol. Two different anti-talin antibodies (2.2.1.1) were used to detect talin cleavage by Western blotting. The anti-talin tail (Sigma, 8d4) mAb recognizes an epitope C-terminal to the talin head domain (Figure 1.1), and detects both full-length talin as well as the talin tail fragment generated by calpain-mediated proteolysis. Anti-talin head (Santa Cruz, H-300) polyclonal antibody recognizes an epitope within the N-terminus (Figure 1.1), and detects both the full-length polypeptide as well as the talin head fragment. 2.6.2 Cleavage of talin by purified p and m-calpain To immunoprecipitate talin, 200 pi Protein G beads in wash buffer (10 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.8% NaCl) were incubated with 50 pi goat anti-talin polyclonal antibody (C-20, Santa Cruz) at 4°C for 30 minutes. The antibody-coated beads were then washed three times with wash buffer to remove any unbound antibody. A single cell suspension of murine splenocytes was prepared as described previously (2.1.2), centrifuged, and resuspended in 500 pi resuspension buffer: 10 mM Tris-HCl pH 7.5, 1% BSA, 150 mM NaCl. Cells were lysed by the addition of 500 pi 2X lysis buffer (10 mM Tris-HCl pH 7.5, 2% Triton-X 100, 1% BSA, 0.8% 31 NaCl) and incubated on ice 10 minutes. The lysate was centrifuged for 20 minutes at 13,000 rpm to remove insoluble debris, and the supernatant was incubated with the bead-antibody mixture for 1 hour at 4°C. Beads were washed four times with wash buffer to remove any unbound proteins. After the addition of the fourth wash, the bead suspension was divided in half, and subjected to microcentrifugation. One half was resuspended in 180 pi u-calpain cleavage buffer (10 mM Tris-HCl pH 7.6, 10 uM CaCb), while the other half was resuspended in the same volume of m-calpain cleavage buffer (10 mM Tris-HCl pH 7.6, 2 mM CaCla). Each of the samples was then split equally into 6 microfuge tubes. Reactions were started by the addition of the indicated amount of p-calpain (from human erythrocytes) or m-calpain (from rat recombinant; both from Calbiochem) enzymes. Tubes were mixed at room temperature for one hour and 30 pi reducing SDS-PAGE sample buffer was added to each tube to stop the reactions. The samples were boiled for 5 minutes and supernatants from 1 x 106 cell equivalents were loaded onto 5% SDS-PAGE or Ready Gel Precast 4-15% gradient gels (Bio-Rad). Membranes were probed with anti-talin tail or anti-talin head antibodies respectively. 2.6.3 Analysis of talin cleavage in whole cells 2.6.3.1 Anti-talin head blots Resting T cells (2.1.2) or T28 cells were suspended to 1 x 106 cells/ml in RPMI + 2% FCS, then stimulated with the indicated concentrations of ionomycin or 50 ng/ml PMA for 30 minutes at 37°C. Cells were pelleted by centrifugation and lysed by the addition of reducing sample buffer followed by boiling for 5 minutes. Lysates (5 x 105 cell equivalents per lane) were loaded on SDS-PAGE gradient gels, transferred to membranes, and then probed with anti-talin head antibody. 32 2.6.3.2 Anti-talin tail blots LNCaP cells were seeded at 2.0 x 105 cells per well in 6-well plates. Seventy-two hours later, cells were pretreated with 20 pM calpeptin as indicated in fresh media for 15 minutes at 37°C. Cells were then stimulated with 10 pM ionomycin at 37°C for 20 minutes as indicated. The media was gently removed, and cells were scraped into lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5% Igepal, plus protease inhibitors) to harvest. T28 cells or CTLs were washed once and resuspended tol x 106 cells/ml in phenol red free RPMI + 5% FCS, and stimulated with 500 ng/ml (T28 cells) or 750 ng/ml (CTLs) ionomycin for 20 minutes at 37°C. Cells were pre-incubated with 500 pM calpeptin at 37°C for 15 minutes prior to stimulation with ionomycin as indicated. Following stimulation, cells were subjected to microcentrifugation and the media was decanted. The cells were lysed with lysis buffer for 1 hour on ice, and centrifuged at 13,000 rpm for 20 minutes at 4°C. Supernatants were recovered, and total protein was quantitated using a bicinchoninic acid (BCA) Protein Assay kit (Pierce, Rockford, IL). Fifteen micrograms of protein per sample were loaded onto 5% SDS-PAGE gels, transferred, and then probed with the anti-talin tail monoclonal antibody. The positive control for calpain cleavage of talin was immunoprecipitated talin incubated with p-calpain (10 ng, 2.6.2). 2.6.3.3 Immunoprecipitating talin from stimulated cells T28 cells or resting T cells (2.1.2) were washed once and resuspended to 1 x 106 cells/ml in HBSS + 2% FCS. Cells (4 x 106) were stimulated with 500 (T28 cells) or 1000 (resting T cells) ng/ml ionomycin or with 50 ng/ml PMA for 30 minutes at 37°C. While cells were being stimulated, anti-talin antibody coated beads were prepared as described previously (2.6). For each condition, 20 pi of Protein G beads was incubated with 5 pi of anti-talin tail antibody. Following stimulation, cells were pelleted by centrifugation and each aliquot of 4 x 106 cells 33 were lysed as in 2.6 by the addition of 500 pi resuspension buffer followed by 500 pi lysis buffer. The cleared supernatants were incubated with 25 pi of talin antibody-bead mixture for 1 hour at 4°C. Immunoprecipitated talin protein was eluted from the beads by adding 60 pi of reducing SDS-PAGE sample buffer and boiling for 5 minutes. Samples (1 x 106 cell equivalents per lane) were loaded on a 5% SDS-PAGE gel, transferred, and then probed with anti-talin tail mAb. The negative control for calpain cleavage of talin was immunoprecipitated talin (0 ng u-calpain sample, 2.6.2), and positive control was immunoprecipitated talin incubated with p-calpain (80 ng, 2.6.2). 2.7 Talin co-immunoprecipitation with L F A - 1 T28 cells (1.1 x 107 per condition) were suspended at 1 x 106 cells/ml in HBSS + 2% FCS. Cells were stimulated with 500 ng/ml ionomycin for 30 minutes at 37°C, centrifuged, and the media was decanted. The cell pellets were lysed with either 1% CHAPS lysis buffer (10 mM Tris-HCl pH 7.6, 1% CHAPS, 150mM NaCl, ImM CaCl 2, ImM MgCl 2) or 2% Triton-X 100 lysis buffer (HBSS, 2% Triton X-100, 0.2% NP-40/Igepal, ImM MgCl 2 , ImM CaCl2). Lysis reactions were incubated at 4°C for 20 minutes, and the lysates were cleared by microcentrifugation at 13,000 rpm for 15 minutes. Lysates were then incubated with 50 pi TIB213-coupled beads (2.2.3), or 50 pi anti-CD4-coupled beads (previously made by Jose Rey-Ladino) at 4 °C for 2 hours. Beads were washed four times with wash buffers (CHAPS wash buffer: 10 mM Tris pH 7.6, 0.1 % CHAPS, 150 mM NaCl, 1 mM CaCl 2, 1 mM MgCl 2; 2% Triton X-100 wash buffer: same as lysis buffer). Immunoprecipitated proteins were eluted from the beads by adding 30 pi non-reducing loading buffer to each tube. A small aliquot (1.5 pi, 5 x 105 cell equivalents) was removed for anti-LFA-1 Western blot analysis since TIB 218 is unable to recognize reduced LFA-1. Three microlitres of 10X reducing loading buffer was added to the remaining sample (1 34 x 107 cell equivalents), and this was run on a 10% SDS-PAGE gel to probe for talin head co-immunoprecipitation. Immunoprecipitated talin incubated with m-calpain (80 ng, 2.6.2) to generate the talin head fragment was also run as a positive control for the anti-talin head antibody. The non-reduced samples were run on 7.5% SDS-PAGE gels, and probed with TIB218 (1/2 dilution of hybridoma supernatant produced in 2.2.2) followed by anti-rat-HRP (1:5000) to assess efficiency of LFA-1 immunoprecipitation in each reaction. 2.8 Calpain cleavage assay 2.8.1 Cleavage of purified p and m-calpain One hundred nanograms of purified p or m-calpain (2.6.2) was incubated in calpain activation buffer (10 mM Tris-HCl, pH 7.5, 5 mM CaCb) for the indicated times at room temperature. As a control, p or m-calpain was diluted in control buffer (10 mM Tris-HCl, pH 7.5). Reactions were stopped by adding reducing sample buffer and boiling the samples for 5 minutes. The samples were loaded onto 7.5 % SDS-PAGE gels, and transferred for 1.5 hours at 80 V. Membranes were subjected to Western blotting with anti-p/m-calpain antibodies (2.2.1.1). 2.8.2 Calpain cleavage in whole cell lysates L cells were trypsinized to harvest, pelleted, and then lysed with reducing sample buffer. T28 cells were resuspended to 1 x 106/ml in HBSS + 2% FCS, then stimulated with 500 ng/ml ionomycin or 50 ng/ml PMA at 37°C for 30 minutes. Cells were pelleted and lysed with reducing sample buffer. For calpain cleavage controls, the indicated amounts of p-calpain were incubated in control buffer (2.8.1; negative control) or calpain activation buffer (2.8.1; positive control) for 15 minutes at room temperature. Reactions were stopped by the addition of reducing sample buffer, and samples were boiled along with T28 and L cell lysates for 5 minutes. 1 x 106 35 cell equivalents (T28 and L cells) were loaded per lane on a 7.5 % gel, transferred as in 2.8.1, and blotted with anti-p-calpain antibody (2.2.1.1). 2.9 Spectrin cleavage assay LNCaP and T28 cells were stimulated and lysed as in 2.6.3.2. Thirty micrograms of protein per sample was loaded onto 5% SDS-PAGE gels, transferred to membranes, and probed with anti-a-spectrin mAb (2.2.1.1). 2.10 Apoptosis Assay T28 cells were resuspended to 1 x 106 cells/ml in HBSS + 2% FCS. Calpain or PI3K inhibitors were added to each condition as indicated, and cells were incubated at 37°C for 30 minutes. Ionomycin (500 ng/ml) was added as appropriate, and the cells were incubated for another 10 minutes at 37°C. Cells were then stained for FACS analysis using reagents in the Annexin V-PE Apoptosis Detection Kit (BD Pharmingen) following the manufacturer's protocol and analysed by flow cytometry. The percentage of cells undergoing early and late apoptosis was determined by the percentage of cells that were stained with Annexin V-PE (early apoptosis) and Annexin V-PE plus 7-amino-actinomycin (7-AAD; late apoptosis). To calculate apoptosis induction by various inhibitors (% apoptosis), the following equation was applied: % apoptosis induction: % apoptosis (+inhibitor) - % apoptosis (unstimulated) 2.11 Rap Activation Assay 2.11.1 Production of RalGDS Fusion Protein The pGEX-RalGDS(RBD) plasmid encoding a glutathione S-transferase (GST) fusion protein containing the Rapl binding domain (RBD) of the RalGDS protein was a gift from Dr. M.R. 36 Gold (Dept of Microbiology and Immunology, University of British Columbia). The Escherichia coli strain DH5a was transformed with this plasmid, and protein production was initiated by adding 200 pM iso-propyl-p-D-thiogalactopyranoside to the culture. Bacteria were resuspended in sonication buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mg/ml lysozyme, 0.1 mg/ml DNAse I, 10 pg/ml leupeptin, 10 pg/ml soybean trypsin inhibitor, 1 pg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) and lysed by sonication. The lysate was centrifuged at 10, 000 x g for 45 minutes at 4°C. The supernatant was aliquoted and stored at -70°C. For each sample, 20pl glutathione agarose beads (Sigma) was incubated with 20 pi GST-RalGDS bacterial lysate in 500 pi lysis buffer (25 mM Hepes pH 7.5, 150 mM NaCl, 1 % NP-40, 0.25% sodium deoxycholate, 10 % glycerol, 25 mM NaF, 10 mM MgCl 2 , 1 mM EDTA, ImM N a 3 V 0 4 , ImM NaMo04). The mixture was incubated at 4°C for 1 hour, then washed once with lysis buffer. 2.11.2 Rap 1 Activation Assay Cells were suspended in activation buffer (25 mM Hepes pH 7.2, 125 mM NaCl, 5mM KCI, ImM CaCl 2, 1 mM Na 2HP0 4 , 0.5 mM MgS0 4, 2mM glutamine, ImM sodium pyruvate, 1 mg/ml glucose, 1 mg/ml BSA, 50 pM 2-mercaptoethanol) at 2 x 107 cells/ml. The cell suspension was aliquoted at 250 pi per condition into microfuge tubes, then 250 pi activation buffer + 1000 ng/ml ionomycin or 100 ng/ml PMA was added (final cell concentration: 1 x 107/ml, final stimuli concentration: 500 ng/ml ionomycin or 50 ng/ml PMA). Cells were then incubated at 37°C for the indicated times, and the stimulation was stopped by the addition of 500 pi lysis buffer, followed by incubation at 4°C for 10 minutes. Lysates were centrifuged at 13, 000 rpm for 15 minutes, and 300 pi of the cleared lysates were incubated with GST-RalGDS-coated glutathione agarose beads for 30 minutes at 4°C. Beads were washed once with lysis 37 buffer to remove any unbound proteins, then bound proteins were eluted by the addition of 40 pi reducing sample buffer, and boiling the samples for 5 minutes. Five microlitres of cleared lysate or 15 pi of RalGDS pulldown samples for each condition were run on 12.5 % SDS-PAGE gels, and transferred to membrane at 80 V for 1 hour. Membranes were probed with rabbit anti-Rap 1 polyclonal Ab (2.2.1.1). 2.12 Akt phosphorylation assay T28 cells were resuspended to 2 x 106/ml in HBSS + 2% FCS, and stimulated by the addition of HBSS + 2% FCS + 1000 ng/ml ionomycin (final cell concentration: 1 x 106/ml, final ionomycin concentration: 500 ng/ml), and incubated at 37°C for the indicated times. Stimulations were stopped by washing the cells once with ice cold PBS, then cells were pelleted and lysed by the addition of reducing sample buffer. 5 x 105 cell equivalents were loaded per lane on a 10% SDS-PAGE gel, transferred to membrane as in 2.8.1, and blocked with 5 % BSA blocking solution. The blot was probed with rabbit anti-phospho-Akt polyclonal antibody (2.2.1.1) overnight. The blot was re-probed with mouse anti-GAPDH mAb to determine equal loading (2.2.1.1). 2.13 Analysis of talin distribution by confocal microscopy 2.13.1 Slide preparation Polystyrene beads (Polybead® polystyrene 10 micron microspheres, Polysciences Inc., Warrington, PA) were mixed at a 1:1 ratio with 40 pg/ml anti-LFA-1 antibody (TIB213, 2.2.2) or sICAM (2.3) solutions for 1 hour at room temperature, with occasional mixing to prevent settling. Coated beads were washed with 20 mM Tris-HCl, pH 7.0 and incubated with 200 pg/ml BSA for 30 minutes at room temperature, followed by three washes with HBSS + 2% FCS. For confocal microscopy experiments, CTLs were used on Day 6 or 7 without IL-2 38 removal. For each slide, 500,000 beads were mixed with 250,000 CTLs (2.5 x 106 cells/ml in HBSS + 2 % FCS), and incubated together for 5 minutes at 37°C. The mixture was gently resuspended and plated onto a poly-L-lysine coated coverslip, then incubated at 37°C for 20 minutes. The cells were fixed with 4 % paraformaldehyde for 15 minutes, washed twice with PBS, then blocked with PBS containing 1 % BSA for 10 minutes. Cell membranes were permeablized by washing with Hank's Saponin solution (HBSS, 2 % FCS, 5 mM EDTA, 0.5 % Saponin) for 5 minutes. Talin was stained with a 1:200 dilution of anti-talin antibody (Sigma, 2.2.1.1) prepared in Hank's Saponin solution at room temperature for 1 hour. After two washes, cells were incubated with 5 pg/ml Alexa Fluor 488-conjugated anti-mouse IgG antibody (2.2.1.1) in Hank's Saponin for 1 hour at room temperature. After three washes, coverslips were mounted on Vectashield (Vector, Burlingame, CA). 2.13.2 C o n f o c a l M i c r o s c o p y Slides were analyzed with a BioRad Radiance 2000 Multiphoton microscope (Hercules, CA) equipped with Kr and Mai Tai Sapphire lasers and 60x objective lens. Alexa 488 was excited by 488 nm and the emission filter was HQ 515/30. Stacks were collected with 0.2pm z steps, and an iris size of 2 or smaller to maintain confocal conditions. Stacks were 3-D reconstructed using the Velocity software (Improvision, Lexington, MA), and analysed to determine the exact localization of talin in relation to the contact site with the bead. Figures were generated by overlaying a single slice of the fluorescence image over the brightfield image of the conjugate. 39 CHAPTER 3 3.1 The role of calpain activation and talin cleavage in ionomycin stimulation of murine T cells LFA-1 is activated by ionomycin treatment of T cells The effects of ionomycin on LFA-1-mediated adhesion of various T cells were tested. The murine T cell hybridoma line T28 showed low level of adhesion to immobilized ICAM-1, and treatment with ionomycin increased adhesion significantly (Figure 3.1, top panel). Adhesion was mediated by LFA-1 as indicated by the almost complete inhibition by the TIB213 anti-LFA-1 mAb. Peak adhesion occurred at 500 ng/ml of ionomycin, which is consistent with the results reported by Stewart et al. with human T cell blasts (Stewart, McDowall et al. 1998). Similar results were obtained with resting primary T cells (Figure 3.1, middle panel) and CTLs (Figure 3.1, bottom panel), although the highest adhesion was achieved with 250 and 750 ng/ml ionomycin, respectively. Intracellular calcium measurement showed increases in intracellular free calcium in ionomycin-treated primary T cells and T28 cells as expected (Fig. 3.2). The lower concentration of ionomycin necessary to achieve maximal adhesion of resting T cells may be due to the fact that 500 ng/ml ionomycin induced larger calcium increases that remained highly elevated for a longer period than in T28 cells. Treatment with PMA induced LFA-1-9+ mediated adhesion of T cells, but it did not induce Ca -increases in either cell type tested, suggesting that ionomycin and PMA may activate LFA-1-mediated T cell adhesion by separate pathways. 40 Figure 3.1: Ionomycin treatment of T cells activates LFA-1 T28 cells (top panel), primary resting T cells (middle panel) or CTLs (bottom panel) were incubated with the indicated concentrations of ionomycin for 10 minutes at 37°C. LFA-1 -mediated adhesion was measured by incubating cells in plates coated with immobilized soluble ICAM-1, and the percentage of cells adhering was determined. Stimulation with 50 ng/ml PMA was also tested. Ionomycin-stimulated cells treated with anti-LFA-1 antibody (TIB213) showed that the adhesion was mediated by LFA-1 (open bars). For cell adhesion control, BSA was immobilized in place of ICAM-1. The percentage of binding to BSA was less than 1 % for T28 cells and less than 5% for CTLs and resting T cells in all binding assays. The results shown are the means of at least 3 independent experiments for each cell type, each performed in triplicate. Error bars indicate SD. 41 % cell adhesion % cell adhesion % cell adhesion to T28 cells Primary T cells Figure 3.2: Ionomycin induces intracellular calcium flux T28 cells (left panel) or primary T cells (right panel) were stimulated with 500 ng/ml ionomycin (dashed line) or 50 ng/ml PMA (solid line). Intracellular calcium levels were determined using FURA-2. The arrows indicate the time when the stimulus was added. Similar results were obtained in two separate experiments for each cell type. 43 Talin cleavage in response to ionomycin stimulation is undetectable To investigate whether talin is cleaved following ionomycin stimulation, it was necessary to first evaluate the sizes of the cleavage products produced by calpain digestion. This was required because initial blots with anti-talin tail mAb resulted in multiple bands. Additionally, the reported sizes for the tail fragment in the literature range from 190 kDa to 220 kDa, resulting in uncertainty about which was the specific tail fragment band. Talin was immunoprecipitated from murine splenocytes and incubated with various amounts of purified p or m-calpain in a buffer containing Ca 2 + , which would activate calpain proteolytic activity. Two anti-talin antibodies were used to monitor cleavage; both recognize the intact talin protein, and each detects one of the fragments produced by proteolysis (2.6.1). With increasing concentrations of p or m-calpain, there was a gradual decrease in the amount of full-length talin, with a concomitant appearance of the ~220-kDa talin tail (Figure 3.3, upper blot) and ~50-kDa talin head (Figure 3.3, lower blot) fragments. These results also demonstrate that both p and m-calpain are capable of cleaving murine talin in vitro, as there is some confusion in the literature whether p-calpain, m-calpain, or both are responsible for talin cleavage. With proper controls for talin cleavage, we were able to investigate talin cleavage in whole cells. T28 cells and resting T cells were stimulated with a range of ionomycin concentrations, and the cell lysates were probed with the anti-talin head antibody for the appearance of the talin head fragment. In all conditions there was an approximately equal amount of uncut talin, suggesting that cleavage did not occur (Figure 3.4A). Additionally, there were no talin head fragments detected in either blot. There was a reduced amount of full length talin when resting T cells were treated with 1000 ng/ml ionomycin in resting T cells. However, no talin head band was visible, so the apparent decrease was most likely due to unequal loading. 44 Calpain I (ng) Calpain II (ng) 0 5 10 20 40 80 0 5 10 20 40 80 250 — a-talin tail Calpain I (ng) Calpain II (ng) 0 5 10 20 40 80 0 5 10 20 40 80 a-talin head 100 — Figure 3.3: Murine talin is cleaved by both Calpain I and Calpain II Purified talin was incubated with the indicated amounts of Calpain I or Calpain II enzymes. Samples were subjected to Western blot analysis with either anti-talin tail mAb (upper blot) anti-talin head antibody (lower blot). 45 Figure 3.4: Talin is not cleaved following ionomycin stimulation (A) T28 cells (left blot) or primary T cells (right blot) were stimulated with the indicated concentrations of ionomycin or 50 ng/ml PMA for 30 minutes at 37°C. Cell lysates were analyzed by anti-talin head Western blotting. (B) LNCaP, T28 cells or CTLs were preheated with 20 (LNCaP) or 500 (T28 cells, CTLs) pM calpeptin for 15 minutes. Cells were then treated with 7 pg/ml (LNCaP), 500 (T28 cells) or 750 ng/ml (CTLs) ionomycin for 20 minutes. 15 pg of protein extracts were analyzed by anti-talin tail Western blotting. +C: talin cleavage positive control (immunoprecipitated talin cleaved by p-calpain in vitro as described in Materials and methods), U: unstimulated (DMSO control), I: ionomycin stimulated, I+C: calpeptin pre-treated, ionomycin stimulated, P: PMA. FL indicates the full length talin (240 kDa), CP indicates calpain-mediated talin cleavage product (-220 kDa). (C) T28 or primary T cells were stimulated with 500 (T28 cells) or 1000 (primary T cells) ng/ml ionomycin or 50 ng/ml PMA for 30 minutes at 37°C. Talin was immunoprecipitated from lysates as described in Materials and Methods. Samples (T28 cells: 5 x 105, primary T cells: 1 x 106 cell equivalents per lane) were analyzed by Western blotting with anti-talin tail antibody. -C: talin cleavage negative control (immunoprecipitated talin), +C: talin cleavage positive control (immunoprecipitated talin cleaved by p-calpain in vitro as described in Materials and methods), U: unstimulated (DMSO control), I: ionomycin stimulated, P: PMA stimulated. FL indicates the full length talin, CP indicates talin cleavage product generated by incubation with calpain. 46 T28 cells Ionomycin (ug/ml) 0 0.1 0 .25 0.5 0.75 1.0 P M A P r i m a r y T cells Ionomycin (ug/ml) 0 0.1 0 .25 0.5 0.75 1.0 P M A B L N C a P T28 cells C T L s + C U I I+C U I I+C P U 1 I+C P 2 5 0 2 0 0 •^^ ^^^  d^^w^  p^fi^  i^B^  ^ ((j^  f^in^  B^P^  F L C P c Primary T T28 cells c e I l s N - FL k- C P 47 There was a faint 80 kDa band in all of the lanes for both blots, however this was likely a non-specific band, because the size does not match that of the known tail or head fragments (Figure 3.3). The failure to detect the talin head fragment in Figure 3.4A may be due to the fact that the binding of the anti-talin head antibody is relatively weak. Increasing the concentration of antibody in blots resulted in very high background that obscured any bands. Instead, cleavage was monitored using the anti-talin tail antibody. Ionomycin treatment did not decrease the amount of full length talin, nor increase the cleaved tail fragment in T28 cells or CTLs (Figure 3.4B). Ionomycin treatment of the human prostate cancer cell line LNCaP activates calpains and induces cleavage of the calpain targets a-spectrin and E-cadherin (Rios-Doria, Day et al. 2003) (and below). Surprisingly, increased talin cleavage was not detected in ionomycin-treated LNCaP cells (Figure 3.4B). Talin cleavage in response to ionomycin treatment may be limited and at a level that is undetectable by Western blotting of whole cell lysates. Therefore, full length talin and the talin tail fragment were immunoprecipitated from stimulated cells using an anti-talin tail polyclonal antibody (Santa Cruz) that recognizes that C-terminal portion of the protein (Figure 1.1). Small amounts of the tail fragment were constitutively present in both T28 cells and resting T cells (Figure 3.4C), but did not increase significantly with ionomycin treatment. This result confirms that ionomycin stimulation of T cells does not activate the cleavage of talin. Although we have been unable to detect talin cleavage by a number of approaches, there is still the possibility that a small proportion of talin is cleaved to yield a talin head fragment which interacts with the cytoplasmic domain of LFA-1. This was tested by LFA-1 co-immunoprecipitation assay. T28 cells were stimulated with 500 ng/ml ionomycin, lysed, and 48 LFA-1 was immunoprecipitated with anti-LFA-1 coupled beads (2.2.3). Two different lysis buffers were tested in this experiment, and anti-CD4 coupled beads were used as a control for the immunoprecipitation. Although talin head was readily detected in the positive control lane by Western blotting, no talin head was detected in the anti-LFA-1 immunoprecipitates (Figure 3.5, upper blot) using either of the two detergent conditions tested in this experiment. A small aliquot of sample was analyzed for the amount of LFA-1 being immunoprecipitated from the lysates. There are two bands in close proximity at approximately 90 kDa that are difficult to distinguish due to overexposure of the blot (Figure 3.5, lower blot). The upper band is specific to LFA-1 as it is absent from the anti-CD4 control lane. The lower band is due to cross reaction of the secondary antibody, as blots without primary antibody had this band. The presence of the upper band in the anti-LFA-1 IP lanes demonstrates that LFA-1 was immunoprecipitated from the cell lysates. An additional lysis condition (10 mM Tris pH 7.5, 1% Triton X-100, 1% BSA, 0.8% NaCl) was tested in an attempt to identify conditions that maintained the possible talin head-LFA-1 interaction. However, talin head was never detected. These results suggest that talin head fragments that interact with LFA-1 are not generated upon ionomycin stimulation. 49 IP a-LFA-1 a-CD4 detergent T T C C ionomycin - + + 50—«!*** talin head ^ ^ ^ ^ ^ ^ ^ ^ W " W ' V - T Figure 3.5: T a l i n head fragment does not co-immunoprecipitate wi th L F A - 1 T28 cells were stimulated with 500 ng/ml ionomycin for 30 min at 37°C, then lysed with either 2% Triton X-100/0.2% NP-40 or 1 % CHAPS lysis buffers. LFA-1 was immunoprecipitated as described in Materials and Methods, and samples were subjected to Western blot analysis with talin head (upper blot) and LFA-1 (lower blot) antibodies. T: 2% Triton X-100/0.2% NP-40 lysis buffer, C: 1% CHAPS lysis buffer. The first lane of the anti-talin head blot is the talin head positive control (immunoprecipitated talin incubated with p-calpain to generate talin head as described in Materials and methods). 50 Calpain inhibitors provide conflicting evidence of calpain activation by ionomycin The requirement for calpains in ionomycin-induced activation of LFA-1 was tested using the membrane permeable calpain inhibitor calpeptin. Pre-incubation of cells with calpeptin prior to stimulation with ionomycin inhibited LFA-1-mediated adhesion of T28 cells (Figure 3.6A, top panel), primary T cells (Figure 3.6A, middle panel) and CTLs (Figure 3.6A, bottom panel). The inhibition was dose-dependent, with maximal inhibition occurring at 500 pM calpeptin. To evaluate the specificity of calpeptin treatment, its effect on PMA-induced activation of LFA-1 was tested. PMA is thought to activate LFA-1 through the PKC pathway (Bazzoni and Hemler 1998), independent of Ca 2 + flux (Figure 3.2) and calpain activation (Rock, Brooks et al. 1997). Unexpectedly, calpeptin also inhibited PMA-induced LFA-1 activation in all three cell types (Figure 3.6). These results suggest that the inhibition of adhesion by calpeptin may be due to a non-specific effect. To further investigate whether calpain mediates ionomycin-induced LFA-1 activation, additional calpain inhibitors were tested. Calpeptin, ALLN and calpain inhibitor III are modified peptides that compete for the active sites of calpains (Wang and Yuen 1994), whereas 9-1-PD 150606 targets the Ca binding sites of the protease (Wang, Nath et al. 1996). Calpeptin, ALLN and calpain inhibitor III have been reported to prevent calpain-mediated processes in whole cells at concentrations up to 100 pM (Canu, Dus et al. 1998; Patel and Lane 1999; Lee, Kwon et al. 2000; Rios-Doria, Day et al. 2003; Sedarous, Keramaris et al. 2003), while PD150606 is effective at 50 pM (Sedarous, Keramaris et al. 2003). ALLN and calpain inhibitor III failed to inhibit ionomycin-induced activation of LFA-1 whereas calpeptin and PD 150606 inhibited LFA-1 activation in T28 cells (Figure 3.7). Inhibition of PMA-induced LFA-1 activation by PD 150606 was tested in two separate experiments, and was found not to prevent 51 LFA-1-mediated adhesion (data not shown). Thus, the results with multiple calpain inhibitors are do not yield a clean answer as to whether calpains are responsible for ionomycin-induced activation of LFA-1. 52 Figure 3.6: Calpeptin inhibits both ionomycin and P M A induced LFA-1 activation (A) T28 cells (top panel), primary resting T cells (middle panel), or CTLs (bottom panel) were incubated with the indicated concentrations of calpeptin, then stimulated with 500 (T28 cells), 250 (resting T cells) or 750 (CTLs) ng/ml ionomycin for 10 minutes at 37°C. LFA-1-mediated adhesion was analysed as in Figure 3.1. Percentage inhibition of adhesion was calculated as described in Materials and Methods. Pre-treatment with blocking antibody (TIB213) resulted in less than 6% (T28 cells and CTLs), or less than 3% (resting T cells) adhesion in all binding assays. Results shown are the means of three independent experiments for each cell type, each performed in triplicate. Error bars indicate SD. (B) T28 cells (top panel), primary resting T cells (middle panel), or CTLs were incubated with the indicated concentrations of calpeptin as in Figure 3.6A, followed by stimulation with 50 ng/ml PMA for 10 minutes. Adhesion to immobilized ICAM-1 was analysed as in Figure 3.1. Results shown are the means of at least three independent experiments for each cell type, each performed in triplicate. Error bars indicate SD. 53 % inhibition of adhesion % inhibition of adhesion n -5" T3 3. 3 SS H « •a" 5* % inhibition of adhesion 100 80 60 i § 4 0 5 ^ 20 -20 -40 T calpain inhibitor III ALLN calpeptin PD150606 Figure 3.7: Not all calpain inhibitors prevent ionomycin-induced activation of LFA-1 T28 cells were incubated with 25 (dotted bars), 50 (grey bars) or 100 (open bars) pM calpeptin, ALLN, calpain inhibitor III or PD150606 then stimulated with 500 ng/ml ionomycin as in Figure 3.6A. Percentage inhibition was calculated as in 3.6A, negative values indicate increased adhesion. Results shown are the means of three independent experiments, each performed in triplicate. Error bars indicate SD. 55 Calpain auto-cleavage is undetectable in whole cells Calpains autolyse when incubated with Ca 2 + , cleaving the 80-kDa catalytic polypeptide into a 78-kDa fragment (Goll, Thompson et al. 2003), and this process can be used as a sign of calpain activation. Since the size differences between intact and autolysed calpains are difficult to distinguish by Western blotting, purified p and m-calpain were incubated in Ca containing buffer to be used as cleavage controls. Calpain I (p-calpain) was rapidly cleaved to the 78-kDa fragment (Figure 3.8 A, upper blot), while cleavage of calpain II (m-calpain) was not detected (Figure 3.8A, lower blot). Since a control for m-calpain autolysis was not obtained, only p-calpain activation in whole cells was investigated. T28 cells were stimulated with 500 pg/ml ionomycin or 50 ng/ml PMA and the cell lysates were probed with the anti-p-calpain antibody. The murine fibroblast L cell line was loaded as a control, since adherent fibroblasts have constitutively active calpains (Dourdin, Bhatt et al. 2001). Whole cell lysates produced multiple bands with very high background (Figure 3.8B). None of the bands in the T28 or L cell lanes are of the same size as those produced by purified p-calpain. Both calpains are ubiquitously expressed in mammals and various tissues (Croall and DeMartino 1991). However, calpain is present in extremely low levels in human T lymphocytes (Deshpande, Goust et al. 1995). The titration of purified p-calpain in Figure 3.8B demonstrates that the antibody has limited sensitivity for p-calpain. Ten nanograms of recombinant p-calpain produced faint bands that may not be visible above the high background seen in blots of whole cell lysates. Therefore, the inability to detect calpain autolysis in whole cells may be due to low p-calpain expression in T28 cells as well as the limited sensitivity of the antibody. 56 A C 5 15 30 100 — I1*** mm mm a-Calpain I 64 —I a-Calpain II 64 —I B 100 ng 10 ng 1 ng T28 cells - + - + - + L U I P a-Calpain I Figure 3 . 8 : Calpain cleavage in murine cells could not be detected (A) 100 ng of calpain I (upper blot) or calpain II (lower blot) was incubated for the indicated times (in minutes) in calpain activation buffer. As a control, calpain I or II was incubated in control buffer (C). Samples were subjected to Western blot analysis with anti-p-calpain (left panel) or anti-m-calpain (right panel) antibodies. (B) The indicated amounts o f calpain I were incubated in calpain activation buffer (+) or control buffer (-) for 15 minutes at room temperature. T28 cells were stimulated with 500 ng/ml ionomycin or 50 ng/ml P M A for 30 minutes at 37°C. Samples (1 x 106 cell equivalents per lane) were analyzed by anti-p-calpain Western blotting. L : L cell lysate, U : unstimulated ( D M S O control), I: ionomycin stimulated, P: P M A stimulated. 57 Alpha-spectrin is not cleaved by ionomycin stimulation Spectrin is a well-documented calpain substrate that is used as an assay for calpain activity in intact cells (Wang, Nath et al. 1996; Dourdin, Bhatt et al. 2001; Rios-Doria, Day et al. 2003). Active calpain cleaves a-spectrin into two fragments of 145 and 150 kDa. Western blotting with an anti-a-spectrin antibody that recognizes full length spectrin (240 kDa) and the 150-kDa fragment showed spectrin cleavage in control LNCaP cells treated with ionomycin, as well as inhibition of cleavage by calpeptin. In contrast, spectrin cleavage was not detected in T28 cells treated with 500 ng/ml ionomycin (Figure 3.9), indicating that calpains are not activated by ionomycin stimulation in T cells. Unexpectedly, treatment of T28 cells with ionomycin plus 500 ng/ml calpeptin resulted in a-spectrin cleavage. Figure 3.9: Spectrin is not cleaved in response to ionomycin stimulation in T28 cells LNCaP or T28 cells were treated as in Figure 3.4B. Thirty micrograms of protein extracts were analyzed by Western blotting with anti-a-spectrin antibody. U: unstimulated (DMSO control), I: ionomycin stimulated, I+C: calpeptin pre-treated, ionomycin stimulated. FL indicates full length a-spectrin (240 kDa), CP indicates cleavage product of a-spectrin (-150 kDa). 58 Calpeptin and PD150606 induce apoptosis following ionomycin stimulation Since spectrin can also be cleaved by caspases to yield a 150-kDa fragment (Nath, Raser et al. 1996), the above results led us to suspect that calpeptin may be inducing apoptosis in cells at high concentrations. Therefore, cells treated with calpain inhibitors plus ionomycin or PMA were stained with the apoptosis indicators Annexin V-PE and 7-AAD and then analysed by FACS. Ionomycin alone induced apoptosis in T28 cells (Figure 3.10), as has been previously described for other calcium mobilizers (Orrenius, Zhivotovsky et al. 2003). Both calpeptin and PD150606 also induced apoptosis in a dose-dependent manner. As expected, calpeptin induced apoptosis of both ionomycin and PMA treated cells at high concentrations. Although PD150606 did not significantly inhibit PMA-activated adhesion, which suggests that the inhibition was specific, it also induced apoptosis in ionomycin-stimulated cells. PD 150606 is a more potent inhibitor of ionomycin-induced LFA-1 activation, and also induced apoptosis more strongly than calpeptin at the same concentration (100 pM). These results suggest that the inhibition of LFA-1 with these two calpain inhibitors may be due, at least in part, to the rapid induction of apoptosis. 59 Figure 3.10: Calpeptin and P D 1 5 0 6 0 6 induce apoptosis in a dose-dependent manner T28 cells were pre-treated with the indicated concentrations of calpeptin or PD 150606 for 30 minutes at 37°C, then stimulated with 500 ng/ml ionomycin (black bars) or 50 ng/ml PMA (grey bars) as in Figure 3.6A . Cells were stained with Annexin V-PE and 7-AAD, and analysed by FACS. Percentage apoptosis was calculated as described in Materials and Methods. Results are the means of three independent experiments. Error bars indicate SD. 60 3.2 Potential mediators of ionomycin-induced activation of LFA-1 Rapl is not activated by ionomycin-induced calcium fluxing Rapl is an important mediator of P2 integrin function (Katagiri, Hattori et al. 2000; Katagiri, Hattori et al. 2002) through its effector RapL (Katagiri, Maeda et al. 2003), and is activated by ionomycin treatment of human T lymphocytes (Reedquist and Bos 1998; Sebzda, Bracke et al. 2002). To test whether Rapl is activated in murine T cells by ionomycin treatment, I carried out Rapl activation assays. This assay is based on the higher affinity of the RalGDS protein for active Rapl-GTP than for the inactive GDP-bound form of Rapl (Herrmann, Horn et al. 1996). Activated Rapl can be detected by immunoblotting blots of GST-RalGDS pull-downs with an anti-Rap 1 antibody. Using this method we determined that T28 cells have low levels of constitutively active Rapl, and that ionomycin treatment (500 ng/ml) did not stimulate Rapl (Figure 3.11, upper blot). Stimulation with 50 ng/ml PMA for 5 minutes was tested as a control for the assay, and resulted in the detection of high levels of active Rapl, as previously described for primary T cells and other cell lines (McLeod, Ingham et al. 1998; Reedquist and Bos 1998; Arai, Nosaka et al. 2001; Liu, Schwartz et al. 2002; Sebzda, Bracke et al. 2002; McLeod, Shum et al. 2004). Whole cell lysates from each condition were run as a control for equal starting cell material in the assay (Figure 3.11, lower blot), and indicate that each condition received approximately equal amounts of protein for the affinity purification. Rapl activation by ionomycin in primary T cells peaks at 1 minute (Reedquist and Bos 1998), suggesting that the timepoints tested in my assay may have missed Rapl activation. However, 1 and 2 minute time points were tested in separate trials and no increase in Rapl activation was detected. These results demonstrate that ionomycin stimulation does not increase the amount of activated Rapl in T28 cells, consistent 61 with previous reports that Rapl is not activated by ionomycin stimulation of the Jurkat T cell line (Reedquist and Bos 1998) or in a number of B cell lines (McLeod, Ingham et al. 1998). 0 5 10 15 30 P M A Figure 3.11: R a p l is not activated by ionomycin stimulation in T28 cells T28 cells were stimulated with 500 ng/ml ionomycin for the indicated times (in minutes), or PMA for 5 minutes at 37°C. Activated Rap (Rap-GTP) was pulled down using Ral-GDS coated agarose beads as described in Materials and Methods. Pull-down samples (upper blot) and whole cell lysates (lower blot) were analyzed by Western blotting with anti-Rap 1 antibody. 62 Effects ofpharmacological inhibition of Protein kinase C's (PKCs) and PI 3-kinases (PI3Ks) on ionomycin-induced LFA-1 activation P K C s are involved in L F A - 1 activation by chemokines (Giagulli , Scarpini et al. 2004) and T C R stimulation (Dustin and Springer 1989; Hauss, Mazerolles et al. 1993), and can activate L F A - 1 when expressed in constitutively active forms (Katagiri, Hattori et al. 2000). P K C s were therefore studied as possible mediators of ionomycin-induced activation of L F A - 1 . Bisindolylmaleimide I ( B I M I) inhibits the conventional P K C isoforms a, pi, pil, and y, the novel P K C isoforms 8 and 8, and the atypical P K C C,. Preincubation with B I M I almost completely abrogated PMA-induced adhesion of T28 cells (Figure 3.12), while having a negligible effect on ionomycin-induced adhesion. This supports previous findings that activation of P K C s is responsible for L F A - 1 stimulation by P M A (Dustin and Springer 1989; Hauss, Mazerolles et al. 1993). The minimal inhibition of ionomycin-induced adhesion also further supports the idea that P M A and ionomycin act through distinct pathways. 100 5 10 20 Bisindolylmaleimide I (pM) Figure 3.12: P M A activation of L F A - 1 is through a PKC-dependent pathway T28 cells were incubated with the indicated concentrations of B I M I for 30 minutes, then stimulated with 500 ng/ml ionomycin (black bars) or 50 ng/ml P M A (grey bars) for 10 minutes at 37°C. Percentage inhibition of adhesion was analyzed as in Figure 3.6A. Results are the means of three independent experiments, each performed in triplicate. Error bars indicate SD. 63 PDKs are involved in the stimulation of LFA-1 dependent adhesion by chemokines (Constantin, Majeed et al. 2000) and by TCR stimulation (O'Rourke, Shao et al. 1998; Mueller, Daniels et al. 2004). Additionally, expression of a constitutively active form of PI3K activates LFA-1-mediated adhesion in a pro-B cell line (Ba/F3) (Katagiri, Hattori et al. 2000). Therefore, PI3K activation was chosen for further study as a potential mediator of ionomycin-induced LFA-1 activation. Cells were pre-treated with the irreversible PI3K inhibitor wortmannin and the specific but reversible PI3K inhibitor Ly294002 prior to stimulation with ionomycin or PMA. These agents inhibited ionomycin-induced LFA-1 adhesion in a dose dependent manner in T28 cells (Figure 3.13A, upper panel). Neither PI3K inhibitor induced apoptosis above the level seen with ionomycin alone (Figure 3.13B), indicating that the inhibition of adhesion is not due to induction of apoptosis. The inhibitors also failed to significantly inhibit PMA-induced LFA-1 activation, further supporting their specificity. These results suggest that PI3K signalling is at least partially responsible for the activation of LFA-1 by ionomycin. However, these findings were not fully reproduced in primary T cells or CTLs. Ly294002 pre-treatment resulted in significant inhibition of ionomycin stimulation of primary T cells (Figure 3.13 A, middle panel), with minimal inhibition of PMA. High variability in the degree of inhibition between experiments resulted in non-significant inhibition with wortmannin. Since only one of two PI3K inhibitors tested significantly inhibited ionomycin-induced LFA-1 activation, it is difficult to conclude whether PI3K is responsible for the ionomycin response in resting T cells. Both inhibitors significantly inhibited CTL adhesion stimulated by ionomycin (Figure 3.13A, bottom panel). However PMA-induced adhesion was also significantly inhibited. As a result, non-specific effects of these inhibitors cannot be ruled out in the case of CTLs. 64 Figure 3.13: PI3K inhibitors prevent ionomycin but not P M A induced LFA-1 activation in T28 cells (A) T28 cells (top panel), primary T cells (middle panel), or CTLs (bottom panel) were incubated with the indicated concentrations of Ly294002 and Wortmannin and then stimulated with ionomycin or PMA as in Figure 3.6A. Percent inhibition of adhesion was calculated as in Figure 3.6A. Results shown are the means of at least three independent experiments, each performed in triplicate. Error bars indicate SD. (B) T28 cells were pre-treated with Ly294002 (50 pM) or Wortmannin (50 nM) then stimulated with 500 ng/ml ionomycin as in Figure 3.6A. Apoptosis induction was analysed as in Figure 3.10. Results are the means of three independent experiments. Error bars indicate SD. 65 Since PI3K inhibitors blocked ionomycin-induced LFA-1 adhesion, we wished to confirm whether ionomycin activates PI3K. Akt/protein kinase B (PKB) is a kinase that is activated by phosphorylation following PI3K signaling. Therefore, Akt activation can be detected with a phosphorylated (Ser473) Akt-specific antibody (2.2.1.1). We found that in T28 cells Akt was phosphoryated without stimulation, and that the level of phosphorylation was not altered by ionomycin treatment at any of the time points tested (Figure 3.14, top panel). The blot was re-probed for GAPDH as a control for equal loading (Figure 3.14, bottom panel). These results demonstrate that ionomycin does not increase Akt phosphorylation in T28 cells, suggesting that PI3Ks are not activated by ionomycin. 0 2 5 10 15 64-*<gfc < * » j f c ^ ^ _ P . A k t Figure 3.14: Phosphorylation of Akt is unchanged by ionomycin stimulation T28 cells were stimulated with 500 ng/ml ionomycin for the indicated times (in minutes). 5 x 105 cell equivalents were loaded per lane and subjected to Western blotting with a phospho-Akt specific antibody (upper blot). The blot was reprobed with anti-GAPDH antibody for loading control (lower blot). 67 CHAPTER 4 DISCUSSION The head domain of talin has been implicated as an important mediator of integrin activation. Recombinant talin head fragments bind to the cytoplasmic P chains of integrins, and overexpression of these fragments is sufficient to activate allbp3 and LFA-1 integrins in CHO (allbp3) and K562 (LFA-1) cell lines (Calderwood, Zent et al. 1999; Calderwood, Yan et al. 2002; Vinogradova, Velyvis et al. 2002; Kim, Carman et al. 2003; Tadokoro, Shattil et al. 2003). However, the integrin-binding site within the talin head is thought to be masked by interactions with the talin tail domain in intact talin, in a manner analogous to other ERM proteins. Cleavage of talin by calcium-dependent calpain proteases in vitro produces a fragment containing the talin head domain that has substantially higher affinity for integrin P tails than intact talin (Yan, Calderwood et al. 2001). LFA-1-mediated adhesion of T cells blasts can be activated by calcium mobilizers, and activation is abrogated by pre-treatment with the calpain inhibitor calpeptin (Stewart, McDowall et al. 1998). Together, these findings suggest that increases in intracellular calcium activate calpains, inducing talin cleavage to generate free talin head, which binds to the cytoplasmic tail of LFA-1, inducing conformational changes and activating its ligand-binding function (Dustin, Bivona et al. 2004). The results presented above dispute the view that calpain-mediated cleavage of talin is important for the activation of LFA-1 by calcium ionophores. Ionomycin-induced calcium increases activate LFA-1 in the helper T cell hybridoma line T28, primary splenic T cells, and CTLs, consistent with previous reports (Stewart, McDowall et al. 1998). However, ionomycin stimulation did not activate talin cleavage in T cells. The calpain inhibitors calpeptin and PD 150606 inhibited ionomycin-induced LFA-1 activation; however, the effects of these inhibitors were non-specific. First, calpeptin also inhibited LFA-1 activation by PMA, which did 68 not cause increases in intracellular calcium, and is therefore unlikely to activate calpains. Second, calpeptin and PD 150606 rapidly induced apoptosis in ionomycin-stimulated cells, particularly at high concentrations. Third, two additional calpain inhibitors, ALLN and calpain inhibitor III, did not inhibit ionomycin-induced LFA-1 activation. Furthermore, we could not detect spectrin cleavage in response to ionomycin stimulation in T cells. Therefore, these results suggest that ionomycin-induced activation of LFA-1 is not mediated by the calpain-mediated cleavage of talin. Calpain-mediated cleavage of talin is not required for the activation of integrin-mediated adhesion. Disruption of the common subunit calpain gene eliminates both p and m-calpain activity, and results in embryonic lethality (Arthur, Elce et al. 2000). Fibroblasts derived from these mice maintain the ability to adhere to fibronectin even though talin cleavage is completely abrogated in these cells (Dourdin, Bhatt et al. 2001). Additionally, calpain inhibitors prevent talin cleavage in endothelial cells, but do not affect initial cell adhesion (Kulkarni, Saido et al. 1999). Integrin-mediated signalling has been shown to induce calpain activation and talin cleavage in platelets (Fox 1985; Schoenwaelder, Yuan et al. 1997), fibroblasts (Dourdin, Bhatt et al. 2001; Franco, Rodgers et al. 2004), and endothelial cells (Fujitani, Kambayashi et al. 1997). Calpain activity is important for the spreading and motility of a number of cells types (Huttenlocher, Palecek et al. 1997; Potter, Tirnauer et al. 1998; Croce, Flaumenhaft et al. 1999; Kulkarni, Saido et al. 1999; Bhatt, Kaverina et al. 2002), and appears to involve the turnover of focal adhesions at attachment sites at both the leading and rear edges of cells (Glading, Lauffenburger et al. 2002). Calpains localize to focal adhesions (Beckerle, Burridge et al. 1987), and cleave a number of proteins involved in cytoskeletal linkages, including talin (Glading, 69 Lauffenburger et al. 2002). Recently, calpain cleavage of talin has been identified as a critical step in focal adhesion disassembly (Franco, Rodgers et al. 2004). A mutant form of talin containing a point mutation rendering it resistant to proteolysis by calpain was generated by this group and expressed in a talin deficient cell line. This experiment demonstrated that calpain-mediated cleavage of talin is a rate-limiting step in the disassembly of talin from focal adhesions. Additionally, the ability of calpain to cleave talin is required for the disassembly of other adhesion components, including paxillin, vinculin and zyxin. These findings identify calpain-mediated proteolysis of talin as a key mechanism by which adhesion dynamics are regulated. Therefore, calpain activation and cleavage of talin appears to be critical for integrin-mediated adhesion events such as cell spreading and migration, but not for initial integrin activation events. It is unclear whether ionomycin treatment of T cells efficiently activates calpains. Cleavage of talin and spectrin, two well-characterized calpain substrates, was not detected in this study. Although calpains were initially thought to be regulated by increases in intracellular calcium, the calcium concentrations required for calpain activation in vitro are not readily achieved in intact cells, leading to the proposal that other mechanism exist to lower the calcium requirement of calpains. Calpain activation and proteolytic activity has been detected in aggregated platelets and adherent endothelial cells treated with the calcium ionophore A23187 (Fujitani, Kambayashi et al. 1997; Schoenwaelder, Yuan et al. 1997; Croce, Flaumenhaft et al. 1999). Substrate cleavage is not detectable until well after platelet aggregation has occurred (Schoenwaelder, Yuan et al. 1997), suggesting that integrin-mediated signaling is required for calpain activation by calcium ionophores. However, calpain proteolytic activity has been reported to be activated in platelets by high concentrations of A23187 (lpM) in the absence of 70 stirring, which minimizes aggregation (Oda, Druker et al. 1993). Therefore calcium fluxing induced by high concentrations of calcium ionophore may be sufficient to activate calpains in platelets in the absence of ligand binding. In addition to factors required for activation, the proteolytic activity of calpains may also be regulated by the subcellular location of activated calpains relative to the substrate proteins (Goll, Thompson et al. 2003). Calpain activity translocates to the cytoskeletal/membrane fraction isolated by 0.1% Triton X-100 lysis after ionomycin stimulation of T cells, resulting in cleavage of phosphotyrosine phosphatase 1-B (PTP-1B) (Rock, Brooks et al. 1997). However, CD3 cross-linking of T cells activates calpain-mediated proteolysis of a-actinin, but not talin (Selliah, Brooks et al. 1996). Therefore, talin may not be in the correct location for cleavage by activated calpains in stimulated T cells. The exact mechanism of inhibition by which calpeptin and PD150606 inhibit ionomycin-induced adhesion is unclear, but is likely due to non-specific effects. Calpeptin also inhibits PMA-induced LFA-1 activation, and both inhibitors induce apoptosis in ionomycin treated cells, particularly at high concentrations. Both inhibitors have been reported to have non-specific effects. Calpeptin inhibits PTP-1B (Schoenwaelder and Burridge 1999), cathepsin L (Sasaki, Kishi et al. 1990), and papain (Tsujinaka, Kajiwara et al. 1988). PD150606 is more potent than the active-site inhibitors, but it also is not entirely specific for calpains as it can also inhibit calmodulins (Wang, Nath et al. 1996), and has been reported to have calpain-independent effects in motor neurons (Van den Bosch, Van Damme et al. 2002). Toxicity after incubation with calpeptin has also been described in the Jurkat T cell line at similar concentrations (-140-280 pM) (Vanags, Porn-Ares et al. 1996). The non-specific effects reported here have important consequences for studies that have identified a role for calpains in LFA-1-mediated adhesion 71 using high concentrations of calpeptin (Stewart, McDowall et al. 1998; Constantin, Majeed et al. 2000), and further investigation is required. Talin accumulates in the junctions between T cells and target cells (Kupfer, Singer et al. 1986; Kupfer and Singer 1989; Kupfer, Monks et al. 1994; Monks, Freiberg et al. 1998; Somersalo, Anikeeva et al. 2004), suggesting that talin is important for LFA-1 function. However, whether talin is required for the initial activation of integrins is controversial. Knock down of talin 1 expression by small interfering RNAs decreases allbp3 integrin activation in embryonic stem-cell derived megakaryocytes by multiple agonists (Tadokoro, Shattil et al. 2003). Additionally, the phenomenon of trans-dominant inhibition has been proposed to be mediated by competition for talin (Calderwood, Tai et al. 2004). Expression of a membrane-localized P3 cytoplasmic domain (Tac-P3) inhibits the activation of transfected and endogenous pi and P3 integrins in CHO cells. Tac-p3 constructs with mutations that disrupt talin binding are no longer able to mediate trans-dominant inhibition, while the expression of talin head fragments reverses the inhibitory effect of Tac-p3. Conversely, cells from talin-deficient Drosophila display normal adhesion to extracellular matrix proteins (Brown, Gregory et al. 2002). Embryonic fibroblasts derived from talin 1-deficient mice also show normal integrin-mediated adhesion to extracellular matrix proteins (Giannone, Jiang et al. 2003; Jiang, Giannone et al. 2003). The reason for the discrepancy in these results is not known. My studies indicate that talin consistently accumulates at the contact site with activated integrins only after ligand binding (Supplemental Figure, Appendix). This is consistent with previous reports that talin interaction with integrins requires integrin occupancy (Miyamoto, Akiyama et al. 1995; Vignoud, Albiges-Rizo et al. 1997; Mattel, Racaud-Sultan et al. 2001), and that talin accumulation at the contact site with APCs is dependent on interactions with LFA-72 1/ICAM-l interactions (Sedwick, Morgan et al. 1999). These results suggest that the interaction of talin with LFA-1 is induced by post-adhesion events, and that talin is not required for integrin activation. Talin has been shown to be critical for establishing a stable connection between integrins and the cytoskeleton, and in the formation of focal adhesion complexes (Priddle, Hemmings et al. 1998; Brown, Gregory et al. 2002; Giannone, Jiang et al. 2003; Jiang, Giannone et al. 2003). Therefore, talin may influence adhesion-dependent integrin function, by regulating integrin interactions with other components of focal adhesions as well as with the actin cytoskeleton. The accumulation of full-length talin at the contact site with sICAM-1 coated beads also suggests that calpain-independent events are involved in inducing the interaction of talin with integrins. As discussed in the introduction, talin binding to integrin cytoplasmic tails is also regulated by PIP2 binding, and PIP2 is required for integrin localization in focal adhesions. PIP2 accumulates with talin only after integrin adhesion in vitro (Martel, Racaud-Sultan et al. 2001), which may explain the ligand-binding requirement of talin accumulation at the contact site. Further experimentation is required to explore PIP2-talin interactions in whole cells, and whether phosphorylation of talin is also involved in full activation, as has been seen in other ERM proteins. Ionomycin activation of LFA-1 is independent of calpain-mediated talin cleavage; therefore additional signalling mechanisms were investigated for involvement. The PI3K inhibitors wortmannin and Ly294002 inhibited ionomycin-induced adhesion of T28 cells in a dose-dependent manner, implicating PI3Ks in ionomycin activation of LFA-1. To confirm that ionomycin activates PI3K, Akt/PKB activity was studied. An increase in Akt phosphorylation was not detected following ionomycin stimulation of T28 cells, which refutes the conclusion 73 that PI3K is at least partially responsible for ionomycin activation of LFA-1 since it argues that ionomycin does not activate PI3K. However, expression of a constitutively active form of Akt does not activate LFA-1-dependent adhesion, while expression of a constutively active PI3K does (Katagiri, Hattori et al. 2000). Therefore, an effector other than Akt may be important in LFA-1 activation by PI3Ks. Ionomycin treatment of fibroblasts fails to stimulate Akt S6rC phosphorylation, but does strongly activate the serine-threonine kinase p70S6 kinase (p70 ) in a PI3K-dependent manner (Conus, Hemmings et al. 1998). p70 is activated following integrin ligation (Malik and Parsons 1996; Balasubramanian and Kuppuswamy 2003; Levy, Ronen et al. 2003), however its involvement in integrin activation has not been demonstrated. Erk2 and Itk have been implicated as PI3K downstream effectors activated by TCR stimulation (Von Willebrand, Jascur et al. 1996; Woods, Kivens et al. 2001). The MEK/ERK pathway and Itk are both are involved in the activation of pi integrins (Ashida, Arai et al. 2001; Woods, Kivens et al. 2001), and the MEK/Erk pathway has been implicated in the activation of LFA-1 by TCR stimulation (O'Rourke, Shao et al. 1998). Therefore, there are a number of PI3K downstream effectors that may be involved in ionomycin-induced activation of LFA-1. Further experimentation is required to investigate each of these candidates. 74 Conclusion: The aim of this thesis was to test the hypothesis that ionomycin stimulation of T cells activates calpain-mediated cleavage of talin to yield a talin head fragment that activates LFA-1 function. The results presented here demonstrate that cleavage of talin by calpains is not the mechanism of LFA-1 activation by ionomycin. Calpains may not be efficiently activated by calcium increases induced by ionomycin, and may require additional signals for activation. However, it is possible that calpains are activated, but that talin in not in the correct subcellular localization to be susceptible to cleavage. Calpain-mediated proteolysis of talin has been shown to be important for the disassembly of focal adhesions. It should be noted that LFA-1 does not form focal adhesions, therefore whether calpain activity has a similar role in T cell migration mediated by LFA-1 remains to be determined. Preliminary results suggest that talin is not involved in the initial activation of L F A - 1 , as talin only accumulates at the contact site with active integrins after ligand binding. Therefore talin may be involved in formation of stable linkages with the actin cytoskeleton, as has been demonstrated with other integrins. These results also suggest that talin interaction with integrins may be regulated by a mechanism other than calpain-mediated cleavage; phosphorylation-induced conformational change in the presence of PIP2 is a strong candidate for this role. Integrating the data presented in this thesis, as well as evidence from the literature, a model of the regulation of LFA-1 function can be proposed: physiological stimulation of LFA-1 such as through the T C R or chemokine receptors activates R a p l , which subsequently binds to and regulates the subcellular location of RapL. RapL binding to the (XL subunit of LFA-1 induces 75 a separation of the a and P cytoplasmic tails, initiating conformational changes in the extracellular domain that increases affinity for I C A M - 1 . The interaction between RapL and LFA-1 may also direct the re-distribution of LFA-1 on the plasma membrane to increase avidity for ligand, although the requirement of clustering prior to ligand engagement remains controversial. LFA-1 in this intermediate affinity conformation is capable of binding to ligand. Ligand binding induces conformational changes in the integrin head domains that result in additional separation of the cytoplasmic tails, and further increases the affinity of LFA-1 for ligand. Signalling processes initiated by ligand binding stimulates phosphorylation of talin and subsequent PIP2 binding, which induces conformational changes that unmask the integrin binding domain in talin head. Talin then binds to the P2 subunit of L F A - 1 , and this interaction may stabilize the separation of the cytoplasmic tails to maintain LFA-1 in the high affinity state. The actin- and vinculin- binding domains throughout the talin protein also provides a firm link between LFA-1 and the actin cytoskeleton, further stabilizing the integrin. 76 APPENDIX The distribution of full length talin in ligand-bound and antibody-bound CTLs Talin distribution in cells was evaluated by confocal microscopy using an antibody (Sigma, 8d4) that recognizes full length talin, but not the talin head fragment. Although the antibody used for visualizing talin is capable of detecting the talin rod fragment, the major integrin-binding site is within the head domain (Yan, Calderwood et al. 2001). This suggests that it is the full length talin protein that is detected at the contact site, however further experimentation is required to confirm this. The requirement for ligand binding was also explored in this experiment by incubating cells with either sICAM-l-co'ated or anti-LFA-1 coated beads. Protein coated beads were used to avoid any interference from multiple protein-protein interactions that occur when target cells are used. Talin localized at the contact site with ICAM-1-coated beads in 96% of conjugates analyzed (n=25; Fig 12, rows 1 and 2). LFA-1 distribution was not evaluated in this experiment because LFA-1 molecules interacting with ICAM-1 or TIB 213 may have been inaccessible to antibody staining. However, areas of the cellular membrane interacting with beads would presumably contain LFA-1. Therefore, these results suggest that talin co-localized with LFA-1 at the contact site after CTLs bound to ICAM-1. Talin localized to the contact site in only 25% of conjugates with anti-LFA-1 coated beads (n=19; Figure 12, rows 3 and 4). Talin remained evenly distributed throughout the cell in 32% of the conjugates, and talin accumulated at a site other than the contact site in the remaining conjugates (42%). This suggests that CTL interaction with antibody coated beads is insufficient to consistently initiate talin accumulation with LFA-1, and that ligand binding is required for this phenomenon. As a control, CTLs that had been 77 incubated with poly-L-lysine-coated beads were imaged (Figure 12, row 5) to demonstrate that integrin-mediated adhesion to beads is necessary for talin redistribution. These results suggest that full-length talin consistently co-localizes with LFA-1 only after ligand binding. Talin association with LFA-1 was not directly tested in this experiment, however, full-length talin has been co-immunoprecipiated with P2 from neutrophils (Sampath, Gallagher et al. 1998) and P2 cytoplasmic peptides from T cells (Valmu, Fagerholm et al. 1999). 78 Supplemental Figure: Talin consistently localizes to the contact site with sICAM but not a-LFA-coated beads CTLs were incubated with sICAM-(rows 1 and 2) or anti-LFA-l-(rows 3 and 4) coated beads as described in the Materials and methods to initiate adhesion. Cells were then fixed, permeabilized, and stained with anti-talin (Sigma) mAb, followed by Alexa-488-conjugated anti-mouse secondary antibody (green). The stained cells were analyzed by confocal microscopy. Stacks were 3D reconstructed to analyze for cell-bead interaction, and to determine the localization of talin relative to the contact site. Images shown are a single slice that is representative of talin distribution at the CTL-bead contact site. 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