Biological aspects of signal transduction by cell adhesion receptors

Biological Aspects of Signal Transduction by Cell Adhesion Receptors SureshK. Alahari,PeterJ. Reddig,and R. L. Juliano Department of Pharmacology,School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599

Cell adhesion receptors such as integrins, cadherins, selectins, and immunoglobulin family receptors profoundly modulate many signal transduction cascades. In this review we examine aspects of adhesion receptor signaling and how this impinges on key biological processes. We have chosen to focus on cell migration and on programmed cell death. We examine many of the cytoplasmic signaling molecules that interface with adhesion receptors, including focal adhesion kinase (FAK), phosphatidylinositol-3-kinase (Pl3K), and elements of the Erk/MAP kinase pathway. In many cases these molecules impinge on both the regulation of cell movement and on control of apoptosis. KEY WORDS: Signal transduction, Adhesion receptors, Cell migration, Apoptosis, Integrin, Cadherin, Selectin, IS-CAM. 0 2002. Elsevier Science (USA).

I. Introduction Over the past several years it has become clear that integrins and other cell adhesion receptors play key roles in regulating several signal transduction pathways. These actions of adhesion receptors can thus impinge on many important biological processes. The literature in this area has grown rapidly and is now difficult to survey within the confines of a single article. This review recapitulates current understanding of several families of adhesion receptors and their effects on signaling, with particular emphasis on integrins. It then focuses on how the actions of adhesion receptors and their associated proteins regulate certain key cellular processes. In this article we have chosen to focus on cell motility and on regulation

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Copyright 2002, Elsevier Science (USA). All rights reserved.

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of programmed cell death. In particular, we seek to connect the regulation of signal transduction by adhesion molecules to the control of downstream phenomena involved in motility and apoptosis. However, remember that signals regulated by adhesion receptors also affect many other processes such as cell cycle traverse and ceil differentiation that are not reviewed here.

II. Structure Receptor

and Function Familes

of Adhesion

A. lntegrins The integrins comprise a family of cell-surface glycoproteins that functions as receptors for extracellular matrix (ECM) proteins, or for transmembrane counterreceptors on other cells. Integrins are heterodimers that include c+ and /?-subunits with each subunit having an extracellular domain, a single transmembrane region, and (other than PA),a rather short cytoplasmic domain (Rosales et al., 1995; Hynes, 1999). The vertebrate integrin family includes at least 18 distinct a-subunits and 8 or more B-subunits; these can associate to form over 20 distinct integrins. The ligand binding abilities of the integrin heterodimers are determined by the U/B pairings. The ligands for integrins are usually large extracellular matrix proteins including collagen, laminin, vitronectin, and fibronectin (Kuhn and Eble, 1994); however, some integrins recognize short peptide sequences within the larger protein, for example the RGD (Arg-Gly-Asp) sequence. Thus there has been interest in the pharmaceutical industry in developing short peptides or peptidomimetics that can block integrin functions in disease processes including coagulation disorders, inflammation, and cancer (Ruoslahti, 1996; Arap et al., 1998). Some integrins, such as a@~, the “classic” fibronectin receptor, interact only with a single ECM protein; however, more commonly, an integrin will recognize several distinct matrix proteins (Rosales and Juliano, 1995). In addition, most cells express several distinct integrins and are thus capable of interacting with multiple ECM proteins. Some integrin subunits are subject to alternative splicing of their cytoplasmic domain regions; these alternatively spliced versions can have quite distinct biological roles (Fomaro and Languino, 1997). The relationships between integrin structure and the various functions of integrins are an active area of investigation. In terms of ligand binding, it seems clear that both w and B-subunit extracellular domains contribute to the formation of the binding site. For the subset of integrins that contains an inserted domain (I or A domain) in the a-subunit, this domain clearly plays an important role in ligand binding, and key insights have been gained from X-ray work (Emsley et aZ.,2000). In an exciting recent development, the crystal structure of the extracellular portion of the (II& integrin has been solved at 3.1 A resolution (Xiong et al., 2001).

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SlGNALTRANSDUCTlONEJYCELLADHESlONRECEPTORS Myosin

Endonexin

J*Bl
FAK

a-actinin

-Paxillin

_ ,Nischarin

TAP20 WAIT1

d

4 -

-1AP

50

Caheticulin FIG. 1 Proteins associated with integrins. Tbe figure represents a compilation of recent reports on interactions between various integrins with other proteins. In most cases the binding is via the integrin cytoplasmic domain. However, IAP50, uPAR, and TM4 proteins are transmembrane proteins that interact with other integrin regions.

Although this X-ray analysis confirms many of the previous concepts of integrin structure, such as the presence of a seven repeat j?-propeller structure in the a-subunit (Humphries and Newham, 1998), it also provided some surprises, such as the flexibility of the “stalk” regions of both subunits. A large number of cytosolic proteins bind directly to integrin a! or j3 cytoplasmic domains; many of these are illustrated in Fig. 1. In most cases the roles of these interacting proteins are only poorly defined. However, it seems likely that many of them play a part in linking integrins to the cytoskeleton, and in integrindependent signaling. Integrins also associate with a number of other proteins via their external and transmembrane domains, including caveolin, several tetraspannins, IAP, urokinase plasminogen activator receptor, several metalloproteases, and proteases involved in transforming growth factor-p (TGF-fi) processing (Hemler, 1998; Porter and Hogg, 1998). The short cytoplasmic domains of integrins provide a key link between the ECM, intracellular structures, and signaling cascades. The o- and B-subunits both make important contributions to various aspects of overall integrin function including signal transduction, cell motility, cytoskeletal organization, and control of integrin

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affinity for ligands (integrin activation). The /3 cytoplasmic domain is critical for recruitment of integrins to focal contacts (the specialized sites of cell-matrix adhesion), as its truncation/mutation impairs this process (LaFlamme et al., 1992; Reszka et al., 1992). As is true of other types of receptors, integrins can exist in different affinity states with respect to ligand binding. Integrins can be placed in a high-affinity (“activated”) state in response to certain agents that bind the extracellular domain and influence its conformation (divalent cations, antibodies). Integrins can also respond to signals generated within the cell that presumably impact on the cytoplasmic domain (“inside-out signaling”) (Humphries, 1996; Keely et al., 1997). Thus, activation of H-Ras-driven cascades reduces integrin affinity, whereas activation of the Rap-l or R-Ras small GTPases tends to enhance integrin aftinity (Parise et al., 2000). The contributions of the cytoplasmic domains to integrin activation are quite complex. For example, partial truncation of ~4, ~2, or ,62 cytoplasmic domains prevents integrin activation, whereas truncation of the aIIb subunit activates the fibrinogen-binding integrin crtt& (O’Toole etal., 1994). A rather coherent model of how cytoplasmic domains influence integrin activation is now widely accepted (Hughes et al., 1996). This model suggests that the a-subunit cytoplasmic domain inhibits certain functions of the /3 cytoplasmic domain (e.g., recruitment to focal contacts), but that binding of a ligand to the integrin relieves this inhibition, possibly by allowing the subunits to swing apart like a hinge. The membrane-proximal regions of all (Y-and B-subunits are highly conserved: the conserved u sequence is GFFKR and the conserved B sequence is LLv-iHDR. Deletion of either of these sequences activates the integrin, “locking” it in a high-affinity state independent of cellular activity, whereas mutation C-terminal to these conserved sequences can affect activation that depends on intracellular functions (Hughes et al., 1996). The relative motions of the a- and B-subunits can also affect integrin affinity, allowing the “hinge” to swing, thus opening up the extracellular ligand-binding site (Hughes et al., 1996; Keely et al., 1998). This concept of regulatory interactions between the cytoplasmic tails of the CX-and #?-subunits has been supported by recent nuclear magnetic resonance (NMR) studies (Vinogradova et al., 2000).

B. Cadherins The cadherins are a family of transmembrane proteins that shares an extracellular domain comprised of repeats of an approximately lOO-amino acid module (Chothia and Jones, 1997; Humphries and Newham, 1998). The “classic” cadherin subfamily includes the N, P, R, B, and E cadherins as well as several other members (Takahashi et al., 1995). Members of this subfamily contain five repeat modules and these proteins serve primarily as calcium-dependent cell-cell adhesion molecules. The classic cadherins are vitally important in early developmental

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processes (Vleminckx and Kemler, 1999). They localize at specialized sites of cell adhesion that are termed adherence junctions; at these sites cadherins can establish linkages with the actin-containing cytoskeleton. Another important subfamily of cadherins involved in adhesion is represented by the desmogleins and desmocollins, a group of desmosome-associated cadherins that forms intracellular linkages to intermediate filaments rather than actin filaments (Cowin and Burke, 1996; Hynes, 1999). Much progress has been made recently using NMR and X-ray crystallography to determine the structure of the classic cadherin repeat module, which comprises a seven-stranded /?-barrel and a short a-helix (Humphries and Newham, 1998). Classic cadherins consist of an amino-terminal external domain having five tandem repeats, a single transmembrane segment, and a cytoplasmic carboxyterminal domain of about 150 amino acids. The binding functions of the cadherin are localized in the external domain, which functions as a cis-dimer and interacts in trans with a similar dimer on a neighboring cell. The repeats are bridged by calcium-binding sites that impart rigidity to the cadherin molecule. Recent evidence suggests that a relatively short peptide sequence flanking an HAV motif in the amino-terminal module plays a vital role in cadherin homotypic adhesions (Humphries and Newham, 1998); however, other repeat units also contribute to the interaction (Chappuis-Flament et al., 2001). In summary, cadherins on one cell surface form a series of rigid dimers that presents several cadherin repeats to equivalent dimers on the opposing cells; lateral motion of these complexes allows the cell junction site to “zip up” to form a stable adhesion (Vasioukhin et al., 2000). The cytoplasmic domains of cadherins interact with a group of intracellular proteins termed catenins. These proteins are critical for cadherin function, as deletion of catenin binding sites leads to a reduction in cadherin-mediated adhesion (Gumbiner, 1996; Vleminckx and Kemler, 1999). /I-Catenin binds directly to the cadherin cytoplasmic domain whereas a-catenin binds to /I-catenin and serves to link the complex to the actin cytoskeleton by direct interactions with actin and a-actinin (Cowin and Burke, 1996). Another protein termed ~120”” binds to the membrane proximal region of the cadherin cytoplasmic domain and plays a role in cadherin clustering and in signal transduction (Vleminckx and Kemler, 1999; Noren et al., 2000). The regulation of adherens junction formation by cadherins is a complex process that involves Rho-family GTPases, as well as a variety of cytoskeletal proteins (Gumbiner, 2000). Disruption of cadherin expression or function leads to serious pathophysiological consequences. For example, in epithelial tumors progression toward an invasive, malignant phenotype is associated with loss or mutation of E-cadherin, or with the disruption of cadherin-catenin complexes (Birchmeier, 1995). In addition to a structural role in organizing adherence junctions, cadherins are also clearly involved in signaling, especially in connection with the Wnt pathway (Peifer, 1996; Aplin et al., 1998). Thus the cadherins play a key role in both tissue organization and in regulation of signaling cascades.

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C. Ig-CAM Superfamily The immunoglobulin (Ig) superfamily of cell adhesion molecules (Ig-CAMS) represents an extraordinarily diverse group of cell-cell adhesive receptors. Members of this family are defined by the presence of one or more copies of the Ig fold, a globular domain having two cysteine residues separated by 55-75 amino acids (Vaughn and Bjorkman, 1996). In addition, Ig-CAMS often contain one or more copies of a fibronectin type III repeat domain. Typically Ig-CAMS have a large amino-terminal extracellular domain containing the Ig modules, a single transmembrane domain, and a cytoplasmic tail. Members of the Ig-CAM family function in a wide variety of cell types and are involved in many different biological processes. There has been substantial progress in structural analysis of Ig-CAMS, especially with regard to a subfamily of Ig-CAMS that serves as counterreceptors for certain integrins. This includes ICAMs 1-3, VCAM- 1, and MadCAM- 1, where crystal structures of two Ig domains have been determined in each case, and where the integrin-binding site has been mapped for ICAM- (Casasnovas et al., 1999). One important biological context for Ig-CAMS is the immune system. In fact, integrins, selectins, and Ig-CAMS are all critically involved in multiple aspects of immune function (Rosales and Juliano, 1995; Springer, 1995). Several Ig superfamily receptors are expressed on T lymphocytes, including CD2, CD4 or CD8, ICAMs 1 and 2, and the T cell receptor (TCR) itself. These Ig-CAMS play important roles in antigen recognition, cytotoxic T cell functions, and lymphocyte recirculation. In contrast to the situation with neural Ig-CAMS (see below), Ig family proteins in the immune system primarily engage in heterotypic interactions. Thus, CD2 on T cells interacts with LFA-3 expressed on target cells, the TCR interacts with MHC class II proteins on antigen-presenting cells (all of these Ig superfamily), whereas ICAMs on endothelial cells are recognized by ,& integrins on leukocytes. Other Ig-CAM receptors are found on vascular endothelial cells and play an important role in leukocyte behavior during inflammation. Thus, VCAM-1 is an endothelial cell counterreceptor for the integrin @i found on leukocytes, whereas PECAM-1 is an endothelial cell Ig-CAM that is important in maintaining contacts between adjacent vascular endothelial cells and in supporting the extravasation of leukocytes (Newman, 1999). In nervous tissue many different members of the Ig-CAM family are involved in axon guidance and in the establishment and maintenance of neural connections (Murase and Schuman, 1999). An important and well-studied example is NCAM, which contains five Ig folds in its extracellularportion (Crossin and Krushel, 2000). NCAM serves as a homotypic, calcium-independent cell-cell adhesion receptor. There are three forms of NCAM, two with transmembrane domains and one having a GPI link to the membrane, but all seem to be involved in cell interactions. Other neural cell adhesion molecules that are Ig-CAMS include Ll, NgCAM, TAGl, contactin, and fasciclin II (Tessier-Lavigne and Goodman, 1996; Crossin

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and Krushel, 2000). The netrin receptors, such as DCC, are another group of Ig-CAMS important in neural development. These proteins interact with lamininlike netrins in the extracellular matrix to generate specific guidance cues to migrating axons (Culotti and Merz, 1998). The dozen or so members of the Eph subfamily of transmembrane tyrosine kinases that bind their cognate ligands (ephrins) on neighboring cells are another group of key Ig-CAM receptors involved in neural development (Bruckner and Klein, 1998). An important group of signaling proteins that overlaps with the Ig-CAM superfamily are the receptor protein tyrosine phosphatases (RPTPs) (Neel and Tonks, 1997; Li and Dixon, 2000). The RPTPs typically have a large external domain (often having Ig motifs), a single transmembrane helix, and a cytoplasmic domain containing two signature tyrosine phosphatase domains flanked by a variety of noncatalytic sequences. RPTPs function conversely to receptor tyrosine kinases (RTKs); that is, ligand binding results in dimerization of RPTPs but inhibits enzyme activity (Jiang et al., 1999). It is clear that several RPTPs can engage in homotypic or heterotypic cell adhesion through their extracellular domains (Neel and Tonks, 1997). Not much is known about the interactions of Ig-CAMS with cytoplasmic proteins. A linkage between Ll and actin mediated by ankyrin has been suggested (Crossin and Krushel, 2000). The ICAMs interact with ezrin, a member of the ERM family of proteins that serves to directly link certain membrane receptors to the actin cytoskeleton (Bretscher, 1999). It seems likely, however, that multiple other interactions exist and contribute to the functions of Ig-CAMS.

D. Selectins There are three selectins, L, E, and P, that comprise a small family of lectin-like cell-cell adhesion molecules that are primarily involved in leukocyte trafficking (La&y, 1995). Selectins have an amino-terminal domain homologous to calciumdependent animal lectins, followed by an epidermal growth factor (EGF)-type domain, several complement regulatory protein repeats, a transmembrane segment, and a cytoplasmic tail. Selectins employ calcium-dependent recognition of sialylated glycans to mediate heterotypic cell-cell interactions. Of necessity, there is tight regulation of the expression and function of selectins, so as to come into play only when leukocytes need to stick to the vessel wall during normal cellular trafficking or during inflammation. The ligands for selectins are tetrasaccharide residues of the sialyl-Lewisx type; such motifs appear on glycolipids as well as on glycoproteins. However, the binding affinities of selectins for isolated sialyl-Lewisx saccharides are very poor, whereas selectins promote high-affinity cell-cell binding. Thus, physiological ligands for selectins must likely include sialyl-Lewisx saccharides in the context of a macromolecular scaffold (Varki, 1997). The best documented high-affinity

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counterreceptor for a selectin is PSGL- 1, a mucin-like transmembrane glycoprotein found on leukocytes and lymphoid cells (Yang et al., 1999).

III. Overview Receptor

of Upstream Signaling

Events

in Adhesion

Understanding how adhesion receptors affect signaling is most advanced in the context of the integrin family. However, as will be seen below, much of integrindependent signaling involves the actin cytoskeleton. Because it is clear that cadherins, selectins, and Ig-CAMS can also interdigitate with the cytoskeleton, it seems likely that many of the effects of integrins will be paralleled in effects mediated by other families of adhesion receptors. Nonetheless, there will also clearly be opportunities for specificity. Here we focus on how integrins trigger and modulate signaling processes.

A. Direct Signaling by lntegrins It is now quite clear that integrins can directly activate intracellular signaling processes in the absence of soluble growth factors. Thus integrin engagement with ligand, along with integrin clustering, can recruit a number of structural and signaling components leading to activation of important signal transduction cascades, especially the MAP kinase pathway (Miyamoto et aZ., 1995). The literature on direct integrin signaling has been recently reviewed (Aplin et al., 1998; Giancotti and Ruoslahti, 1999). Here we will simply outline some of the basic ideas. An early insight into the possibility of integrin signaling was provided by the observation that integrin-mediated adhesion and (or) clustering could lead to enhanced tyrosine phosphorylation (Kornberg et al., 1991). It soon became clear that adhesion was activating a cytoplasmic nonreceptor tyrosine kinase currently known as focal adhesion kinase (FAK) (Hanks et al., 1992; Schaller et aZ., 1992). This protein has a central kinase domain and extensive N- and C-terminal regions that provide docking sites for other molecules and that are involved in FAK’s subcellular localization. In particular, the C-terminal has a “focal adhesion targeting” sequence that is involved in bringing FAK to integrin-rich focal contacts. FAK binds to a number of other signaling and structural proteins including c-Src, phosphoinositide 3-kinase (P13K), GRAF (a Rho-GAP), paxillin, and ~130 Cas. Activation and tyrosine phosphorylation of FAK accompany integrin-mediated cell attachment, and dephosphotylation takes place rapidly when cells are detached (Aplin et al., 1998; Parsons et al., 2000). The mechanisms underlying these events are poorly defined. There is little evidence for a direct association of FAK with integrins; indeed, at least in some cell types, FAK phosphorylation seems to

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be a relatively late event that is downstream of actin filament assembly (Gao et aZ., 1997). FAK is postulated to play a role in cell migration and apoptosis (see below). It has also been linked to integrin-mediated activation of the ERK-MAP kinase cascade, at least in some situations. Several quite different mechanisms for adhesion-triggered activation of ERK have been proposed. One model suggests a central role for FAK. Thus, upon integrin engagement with ECM proteins, FAK is recruited to focal contacts and autophosphorylated at Y397. This provides a binding site for the c-Src SH2 domain and recruits Src, which then phosphorylates FAK at additional sites. One site, Y925, provides a binding site for the SH2 domain of the adaptor protein Grb-2. Recruitment of the Grb-2/Sos complex sets the stage for activation of Ras by the exchange factor activity of SOS.Ras activation is followed by activation of the downstream kinase cascade comprised of Raf-1, MEK, and Erk (Aplin et al., 1998; Schlaepfer et al., 1999). Although there is substantial evidence in support of this mechanism, there are also studies that indicate that integrin-dependent Erk activation can take place independently of FAK activation (Wary et al., 1996; Lin et al., 1997b). Another interesting model for integrin activation of the ERK cascade involves the transmembrane protein caveolin-1, the Src-family kinase Fyn, and the adaptor protein She. A subset of integrin a-subunits is proposed to be able to activate Fyn thus causing tyrosine phosphorylation of She and subsequent recruitment of the Grb-2/Sos complex. This then triggers Ras and the downstream kinase cascade leading to Erk activation (Wary et al., 1998; Giancotti and Ruoslahti, 1999). In both of these models Ras plays a key role in signaling to ERK, however, there is also evidence for Ras-independent mechanisms of integrin signaling to ERK (Howe and Juliano, 1998). The existence of several somewhat contradictory models for integrin-mediated Erk activation seems troubling at first. However, this may reflect the fact that signaling events are often highly cell-context dependent. One recently elucidated possibility is that the relative roles of the FAK-dependent and She-dependent pathways may reflect the levels of Raf-1 and B-Raf in different cell types. In addition to the postulated FAK/Grb-2/Sos/Ras assembly, FAK binds pl30Cas, which, when phosphorylated, creates binding sites for the C&II-C3G complex (Vuori, 1998). The C3G exchange factor can activate the Rap1 GTPase, which can activate B-Raf and consequently MEK and Erk. Thus, depending on the ratio of Raf isoforms, integrin signaling to Erk might be predominantly through She (when Raf-1 is high) or through FAK (when B-Raf is high) (Barber-is et al., 2000). The biological significance of direct integrin activation of the ERK cascade remains obscure. Integrin-mediated Erk activation is insufficient to trigger mitogenesis; additional signals provided by growth factors are still required. One possibility is that integrin modulation of the Erk pathway provides a mechanism for localized control of actinomyosin contractility and cell locomotion (see below). Recent research has indicated there is an intricate bidirectional pattern of communication between integrins, small GTPases, and the actin cytoskeleton. Members

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of the Rho family of small GTPases influence many key cellular processes, but are particularly important in regulation of actin filament assembly (Kjoller and Hall, 1999; Schoenwaelder and Burridge, 1999). Several studies have shown that integrin engagement activates the Rat and CDC42 GTPases (Bourdoulous et al., 1998; Clark et aZ., 1998; Price et al., 1998; Ren et al., 1999; O’Connor et&., 2000). Activated Rat and Cdc42 can couple to a variety of downstream effecters through processes that are also influenced by integrins. Thus constitutively active Rat is not sufficient to activate PAK in nonanchored cells, whereas in adherent cells, Rat translocates to the membrane and PAK is activated. These results indicate that integrin engagement with the ECM enables membrane association between Cdc42 and PAK, thus allowing kinase activation (de1Pozo et al., 2000). The precise mechanism(s) of integrin-mediated activation of Rat and Cdc42 have yet to be worked out, but no doubt exchange factors and/or GTPase-activating proteins are involved. It is clear that integrin activation of Rho GTPases likely plays a significant role in cytoskeletal organization and cell motility.

B. lntegrin and Cytoskeletal Modulation RTK/Ras/MAPK Cascade

of the

Integrin-mediated cell anchorage and the formation of cytoskeletal complexes can regulate signaling involving polypeptide growth factors and receptor tyrosine kinases (RTKs) via the RTK/Ras/MAPK pathway; this occurs in at least three distinct ways. First, there is modulation at the level of activation of the RTK. Second, the coupling between upstream and downstream events in the pathway is affected. Third, the traffic of signaling components between the cytoplasm and nucleus can be affected. These topics have recently been reviewed in detail (Juliano, 2002) and will be only briefly outlined here. A key role for integrins in the effective activation of RTKs was demonstrated several years ago (Miyamoto et al., 1996). In fact, there are at least two examples of integrin activation of RTKs that take place in the absence of growth factors, one involving the PDGFj3 receptor (Sundberg and Rubin, 1996) and the other EGFR (Moro et al., 1998). More commonly, both integrin engagement and the presence of growth factor are required to promote efficient RTK activation. Direct associations between RTKs and integrins have been observed for the PDGFBR, insulin receptor, and VEGFR2 (Schneller et al., 1997; Soldi et al., 1999). It seems obvious that formation of direct or indirect complexes between the RTKs and the integrins could lead to enhanced opportunities for RTK dimerization and crossphosphorylation. Recent evidence suggests that integrin-associated cytoskeletal components may support these putative complexes. Thus an association between the Neu RTK and actin has been observed in mammary carcinoma cells (Li et al., 1999), and a coupling between integrins and RTKs via FAK has also been described (Sieg et al., 2000).

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A second aspect of integrin regulation of the RTK/Ras/Erk cascade involves the coupling between upstream and downstream pathway components. Our group has found that the absence of integrin-mediated cell anchorage blocks the propagation of the mitogenic signal from Ras to Raf-1 (Lin et al., 1997a); another group placed the block at the level of MEK (Renshaw et al., 1997). Anchorage regulation of Erk activation clearly involves the actin cytoskeleton (Chen et al., 1994). A detailed analysis suggests that it is cortical actin filaments rather than focal contacts and stress fibers that are important (Aplin and Juliano, 1999). Consistent with this, expression of active Cdc42, which promotes cortical actin assembly, partially rescued Erk activation in suspended cells (Aplin and Juliano, 1999). Thus, there is solid evidence that integrin-mediated cell anchorage and the actin cytoskeleton can regulate cytoplasmic aspects of the Ras/Raf/Mek/Erk signaling cascade. However, the precise site of regulation has not been consistently defined. A third aspect of anchorage-dependent regulation of the RTK/Ras/Erk cascade concerns the traffic of the signaling components from cytoplasm to nucleus. A strong hint as to the existence of this aspect of regulation came from studies showing that forced activation of Erk is insufficient to drive cells into the cell cycle (Le Gall et al., 1998). This led to studies of the role of integrin-mediated anchorage in the trafficking of Erk between the cytoplasm and nucleus (Aplin et al., 2001). Thus, in suspension cells, or in cells treated with cytochalasin D, the normal trafficking of Erk is disrupted. Even when activated, Erk fails to enter the nucleus and thus cannot phosphorylate its key immediate targets such as the transcription factor Elk-l (Aplin et aZ., 2001). This observation supports a number of other studies showing that there is a close relationship between cytoskeletal organization and intracellular trafficking, with a number of proteins displaying movement between focal contact sites and the nucleus (Aplin et al., 2001).

IV. Signaling

Affecting

Cell Motility

Cell migration is an important component of various biological events, including wound healing, embryonic development, oncogenic progression, metastasis, and angiogenesis (Lauffenburger and Horwitz, 1996; Stupack et al., 2000). Cell motility is regulated by actin cytoskeleton, which in turn is regulated by Rho GTPases that link integrins to the actin cytoskeleton. Migration involves a systematically orchestrated series of events that allows cells to attach and detach in order for cells to protrude projections and directionally advance. The main characteristics of migratory cells include leading lamellipodium with membrane ruffles, decreased focal contacts, and loss of actin stress fibers. Cell motility consists of four essential events: extension of the lamellipod, formation of new focal adhesions at the leading edge, breaking of adhesions at the rear end, and translocation of the cell

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-

CELL MIGRATION

FIG. 2 A model showing the regulation of cell migration through different signaling cascades. Receptor tyrosine kinases (RTK) and integrins can activate Rat to regulate cell migration through the pathways regulated by PAK, MEKKl, or PI3K. PAK can affect cell migration through the activation of MLCK, the activation of LIMK, or the modulation of the ERK cascade. Moreover, Rat can govern cell migration through the MEKKl-INK cascade as well. Further, integrins also regulate cell migration through the activation of the FAK-Cas-Crk-Rat cascade, or through the activation of the ERK cascade. In addition, RTKs control cell migration through the activation of the Ras-ERK cascade, or by directly activating PI3K. Cell migration involves both random and directional components, with the Ras-ERK cascade primarily affecting random motion and Rac/PAK-mediated events primarily involving directional migration.

mass. Many of the events of cell motility involve adhesion receptors, particularly integrins, both in terms of structural organization and in terms of signaling. The following discussion is assisted by reference to Fig. 2.

A. She Regulation

of Motility

She is an adaptor protein and tyrosine phosphorylated she recruits Grb-USos to the membrane resulting in the activation of Ras signaling cascade. She can be activated by growth factor stimulation (Ravichandran, 2001) and by integrins (Wary et al, 1996). She and integrins have been linked through caveolin, a membrane adaptor (Wary et al., 1996). She has been shown to activate ERK signaling independently

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of FAK by associating with Src family kinase Fyn (Giancotti, 2000). In addition, She plays a critical role in cell migration, and it is essential for migration in certain metastatic tumor cells (Collins et al., 1999). In an elegant study, Yamada and colleagues (Gu et al., 1999) reported that integrins regulate two different pathways of cell migration, one through She and the other through FAK, leading to random and directional migration, respectively. She-regulated migration goes through the MAPK pathway, and FAK migration goes through the CAS cascade (Gu et al., 1999).

B. FAK Regulation

of Cell Migration

FAK is a nonreceptor tyrosine kinase that associates with focal adhesions and becomes phosphorylated in response to integrin-mediated cell attachment to the ECM. FAK has a significant role in cell migration. In Chinese hamster ovary (CHO) cells, overexpression of FAK increases cell migration dramatically (Guan and Chen, 1996; Guan, 1997; Cary and Guan, 1999), whereas FAK inhibition results in retarded cell motility (Gilmore and Romer, 1996). Furthermore, FAKdeficient cells showed a decrease in cell migration with an augmented number of focal adhesions (Ilic et al., 1995). Also, it was shown that FAK kinase activity, the SH2 domain-binding site, and the SH3-binding region are all essential for FAK-mediated cell migration (Sieg et al., 1999). FAK has been shown to associate with PDGF (Sieg et al., 2000) and EGF (Lu et aZ., 2001), and FAK needs to be targeted to sites of integrin-receptor clustering for an efficient EGF-stimulated cell motility (Sieg et aZ., 2000). However, in contrast, several carcinoma cells that have up-regulated EGFR expression showed a down-regulation of FAK activity, and increased cell motility, invasion, and metastasis (Lu et aZ., 2001) thus indicating a negative role for FAK in migration of these cells. FAK directly associates with the adaptor proteins ~130 Cas and Grb-2. This association is crucial for certain FAK-regulated signaling cascades. Although Grb-2 binds to FAK, it does not have a role in FAK-promoted cell migration; however, p 130 Cas has been implicated in this process. Upon cell attachment to the matrix, ~130 Cas is phosphorylated (Cary et al., 1998). In CHO cells, coexpression of FAK and ~130 Cas had a synergistic effect in promoting cell migration, suggesting that ~130 Cas is a downstream mediator in FAK-regulated cell migration (Cary et aZ., 1998). Crk is an adaptor protein that interacts with phosphorylated ~130 Cas. This complex drives cell migration and invasion in carcinoma cells through Rae-mediated pathways (Klemke et al., 1998; Cho and Klemke, 2000). CaslCrk association activates Rat through its interaction with the Rat-activating protein DOCK180, which binds to the amino-terminal SH3 domain of Crk (Cheresh et al., 1999). Activation of Rat is known to regulate actin polymerization and membrane ruffling (Ridley et al., 1992). Although the exact mechanism of Cas/Crk regulation of cell migration is unknown, the binding of Cas to Crk seems to serve as

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a switch to initiate cell migration (Klemke et al., 1998). In addition, Crk-family proteins activate several effector proteins including guanine nucleotide-releasing proteins such as C3G and the small GTPase Rap- 1. The Cbl protein has also been shown to regulate cell migration by complexing with Crkl and C3G (Uemura and Griffin, 1999; Feller, 2001). C3G is required for Rap-l activation, which in turn activates some integrins (Ohba et al., 2001), including o&z (Caron et al., 2000) and LFA-1 (Katagiri et aZ.,2000). Overall, the Cas/Crk/DOCKlSO complex seems to induce cell migration invasion through a Rat-dependent mechanism, whereas the Crk- l/Cbl/C3G complex activates cell migration through a Rap- 1-dependent mechanism.

C. Other FAK and Cas-Related

Migratory

Responses

Regulation of p 130 Cas by other signaling molecules can also impinge on cell migration. Laminin promotes ~130 Cas phosphorylation and ~130 Cas/Crk coupling through the (~7integrin, leading to an increase in cell migration. Mutation in the ~7 cytoplasmic domain inhibited Cas phosphorylation and lamellipodia formation, suggesting that the inhibition of cell migration is likely due to a reduction in Rat activation (Mielenz et al., 2001). Abl family kinases have been referred to as negative regulators of cell migration through their ability to regulate the association of Cas and Crk proteins (Kain and Klemke, 2001). Also, it was suggested that Crk is the specific target for Abl kinase, whereas Cas phosphorylation is unaffected by Abl kinases (Kain and Klemke, 2001). Paxillin is a scaffolding protein involved in integrin signaling that binds to several cytoskeletal proteins, including vinculin, FAK, and Src (Aplin et al., 1998). Interestingly, tyrosine phosphorylation of paxillin and tyrosine phosphorylation of ~130 Cas have been shown to exert opposing effects in integrin-mediated signaling cascades; in particular, paxillin reduces cell migration, whereas overexpression of ~130 Cas increases it (Yano et al., 2000). PI3K associates with FAK upon integrin activation, and it is required for FAKpromoted cell migration (Reiske et al., 1999). Because PDK, Src, and Cas all seem to be required for cell migration, it has been suggested that coordinated regulation of these signaling events is essential for FAK-promoted cell migration (Reiske et al., 1999). FAK also interacts with Grb-7 directly, and this association promotes cell migration, and is independent of FAK-Src, FAK-PI3K complexes (Han and Guan, 1999). Src family kinases that are upstream of FAK regulate cell migration as well; cells that lack Src kinases (Src, Yes, Fyn) display impaired cell migration, probably due to dramatic reduction of FAK tyrosine phosphorylation (Klinghoffer et al., 1999). Stat-l, another downstream mediator of FAK, interacts directly with FAK, and has also been implicated in regulating FAK migration (Xie et al., 2001). Thus FAK seems to influence multiple pathways associated with cell migration.

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159

by Rho GTPases

A role for Rho GTPases in integrin-mediated motility has also been widely reported. As discussed, Cas/Crk/DOCK180 molecules enhance cell migration, and this migration is inhibited by dominant-negative Rat-1, indicating that Rat is downstream of these complexes. Tiam-1 functions as an activator of Rat and regulates the invasive capacity of tumor cells (Michiels et al., 1995). For example, it was shown that Tiam-1 increases cell motility and invasion in murine breast cancer cells, where it forms a complex with CD44, a transmembrane glycoprotein (Bourguignon et al., 2000a,b). In contrast, Tiam-1 has been shown to inhibit migration of NIH 3T3 cells (Sander et al., 1999). Activated forms of Rat and Cdc42 induce integrin-dependent cell motility and invasion in T47D cells; PDK inhibitors can block this. These effects have been shown to be independent of Pak, JNK, and S6 kinase (Keely et al., 1997). Furthermore, activation of the (Y&4 integrin in the MDAMB-435 breast carcinoma cell line enhances cell invasion in a Rat- and PI3K-dependent manner (Shaw et al., 1997). In another study, Zetter and colleagues showed that dominant-negative forms of Rho GTPases inhibit cell invasion, indicating that these GTPases are required for this process. They also showed that constitutively active Rat stimulated basal Rat fibroblast invasion independent of PI3K activity (Banyard et al., 2000). Thus mounting evidence strongly suggests that Rho GTPases, especially Rat, play a major role in cell migration and invasion. P21-activated kinase family members are effecters of Rat and Cdc42 that regulate the actinomyosin cytoskeleton (Manser and Lim, 1999), which in turn regulates cell migration. PAK phosphorylates and inactivates myosin light chain kinase (MLCK), thus leading to a reduction in myosin light chain (MLC) phosphorylation and disassembly of focal adhesions and stress fibers (Sanders et al., 1999). Also, PAK phosphorylates LIM kinase, which in turn phospholylates and inactivates the actin-severing protein cofilin (Arber et al., 1998; Yang et al., 1998). In addition, PAK interacts with PIX and forms a complex with Nck, PKL, and paxillin; this complex stimulates focal complex formation (Turner, 2000). Further, PIX interacts with G-protein-coupled receptor kinase-interacting protein (GITl) and separates paxillin from focal contacts to promote migration (Zhao et al., 2000). Thus, PAK and associated proteins have a significant role in regulating cell migration.

E. The Role of ERK in Call Motility Integrin ligation leads to the activation of the mitogen-activated protein (MAP) kinases ERKl and ERK2. These signals impact on the actin-myosin cytoskeleton that is critical for cell migration. Assembly of actin into functional myosin motor units enables the generation of a contractile force that is required for cell migration (Lauffenburger and Horwitz, 1996). MAP kinase activation is critical

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for integrin or growth factor receptor-induced cell migration. It is known that MAP kinase phosphorylates and activates MLCK, which in turn phosphorylates MLC; these phosphorylation steps are essential for cell contraction, which regulates cell migration (Klemke et al., 1997). As discussed above, Cas/Crk coupling enhances cell migration by an increase in membrane ruffling, whereas ERK regulates MLC phosphorylation leading to actin-myosin contraction (Cheresh et al., 1999), indicating that ERK signaling and Cas/Crk signaling are separate events required for cell migration. In summary, ERK activity is required for actin-myosin assembly and cell contraction, but not Cas/Crk coupling. However, both ERK activation and molecular coupling of Cas and Crk coordinately regulate cell migration (Cho and Klemke, 2000).

F. The Role of Nischarin

in Migration

Recently, we discovered a novel protein Nischarin that specifically interacts with os integrin. Overexpression of Nischarin affects the actin cytoskeleton and inhibits cell migration. In addition, Nischarin exclusively regulates Rat driven c-fosmediated pathways, but not ERK driven c-fos induction. Moreover, Nischarin inhibits Rat-induced lamellipodia formation, suggesting that Nischarin affects several Rat-mediated signaling events (Alahari et al., 2000). Our unpublished data indicate that Nischarin inhibits Rat-induced cell migration and invasion in breast and colon carcinoma cells by affecting signaling events associated with PAK activation. Although both PAK and ERK stimulate motility, Nischarin selectively affects only PAK signaling events. This suggests that Nischarin is an important player in cell migration, possibly affecting MLCK or LIMK, as downstream effectors of PAK (S. Alahari, unpublished observations).

V. Regulation

of Apoptosis

by Cell Adhesion

An important development in the cell adhesion field has been the realization that both cell-ECM and cell-cell adhesion can strongly affect programmed cell death. In this section we examine the interconnections between adhesion and deathsurvival pathways, placing particular emphasis on integrins and integrin-associated signaling cascades.

A. Apoptosis

Overview

Apoptosis is a well-regulated means of removing extra, damaged, or misplaced cells from tissues without inducing an inflammatory response. Apoptotic cells

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are systematically dismantled with cleavage of several structural proteins (i.e., laminin, actin, keratins), signaling molecules (MEKKl, PKCS, FAK), and the chromatin. The cell is eventually broken into small membrane-enclosed particles that are phagocytosed by neighboring cells or professional phagocytes. Central to the induction of apoptosis are cysteine proteases, known as caspases, which cleave at conserved aspartic acids. These molecules exist as proenzymes in the cell with very low activity. Their activity can be induced by treatment of the cell with ligands for death-inducing receptors such as Fas or tumor necrosis factor (TNF) or cellular stress such as mitogen deprivation, loss of anchorage, or cytotoxic chemicals. If the molecules are brought in proximity to each other, the low activity of these molecules is enough to stimulate an activating cascade of cleavage. The apoptotic caspases can be separated into a hierarchy of initiators (caspase-2, -8, -9, and -10) and executioners (caspase-3, -6, and -7). Clustering of the initiator caspasesis the event that leads to the stimulation of the caspase-activating cascade. Receptor-mediated activation of apoptosis leads to the association of a death adapter protein with the receptor and the recruitment of a procaspase to the adapter protein. In the case of the Fas death receptor, after binding of Fas ligand (Fas-L), the adapter Fas-associated death domain (FADD) binds to Fas through death domains (DD) on the two molecules. FADD can then recruit procaspase-8 in complex with Flice-associated huge protein (FLASH) through death effector domains (DED) on FADD and procaspase-8 and DED-recruiting (DRD) domain on FLASH molecules, which leads to activation of procaspase-8 and initiation of the caspase cascade. Caspase-8 can also cleave the BH3 only protein Bid to form t-Bid, which leads to the release of cytochrome c from the mitochondria (described below) and amplification of this caspase cascade (Imai et al., 1999; Medema, 1999; Porter, 1999; Wolf and Green, 1999). The proposed initiating event in cell stress-induced apoptosis is the release of cytochrome c from the mitochondria. Upon release, cytochrome c interacts with apoptosis activating factor (Apaf-1) to activate caspase-9, which in turn activates caspase-3, stimulating the cleavage cascade. Translocation of proapoptotic proteins such as the Bax family (Bax, Bak, and Bok) proteins or BH3 only proteins (Bad, Bim, Bmf, Bid, Noxa, and Puma) to the mitochondria leads to the release of cytochrome c. Bax family proteins may stimulate the release of cytochrome c by either homooligomerization or heterodimerzation with proteolytically cleaved Bid (t-Bid). This induces a conformational alteration in Bax. This conformational change in Bax may help to induce pore formation in the rnitochondrial membrane allowing the release of cytochrome c. The closely related proteins Bcl-XL and Bcl-2 block apoptosis, in part, by binding and interfering with the activity of Bax or BH3 only proteins. The mitochondria also releases repressors of inhibitors of apoptosis (IAPs) known as SMAC or DIABLO and an apoptosis-inducing factor (AIF). AIF may be able to induce apoptosis independently of caspases activity. In spite of this evidence, a central role for the mitochondria in apoptosis is not certain. In Caenorhabditis elegans the mitochondria does not appear to have a

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A

ETAL.

-

B&2 Cytochrome e release Caspase Activity GSK3f3 Activity Bad/Bcl-2 Interaction Bak Expression

B

Detachment

from ECM

Fast c-Flip

i

1

f

Anoikis

t

i Caspase Activation

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prominent role. The penultimate caspase, CED-3, is activated by CED4. CED-4 is repressed by CED-9 until an activating signal is received, with no obvious role for the mitochondria. It is also not certain if mitochondrial disruption is the initiating event in non-death-receptor-mediated apoptosis in mammalian cells or if it acts secondarily to the activation of caspases by a CED4-like activator. Thus, many potential players have been defined as having a role in apoptosis, but the order and relative importance of each are still debated (Green, 1998; Medema, 1999; Porter, 1999; Wolf and Green, 1999; Desagher and Martinou, 2000; Huang and Strasser, 2000; Finkel, 2001; Hunot and Flavell, 2001).

6. Signaling Cascades Survival

Regulating

Anchorage-Dependent

This discussion of the relationship between cell anchorage and signaling is assisted by reference to Fig. 3.

1. Jun Kinase (JNK) Early studies on the importance of the extracellular matrix (ECM) in cell survival demonstrated that detachment of epithelial Madin-Darby canine kidney (MDCK) cells from the ECM induces apoptosis. This suspension-induced cell death was denoted anoikis (Frisch and Francis, 1994). Suspension of MDCK induced JNK

FIG.3 (A) Signaling pathways

regulating adhesion-dependent apoptosis. The activity of several pathways acts positively or negatively to regulate apoptosis in response to changes in the cellular adhesion status. The activity of FAK stimulates survival signals from focal complexes associated with sites of integrin contacts with the ECM. FAK activation of the PI3K, Ras, or JNK cascades can protect cells from apoptosis by acting on a variety of downstream components. Disruption of this signal by cleavage of FAK by caspases-3, -6, and -7 appears to be an important component of the apoptotic process. Generation of the carboxy-terminal FAT/FRNK from FAK by its cleavage further disrupts FAK signaling by interfering with FAK localization and effector interactions. Stimulation of the PI3K pathway via multiple inputs is critical for anchorage-independent survival. Most of the activities of PI3K appear to be mediated by activation of PKBlAkt via PIP3 and PDK-IIPRK-2. Activated Ras imparts anchorage-independent survival, in part by activating the PI3K cascade and inhibiting cytochrome c release and caspase activation. Activation of the Raf/MAPK cascade contributes to Ras-mediated survival upstream of caspase activation. Stimulation of the INK pathway by FAK or MEKK signaling may be anti- or proapoptotic, depending on the cellular context. (B) Stimulation of anoikis via death receptor activation. The activation of a death receptor family member such as Fas by its ligand induces the formation of the death-induced signaling complex (DISC). The recruitment of procaspase-8 by FLASH and FADD through their death domain interactions with the receptor leads to cleavage and activation of the caspase cleavage cascade. Introduction of SODD or dn-FADD into cells, which block death receptor signaling, also inhibits anoikis. Additionally, detachment of cells from the ECM elevates Fas expression and reduces the levels of c-Flip. The reciprocal regulation of these regulators of death receptor signaling stimulates anoikis by activation of the death receptor-regulated caspase-8.

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and caspase-7 activity. The JNK signaling cascade is an important cytoplasmic mediator of cellular stress response signals to the nuclear transcription machinery (Garrington and Johnson, 1999). Overexpression of Bcl-2 and inhibition of caspase activity blocked the suspension-induced JNK. These studies indicate that JNK activation downstream of caspase activation is important in anoikis (Frisch et al., 1996a). Studies demonstrating that the upstream kinase in the JNK signaling pathway, MEKKl, is cleaved during anoikis in a caspase-dependent manner, a dominant-negative MEKKl inhibits anoikis, and the MEKKl C-term 80-kDa cleavage product can induce apoptosis in MDCK cells support JNK’s role in anoikis (Cardone et al., 1997). Other investigators found that detachment of MDCK cells from the ECM induces JNK. However, in this study neither activity positively correlates with the induction of anoikis. A dominant-negative SEKl, which depresses JNK activity upon suspension, fails to inhibit anoikis. Constitutively active PDK, Akt/PKB, and the caspase inhibitor z-VAD-fmk also inhibit anoikis without affecting JNK activity (Khwaja and Downward, 1997). An additional report questions the involvement of JNK in mediating anoikis. Three different epithelial (HUVEC, IEC-18, and MDCK) cell lines were examined for the relationship between activation of JNK and anoikis. In spite of all three lines being sensitive to anoikis, no temporal relationship exists between JNK activation and anoikis in any of these cells (Krestow et al., 1999). Thus, the involvement of JNK in mediating the signal for anoikis remains uncertain, with its importance depending on experimental conditions. 2. PI3K PI3K activation allows the generation of 3’-phosphorylated phosphoinositides (PI3,4P and PI3,4,5P), which stimulates the recruitment of PKB/Akt to the membrane through its PH domain. At the membrane, phosphorylation of PKB/Akt at Thr-308 and Ser-473 by the PDK- l/PRK-2 complex activates PKB/Akt. Activated PKB/Akt may mediate cell survival through phosphorylation of several substrates including Bad. The phosphorylation of Bad induces dissociation of Bad from Bcl-2 or Bcl-XL and association of Bad with cytoplasmic 14-3-3 proteins. This switch in Bad partners inhibits Bad’s proapoptotic activities (Datta et al., 1999). PKB/Akt phosphorylates procaspase-9 on Ser-196 in vitro, which inhibits its protease activity (Cardone et aE., 1998). PKB/Akt also phosphorylates the Forkhead family of transcription factors, inhibiting their ability to activate proapoptotic genes such as Fas-L. PKB/Akt also enhances the degradation of IKB, thereby activating the prosurvival transcription factor NF-KB (Datta et al., 1999). As implicated in the anoikis study, PI3K has an important role in mediating anchorage-dependent survival. One indication of this is that a constitutively active PI3K mutant was found in several small cell lung cancer cells that grew independently of anchorage (Moore et al., 1998). Additionally, the survival of intestinal

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epithelial cells after serum deprivation is enhanced by the integrin ~5 via the PI3K, PKBIAkt pathway (Lee and Juliano, 2000). Furthermore, the ability of aspi and a+?3 to inhibit apoptosis in serum-deprived cells correlates with their ability to enhance Bcl-2 gene expression. This regulation is dependent on FAK activation of Ras and the subsequent activation of PI3K and PKBIAkt (Matter and Ruoslahti, 2001). One direct connection between integrins, PI3K, and anoikis is the serine/ tbreonine integrin-linked kinase (ILK- 1). ILK- 1 binds the fi i cytoplasmic domain and is activated after adhesion to fibronectin (FN) in a PI3K-dependent manner. ILK- 1 phosphorylates PKB/Akt on Ser-473 to stimulate its activity. Overexpression of ILK-l suppresses anoikis. This can be reversed by a dominant-negative (dn) ILK or dn-Akt, but not dn-FAK. Thus, ILK- 1 activation of PKB/Akt may be important for Bt integrin-mediated survival (Delcommenne et al., 1998; Attwell et cd., 2000). Further evidence for the role of PI3K in anchorage-dependent survival is in the Ras-mediated inhibition of anoikis in mammalian intestinal epithelial cells. The inhibitory activity of Ras correlates with the down-regulation of Bak and stabilization of Bcl-XL. Blocking PI3K activity inhibits the suppression of Bak levels by Ras, whereas the Ras regulation of Bcl-XL is independent of PI3K activity. Overexpression of Bak enhances anoikis in Ras-transformed intestinal epithelial cells and reduces their tumorigenicity. Hence, regulation of Bak levels may be an important aspect of PDK-mediated survival (Rosen et al., 1998,200O). Interestingly, in addition to PKB/Akt regulation of caspase activity, caspases may be able to regulate the level of PKB/Akt. Activation of integrin a#!?4 stimulates ~53’s transactivating function and induces apoptosis in carcinoma cells. In the absence of ~53, the integrin &j/l4 stimulates survival by activating PKB/Akt. In the presence of p53, c&$4 clustering reduces PKB/Akt levels in a caspase-3-dependent manner, elevates Bax, and induces apoptosis (Bachelder et aZ., 1999a,b). This demonstrates the importance of the genetic context in the regulation of survival by adhesion receptors. A more circuitous relationship for PKB/Akt and anoikis involving insulin-like growth factor- 1 (IGF- 1) has recently been defined. In PC 12 and MCFl OA epithelial cells, anoikis correlates with the loss of IGF-1 expression (-16 hr after detachment from the matrix). IGF-1 can stimulate PKB/Akt phosphorylation and activation in suspended cells. The activated PKB/Akt in suspended cells can still phosphorylate the proapoptotic protein Bad. However, PKB/Akt in suspended cells is unable to phosphorylate and inactivate glycogen synthase kinase 3/I (GSK-3/?). This results from the relocalization of active PKB/Akt away from the plasma membrane and GSK-38. GSK-3B phosphorylates cyclin Dl, stimulating cyclin Dl turnover. As might be expected, cyclin Dl is rapidly lost in detached cells. This loss of cyclin Dl correlates with the loss of cdk4 activity and the induction of hypophosphorylated Rb. Hypophosphorylated Rb acts in combination with E2F and histone deacetylase to repress the IGF-1 promoter. This eventually leads to the loss of sufficient IGF-1

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expression, reduced PKB/Akt activity, and the activation of proapoptotic proteins. This progression of events may prevent the induction of apoptosis by transient cell detachment, while allowing the induction of apoptosis after prolonged detachment (Yu et al., 2001). PTEN is a dual-function lipid and protein phosphatase that can dephosphorylate PIP3, thus inhibiting PI3K signaling (Maehama and Dixon, 1998; Di Cristofano and Pandolfi, 2000). PTEN enhances anoikis in breast epithelial and glioma cells. The enhancement of anoikis correlates with reductions in PKB/Akt and Bad phosphorylation (Davies et al., 1998; Lu et al., 1999). These activities of PTEN reinforce the importance of the PI3K signaling pathway in adhesion-dependent survival. These reports indicate that PI3K plays a central role in anchorage-dependent survival. Survival signals from integrins in growth factor-deprived cells depend on this pathway. Activation of this pathway is necessary for inhibition of anoikis by several prosurvival signaling molecules with connections to integrins including FAK, Ras, and ILK- 1. Additionally, constitutive activation of members of the PBK cascade blocks anoikis. Thus, although it is not always needed for anchorageindependent survival (McFall et al., 2001), activation of PI3K signaling may be a key step in stimulating anchorage-independent survival.

3. Ras/Raf/MAPK The ability of oncogenic Ras to inhibit anoikis in epithelial cells is thought to be mediated, in large part, by its activation of PI3K and to be independent of Raf activation (Khwaja et al., 1997; Rosen et al., 1998). However, in studies using the lung fibroblast line CCL39 cells stably expressing an estrogen inducible Raf-1 ( ARaf-ER) anoikis can be inhibited upon Raf activation. Raf signaling to MEKl was necessary for Raf-l’s activity because inhibition of MEKl with the MEKl inhibitor PD98059 blocks the ability of ARaf-ER to inhibit anoikis. Activation of ARaf-ER did not stimulate the PKB/Akt phosphorylation, so the PI3K pathway did not appear to be necessary for enhanced survival (Le Gall et al., 2000). Introduction of an activated Ras V12 or a membrane-targeted PKB/Akt into MDCK can block anoikis upstream of cytochrome c release and caspase activation. The protection afforded the suspended MDCK cells by the PKB/Akt is less than that of activated Ras. So, other Ras effecters are probably involved. The use of the strong, inducible ARaf-ER mutant also prevents anoikis in this system. Unlike the PKB/Akt inhibition of anoikis, Raf inhibits anoikis upstream of caspase activation, but downstream of cytochrome c release. The discrepancy in Raf’s importance for inhibition of anoikis likely results from the different levels of potency of the Raf mutants employed (Rytomaa et al., 2000). Thus, Ras likely blocks anoikis by activation of PKB/Akt and Raf. Activation of these pathways enables Ras to block activation of the death pathway at different levels of regulation.

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4. FAK, Adhesion, and Apoptosis FAK plays an important role in stimulating cell growth and inhibition of apoptosis by its maintenance of survival signals. Early work demonstrated the importance of sustained FAK signaling for cell survival especially in tumor cells. In tumor lines with high levels of FAK, depletion of endogenous FAK with antisense oligonucleotides causescell detachment and apoptosis (Xu et al., 1996). In chicken embryo fibroblasts (CEF), the microinjection of peptides corresponding to the FAK binding region of the pi cytoplasmic domain or anti-FAK antibodies that bind to the focal adhesion targeting (FAT) domain of FAK inhibits the localization of FAK to focal adhesions. Treatment of cells with these reagents prior to cell spreading and focal contact formation kept the cells rounded and led to apoptosis within 4 hr of treatment. However, these reagents had no effect in well-spread, adherent cells (Hungerford et al., 1996). In MDCK cells, the ability of activated FAK to prevent anoikis plays an important role in the ability of FAK to transform these cells (Frisch et al., 1996b). The importance of FAK activity for the prevention of apoptosis is reinforced by its early destruction by proteolysis during induction of apoptosis. Induction of cell death by several stimuli including serum withdrawal, treatment with toxins, Apo-2VTRAIL, or Fas-L induces the cleavage of FAK. This cleavage of FAK appears to be mediated by caspase-3, -6, and -7. Caspases-3 or -7 initiate the cleavage of FAK at Asp-772. This is followed by cleavage by caspased at Asp-704. The cleavage of FAK occurs prior to cell detachment from the ECM. However, the cleavage of FAK may occur prior to or after loss of FAK from focal adhesions. Binding of paxillin to the N-terminal FAK cleavage fragment is lost, but pp130 Cas and vinculin binding is not. Caspase cleavage of FAK also generates carboxyterminal fragments that contain the FAT domain. This cleavage of FAK and the generation of protein fragments that suppress endogenous FAK may act to amplify the apoptotic signal by suppressing any remaining FAK survival signals (Crouch et al., 1996; Wen et al., 1997; Gervais et al., 1998; Levkau et al., 1998; van de Water et al., 1999). The importance of the carboxy-terminus of FAK in propagating the apoptotic signal has been investigated further. In rat synovial fibroblasts (RSF), FAK is an important mediator of the FN survival signal. The carboxy-terminal fragments of FAK, FRNK and FAT, displace endogenous FAK from focal contacts. FAT contains only the focal adhesion-targeting sequence, whereas FRNK also contains the proline-rich domain-l (PR-1). Only the FAT mutant induces apoptosis. However, the deletion of the FAT domain from FRNK or mutation of prolines in the PR-1 also stimulates apoptosis. These mutants of FAK may act to disrupt the FAK survival signal by prevention of the proper localization or activation of signaling molecules downstream of FAK. One of these molecules is the adapter protein ~130 Cas, which binds the proline-rich domain-l of FAK via its SH3 domains. Ras also appears to be downstream of FAK in mediating the FN survival signal, but not

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the Raf-MEKl-ERK pathway. The small Rho GTPase Rat-1, the Rat effector PAK-1, MKK4, and INK1/2 may form a survival signaling cascade downstream of FAK, ~130 Cas, and Ras in RSFs. Inhibition of PI3K does not affect survival on FN in these cells (Almeida et al., 2000). Furthermore, a deletion mutant of human FAK (FAK-CD) containing only the region found in FRNK can localize to focal adhesions, instigate cell rounding, reduce endogenous FAK phosphorylation, and eventually lead to death in human breast cancer cells and human melanoma cells, but not normal human breast cells (Xu et al., 1998,200O). The disruption of the FAK survival signal stimulates apoptosis through several classic apoptotic pathways. The importance of ~53 in activating apoptosis in the absence of FAK signals was demonstrated using FAK and/or p53-deficient embryonic stem cells. The induction of apoptosis in FAK-deficient cells by serum deprivation is strictly dependent on the presence of ~53. Also, Bcl-2 was shown to enhance survival after the loss of FAK (Ilic et al., 1998). The proapoptotic Bcl-2 family member Bax helps to mediate the apoptotic signal after disruption of FAK signaling in mammary epithelial cells. The conformational change that may be permissive for the movement of Bax to the mitochondria was stimulated by disrupting FAK signaling with the FAT domain. Activated versions of PI3K kinase and c-Src prevented apoptosis induced by defective pp125 FAK signaling in these cells. Interestingly, the induction of apoptosis by this FAT protein in mammary epithelial cells appears to occur without the disruption of focal adhesions or cell attachment (Gilmore et al., 2000). Several investigators have observed the importance of PI3K in the transmission of the FAK survival signal. FAK can interact with the catalytic ~85 subunit of PI3K via Tyr-397 of FAK in an adhesion-dependent manner and tyrosine phosphorylate the ~85 subunit (Chen and Guan, 1994; Chen et al., 1996). Substitution of alanine for aspartate at amino acid 395 suppresses PI3K association with FAK, but not association with Src (Reiske et al., 1999). This mutation inhibited the ability of FAK to enhance survival in MDCK exposed to UV radiation. This strongly implicates PI3K as a downstream mediator of FAK survival signaling (Chan et al., 1999). Further evidence for the interplay between FAK and PI3K signaling comes from studies with the tumor suppressor PTEN. PTEN interacts with FAK at Tyr397. PTEN-deficient cells in suspension maintain FAK phosphorylation, PDK activity, PIP3 levels, and Akt/PKB phosphorylation, and are resistant to apoptosis. Introduction of wild-type PTEN reversed all of these cell alterations. Introduction of exogenous FAK in the PTEN-expressing cells reverts the cells toward the PTEN null phenotype. Thus, PTEN can suppress PI3K signaling by suppressing the levels of PIP3 and FAK activity (Tamura et al., 1999). Thus, maintenance of FAK signaling is important for cell survival. The signaling cascade stimulated by FAK for survival may include PDK, ~130 Cas, Rat, and the JNK pathway. Caspase-mediated cleavage of FAK to dampen the FAK survival signal appears to be important for the initiation of apoptosis. The carboxy-terminal

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fragments generated by FAK cleavage may amplify the death signal by inhibiting the remaining intact FAK.

C. Anoikis, Death Receptors,

and Cytochrome

c

A recent and interesting development in the study of anoikis is the identification of a role for the death receptor signaling pathway. Direct study of the involvement of death receptor signaling in anoikis involved using inhibitors of death receptor signaling. Silencer of death domains (SODD) (which binds and inhibits the death domains of TNFRl and death receptor 3, but not FAS or FADD) and a dominant-negative FADD (containing the death domain but lacking the death effector domains) block anoikis in several different epithelial cell lines. Cleavage of caspase-8, a hallmark of death receptor-induced apoptosis, and Bid also occurs during anoikis. Caspase-8 cleavage during anoikis is independent of other caspases, but was inhibited by the overexpression of dominant-negative FADD, Bcl-2, or Bcl-XL. Dominant-negative FADD and caspase inhibitors block cytochrome c release in suspended MDCK cells. This suggests that caspase activation is upstream of cytochrome c release during anoikis, which agrees with the ability of dn-FADD to inhibit anoikis. However, full activation of caspase-8 follows cytochrome c release suggesting a feedback loop (Frisch, 1999; Rytomaa et al., 1999, 2000). A study in anoikis-sensitive human umbilical vein endothelial cells (HUVECs) also implicates the death receptor pathway. In HUVECs matrix attachment regulates the level of Fas and c-Flip, an inhibitor of caspase-8. Cell detachment induces activation of the Fas death pathway by elevating the expression of the Fas receptor. This elevation of Fas increases the Fas-Fas-L interaction. Additionally, the level of c-Flip is reduced upon detachment from the ECM. The reduction of c-Flip levels may facilitate the activation of caspase-8. The induction of the Fas-FADD complex by Fas-L and caspase-8 activation precedes the induction of anoikis. Blocking any of these death receptor events inhibits anoikis (Aoudjit and Vuori, 2001). Thus, stimulation of anoikis may be dependent on death receptor activation and caspase-8 cleavage for its initiation. As might be expected, two important cleavage targets downstream of FADD are PKB/Akt and FAK. The elimination of these survival molecules appears to be important for death receptor-induced anoikis (Xu et al., 2000; Bachelder et al., 2001).

D. Adhesion

Receptors

That Regulate

Apoptosis

Cell growth and survival are dependent not only on simple attachment of the cell to a substrate, but on the nature of the substrate and the receptors mediating the attachment. Early work in this area demonstrated that the integrin asp t promotes

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survival in CHO and I-IT29 colon carcinoma cells after serum withdrawal. The cytoplasmic domain of as was necessary for this effect. This (;y5effect was mediated through increased levels of Bcl-2 and increased FAK tyrosine phosphorylation (Zhang et al., 1995; O’Brien et al., 1996). The specificity of the u5 subunit in cell survival was demonstrated by overexpression of the integrin subunits os and (112in rat intestinal epithelial cells. Elevation of ~5 levels, but not a2, conferred resistance to several apoptotic stimuli including serum starvation, etoposide, and staurosporine. The cytoplasmic domain of (1/swas not essential for this process. The survival of the ~5 cells appeared to be mediated through the PI3K, PKB/AKT pathway (Lee and Juliano, 2000). Elevation of as/31 levels can also inhibit cell growth and tumorigenicity. The switch from prosurvival to growth inhibitory appears to occur when 05p1 is not ligated to its substrate (Giancotti and Ruoslahti, 1990; Vamer et al., 1995). Elevation of the integrin 83 subunit also stimulates apoptosis when cultured in the absence of a& ligation, such as in collagen gel. This growth inhibitory activity may result from the ability of the /lt and /Is cytoplasmic domains of unligated integrins to recruit caspase-8 to the membrane and activate it in a FADD-independent manner (Stupack et aZ., 2001). A negative correlation between cell survival and integrin levels was also observed for the integrin CX~ subunit during the induction of anoikis by ~16~~~~. Anoikis positively correlates with the transcriptional elevation of 45 levels in cells expressing elevated levels of ~16’~~~~.The induction of anoikis by P16INK4acan be reversed by soluble FN, inhibitory ~5 antibodies, and antisense down-regulation of a5 mRNA (Plath et al., 2000). Again, increased levels of an unligated integrin is proapoptotic. Thus, the status of integrin ligation with appropriate substrates may be critically important for the decision between cell survival or death. Integrin ~$3 has been shown to be protective in several cell systems including melanoma, glioblastoma, and endothelial cells (Montgomery et al., 1994; Scatena et al., 1998; Uhm et al., 1999). The most extensive study of CX& and cell survival has been in angiogenesis. Treatment of tumors with antibodies to integrin a$3 induces apoptosis in angiogenic blood vessels and tumor regression, however antibodies to ,!?t integrins do not (Brooks et al., 1994). The @s-mediated survival of angiogenic blood vessels correlates with inhibition of p53 activity, reduction of p2 1Wafl’Cipl,and increases in the Bcl-2/Bax ratio in endothelial cells. Activation with integrin-specific antibodies for cQ5 or /3t does not have this effect (Stromblad et al., 1996). Surprisingly, homozygous deletion of all a, integrin isoforms from mice does not interfere in a detectable way with developmental angiogenesis (Bader et al., 1998). Thus, other integrins such as a$~, @t, and asp1 may also contribute to endothelial cell survival (Senger et al., 1997; Kim et al., 2000). The in viva importance of /3 t signaling for cell survival was demonstrated using the ,i3, cytoplasmic domain linked to the CD4 extracellular domain. Expression of

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this construct has the ability to disrupt intracellular /3 1 signaling. Expression of this construct in the mammary epithelia via the mouse mammary tumor virus results in abnormal mammary gland development. The infected glands exhibit stunted development during lactation with decreased proliferation rates and increased apoptosis. Disruption of INK and MAPK activation, decreased phosphorylation of She, and decreased phophorylation of PKB/Akt and its substrate Bad are observed in the j3i-CD4 expressing glands, although FAK signaling is not affected (Faraldo et al., 1998,200l). Survival of cells such as mammary epithelial cells requires the intact basement membrane, not simply fibronectin or collagen I (Boudreau et al., 1995). The basement membrane for breast epithelia is a complex ECM composed of laminin, collagen IV, nidogen, and perlecan that is critical for cell survival. Laminin and the og and pi integrin subunits contribute to inhibition of apoptosis in primary mammary epithelial cells. In mammary epithelial cells the insulin receptor provides the additional signal necessary for survival. Autophosphorylation of the insulin receptor is independent of the basement membrane, but tyrosine phosphorylation of the insulin receptor substrate-l, its association with PBK, and phosphorylation of PKB depend on the presence of laminin in the ECM. Thus, control of survival by the insulin receptor and the ECM lies in the coupling of downstream effecters (Pullan et al., 1996; Farrelly et al., 1999). The survival of cells depends not only on the types of integrins and ECM present, but on the degree of cell spreading and the rigidity of the cell contacts. When using FN-coated beads or FN-coated micropatterned surfaces to regulate cell spreading without altering the contact surface, cells exhibit a strong dependence on the degree of cell spreading for survival. Increasing the surface area covered by the cell allows for greater survival. However, some integrin specificity was still observed showing increased survival with o,#?s ligands over fli ligands with the endothelial cells used (Chen et al., 1997). Other investigators used NM 3T3 cells plated on collagen I-coated polyacrylamide to determine the importance of ECM flexibility in controlling growth and survival. In normal cells increased flexibility of the substrate led to decreased cell growth and increased apoptosis. However, Ha-Ras-transformed cells were not affected by the flexibility of the ECM. This loss of ECM flexibility dependence may be important for cell transformation (Wang et al., 2000). Thus, cell survival is not simply dependent on cell attachment to a generic surface, but the specific receptors mediating the attachment and the nature of the substrate are key in determining the decision between life and death for individual cells. An important aspect of apoptosis is the rapid removal of the cell fragments from the tissue before they can start an inflammatory response. Several recent studies have demonstrated that integrins have a role in the removal of these apoptotic fragments. In professional and nonprofessional phagocytes, an activated Rat- 1 or overexpression of C&II enhances the engulfment of apoptotic cell corpses. The elevated

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uptake of apoptotic bodies was blocked by dominant-negative forms of Rat- 1, but not Rho A (Tosello-Trampont et al., 2001). Importantly, signaling through the c+, integrin, in addition to mediating the binding of apoptotic cell fragments, sets up the p 130 Cas-CrkI-DOCK1 80 signaling complex, stimulating Rat activation of phagocytosis. This makes signaling through C&II-DOCK1 80-Rac- 1 functionally analogous to the C. elegans CED-2-CED-5-CED-10 complementation group that has been shown to be important for apoptotic body engulfment (Albert et al., 1998, 2000). Other investigations have demonstrated that in macrophages the uptake of apoptotic bodies via the integrin ~5s occurs in a Rho GTPase and PI3K-dependent manner. Inhibition of Rat, Cdc42, and PDK block apoptotic body uptake; conversely inhibition of Rho A enhances apoptotic engulfment (Leverrier and Ridley, 2001). The integrins a&i and ai& also influence the uptake of apoptotic cell fragments by professional macrophages (Erwig et al., 1999). Thus, several different integrins have the ability to influence the uptake of apoptotic fragments, likely via Rho GTPase family members. Regulation of phagocytosis adds another important function to the integrin’s repertoire of functions in regulating cellular homeostasis.

E. Actin Cytoskeleton

A hallmark of apoptosis is the disruption of the actin cytoskeleton. Actin cleavage occurs in apoptotic neutrophils and neurons and this activity depends on active calpain, but not caspases (Brown et al., 1997; Squier and Cohen, 1997; Villa et al., 1998). However, actin can be cleaved by caspase-3 in vitro (Kayalar et al., 1996). Additionally, actin cleavage is observed in solid tumors and inhibition of caspase-3 prevents apoptosis and actin cleavage in U937 cells (Mashima et al., 1997, 1999). Interestingly, an actin-disassembling region of the Ste20-related kinase SLK, which is released by caspase-3 during UV, W-a, and c-Myc-induced apoptosis, is able to initiate apoptosis in myoblasts (Sabourin et al., 2000). Other actin cytoskeleton-associated proteins such as cz-adducin, filarnin, gas-2, and gelsolin are also cleaved during apoptosis caspase-dependent and -independent manners. Gelsolin, which severs actin and caps the growing ends in a regulated manner and is important in fibroblast motility, is cleaved during apoptosis by caspase-3. The cleaved gelsolin constitutively cleaves the actin cytoskeleton and induces apoptosis in several cell types (Kothakota et aZ., 1997; Kwiatkowski, 1999). However, others have observed that gelsolin may also inhibit apoptosis or have no effect on it (Ohtsu et al., 1997; Posey et al., 2000). Regulation of cytoskeletal architecture appears to be important during apoptosis. However, it is unclear whether assembly or disassembly of the actin cytoskeleton is the important event in apoptosis. Disruption of the actin cytoskeleton with cytochalasin D alone or in conjunction with other stimuli can induce apoptosis in some

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cell types (Korichneva and Hammerling, 1999; Flusberg et al., 2001; White et al., 2001). Paradoxically, stabilization of the actin cytoskeleton with jasplakinolide enhances apoptosis induced by cytokine deprivation (Posey and Bierer, 1999). This suggests that it may be the dynamic rearrangement of the actin cytoskeleton that plays an important role in apoptosis. The importance of actin dynamics is implied in W irradiation, TNF-a, and etoposide-induced apoptosis. These agents induce actin depolymeriztaion during the induction of apoptosis. However, membrane blebbing requires new actin filament polymerization. So, both polymerization and depolymerization of actin may be important at different times during apoptosis (Levee et al., 1996; Suarez-Huerta et cd., 2000). Molecules have been identified that may be important for transducing the signal between cytoskeleton disruption and apoptosis. Activation of JNK may be important for the induction of apoptosis by alterations in the actin cytoskeleton. Disruption of the actin cytoskeleton activates JNK signaling and stimulates apoptosis independently of MEKKl (Yujiri et al., 1999). Disruption of the cytoskeleton may also inhibit PBK signaling, thus enhancing cell death (Flusberg et al., 2001). The disruption of the cytoskeleton may also release proapoptotic proteins that are sequestered by it under normal growth conditions. The BH3 only protein Bmf binds to dynein light chain 2, which localizes it to the myosin V motor. Treatment of cells with cytochalasin D or induction of anoikis initiates the release of Bmf from the cytoskeleton. The free Bmf can associate with Bcl-2, potentially disrupting its survival functions. Another BH3 only protein Bim may act similarly, but it may be associated with the microtubules and respond to alterations in this network (Puthalakath et al., 2001). Thus, the role of the actin cytoskeleton in apoptosis is complex. The dynamic deconstruction and reconstruction of portions of this network may act as a signal for apoptosis and also be important for its execution. This dual role may lie behind the contradictory nature of some of the studies. Further definition of the molecular actions of proteins such as Bmf may help to clarify the role the actin cytoskeleton plays in apoptosis.

VI. Summary The relationships between adhesion receptors, signaling cascades, and their downstream consequences seem to grow evermore complex. As we have indicated above, the same molecules and signaling pathways often impinge on several important biological processes, here focusing on motility and cell death. Further, the dominant players in a particular biological process seem to vary from one cell type to another. A case in point is the controversy and complexity that surrounds anchorage regulation of programmed cell death, as we have described above. Investigators

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will need to address this complexity and begin to define quantitative relationships among signaling molecules and downstream effecters in the context of individual types of cells and tissues.

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