Cytoskeleton and cell adhesion molecules in cell shape, growth regulation, and neoplasia

CYTOSKELETON AN D CELL ADH ESION MOLECULES IN CELL SHAPE, GROWTH REGULATION, AND NEOPLASIA

R. Rajaraman

I. II.

III.

IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Extracellular Matrix and Cell Adhesion Receptors . . . . . . . . . . . . . . . A. Integrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

110 111 112

B. C. D.

114 114 114

I m m u n o g l o b u l i n Superfamily . . . . . . . . . . . . . . . . . . . . . . . Selectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cadherins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

115

A. B. C.

M e m b r a n e Skeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microfilaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

115 116 119

D. E.

Intermediate Filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

120 121

Cell Shape and Cell Proliferation . . . . . . . . . . . . . . . . . . . . . . . .

122

A.

126

Mitogen Mediated Signal Transduction

Advances in Structural Biology, Volume 4, pages 109-149. Copyright 9 1996 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-967-2.

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B. AdhesionActivated Signal Transduction . . . . . . . . . . . . . . . . . . 132 C. Crosstalk Between Mitogen and Adhesion Induced Signal Cascades . . . 137 V. Cytoskeleton,Growth Regulation, and Neoplasia: A Hypothesis . . . . . . . . 139 VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

I.

INTRODUCTION

The term cytoskeleton conventionally refers to the system of fibrillar structures in the cytoplasm of eukaryotic cells consisting of microfilaments, microtubules, and intermediate filaments (Bershadsky and Vasiliev, 1988). By definition, then, "cyto" in "cytoskeleton" refers to cytoplasm. Our knowledge about the structure and function of cellular filamentous structures has expanded considerably in recent years. Fibrillar mesh work is not limited to the cytoplasm but spans contiguously from the cytoplasmic face of the plasma membrane, through the cytoplasm, to the nucleoplasm including the nucleolus. Therefore, from the context of the cell ("cyto" meaning cell), the term cytoskeleton should be extended to encompass all the various filamentous structural systems that pervade the entire cell. Thus, the new definition of cytoskeleton should include (1) the spectrin network of membrane skeleton, (2) the actin filaments, (3) the microtubules in the cytoplasm, (4) the intermediate filaments in the cytoplasm and the nucleus, and (5) the fibrillar nuclear and nucleolar matrices consisting of unidentified structural proteins. Cells interact with the extracellular matrix (ECM) components and respond to stimuli via a variety oftransmembrane receptors and the cytoskeletal system. While the cytoplasmic filament systems appear to be involved in the maintenance of the cell shape, intracellular organelle transport, signal transduction, nucleokinesis, and cytokinesis, the nuclear matrix appears to be engaged in the maintenance of function and order in terms of DNA and RNA syntheses and in the nucleo-cytoplasmic translocation ofmacromolecules. The present communication summarizes recent advances in the knowledge of the structure and function of the various cytoskeletal systems and their role in cellular responses to various environmental stimuli. The recent advances in the understanding of the mechanisms of signal transduction in relation to mitogenesis, cell shape, and the cellular skeletal system are reviewed. The regulatory communications between the signal transduction cascades and the various cytoskeletal systems related to cell division, differentiation, and neoplasia are discussed. Finally, it is hypothesized that the ligand-induced mitogenic cascade and the novel ligand-induced adhesion cascade are required for normal cell proliferation, extensive crosstalk between these two cascades are envisaged to function in a coordinate fashion, and that perturbations in the crosstalk between these two cascades might result in anchorage independent growth and neoplasia.

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EXTRACELLULAR MATRIX AND CELL ADHESION RECEPTORS

Cells secrete numerous macromolecules that form the extracellular (interstitial) matrix which holds the cells together in the multicellular organisms. Arole for ECM components in histogenesis was suggested by several investigators (Hay, 1981). ECM molecules fall into three major categories: collagens, glycoproteins, and proteoglycans. So far about 20 different genetic types of collagen chains have been identified (Alberts et al., 1989). These form triple helical macromolecules of one or more of the different gene products giving rise to about 14 different combinations, expressed in different tissues. They form a wide range of structures. The best defined collagens include fibrillar collagen types I, II, III, V, and XI, and amorphous collagen IV found in basement membrane and type VIII collagen in Descemet's membrane. The glycoproteins include entactin, fibulin, fibronectin, the laminin family of proteins, tenascin, thrombospondin, vitronectin, epilegrin, and others. Proteoglycans are proteins with one or more covalently bound glycosaminoglycan side chains. These are highly heterogeneous, owing to the size and composition of the core proteins and to the size, number, and nature ofpolysaccharide side chains, Most of them are ECM components, while some are transmembrane molecules (Rajaraman, 1991; Argraves et al., 1990; Ekblom et al., 1986; Ruoslahti et al., 1985; Ruoslahti, 1989). The basement membrane is a special type of ECM that is the substrate for endothelial and epithelial cells. The various basement membranes are not identical in composition and structure and there are developmental changes in basement membrane components (Kleinman and Schnaper, 1993). ECM provides attachment sites for the cells and cell-matrix interactions are required for the maintenance of the proper tissue architecture. Many ECM proteins consist of a variety of domains or modules brought together through evolution by exon shuffling; these modules usually carry out a particular function and have a common structural framework. This modular organization helps fine-tuning of various defined binding interactions between functionally diverse proteins. Thus, most of the ECM components possess the property of self-aggregation in addition to having binding sites for other ECM matrix components and form fibrillar interstitial matrices; they also interact with specific cell surface receptors. For example, fibronectins can interact with cell surfaces at several locations through receptors of at least two distinct classes: integrins and membrane associated proteoglycans (Rajaraman, 1991; Bemfield et al., 1992; Yamada, 1991). The collagen types I, II, and III form fibrils and fibers. Type IV collagen is amorphous and is found in the basement membrane in association with fibronectin, laminins, and entaetin. Fibronectin binds with collagens and proteoglycans; proteoglycans form preeipitable associations with fibronectin, laminin, and vitronectin. These interactions of various ECM proteins among themselves and others result in heteroand homopolymeric structures in the matrix, the composition of which may vary

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in different tissues and is influenced by the physiological stage of the cell such as cell proliferation, wound healing, differentiation, or neoplasia. ECM components give tensile strength to tissues (e.g., the fibrillar interstitial collagens), confer elasticity to tissues (e.g., elastin), maintain the aggregation of cells into tissues (e.g., aggregating proteoglycans), act as sheets to separate planes of cells and to filter molecules (e.g., the collagens and glycoproteins of basal lamina and basement membranes), and serve as the nidus for calcification (e.g., bone matrix macromolecules; Mosher et al., 1992). The characteristics of the ECM are dynamic and are dictated by various cell types in the tissue. The composition of ECM can affect the behavior of cells and varies during embryonic development. In addition, various growth factors appear to be lodged in an inactive form in the ECM and are activated when required such as during wound healing (Rajaraman, 1991; Ruoslahti et al., 1985; Mosher et al., 1992). Growth factors and cytokines sequestered in the extracellular matrix seem to play a role in cellular differentiation; these may be activated by neoplastic cells for neovascularization (Rogelji et al., 1989; Masumoto and Yamamoto, 1991; Vlodavsky et al., 1990, 1991). The influence of ECM on cellular behavior is brought about by various cell surface receptors that fall into four major groups called the integrins, immunoglobulin super family, selectins, and cadherins. A.

Integrins

Integrins are family of heterodimeric transmembrane receptors with an a and 13 subunit, and are involved in various aspects of cell-cell and ceI1-ECM interactions. They are divided into three subclasses according to the identity of the 13 chain (Hynes, 1987). At least 14 different ct subunits and eight different 13 subunits are known to date (Hynes, 1992; Springer, 1990; Ruoslahti, 1991; Rajaraman, 1991) (Figure 1). Each 13 chain associates with several different ct chains and some Gt chains also can associate with more than one 13 chain. The 131-integrins and the 133-integrins function as receptors for various ECM components such as fibronectin, laminin, collagen and vitronectin. The 132 integrins are expressed in leukocytes where they mediate various types of cell-cell interactions via cell surface counter receptors (Springer, 1990). Usually a given cell type may express several types of integrins. The ligand specificities of a given integrin may vary depending upon the cell type (Rajaraman, 1991). Anchorage independent growth as well as tumorigenic transformation results in the differential expression of integrins. When human osteosarcoma MG-63 cells are maintained in suspension, several integrins are upregulated. Within four hours after layering on agarose-coated petri plates, the mRNA levels for both the a2 and ~4 are increased four and sixfold, respectively. In several differentiated cell lines ot2, a4, and av are upregulated under anchorage independent conditions; but there was no change in a5 subunits. However, there is no induction of new integrin subunit expression due to the change in growth conditions. Studies with

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PROMISCUOUS i~ SUBUNITS fl subunits

131,CD29

132,CD18 133,Gpilla [37

Ligands

a subunits or1, CD49a or2, CD49b or3, CD49c or4, CD49d c~5, CD49e c~6, CD49f o~7, CD49g c~8, CD49h av, CD51 OiL, CD11 a, LFA-1 C~M,CD11b, Mac-1 otx, CD11 c, (p150/95) C~llb, Gpllb ow, CD51 @4, CD49d O~HML-1

Coll., LM. Coll., (LM) EL, FN, Coll., LM FNalt, VCAM-1 FN LM LM ? FN, VN ICAM-1, ICAM-2, ICAM-3, ICAM-R ICAM-1, iC3b, FB, FX FB, iC3b? FB, FN, vWF VN, FB, vWF, FN, TSP etc. FNalt, VCAM-1 ?

PROMISCUOUS ~ SUBUNITS fl subunits

Otsubunits 131,CD29 1133,Gpllla av, CD51

o~4, CD49d or6, CD49f

135 136 138 131,CD29 137 131,CD29 134

Ligands FN, VN VN, FB, vWF, FN, TSP VN FN ? FNalt, VCAM-1 FNalt, VCAM-1 LM (LM?)

Figure 1. Integrin (z and 13 subunit nomenclature, associations between different ot and 13integrin subunits and their ligands. (Coll., collagen; EL, epiligrin; FB, fibrinogen; FN, fibronectin; FNalt, fibronectin alternative cell binding site; ICAM, intercellular cell adhesion molecule; LM, laminin; VCAM-1, vascular cell adhesion molecule-I; VN, vitronectin; vWF, von Willibrand factor; TSP, thrombospondin.)

cytochalasin B indicate that this change is probably due to lack of cytoskeletal elements (Chen et al., 1992). Changes in integrin expression observed in malignant cells include reduced expression of ct5 and increased expression of t7'6 (Dedhar, 1990; van Waes et al., 1991). Increased expression of integrin ot2, ot6, and avl33 integrins may favor metastasis (Hart et al., 1991; Chan et al., 1991).

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The immunoglobulin superfamily of more than 37 proteins (Williams and Barclay, 1988; Springer, 1990; Obrink, 1991) includes a diverse group of receptors involved in equally diverse functions such as cell-cell recognition (Williams and Barclay, 1988), cellular immunity, neuronal development, leukocyte trafficking (de Fougerolles and Springer, 1992; Fawcett et al., 1992), and receptor protein tyrosine kinase function (Albelda, 1993). The common feature of these groups of proteins is the variable number ofimmunoglobulin loops of about 100 amino acids arranged in 13pleated sheets stabilized by disulfide bond. Amino acid homology is limited to the immunoglobulin loops; the functional domains between different molecules do not show sequence homology.

C. Selectins Selectins are a family of carbohydrate-binding proteins. The three known selectins are L-selectin (L for lymphocyte; synonyms: Mel-14, Lam-1, Leu-8, gp-90, peripheral lymph node homing receptor; TQ 1, DREG56, LEC-CAM- 1), E-selectin (E for endothelial cell; synonyms: ELAM-1, vascular selectin), and P-selectin (P for platelets and endothelial cells; synonyms: GMP 140, CD62, PADGEM). Like other Ca 2+dependent (C-type) lectins, selectins have homology in the carbohydrate recognition domain; they are also Ca 2§ dependent for their activity. These are transmembrane molecules that display five common structural domains: (1) a lectin domain at the N-terminus, that specifically binds to carbohydrates; (2) an epidermal growth factor (EGF)-like domain; (3) a domain of variable length containing a series of repetitive sequences of about 60 amino acid long, sharing extensive sequence homologies to complement binding proteins; (4) a transmembrane domain of approximately 25 amino acids; and (5) a short cytoplasmic, C-terminal tail. Selectins are involved in lymphocyte adhesion to high endothelial venules and lymphocyte recirculation between blood and lymph (Albelda, 1993).

D. Cadherins Cadherins are calcium-dependent transmembrane cell-cell adhesion receptors with 50% sequence homology with each other and bind by means of homophilic interactions. Four different subclasses of the members of this family, E-cadherin (epithelial cadherin or uvomorulin, also found in Langerhans cells; Tang et al., 1993), N-cadherin (neural cadherin), P-cadherin (placental cadherin), and L-CAM (liver cell adhesion molecule) have been cloned and sequenced. Novel cadherinlike molecules have been identified that share consensus sequences with cadherins, but display different overall structures. They selectively bind to identical cadherin types causing homotypic cell aggregation, partially explaining preferential adhesion between homotypic cells. Expression of cadherins is developmentally regulated. The turning on and off of cadherin expression correlates with a variety of

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morphogenic events that involve cell aggregation and disaggregation (Takeichi, 1988, 1991). Cadherin dependent cell adhesion is elaborated by Collins in this volume.

I!1. CYTOSKELETON A. Membrane Skeleton

The shape and structural integrity of the mammalian cells is maintained by a submembranous cytoskeletal network known as the membrane skeleton. The major component of this network is spectrin, a dimer of heterodimeric non-covalently associated tx-spectrin (M r 240 kD), and [3-spectrin (M r 220 KD) found as an (a,~)2 tetramer interconnected by polymeric actin links. This spectrin network is linked to the transmembrane polypeptide the anion transporter (band 3) via ankyrin, bridging the [3-spectfin and the anion transporter (Marchesi, 1979). Another spectrin-like protein called fodrin was identified in the membrane skeleton of brain in association with a 235 kD 13spectrin (Levine and Willard, 1981). Similarly, in the terminal web of intestinal brush border, a 240 kD ct-spectrin-like protein was found associated with a 260 kD protein in a similar fashion; this was termed TW260/240. These molecules (spectrins, fodrins, TW260/240) show similar properties such as (a) association with the cytoplasmic side of the cell membrane; (b) physical, structural, and positional similarity to erythroid spectrins; (c) capable of crosslinking and gelling F-actin; (d) binding to cell ankyrin; and (e) binding with calmodulin; and (f) show high immunologic cross-reactivity with anti-spectrin antisera. Furthermore, the different types of spectrins show preferential distribution in different cell types and also in a developmental stage-dependent manner (Lazarides et al., 1984). In nucleated cells, during the patching and capping of ligand occupied receptors, the submembranous spectrin network also coordinately undergoes capping phenomenon (Rajaraman and Faulkner, 1985). Through interactions with additional actin crosslinking proteins such as filamin or a smaller related protein, ABP-120 (Noegel et al., 1989), actin filaments near the membrane are incorporated into the cytoplasmic actin cytoskeleton. Spectrins bind directly or indirectly (via ankyrins) to diverse plasma membrane proteins, including ion pumps, channels, and adhesion receptors, and are believed to play a role in segregating the various receptors into distinct domains on the plasma membrane (Bennett, 1990). However, fodrin-like molecules in neurons were shown to be involved in memory (Bums, 1985). In erythrocytes, spectrin functions to regulate cell shape. During mitosis, cells round up from the substratum, cell substratum-adhesive contacts are lost, actin filaments undergo extensive reorganization, and the nuclear membrane is disorganized by the phosphorylation of lamin proteins. Similarly, the 13subunit of spectrin is phosphorylated at serine residues and spectrin redistributes to the cytosol. This could be functionally related to the restructuring of the actin filament network from

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focal adhesion to the microvilli and at the cleavage furrow contractile ring for cytokinesis (Fowler and Adam, 1993). B. Microfilaments

Microfilaments (actin filaments or F-actin) are polymerized from globular actin monomers or G-actin. The primary structure of actins from different cell types and organisms is highly conserved through evolution. Some lower eukaryotes have only one actin gene encoding a single protein. Actins of vertebrates have several forms ofactin coded by families ofactin genes; they fall into three electrophoretic classes, (the most acidic), 13,and ~. Each vertebrate species expresses at least six different actins: two non-muscle actins (13 and T), cardiac muscle a-actin, skeletal muscle a-actin, and two smooth muscle actins (or and ~). Several actin types can be expressed in a given cell, but localized in different cellular compartments. Thus, actins of higher vertebrates are tissue-specific and highly conserved through species in a given cell type. Specific interaction of either F-actin and G-actin with actin binding proteins (ABPs) determines the different organizations and cellular localizations of actin filaments. ABPs containing actin binding domains (ABDs) consisting of 100-250 residues, are able to interact with actin. Like other multifunctional proteins, ABPs are assembled from various domain modules, each with specific properties. Often, an ABP can contain more than one ABD, as in the case of F-actin cross-linking proteins and the gelsolin family (Matsudaira, 1991). ABPs can be classified according to their degree of sequence homology into about nine subfamilies: (1) the profilin family (+/-17 kD); (2) the cofilin family (15--20 kD); (3) the ct-actinin family (variable in size); (4) the myosin family; (5) the caldesmon family; (6) the gelsolin family (variable sizes); (7) the tropomyosin family; (8) the neuronal synapsins; and (9) the Capz36-related ABPs (Vandekerckhove and Vancompernolle, 1992). There are other proteins that interact with actin, but do not fall into any related subfamilies. The ABPs can also be classified according to their functions into five classes: (1) ABPs that interact with monomeric actin and control the pool of unpolymerized actin in the cell (e.g., profilin and thymosin 134); (2) ABPs that either induce or inhibit early events in actin polymerization (e.g., gelsolin and actobindin); (3) ABPs that bind to one of the ends of the actin filaments and that determine the direction of filament growth or that anchor the filament ends to other proteins or structures (e.g., calz36/32, gcap39, and acumentin); (4) the ABPs that bind laterally to actin filaments; these proteins cross-link actin filaments, link actin filaments to other proteins, or regulate the interaction of other ABPs with filamentous actin; these include two major groups of proteins termed the bundling proteins (tropomyosin, 30 kD protein, ABP-50/EFla protein, fascin, 55 kD protein, protein 4.9/dematin, synapsin I, fimbrin/plastin/ABP-67, ot-actinin) and gelation proteins (ABP-120, spectrins and fodrins [the membrane skeletal components] and ABP/filamin); and

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(5) ABPs that sever actin filamems by disturbing the interaction between adjacent F-actin protomers (e.g., gelsolin). Finally, some ABPs combine several actin-binding properties. For instance, gelsolin combines actin nucleation, F-actin severing, and F-actin capping activities (Vandekerckhove and Vancompernolle, 1992). The cross-linking APBs have at least two actin-binding sites. The actin-binding proteins share a common actin-binding domain but differ in the location of actin-binding site, spacer domains, oligomerization domains, and calcium-binding domains, resulting in different actin-binding properties. Thus, these proteins regulate the degree of actin polymerization, the integrity, length, orientation, distribution, and the function of actin filaments (Matsudaira, 1991). Bundles or meshworks of cytoplasmic actin filaments form a continuum of structures that act as a structural scaffold anchoring organelles and the protein synthetic machinery, support the plasma membrane, and anchor the cell to the ECM or to adjacent cells. The physical state of actin seems to be regulated by three different mechanisms. The first system is the basic reversible microfilament cycle of polymerization of G-actin to the filamentous form, F-actin. This polymerizationdepolymerization cycle is regulated by proteins which sequester actin monomers (e.g., profilin), control nucleation of the filament (villin), block the barbed end of the filament (fragmin), block the pointed end of the filament (13-actinin), sever the filament (gelsolin), and "nibble" at the filament (depactin). Second, the F-actin can be crosslinked to form structures of higher complexity such as bundles and networks. These are found in structures such as filopodia and microvilli, the structural features of cell surface. This organization is regulatedby at least four classes of actin crosslinking proteins that form tight actin filament bundles (villin), loose actin filament bundles (tx-actinin), orthogonal networks (ABP120), and cross-link actin oligomers (spectrin/fodrin; Matsudaira, 1991). In the third system, actin structures are anchored to the membrane, facilitated by another class of actin-binding proteins, which include MARCKS (myristoylated, alanine-rich C Kinase substrate) and ABP50 (Aderem, 1992). Ponticulin is a transmembrane ABP involved in nucleation of actin filaments (Chia et al., 1993). Erzin is involved in attaching actin filaments to the cell membrane. Other erzin-related proteins meosin, radixin, and merlin seem to have similar functions. The recently characterized merlin is coded by NF2 gene and its deficiency causes neurofibromatosis 2; therefore, this is a candidate tumor suppressor gene localized in chromosome 22q 12 (Trofatter et al., 1993). Actin bundles of uniform polarity usually do not contain myosin. These form bundles of parallel arrays of closely packed actin filaments with the +ends, where actin polymerization or depolymerization occurs, facing the membrane. Such bundles are found in the microvilli, filopodia, and lamell~podia. The actin crosslinking proteins in these bundles include the 68 kD fimbrin, human fimbrin isoforms, T-plastin (in epithelial and mesenchymal cells) L-plastin in leukocytes, ABP-76, and a 55 kD protein, villin, fascin, the ABP-50 (EF-la), and a 30 kD protein (Matsudaira, 1991).

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Actin filament bundles with alternative polarities usually contain myosin. These include the myofibrils of striated muscle cells, the stress fibers in fibroblasts and other cultured cells, the contractile belt, and the contractile ring. Filaments in these bundles are crosslinked by a actinin (102 kD), fimbrin, and the 55 kD protein. Myosins (480 kD, consisting of two heavy chains of 210 kD and two pairs of light chains of 20 kD) are a large family of diverse proteins capable of binding to actin and have ATPase activity, stimulated by actin binding. A wide variety of myosinrelated proteins serve as intracellular molecular "motors" (Kiehart, 1990). These myosin-like proteins share a common "head" domain capable of interacting with actin (Kom and Hammer, 1988), but have a variety of COOH-termini that specify interaction with the transported component (Kiehart, 1990). The conventional myosin molecules such as the one found in muscle cells belong to myosin-II class and have a COOH terminus that exhibits high degree of a helix. This a-helical region can adopt a coiled-coil configuration and favor self-assembly and filament formation. In such instances, the transported component specified by the COOH terminal domain interactions is the myosin filament itself, and associated structures. Such myosin systems play a role in cytokinesis and probably also in nuclear migration (Watts et al., 1987; De Lozanne and Spudich, 1987; Knecht and Loomis, 1987). Another, more heterogeneous class of unconventional myosins are termed myosin I and these also contain characteristic myosin-head domain but possess unique and diverse COOH terminal domains. The COOH terminal domains of myosin I can interact with a wide variety of cellular targets, such as actin, cytoplasmic organelles, and secretary vesicles or plasma membrane, to effect intracellular transport (Johnston et al., 1991). Myosin and tropomyosin contribute to the ATP-dependent movements of actin filaments or microfilament associated cellular organelles. Myofibrils of striated muscle cells have the highest order of organization consisting of repetitive structural units, the sarcomeres. Each sarcomere has two arrays of parallel actin filaments with opposite polarities. The barbed ends of filaments of each polarity are attached to the structures called Z disks and contain actin binding protein, a-actinin. Actin and myosin filaments interdigitate in the central zone and they slide along one another during muscle contraction in an energy dependent fashion. Three-dimensional actin networks consist of filaments crossing one another at various angles more or less in an orthogonal fashion. This network provides a cytoplasmic skeleton for anchoring cell organelles and protein complexes involved in various cell metabolic activities. The actin-bundling protein ABP-50 is identical to EF-la and appears to have evolved independently in relation to the protein synthetic machinery. It appears that 97% of the active mRNA, in the form of polyribosomes, is associated with the microfilament framework; protein synthesis is inhibited by depolymerization of microfilaments with cytochalasin D in direct proportion to the release ofmRNA (Fey et al., 1986; Singer, 1992). Large V-shaped actin crosslinking proteins such as filamin or ABP-120 are found in these areas (Gorlin et al., 1990). Proteins such as vinculin (130 kD) and talin (215 kD) are

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found in focal contact areas of cell attachment. Talin binds to vinculin in vitro and also binds to transmembrane cell adhesion receptors, integrins (Clark and Brugge, 1995).

C. Microtubules Microtubules are hollow cylinders of about 25 nm in diameter, with a wall of about 5 nm. The building block of microtubules is tubulin, which is a heterodimer of Gt-tubulin and 13-tubulin (both about 55 kD). Both subunits bind GTP, and tubulin is considered to be evolutionarily related to the GTPase family of proteins (Bourne et al., 1991). The wall of a microtubule is made of 10--16 (most commonly 13) parallel protofilaments of tubulin linear polymers. Each protofilament is a polar structure. Microtubules depolymerize in cold temperatures (4~ or in the presence of colchicine or calcium ions. Other drugs including colcemid, podophyllotoxin, and nocodazole bind to tubulin in the same site as does colchicine and have a similar inhibitory effect on tubulin polymerization. Vinblastine and vincristine also inhibit microtubule assembly by binding to a site other than the one colchicine binds to. Another drug taxol inhibits depolymerization and stabilizes the microtubules. GTP and magnesium ions favor tubulin polymerization at 37~ In steady state conditions in vitro, net tubulin addition onto the microtubule occurs at one end of the polymer, and net tubulin loss occurs at the opposite end. Thus, a unidirectional flux of tubulin from one end of the microtubule to the other, is often called "treadmilling" (Margolis and Wilson, 1981). Several proteins are usually found attached to the outer surface ofthe microtubule wall and are called microtubule-associated proteins (MAPs). These include MAP- 1 (A, B, and C) (300-350 kD), MAP-2 (A and B) (270-285 kD), tau proteins (about 20) (60kD), and several others. Most MAPs control the stability of microtubules and act as the targets ofintracellular regulatory signals, such as cAMP and calcium. Kinesin (110 kD), dynein (MAP- 1C) a multi-subunit complex consisting of several proteins ranging in size from 53 kD to 410 kD, and dynamin (about 100 kD) have been well characterized; all of them have microtubule-associated ATPase (or other nucleotidase) activity and are capable of producing force in an ATP and Mg 2+ or Ca 2+ dependent fashion (Vallee and Shepmer, 1990). Kinesin is a motor protein associated with microtubules capable of anterograde (away from the cell body) organelle transport. Dynein, on the other hand, moves microtubules in a retrograde fashion. Dynamin cross-links microtubules with a periodicity of 13 nm and is involved in longitudinal mass movement of microtubule bundles. The recent addition to the list of MAPs is histone H1 found associated with axonemal microtubules in sea urchin sperm flagella (Multigner et al., 1992). The mode of attachment of these various proteins to microtubules, their relationship to organelle transport, and their relationship to the cytoplasmic membrane are not known at present. The functions ofmicrotubules include cytoskeletal function, chromosome

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movements, axonal transport, transport of ions, regulation of cell growth, organelle movements, and morphogenesis. D.

Intermediate Filaments

Intermediate filaments are larger in diameter (about 10 nm) than actin filaments (7 nm) and smaller than microtubules (25 nm) and constitute a dense mesh work of filaments concentrated around the nuclear periphery, radiating toward the cell periphery and are associated with the plasma membrane in all directions. They also interact with other cytoskeletal components including microtubules and microfilaments (Goldman et al., 1986); thus, when microtubules are depolymerized by exposure to colchicine, vimentin intermediate filaments collapse around the nuclear periphery. Intermediate filaments interact with the plasma membrane via desmosomes and hemidesmosomes and with the nuclear surface. The inner surface of the nuclear membrane is anastomosed with the nuclear intermediate filament proteins, lamin A, B, and C. Intermediate filaments are a family of self-assembling polymorphic proteins with a highly conserved central helical rod domain of 310 residues, flanked on both ends by hypervariable and non-helical NH 2 and COOH termini. The filaments consist of seven or eight tetramers per diameter, with double stranded coiled coils arranged with one-half unit length stagger. Since the dimers are antiparallel in arrangement, these filaments are non-polar (Osborn and Weber, 1986). Intermediate filaments are classified according to the degree of homology in structure and composition or their tissue-specific expression (Steven, 1990). The various polymorphic forms include: Keratin types I and II (40-56.5 kD, 53--67 kD; epithelia), vimentin (type III; 57 kD; mesenchymal cells), desmin (type III; 53-54 kD; myogenic cells), glial fibrillary acidic protein (GFAP) (type III; 50 kD; glial cells and astrocytes), peripherin (type III, 57 kD, peripheral neurons), neurofilament proteins: NF-L, NF-M, and NF-H (type IV; 62 kD, 102 kD, and 110 kD respectively; neurons of central and peripheral nerves); lamins: lamin A, B and C (Type V, 70 kD, 67 kD, and 60 kD, respectively, all cell types), and nestin (type VI, 240 kD, neuroepithelial stem cells). Cells contain more than one type of intermediate filaments, with great diversity in their expression and tissue distribution. They are phosphorylated usually at serine residues. Phosphorylated proteins disassemble and the dephosphorylated state favors self-assembly. Thus, during mitosis, phosphorylation oflamins by the cdc2 kinase results in disassembly of the nuclear lamin structure resulting in the disassembly of the nuclear membrane, an event that occurs before mitosis. On the other hand, vimentin filaments reorganize prior to mitosis. Intermediate filaments are highly dynamic structures that play important roles in the cells as indicated by the dramatic changes in the composition of intermediate filaments during neuronal development (Eriksson et al., 1992). A heterogeneous poorly characterized family of proteins called intermediate filament associated proteins, IFAP, have been observed in different tissues. These

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include BAPP-IFAP (Durham et al., 1994), plectin (Seifert et al., 1992), restin (Hilliker et al., 1994), G3.5 antigen (Price et al., 1993), filaggrin (Haydock and Dale, 1990), IFAP-300Da (Skalli et al., 1992; Yang et al., 1993), trichohyalin (Lee et al., 1993), IFAP-70/280 (Yang et al., 1992), NF-66 (Vickers et al., 1992), filamin (Brown and Binder, 1992), IFAPa-400 (Vincent et al., 1991), NFPA-73 (Ciment, 1990), p240 (Brown and Binder, 1990). These proteins are found in association with various intermediate filaments in a tissue or developmental specific fashion and may also be cross-linking intermediate filaments with other cytoskeletal structures. E. Nuclear Matrix

Selective removal of chromatin leaves a residual nuclear material, composed largely of protein composed of a nuclear network of matrix, heterogenous nuclear RNA, nuclear pores, the proteins of the nuclear lamina, DNA replication sites, and transcriptionally active genes (Berezney and Coffey, 1974; Cook, 1991). Whole mount studies show that the nuclear matrix, bound by an outer nuclear lamina, is linked to the cytoplasmic filamentous framework, as well as to an inner three dimensional anastomosing network in which nucleoli are enmeshed (Pienta and Coffey, 1984). Nuclear pore complex structure has been delineated (Willison and Rajaraman, 1977; Akey, 1991), and a large number of nuclear pore proteins, collectively called NUPs and NSPs, have been identified and some have been cloned and sequenced (Sukegawa and Blobel, 1993). Immunological probes reveal that the nuclear envelope is composed of lamins (intermediate filaments) that undergo cell cycle-dependent phosphorylation (Gerace and Blobel, 1980) and that the nucleolus has fibrillogranular and "fibrillar center" components containing fibrillarin and RNA polymerase I, respectively (Scheer and Rose, 1984; Bouvier et al., 1985). The nuclear matrix is composed of structural domains involved in the various functions of the nucleus such as transcription, replication, and macromolecular transport. The nuclear membrane, the nuclear pore complex, and the nucleolus represent nuclear structural landmarks. Even in the absence of physical compartmentalization comparable to the cytoplasm, the interphase nucleus is organized into domains occupied by the individual chromosomes, centers of mRNA processing, and tRNA synthesis. DNA synthesis loops and transcription loops have been visualized; heteronuclear RNA in the form of ribonucleoprotein is associated with the nuclear matrix; enzymes involved in the nuclear activities such as ligases, topoisomerases, helicases, and polymerases appear to be attached to the nuclear matrix; models involving replication of the nuclear matrix have been proposed (Cook, 1991). It has recently been shown that transcription factors, the ultimate regulators of gene expression, associate with the nuclear matrix. Nuclear matrix protein composition varies in a proliferation or differentiation stage specific manner

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and the matrix contributes to the transcriptional control of gene expression in a tissue specific manner (Stein et al., 1991). The nuclear matrix provides a dynamic structural support for the various nuclear functions. Using fluorescence hybridization with genomic, complementary DNA and intron-specific probes, fibronectin and neurotensin mRNA processing sites in the nuclear matrix have been mapped (Xing et al., 1993). mRNA seems to accumulate in elongated tracks overlapping and extending beyond the site of transcription, mRNA splicing appear to occur within these tracks, as evidenced by spatial separation ofintron-containing and intron-spliced transcripts. Poly-A RNArich domains were localized in discrete, internal nuclear regions that formed a ventrally positioned horizontal array in cell monolayer. Spliceosome assembly factor SC-35 (involved in splicing the introns away) was localized within the center of the individual domains. Thus, it appears that the nuclear matrix contains a specific topological arrangement of noncontiguous centers involved in precursor messenger RNA metabolism, from which RNA transport toward the nuclear envelope radiates (Carter et al., 1993). Chromosomes themselves undergo cyclical condensation and decondensation during the mitotic cycle. Chromatin is a dynamic macromolecular assembly that constantly alters its composition and conformation to accommodate different stages of genetic activity, largely due to the intrinsic properties of arrays of nucleosome cores (Hansen and Ausio, 1992). Chromosomes in a genome are replicated in a fixed order and this order of chromosome replication is altered in neoplastic cells. All these dynamic changes occur within the context of the nuclear matrix.

IV. CELL SHAPE A N D CELL PROLIFERATION Several studies in the past two decades have established that cell shape and cytoarchitecture have a regulatory role on cell metabolic activity, gene expression, cell proliferation, and differentiation (Ben-Ze'ev, 1986, 1989; Puck and Krystosek, 1992). Most normal cells attach and spread on an appropriate substratum for optimal growth and cell division in in vitro model systems. This phenomenon is called anchorage dependency for growth. However, transformed cells and tumor cells have lost this anchorage dependency for growth and are capable of synthesizing DNA and undergoing mitosis under anchorage independent conditions such as soft-agar or methocel culture (Stoker et al., 1968; Rajaraman and Lonergan, 1982; Vasiliev, 1985). When normal cells are grown under anchorage independent conditions, the cells remain spherical in shape, they show a rapid decline in the syntheses ofDNA, RNA, and protein and the cells eventually die by apoptosis in a few days due to physiological starvation (Benecke et al., 1980; Farmer et al., 1978; Ben-Ze'ev et al., 1980; Ben-Ze'ev and Raz, 1981; Rajaraman and Faulkner, 1984; Rajaraman et al., 1994). When cells kept in suspension are replated on adhesive substratum, they settle down, spread on the substratum, and normal cellular metabolism is reacti-

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vated. It was shown that cell attachment (without spreading) to the substratum was sufficient to induce protein synthesis, while nuclear events responded to the change in cell shape due to spreading. One of the early proteins that was synthesized turned out to be actin (Benecke et al., 1980; Ben-Ze'ev et al., 1980). Th.e spherical cell shape in suspension shuts off the metabolic activity of the anchorage dependent cells and the more they spread on the substratum the higher the rate of DNA synthesis and cell proliferation are induced (Folkman and Moscona, 1978; Ingber et al., 1990). Normal cells secrete ECM components such as fibronectin, even in the absence of serum proteins, before they spread on the substratum via filopodia and lamellipodia (Rajaraman et al., 1977, 1983). However, fibronectins secreted by transformed cells are defective and, therefore, are not able to attach and spread under serum free conditions (Rajaraman et al., 1974, Rajaraman, 1991). Primary diploid mouse embryo fibroblasts grow only when they are attached and spread, while the transformed 3T3 cells can proliferate in a spherical configuration in the absence of cell spreading (O'Neil et al., 1986). Cell shape is determined by the interaction of the cell with the substrate attached ECM (Rajaraman and Lonergan, 1982). Integrins transmembranally interact with intracellular actin filaments on the interior and with the extracellular matrix components in the exterior. In vitro, these points of cell substratum contact or focal adhesion complexes are characterized by specific organization of different ECM proteins on the outside of the cell, the various integrins forming the transmembrane link, and various cytoskeletal elements including actin filaments, a-actinin, talin, vinculin, paxillin, and tensin in the membrane-cytoplasm interface (Clark and Brugge, 1995). These ECM-transmembrane integrin-cytoskeletal assemblies also form the framework for the assembly of signaling complexes. Interference with the microtubule or microfilament organization dramatically affects the initiation of DNA synthesis (Crossin and Carney, 198 la; Maness and Walsh, 1982). The transformed cell membrane is locked up in the state of normal mitotic cell membrane in that it displays increased surface proteases, altered surface antigens, and are less adhesive (Fox et al., 1971), and generally display an absence of or reduced ECM elaboration. DNA synthesis in transformed cells is not dependent on the cell shape. Therefore, it follows that the uncoupling of the cell shape-dependent growth potential in transformed cells is related to the altered cytoarchitecture in these cells; the decrease in cell shape dependent growth regulation is accompanied by a gradual loss of cell-shape dependent macromolecular synthesis (Raz and Ben-Ze' ev, 1982; Wittelsberger et al., 1981). In the cell lines ranging from primary diploid fibroblasts to completely anchorage independent neoplastic fibroblasts, the progressive uncoupling of cell shape dependent macromolecular metabolism is accompanied by increased alterations in cell morphology from a well spread state to a non-spread spherical shape; this change in cell shape is probably a reflection of altered cytoskeletal organization (Wittelsberger et al., 1981).

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Table 1. Examples of Crosstalk Between the Mitogenic Cascade and the Adhesion Cascade Nature of Molecule

Phenotype

Scatter factor/ factor v-sis/c-sis

Growth Induces cell proliferation as well as cell hepatocyte factor/Morphogen growth differentiation (Montesano et al., 1991 ). Growth factor c-sis or PDGF causes normal cell proliferation and cell adhesion; v-sis disrupts cell adhesion, alters cell morphology, and favors continuous cell proliferation (Westermark et al., 1993). Dibutyryl cyclic Secondmessenger Induces reverse transformation, microfilament AMP assembly, fibroblastic morphology, and deposition of ECM proteins (Rajaraman et al. 1980). Proteoglycan Extracellular matrix Transfectionof decorin cDNA in CHO cells decorin tumorigenicity reinstates normal cell proliferation and adhesive properties (Yamaguchi and Ruoslahti, 1988). Proteoglycan syn- Transmembrane Transfection of syndecan cDNA and its expression decan proteogl ycan in S115 epithelial cells results in anchorage dependency and normal growth behavior (Leppa et al., 1992). Fibronectin recep- Integrin Transfection of cz5l]l gene in tumorigenic CHO tor, 0~5131 cells suppresses tumorigenicity and anchorage independent growth (Gioncotti and Ruoslahti, 1990). E-Cadherin Cadherin Re-expression of E-cadherin by cDNA transfection in poorly differentiated carcinoma cell lines inhibits invasiveness (Schipper et al., 1991 ; Frixen et al., 1991 ). CMAR NRTK Removal of CMAR protein function by mutagenesis abolishes cell adhesion to collagen. Transfection of CMAR cDNA results in increased expression of integrins. Loss of function mutation leads to metastatic behavior. Therefore, this is a candidate tumor suppressor gene (Pullman and Bodmer, 1992). v-src/c-src NRTK c-src activity results in normal cell proliferation and adhesion. But, v-src disrupts cell adhesion, phosphorylates several cytoskeletal proteins, and favors cell proliferation (see above). Merlin Cytoskeletal protein Behaveslike a tumor suppressor protein (NF2 gene); loss of function mutation or deletion causes central nervous system tumors including multiple meningiomas and bilateral vestibular schwannomas. Favors cell proliferation (Trofatter et al., 1993). a-Actinin Cytoskeletal protein Transfectionof 0t-actinin gene in highly malignant SV-40 transformed mouse cells with sixfold decrease in a-actinin expression restored normal levels of cz-actinin expression and restored normal cell adhesion, loss of anchorageindependent growth, and tumorigenicity (Gluck et al., 1993).

(continued)

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Table 1. (Continued)

Nature of Molecule Profilin

Phenotype

Cytoskeletal protein

Profilin forms a complex with PIP2 and prevents it from hydrolysis by unphosphorylated PLC-~I. In the presence of phosphorylated PLC-~I, it releases PIP2 and catalyzes ATP exchange on Gactin to promote actin filament formation (see above). MARCKS Cytoskeletal protein A serine phosphoprotein that reversibly binds with actin filaments. May be involved in maintaining cell shape and anchorage independent growth (see above). G protein GTPase A signal transduction component involved in metastatic behavior and cell adhesion and motility (see above). Rho and Rac GTPases Signal transduction components involved in reorganization of actin filaments related to focal adhesion and membrane ruffles (see above). Rab family GTPases Signal transducing machinery associated with cytoskeletal system involved in regulation of vesicular transport (see above). MAP kinase Kinase Activated when microtubules are disrupted by colchicine and phosphorylates MAP2. Links mitogenic cascade with microtubute (see above). NDP kinases Kinases Generate GTP from GDP. Associated with microtubules. Loss of expression increases metastatic potential (see above). Focal Adhesion Kinase Activated by integrins and growth factors (see Kinase above). NFkB Transcription factor Activated by cell adhesion resulting in gene expression. Has ankyrin repeat motifs (Juliano and Haskill, 1993). Zyxin Transcription factor? Contains LIM domains found in transcription factors. Localized in focal adhesion (Crawford et al., 1992; Sadler et al., 1992). Homeobox genes Transcription factors Associated with expression of cell-cell adhesion and detachment factors, patterning, and morphogenesis (Edelman, 1988). AP1 Transcription factor Activated by o~5131occupancy in T cells (Yamada et al., 1991). SRE Binding site for SRF Found upstream of start site for c-fos transcription, one of the immediate early genes. Also found in the upstream of the actin promotor gene (Ridley and Hall, 1992; Mohun et al., 1987; Sheng et al., 1988). Notes:

Someof them are recognized by direct studies. Others are recognized by mutational changesor gene transfection studies, which demonstrate phenotypic alterations affecting cell adhesion or cell proliferation.

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These and other extensive studies in this area strongly suggest that cell shape in vitro, maintained by the cytoskeleton, plays a regulatory role in various cellular activities including cell locomotion, proliferation, and differentiation. However, until recently, no satisfactory biochemical explanation on the mechanism of this process was available. In the recent past several avenues of research have yielded evidence for the involvement of the cytoskeletal system in the metabolic activities of the cell and its relationship to the external stimuli-induced signal transduction cascade during mitogenesis or differentiation. Some of these are summarized below. (See also Table 1.)

A. Mitogen Mediated Signal Transduction Protein Tyrosine Kinases The studies on the mechanism of cell transformation by retroviruses have contributed to the discovery of protein tyrosine kinases, which was followed by the discovery of a whole family of oncogenes and proto-oncogenes that belong to the categories of receptor tyrosine kinases--RTKs and non-receptor protein tyrosine kinases--NRTKs. To date, more than 50 RTKs, belonging to 14 different families and several NRTKs and other intermediates in the signal transduction cascade have been identified. Activation of these receptors by specific ligands regulates several physiological processes such as cell proliferation, differentiation, and programmed cell death or apoptosis. Molecular lesions in RTKs and NRTKs that result in constitutive activation of these kinases lead to oncogenesis, while kinase inactivating mutations can lead to developmental disorders (Ullrich and Schlessinger, 1990; Fantl et al., 1993). Binding of ligands such as growth factors with their receptors (RTKs) induces a cascade of signal transducing reactions in the target cell (Cantley et al., 1991), that culminates after several hours, in the initiation of DNA synthesis. These include changes in cell shape and cell membrane ruffling activity (Ridley and Hall, 1992; Ridley et al., 1992). Growth factors and various cytokines induce oligomerization of their receptors (Lemmon and Schlessinger, 1994). Ligand-induced dimerization can take place between two identical receptors (homodimerization); between different members of the same receptor family (heterodimerization); or between a receptor and an accessary protein such as an NRTK (heterodimerization). Conformational changes due to dimerization activates the intrinsic protein kinase function by autophosphorylation (Ullrich and Schlessinger, 1990) and confers a mechanism of expanding the specificity of ligand-receptor interaction and increases the diversity of signaling pathways within the cell.

SH2 and SH3 Modules Autophosphorylation of RTKs provides a binding site for Src-homology domains (SH2 and SH3, first identified in the NRTK pp60 src and not found in RTKs), which

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appear to be involved in modulation of protein-protein interactions (Feller et al., 1994). SH2 domains are conserved sequences of approximately 100 amino acids, while the SH3 domain consists of a stretch of about 50--60 highly conserved residues. The positively charged amino acids in the SH2 domains bind specifically to the negatively charged phosphotyrosyl-containing amino acid motifs (Songyang et al., 1993); SH3 domains bind to specific proline-rich sequences of about ten amino acids in length (Ren et al., 1993). Some proteins may have more than one SH2 or SH3 domains, indicating that these proteins may be involved in intermolecular protein-protein interactions. Thus, c-CRK, CRKL, Nck, Ash/Grb2, and the Ash/Grb homologs Drk and Sem-5 form a growing new class of signal transduction proteins. This class of small SH2-SH3-containing proteins lacks an apparent catalytic domain and may serve as regulatory subunits, coupling different proteins of the signal transduction cascade (Feller et al., 1994). SH2 modules are found in a diverse group of proteins (Koch et al., 1991; Heldin, 1991; Margolis, 1992), some containing enzymatic activities (PI3-kinase, PLC-z, and pp60 csrc) (Cantley et al., 1991) and others without any apparent enzymatic activity (rasGAP, gag-crk, myosinlB of Acanthamoeba, the yeast actin-binding protein, ABP-1, the CDC25 gene product and the FUS-1 product in yeast, nonerythroid spectrin and two neutrophil cytosolic factors NCF47K and NCF65K, and nck) (Mayer et al., 1988, 1992). SH2 modules seem to recognize phosphotyrosine moieties in phosphopeptides such as activated NRTKs and RTKs, thereby facilitating association of kinase substrates with the activated tyrosine kinases. There is structural variability in SH2 domain sequences at likely sites of contacts, which may provide a structural basis for the phosphopeptide selectivity (Cantley et al., 1991; Songyang et al., 1993). SH3 domains are found in a wide variety of proteins including the cytoskeletal proteins, spectrin, myosin, and the yeast actin binding protein, ABP1 (Pawson, 1992) and serve as modules that mediate protein-protein associations. SH3 may bind to membranes or the cytoskeleton (Koch et al., 1991). A common feature of SH3 containing proteins is their association with the cortical membrane associated spectrin-actin cytoskeleton, that regulates cell shape (Drubin et al., 1990). For example, deletion within the SH3 domain of v-src affects the ability of the protein to associate with the detergent-insoluble cytoskeletal matrix. The ATP-insensitive actin-binding site of myosin-1 contains an SH3 module (Jung et al., 1987). Similarly, both NCF47K and NCF65K translocate from the cytosol to the membrane skeleton with activation of the oxidative burst in phagocytic cells (Leto et al., 1990). Many cellular proteins bind to SH3 probes and different SH3 probes bind different sets of proteins in individual cell lysates (Ren et al., 1993). Thus, when platelet growth factor (PDGF) binds to its receptor, PDGF-R, the activated kinase domain of PDGF-R can recruit several signal transducing molecules with SH2 domains such as pp60src, PI 3 kinase, rasGAP, PLCx, Raf protein, phosphotyrosine phosphatase, and other proteins (Figure 2). Specific binding of the SH2 domain of these RTK substrates directly to the phosphorylated tyrosine

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moieties of the activated RTKs provides a common mechanism by which diverse enzymatic and regulatory proteins can interact specifically with RTKs and, thereby, couple growth factor stimulation to multiple intracellular signaling pathways (Koch et al., 1991; Kypta et al., 1990; Heldin, 1991; Margolis, 1992; Pazin and Williams, 1992; Carraway and Carraway, 1994; Clark and Brugge, 1995). Activation of these SH2-containing signaling molecules initiates cellular pathways that culminate in changes in the cytoskeleton, gene expression, and cell division. PI3 kinase, which is a primary target for several tyrosine kinases, may be involved in cell shape and the cytoskeleton (Cantley et al., 1991). PI3 kinase activation by PDGF-R is required for the activation of the downstream component p21Ras, which may directly or via Raf- 1 activate the mitogen activated kinases (MAPKs), that eventually result in the expression of transcription factors c-fos and c-jun. GTPases

The Ras family of proteins consisting of about 50 proteins (ras, rho, rab, and arf) are GTPases, involved in cell proliferation, differentiation, intracellular transport, and cytoskeletal regulation (Downward, 1990; Hall, 1990). Of these p21 rasoccupies a pivotal role in mitogenic signal transduction. The ras proteins function as binary switches, cycling between active and inactive states bound to GTP and GDP, respectively; in addition, their functions depend on their cellular localization. Their activation and inactivation cycle can be regulated by three different classes of proteins, which switch the GTPase on, switch it off, and protect it from switching, respectively. The critical anchoring of GTPases to different cellular membranes is regulated by prenyltransferases and other enzymes (Boguski and McCormick, 1993). The GTP-bound active form is slowly converted to the GDP-bound form by the protein's intrinsic capacity to hydrolyze GTP, a process accelerated by GTPase activating proteins or GAPs. Activation involves the replacement of GDP with GTP, which is mediated by guanine-nucleotide exchange factors, GEFs or guanine-nucleotide-releasing proteins (or guanine-nucleotide-dissociation stimulators, GDSs); the nucleotide exchange can be inhibited by guanine nucleotide dissociation inhibitors or GDIs. The GDIs may also block the action of GAPs. The activators, guanine-nucleotide-exchange factors or GEFs have been recognized for different Ras subfamilies and are designated rasGEF, ralGEF, rapGEF, rho/racGEF, ranGEF, and rabGEF, respectively. While all these GEFs act on non-prenylated forms of their targets, a distinct type of Ras regulator, Smg (small GTP-binding protein) GDS with broad specificity works on isoprenylated forms. Thus, on ligand binding, EGFR dimerizes, autophosphorylates tyrosine residues in the cytoplasmic domain, creating binding sites for SH2 domains of other proteins such as the adapter protein Grb2/Sem5, which in turn binds to the SH3 domain of rasGEF known as Sos 1, thus relocating the later to the membrane from the cytosol. NRTKs activate Ras in a similar way: these phosphorylate the adapter protein Shc, which binds the Grb2-Sos 1 complex bringing the bound Sos 1 close to p21 r a s at the

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plasma membrane (Boguski and McCormick, 1993). This results in p21 r a s dropping GDP and binding to GTP. Ras-GTP is the active form sending signals downstream via Raf-1 to the MAP kinase cascade, before it reaches the inactive GDP bound state (Egan and Weinberg, 1993). Activated Ras recruits Raf- 1 (MAP kinase kinases Or MAPKKK) to the plasma membrane, where it binds to the cytoskeleton and is activated by a yet unknown membrane- and/or cytoskeleton-associated element (Carraway and Carraway, 1994). Activation ofRaf- 1 results in turning on the MAP kinase pathway consisting of three enzymes: MAP kinase kinase (MAPKK or MAP kinase/ERK kinase [MEK]), the mitogen activated protein kinase (MAPK) also referred to as microtubule associated protein-2 kinase or extracellular signal regulated kinase (ERK) and the pp90 ribosomal $6 kinase (pp90rsk). The MAPKK is unique in that it is a serine/threonine/tyrosine kinase, which activates MAPK by phosphorylation at threonine and tyrosine residues. MAP kinase exists in two isofbrms, ERK1 and ERK2, which become phosphorylated at threonine and tyrosine residues following stimulation by growth factor (Ray and Sturgili, 1988; Ahn et al., 1991; Seger et al., 1992; Gomez and Cohen, 1991). The signals delivered by respective ligand binding to various receptors such as EGF-R (Ahn et al., 1991; Seger et al., 1992), NGF-R (Gomez and Cohen, 1991), bradykinin-R (Ahn et al., 1992a), insulin-R (Nakielny et al., 1992), PDGF-R (L'Allemain et al., 1992), progesterone-R (Matsuda et al., 1992), and angiotensin II-R (Ishida et al., 1992) flow through the respective receptors via the combined action of pp21 r a s and pp60 src or directly to raf- 1 kinase (Roberts, 1992), thence to MAP kinase (via MAPKK) to pp90 rsk and finally on to the nuclear protooncogene products c-jun, c-fos, c-myc and p62 tcf (Roberts, 1992; Ahn et al., 1992b; Morrison et al., 1988; Figure 2). In addition to p 120rasGAP,another rasGAP, called neurofibromatosis 1, NF1, has also been identified; mutations in NFlrasGAP results in uncontrolled cell proliferation and malignancy; binding of p21 r a s with p 120rasGAP,favors cell proliferation, while its association with NF lrasGAP inhibits cell division (Chao, 1992; DeClue et al., 1992; Zhang et al., 1991; Bollag and McCormick, 1992). GTPase cascades are also involved in directing cellular behavior in relation to cytoskeletal organization, cell movement, and morphogenesis. In mammalian cells, the actin filaments control movement by the extension of filopodia, lamellipodia, and attachment (Stossel, 1993). Each of these actions are regulated by different GTPases. Activated Cdc42 promotes the formation of filopodia, RacGTP promotes extensions of lamellipodia, and RhoGTP promotes focal adhesion formation. Activated Cdc42 induces filopodia, then lamellipodia, then focal adhesions and cytoskeletal assembly. This is dependent upon activation of Rac and Rho. Activation of Rac produces lamellipodia, followed by focal contacts and actin fibers, and activation of Rho produces only focal adhesions and actin filaments. Thus, Cdc42, Rac and Rho behave as a linear cascade. Different extracellular stimuli activate the Cdc42-Rac-Rho GTPase cascade at different points. PDGF or insulin activate Rac (lamellipodia) followed by Rho activation (focal adhesion and cytoskeletal assem-

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Intqlrin duster ECM with differentadhesivesites

Figure 2. Schematic representation of some crosstalk between the adhesion cascade and mitogenic cascade. Various proteins involved in focal adhesion such as talin, zyxin, paxillin, a-actinin, and others are not shown for reasons of clarity. The known substrates of RTKs include PI3K, PLC-~, PTP, rasGAP, rafl, and src; the substrates of src include PI3K, PLC~, rasGAP, rafl, E-cadherin, and erzin, c-src is also a substrate for pp125 FAK. pp125 FAK can activate the p21 ras-MAP Kinase cascade, pp125 FAK in turn is phosphorylated by occupancy of the serpentine receptor/G protein complex, RTKs or integrins; the figure shows only the integrin activation of pp125 FAK. (See text for details.)

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bly). Lysophosphatidic acid stimulates Rho only (focal adhesion and cytoskeletal assembly) (Ridley et al., 1992; Nobes and Hall, 1995). Similar GTPase cascades involving cytoskeleton have been reported in the yeast cells (Chant and Stowers, 1995).

Crosstalk Between Different Mitogenic Signal Cascades Diverse external stimuli such as growth factors, cytokines, neurotransmitters, phorbol esters, radiation, regeneration, ischemia, seizure, and viruses elicit their effects through one or more of the classical signal transduction cascades that result in cell proliferation. These include the RTK-based signal transduction system, the "serpentine receptor"/G protein system, the nitric oxide-cyclic GMP system, ion channels, and the nuclear receptors (for steroids and related hormones). The principal architecture of the mitogenic signaling cascade consists of a series of ligands, their receptors, GTP-binding proteins, second messenger-generating enzymes, protein kinases, target functional proteins, and regulatory proteins. This system employs three mechanisms for signal transduction: (1) phosphorylation of proteins about the serine, threonine, and tyrosine residues; (2) the GTPases, the molecular switches that are involved in turning on or off the cascade of events; and (3) control of transcription factors that are involved in mitogenesis and differentiation. Receptor occupancy initiates the complex mitogenic cascade. A series of events such as receptor clustering, activation of the kinase function, initiation of the phosphorylation cascade, activation of Na§ § pump, transient increase in intracellular Ca 2§ and pH i, membrane ruffling, focal adhesion formation, activation of MAP kinases, transcription factors, and c-fos/c-jun expressiorv--all culminate eventually in cell proliferation. The significant aspect of these several signal transduction systems is their heterogeneity and the presence of extensive crosstalk at various levels. Such interactions include potentiation, cooperation, synergism, and antagonism as well as co-transmission. A positive signal is frequently followed by negative feedback control. Such crosstalk occurs at the level of the plasma membrane, second messenger generation and degradation, protein kinases and phosphatases, and gene transcription and cell cycle control. For example, Ca §247 homeostasis is maintained by complex interactions between cAMP-mediated signals and signaling cascades initiated by various growth factors, cytokines, and neurotransmitters involving PI3 kinase, PKC, and others (Nishizuka, 1992; Karin and Smeal, 1992). D ~kG produced by hydrolysis of PIP 2 activates PKC, which provides the link between extracellular signals and intracellular responses. Sustaining activation of PKC seems to be a prerequisite for long-term physiological responses such as cell proliferation and differentiation. Crosstalk exists at the transcription level, where various c-jun and c-fos related proteins form various combinations of dimers in regulating the transcriptional activity in relation to the external stimuli. Thus, hormones and growth factors can induce physiological responses via gene expression that persist longer than the initial influx of Ca 2§ and

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alterations in PHi; they also modulate cell adhesion in numerous cell systems by regulating the expression of adhesion molecules over long periods of time (several hours). B. Adhesion Activated Signal Transduction

The major components of the signaling pathways triggered by cell adhesion are also involved in the mitogenic signal transduction pathways. Induction of tyrosine phosphorylation by integrin engagement and clustering (analogous to RTK oligomerization) has been shown by various studies. Integrin occupancy also results in the activation ofserine-threonine kinase families such as protein kinase C (PKC), MAP kinase cascade, increase in the intracellular calcium concentration and elevation ofintracellular pH. Adhesion activated signal transduction can be effected by various types of cell adhesion receptors and can be mediated via different cytoskeletal systems (Schlaepfer et al., 1994; Clark and Brugge, 1995; Richardson and Parsons, 1995).

integrins Integrins are non-tyrosine-kinase transmembrane receptors for extracellular matrix proteins. Occupancy and clustering ofintegrins by the extracellular matrix lead to activation and autophosphorylation of pp 125 FAK and a cytoskeletal component, paxillin. Other cytoskeletal proteins found in the focal adhesion include a-actinin, vinculin, talin, paxillin, and tensin in complex with actin filaments. Pp125 FAK resembles RTKS in that it does not have SH2 or SH3 domains and appears to be activated by autophosphorylation. A COOH-proximal focal adhesion targeting (FAT) domain localizes pp 125FAK tO focal adhesions, while the sequences proximal to amino-terminus binds to the cytoplasmic domains of]3 integrins (Richardson and Parsons, 1995; Clark and Brugge, 1995). As in the case of ligand-bound RTKs, integrin engagement results in the formation of complexes of several proteins with SH2 and SH3 domains. In addition to pp 125 FAK, the kinases that are activated by integrin occupancy include pp60 c-src and related NRTKs, Csk, Syk, PKC, and the kinases in the MAP kinase cascade. Activated pp60 csrc associates with integrin occupancy-activated cytoskeletal complexes in platelets and fibroblasts (Clark et al., 1994; Schlaepfer et al., 1994; Clark and Brugge, 1995; Richardson and Parsons, 1995). Protein tyrosine phosphatases (PTPs) may also participate in the activation of Src family of kinases by dephosphorylating the negative regulatory COOH-terminal phosphotyrosine (Arroya et al., 1994). Similarly, in hematopoietic cells, Syk associates with integrin-dependent cytoskeletal structures. Inhibitors of cytoskeletal assembly and tyrosine kinases inhibit the activation of a number of tyrosine kinases, as well as the formation of focal adhesions and microfilament assembly. Thus, many tyrosine kinases associate with occupied integrins through interactions with cytoskeletal complexes induced by the clustering and cross-linking of integrins (Clark and Brugge, 1995; Richardson and Parsons, 1995).

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Several SH2-SH3 adapter proteins including Crk (Tanaka et al., 1994; Birge et al., 1993), Grb2 (which links activated RTK to the Ras-MAP kinase pathway), and SOS 1 (a guanine nucleotide exchange factor that functions by converting inactive Ras-GDP to active Ras-GTP) have also been implicated in integrin-mediated signal transduction. The association of Grb2 and SOS 1 with pp 125FAKindicates a critical link between the adhesion activated signal transduction and the Ras-MAP kinase pathway of the mitogen activated signal transduction. Ras is activated after engagement of the collagen receptor and the MAP kinase pathway is activated after 3T3 cell adhesion to fibronectin (Clark and Brugge, 1995). Cytoehalasin D blocked the integrin-dependent activation of MAP kinase cascade and the activation of MAP kinase was dependent on cell spreading (Zhu and Assoian, 1995). While growth factor-induced activation of the MAP kinase pathway may be involved in the expression of transcription factors related to initiation of DNA synthesis, integrininduced MAP kinase activation may be involved in the expression of transcription factors related to extracellular matrix components-induced differentiation-related cell spreading. The fact that both cell adhesion and growth factors can activate the major signal transduction cascade MAP kinase pathway indicates a critical link between both those signaling pathways, which, apparently, have been severed in transformed cells. As in the case of the mitogenic cascade (Chant and Stowers, 1995), GTPase cascade involving focal adhesion and cytoskeletal assembly may be expected to be activated by integrin occupancy. However, similar studies with rho and rac GTPases (Ridley et al., 1992; Nobes and Hall, 1995) have not been carried out in relation to integrin. PI3Kinase, a substrate for activated RTKs and NRTKs, is also a substrate for pp 125 FAK.Other second messenger systems such as protein kinase C (PKC) are activated, which may regulate integrin-dependent adhesion and as an integrin-mediated signal transducer (Richardson and Parsons, 1995). Two protein tyrosine phosphatases (PTPs), CD45, a transmembrane PTP in lymphocytes and neutrophils (Arroya et al., 1994), and the cytosolic PTP 1B in platelets have also been identified as components ofintegrin-mediated signaling cascade. These findings indicate that pp125 FAK is a common target for several signal transducing molecules such as integrins (Richardson and Parsons, 1995; Clark and Brugge, 1995), neuropeptide receptors and the associated G proteins (Zachary et al., 1992), non-receptor protein tyrosine kinases c-src (and the src family of NRTKs), the insulin receptor (Pasquale et al., 1988; Hynes, 1992), and the PDGF receptor (Knight et al., 1995). Thus, pp125 FAKmay function as a common down stream element in the signal transduction pathway converging from heterotypic receptors (Figure 2). It would be reasonable to assume that pp125 FAK, paxillin, and p130 are participants in a cluster of phosphorylation events during cell-substrate adhesion and may be related to cell shape and cell motility (Zachary and Rozengurt, 1992). Proteins other than the kinases and GTPases also link actin filament assembly to the mitogenie signal transduction cascade. These include profilin and MARCKS proteins. Profilin binds four to five molecules of phosphatidylinositol 4,5-biphos-

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phate (PIP2). When profilin is bound to PIP2, the cleaving of PIP 2 to inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) is dependent upon phosphorylation of PLCT-1 by activated RTK (Goldschmidt-Clermont et al., 1991). When PIP 2 is cleaved, profilin would be free to interact with actin and influence polymerization. Thus, profilin plays a pivotal role in linking the major mitogenic signal transduction pathway, the phosphatidylinositol cascade, and the actin polymerization-depolymerization cycle (Aderem, 1992). Further, a number of other actinbinding proteins that bind PIP 2 may be expected to have crosstalk with cytoskeleton and mitogenic signal pathway; these include gelsolin, gCap39, severin, protein 4.1, villin, and tensin (Jamney et al., 1992; Yu et. al., 1992). In addition, IP 3 can transiently elevate intracellular calcium, which in turn regulate several actin-binding proteins. DAG can activate and nucleate actin polymerization (Shariff and Luna, 1992) and most isoforms of protein kinase C (PKC).

Immunoglobulin SuperFamily They are involved in various functions such as cell-cell recognition (Williams and Barclay, 1988), cellular immunity (e.g., major histocompatibility antigens, CD4, CD8, and the T cell receptor), neural cell adhesion (N-CAM), leukocyte trafficking (ICAM-1, -II, -III; de Fougerolles and Springer, 1992; Fawcett et al., 1992). The receptor for colony stimulating factor-1 (CSF-1R) and platelet derived growth factor (PDGFR; Albelda, 1993) are typical RTKs involved in signal transduction. A recently discovered member PECAM-1 expressed on platelets, vascular endothelium, monocytes and neutrophils and on T cells, is phosphorylated in response to cellular activation and associates with the cytoskeleton of activated but not resting platelets (Newman et al., 1990). Phosphorylation events and cytoskeletal assembly indicate their involvement in signal transduction cascade. DCC (deleted in colon carcinoma) a transmembrane phosphoprotein with homology to N-CAM appears to be a signal transducing receptor, whose loss confers a growth advantage on evolving tumor cells (Vogelstein et al., 1988, 1989).

5electins L-selectin (90-110 kD) is involved in the binding and extravasation of neutrophils into the inflammatory sites. L-selectin on neutrophils (also found on circulating lymphocytes, monocytes, natural killer cells, thrombocyte precursor cells, and thymocytes) binds with the sialylated oligosaccharide ligands on endothelial cells. These initial week imeractions result in the slow down of neutrophils or the so called "rolling" of neutrophils. During this process the leukocyte integrins are activated; L-selectin molecules on the surface of neutrophils are shed and transendothelial migration is initiated. P-selectin (140 kD) is stored in the a granules of platelets and the Weibel-Palade bodies of vascular endothelial cells. Exposure of endothelial cells and platelets to thrombin or of endothelial cells to oxygen radicals results in the surface expression of P-selectin. P-selectin is an important adhesion

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molecule on platelets, mediating platelet-leukocyte binding in vivo; P-selectin mediated leukocyte adhesion in thrombi results in deposition of fibrin (Palabrica et al., 1992). P-selectin, when released, can inhibit adhesion ofneutrophils to resting endothelium. A newly identified lectin-like protein, NKR-P 1, functions as a signal transduction molecule on natural killer cells (Giorda, et al., 1990). The signal transduction cascade induced by the sequential activation of heterotypic receptors that results in neutrophil migration to sites of inflammation leading to production of oxidative burst seems to be achieved by release of Ca 2+ (Waddell et al., 1991). Cadherins

E-cadherin, a 120 kD transmembrane glycoprotein, is enriched in the adherens junctions of epithelial cells and interacts with the cytoskeleton via associated cytoplasmic molecules, the catenins (Ozawa and Kemler, 1992). E-cadherin expression is frequently downregulated in highly invasive, poorly differentiated carcinomas (Schipper et al., 1991; Frixen et al., 1991). Re-expression of E-cadherin by cDNA transfection in poorly differentiated carcinoma cell lines inhibits invasiveness (Frixen et al., 1991; Vleminckx et al., 1991; Chen and Obrink, 1991; Navarro et al., 1991). Junctional complexes have been shown to be the major sites of tyrosine phosphorylation (Tsukita et al., 1991; Volberg et al., 1992). It has been recently demonstrated that loss of epithelial differentiation and gain of invasiveness correlated with tyrosine phosphorylation of the E-cadherin/13-cantenin complex. This phosphorylation is effected by v-src (Behrens et al., 1993). Microtubules

In the above description, we have been essentially discussing the involvement of microfilaments in the signal transduction pathway. However, microtubules also seem to be involved in crosstalk with the mitogenic signal transduction. Colcemid and other anti-microtubule agents initiate or potentiate DNA synthesis in different cell systems (Vasiliev et al., 1971; Otto, 1987; Crossin and Carney, 1981a, 1981b; Shinohara et al., 1988). In A431 cells, microtubule disruption induces formation of endosome-like intracellular aggregates containing EGF receptors. This seems to favor oligomerization of the EGF receptors and an increase in tyrosine kinase activity in the absence ofEGF (Schlessinger, 1988). This in turn seems to activate MAP kinase pathway. Activated MAP kinase phosphorylates microtubule associated protein 2, the pivotal molecule in the mitogenic signal transduction pathway, which also appears to be the link between the mitogenic cascade and the cytoskeleton. Intact microtubules appear to restrict the motility of growth factor receptor(s), so that they cannot be endocytosed and cross-phosphorylated by oligomerization in the absence of ligand(s). Receptor occupancy may increase the mobility of the receptors, by modulation of the cytoskeleton, which will favor oligomerization and tyrosine kinase activation, and initiate the signal transduction cascade (Willingham and Pastan, 1982; Glenney et al., 1988; Honegger et al., 1987; Chen et al., 1987;

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Moolenar, 1988). Anti-microtubule drugs may create a positive signal for initiation of DNA synthesis by removing the mobility restrictions and favoring endocytosis of the receptors. NUcleoside diphosphate kinases (NDP kinases) are enzymes required for the synthesis of non-adenine-containing nucleoside triphosphates, for example, GTP from GDP (Parks and Agarwal, 1973). Since GTP is required for microtubule polymerization, protein elongation, and intracellular signaling, loss ofNDP kinase activity could lead to defects in mitosis, protein synthesis, and signal transduction. Microtubules assembled in vitro contain NDP kinase activity (Nickerson and Wells, 1984). In Drosophila, the loss of awd gene results in mitotic and developmental abnormalities, demonstrating a critical role for this gene in spindle microtubule polymerization (Biggs et al., 1990). This gene has a high degree of homology to the mammalian nm23 gene (Rosengard et al., 1989), whose expression appears to be greatly reduced in highly metastatic melanoma cells (Steeg et al., 1988; Bevilacqua et al., 1989; Leone et al., 1991). Fusion of normal and metastatic cells resulted in hybrids that were tumorigenic but non-metastatic (Turpeenniemi-Hujanen et al., 1985; Sidebottom and Clark, 1983). These studies strongly indicate a dominant metastatic-suppressor gene associated with microtubule polymerization.

Intermediate Filaments Intermediate filaments associate with the plasma membrane at specialized regions called desmosomes and hemidesmosomes (Garrod, 1993). The potent mitogen adenosine disphosphate (Kartha et al., 1992) as well as phorbol esters (Collard and Raymond, 1992) induce reorganization of intermediate filaments suggesting a role in signal transduction for intermediate filaments. The mitogenic adenosine diphosphate, and not the non-mitogenic purine and pyrimidine nucleotides, induce major reorganization of cytokeratin in African green monkey kidney cells (Kartha et al., 1992). Vimentin is predominantly expressed in proliferating cells. When anti-Ig binds to B lymphocytes, membrane Ig-cytoskeletal interactions occur, in which an increased accumulation of extensive filamentous array of vimentin has been involved (Albrecht et al., 1990), probably involved in anti-Ig mediated signal transduction. The role of IFAPs in signal transduction is not clear at present, although one of them, plectin, has been found at focal contacts and actin stress fibers (Seifert et al., 1992). It is conceivable that these molecules may be involved in stabilizing cytoskeletal assemblies involved in different cellular functions.

Crosstalk Between Different Adhesion Signal Cascades Analogous to the mitogenic cascade, cell adhesion appears to be brought about by integrated (and sequential) activation of heterotypic receptors and there is crosstalk between the signal transduction mediated by various cell adhesion receptors. Adhesion seems to occur by a coordinated sequence of events involving multiple receptors with distinct but inter-related functions. Leukocyte adhesion to

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endothelial cells would be an example of such heterotypic receptor cooperation, where loose contact mediated by L-selectin on the circulating leukocyte surface and the P-selectin or E-selectin on endothelium, results in slowing down or "rolling" of the circulating leukocytes. A chemokine signal released by the endothelial cell is captured by the endothelial cell surface proteoglycan from being washed away and helps trigger the activation of the leukocyte integrins ~L[~2and VLA4 resulting in film attachment via ICAMs and VCAM-1, respectively; the selectins are shed and the process of extravasation is initiated. These interactions are tightly regulated by biochemical mechanisms that control the affinity and avidity of the receptors by various mechanisms (Tanaka et al., 1993; Mackay and Imhof, 1993). Often, these regulatory signals come from a preceding adhesive interaction, which triggers or modulates a second type of adhesive interaction (Springer, 1990; Butcher, 1991). Other examples of the adhesion cascade include activation-dependent adhesion of platelets (Phillips et al., 199 l; Roth, 1992), and T and B cell receptor mediated triggering of integrin activation and cytoskeletal reorganization (Dustin and Springer, 1991). Other cell adhesion molecules such as those of immunoglobulin superfamily and selectins also undergo activation-dependent changes in their binding capacity (O'Rourke and Mescher, 1992; Spertini et al., 1991). In these interactions, the inflammatory cytokines upregulate the expression of endothelial cell adhesion molecules and can also trigger the rapid activation of leukocyte integrins by altering the affinity or function of the existing surface receptors on a rapid time scale (in the order of minutes). These studies reveal the extensive crosstalk between the signals initiated by different cell adhesion receptors.

C. Crosstalk Between Mitogen and Adhesion Induced Signal Cascades At least two signals seem to be required for the initiation cell division in normal cells. For example, in resting T cells, costimulation of integrins and the T cell receptor leads to a proliferative response (Pardi et al., 1992; van Seventer et al., 1990; Burkly et al., 1991). Similarly, an embryonic carcinoma cell line depends on laminin or fibronectin as a second signal after FGF stimulation. Another growth factor called the scatter factor/hepatocyte growth factor (SF/HGF) is a potent mitogen for hepatocytes (Nakamura, 1992) and is also a morphogen responsible for the differentiation of branching tubules in MDCK epithelial cells (Montesano et al., 1991). Thus, the resting normal cells require at least two signals, the mitogenic signaling cascade initiated by a growth factor and the adhesion induced signaling cascade to commit the cell to enter mitosis. This appears to be true in the case of leukocytes and other cell types. In addition to the crosstalk within the various mitogenic signaling systems and within the different adhesion cascades, the data reviewed above (Table 1 and Figure 2) make it apparent that there is extensive crosstalk between the mitogenic cascade and the adhesion cascade at various levels. The initial major links between these

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two main signaling systems are the transient rise in Ca ++ (cell adhesion independent), and pH i (cell adhesion dependent), and the focal adhesion formation. Signals generated by various mitogenic receptors such as the RTKs, the "serpentine" receptors and the adhesive receptors, integrins, seem to converge on the actin filament associated focal adhesion formation and the phosphorylation ofpp 125 FAK (Zachary and Rozengurt, 1992). In addition, crosstalk seems to occur at various levels via profilin/PIP 2 cycle, the cyclic AMP signaling system, the MARCKS protein, the GTPases, and others. The mitogenic cascade culminates in DNA replication following which mitosis completes one cycle of cell division. The cytoskeletal system undergoes a major reorganization during mitosis: the cells round up by dismantling the membrane skeleton, the actin filaments at focal adhesions, the lamin network, and the nuclear membrane; the microtubules take the central stage during chromosomal division, and the circular bundles of actin filaments are formed at the equatorial region to effect cytokinesis. At the completion of mitosis, the process is reversed and the membrane cytoskeleton, actin filaments at the focal adhesion, and the nuclear membrane are reassembled. Albeit not exhaustive, the above examples of crosstalk between the mitogenic cascade and the adhesion cascade make it apparent that the cellular functions are carried out by processes broadly divisible into the reversibly assembled cell adhesion oriented cytoskeletal system and the reversibly phosphorylated soluble cytosolic enzymes related to mitogenesis; the former has restricted boundaries such as the cytoplasm and the nucleus, while the latter can activate gene expression by migrating into the nucleus and phosphorylating certain transcription factors or acting as transcription factors (nuclear receptors of steroid and related hormones) or by translocating activated transcription factors sequestered in the cytoplasm. All these activation reactions are reversible and are regulated by nebulous liaisons between different cascades. As the understanding of the structure and precise functions of other actin-binding proteins, microtubule associated proteins, intermediate filament associated proteins, and nuclear matrix proteins progresses, more and more liaisons between the cytoskeletal system (the "insoluble biochemistry") and the several enzyme-based signal transduction pathways (the "soluble biochemistry") will come to light. Although the finer details of the mechanisms of crosstalk and the resultant phenotypic effects are not yet known, it is suspected that protein-protein interactions facilitated by various protein modules ubiquitously found in different classes of proteins might be responsible for extensive crosstalk between proteins with different and seemingly unrelated functions. These modules or domains include SH2, SH3, and the ankyrin repeat domains. Several components of the mitogenic cascade such as PI 3 kinase, PLC-z, pp60 src and related NRTKs, PTPs, and rasGAP have one or more SH2 domains. Similarly, several cytoskeletal associated proteins of the adhesion cascade including myosinlB, ABP-1, tensin, and non-erythroid spectrin contain SH2 domains. Since SH2 domains interact with phosphotyrosine containing molecules, the SH2 domain containing cytoskeletal components may

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be critical regulatory links between the phosphorylation relays of the mitogenic cascade and the adhesion cascade. SH3 domains are also found in a wide variety of intermediates in the mitogenic signaling cascade including the various NRTKs, and the different cytoskeletal proteins including spectrin, myosin, and ABP 1. Many cellular proteins bind to SH3 probes and different SH3 probes bind different sets of proteins in individual cell lysates (Ren et al., 1993). SH3 module containing proteins are often localized in the submembranous region (Koch et al., 1991), where actin filaments interact with the plasma membrane. For example, deletion within the SH3 domain of v-src affects the ability of the protein to associate with the detergent-insoluble cytoskeletal matrix. In the cytoskeletal protein ankyrin, a 33 amino acid repeat unit functions as a binding site for anion exchanger and interacts with tubulin. Analogous to the SH2 and SH3 modules, the ankyrin repeat modules are also found in a variety of proteins including some transcription factors. These modules are also important in protein-protein interactions, and proteins with such modules are also candidates for crosstalk effector molecules. There are a host of other modules such as EGF motifs, Fibronectin type I, II, and III motifs, and others which facilitate interactions among otherwise unrelated proteins. Cell adhesion related signaling apparatuses such as the focal adhesion complex (consisting of integrin associated cytoskeletal assembly, c-src, pp 125 FAK,and other components) or the junctional complex of epithelial cells (consisting of cadherins, c-src, cytoskeletal proteins, and other proteins) may be related to signal transduction or they may be involved in regulating cell locomotion with respect to their extracellular environments and the morphogenesis of tissues related to differentiation and development. It is highly likely that they are involved in both of these functions. However, at present little is known about the molecular associations and relative organization of the components at these sites.

V. CYTOSKELETON, GROWTH REGULATION, AND NEOPLASIA: A HYPOTHESIS The primary cellular processes are cell adhesion, locomotion, proliferation, differentiation, and death. Cell adhesion plays a central role in locomotion by being reversible, and in proliferation and differentiation by receiving and responding to different environmental stimuli. Therefore, it is imperative for the normal phenotype of cells that the cellular genes (proto-oncogenes) coding for these various factors involved in signal transduction pathways function properly not only during cell proliferation and differentiation, but are required also for the maintenance of the differentiated state. Any one of the steps in the various signal transduction relays can be interrupted and deregulated by various genetic errors. These changes release the proto-oncogenes from the regulatory constraints; they become activated oncogenes and are constitutively expressed, resulting in neoplasia (Bishop, 1991; Cantley et al., 1991; Cleary, 1991; Cross and Dexter, 1991; Lewin, 1991).

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At least two different signals appear to be necessary for the induction of proliferation in resting cells. These are the mitogenic signal and the adhesion signal. When both signals are received, normal cells proceed to synthesize DNA leading to cell division. One without the other does not culminate in cell division. It is likely that cells might require more than one mitogenic stimulus (PDGF followed by EGF or other GFs) as well as more than one adhesive stimulus (different cell adhesion receptors). There are numerous examples where the mitogenic signaling system and the adhesion signaling system intercommunicate at various levels (Table 1). It is hypothesized that this crosstalk between the mitogenic signaling cascade and the adhesion signaling cascade forms the biochemical basis for a link between cell shape and anchorage dependency for growth. A break in communication between the mitogenic signaling cascade and the adhesion signaling cascade at one or more levels would cause variable degree of anchorage independence for growth. Therefore, the degree of autonomy in proliferation and anchorage independence for growth in a neoplastic cell might depend upon the extent of the breakdown of communication between these two cascades. It is further hypothesized that the growth factor induced mitogenic cascade favors cell multiplication, while the adhesive cascade favors G1 arrest and associated differentiation. While the integrin family of receptors may act as a cell division modulators, cadherin family of receptors may be involved in inhibition of cell division and promotion of differentiation. Transformed and malignant cells ot~en show lack of microfilaments and focal adhesions, and junctional complexes, and decreased levels of cytoskeletal proteins; they are anchorage independent for growth and they generally display lack of differentiated properties. It is intriguing to note that NGF which induces differentiation of a PC12 subline, also induces enhanced expression of otll31 integrin accompanied by substantial increase in attachment to collagen (Zhang et al., 1993). It would be interesting to find out if these cells also display increased expression ofneurofibromin, since it is likely that the NF 1 gene might play a central role in diverting the signaling pathway toward differentiation instead of proliferation. Is the type of cell adhesion receptor that mediates cell adhesion a deciding factor between the onset of mitogenic cascade or the differentiation process? For example, DCC (deleted in colon carcinoma) of colon carcinoma is homologous to N-CAM and is a 190 kD transmembrane phosphoprotein with fibronectin type III and C2 immunoglobulin-like domains (Vogelstein et al., 1989; Edelman, 1988). DCC behaves like a tumor suppressor gene in that loss of heterozygosity of DCC at chromosome 18q21.3 confers malignant behavior in colon carcinoma cells and other tumors (Vogelstein et al., 1988; Devilee et al., 1991; Weinberg, 1991). Loss of cell adhesion results in loss of differentiation phenotype and leads to metastatic properties (Pullman and Bodmer, 1992). Thus, cell adhesion related molecules appear to be tumor suppressor genes, favoring differentiation. During wound healing, the normal cells can be turned on to enter the cell cycle, and turned off when the healing process is completed. Thus, under normal circumstances, depending on the cell/tissue type,

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the mitogenic cascade is shut down and the steady state adhesive property and differentiated state is reinstated. But, in transformed cells due to breakdown of communication between the mitogenic and adhesion signaling systems, the mitogenic cascade becomes autonomous and dominant and the adhesion cascade is suppressed by paralysis to varying extent. In fully anchorage-independent cells such as the ascitic cells, the crosstalk between the mitogenic cascade and the adhesion cascade is probably reduced to the minimum and retained only for the purpose of spindle formation and cytokinesis during mitosis. Since anchorage-independent cells can divide without attachment to a substratum, the cell adhesion cascade does not seem to be required for the completion of cell division cycle. Disruption of microfilaments (Rothberg et al., 1978) and micrombules (Shinohara et al., 1988) can short circuit the mitogenic cascade leading to DNA synthesis in the absence of growth factors and cell adhesion. What, then, would be the role of adhesion cascade in normal cells? It is very likely that cell adhesion cascade is initiated almost simultaneously along with the mitogenic cascade (a probable delay of about 2-10 minutes is indicated in in vitro studies; Ridley and Hall, 1992) to effect a feedback inhibition of the mitogenic cascade. Both the transcription of c-fos during serum-induced mitogenesis and the senmainduced transcription of actin are regulated by highly related dyad symmetry elements, DSEs (Treisman, 1992; Mohun et al., 1987); both DSEs are serum inducible and function in a coordinated fashion. The serum induced expression of actin is probably related to a feed back inhibition cascade of the mitogenic cascade. This is further supported by the fact that actin is one of the first proteins that are synthesized by a normal cell, when layered on an adhesive substratum after being kept in suspension for a long time (Farmer et al., 1978). Thus, in normal cells, a positive signal for mitogenesis is followed by negative feed back control. Such controls might occur at the level of the plasma membrane, second messenger generation, and degradation, serine protein kinases and phosphatases, and gene transcription. Tumorigenesis is a complex multistep process, resulting from the accumulation of dominant mutations (gain-of-function mutations), and recessive mutations or deletions (loss-of-function mutations), whose products are involved in cell proliferation or differentiation. PTKs (both RTKs and NRTKs), when mutated at fianctional domains or their regulatory domains, disrupt their normal behavior and subvert the growth and differentiation regulatory pathways. Mutant ot subunit of the G proteins, or mutant small GTP-binding ras-related proteins or their stimulatory or inhibitory proteins are all potentially capable of transforming cells. Similarly, mutations can alter the activity of the transcription factors that could constitutively activate gene expression and may result in unregulated growth. All these mutant proteins would fall under the category of gain-of-function mutation. In addition, a group of genes called tumor suppressor genes that seem to be maintaining the differentiated state by inhibiting cell proliferation, can also be disabled by mutations or deletions (loss of function) that could result in unlimited

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cell proliferation (Marshall, 1991; Sager, 1989; Weinberg, 1991). In a preneoplastic cell in vivo, several gain-of-function mutant proteins accumulate over years before it turns neoplastic; further progression to malignancy is effected by additional loss-of-function mutations. In these instances, the mitogenic cascade becomes progressively autonomous and uninterrupted, while the adhesive cascade is progressively inactivated to varying extent. Under certain circumstances, as in the case of involution or T cell maturation, the mitogenic cascade may be permanently shut off and the cells may not differentiate further; instead, they may be induced to commit suicide by programmed cell death or apoptosis. Even under these circumstances, if a mutation eliminates apoptosis, the cell escapes death and may attain immortality. As further mutational events occur, the benign growth may progress through neoplastic to malignant state (Korsmeyer, 1992; Wyllie, 1992; McDonnell et al., 1989; Vaux et al., 1988). VI.

CONCLUSIONS

This review has brought together the recent advances in several different but related areas of research such as cytoskeletal structural proteins, cytoskeletal regulatory proteins, the components involved in various signal transduction pathways, molecular bases of cell shape, cell proliferation, and neoplasia. This was necessitated by the nature of the relationship between cell shape and growth regulatory events. An attempt has been made to bring together diverse evidence pointing to the relationship between the cytoskeleton and other physiological cellular processes in order to understand the regulatory mechanisms. A start has been made in the understanding of the novel cell adhesion based signal transduction cascades. The knowledge of the ability of various cell adhesion molecules to be able to interact directly with the cytoskeletal system and the classical growth factor-mediated signaling systems is a very significant advancement. The increasing knowledge of the intercommunication between the classical mitogenic signal transduction cascades and the novel cell adhesion receptor-induced signal transduction cascades raises important questions about the signal transduction pathways leading to cell differentiation. These studies also provide, for the first time, a biochemical basis for the role of cell shape in anchorage-independent growth and form liaison to the genetic bases for the origin of neoplastic behavior. These and other observations give credence to the hypothesis that breakage of liaisons between the mitogenic cascade and the adhesion cascade may give rise to the anchorage independent growth displayed by neoplastic cells. Although recent years have seen much progress in the understanding of the molecular basis of cell adhesion, several challenging problems remain yet to be solved. We must learn more about the fundamental cell adhesion receptor based signal cascade; several questions such as the contribution of adhesion cascade to the regulation of the mitotic cascade and the differentiation process should be answered; details of the crosstalk between the mitogenic signal transduction and

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the adhesion signal transduction should be clearly defined. Since ECM components can activate transcription factors via different cell adhesion receptors, the role of different domains in the individual components of the ECM in activating different transcription factors should be understood. We also need to know if the different integrins and other cell adhesion receptors would activate different transcription factors. Is there any tissue specificity of these signal transduction cascades? How many of the cell adhesion receptors would activate pp125FAK? Are other cell adhesion receptors such as immunoglobulin superfamily of proteins, selectins, and cadherin capable of directly activating protein kinases? If so do they have cell adhesion receptor-specific of protein tyrosine kinase(s)? Answers to these questions would be facilitated by a better understanding of the molecular structure and function of numerous actin binding proteins, microtubule associated proteins, intermediate filament-associated proteins, and the nuclear matrix-associated proteins. Another major challenge is the understanding of the generation of differentiation signals in various cell types. In this respect studies on the role of different ras proteins might be rewarding. Studies on the mechanism of downregulating the rasGTP levels leading to cell division or differentiation appears to be important. So some of the most interesting questions in the area of signal transduction are yet to be answered. Finally, how are the various signaling relays coordinated within a given cell? To answer these questions, one must have information on the promotor/enhancer sequences for each one of the ECM components, various cell adhesion receptor molecules, and the tissue specific differentiation antigens. Most of the tools required for asking such questions are available. Some very exciting results can be anticipated in the next few years.

ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada grant No. OGPIN 032. Thanks are due to the following for critically reading the manuscript: Mrs. K. Rajaraman, Drs. L.A. Fernandez, K. Easterbrook, G. Johnston, R. Singer, J.M. MacSween, R. Duncan, and P. Hoffman.

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