Cloning and characterization of tensin

The molecular cloning of the complementary DNA coding for a 90-kilodalton fragment of tensin, an actin-binding component of focal contacts and other submembraneous cytoskeletal structures, is reported. The derived amino acid sequence revealed the presence of a Src homology 2 (SH2) domain. This domain is shared by a number of signal transduction proteins including nonreceptor tyrosine kinases such as Abl, Fps, Src, and Src family members, the transforming protein Crk, phospholipase C-gamma 1, PI-3 (phosphatidylinositol) kinase, and guanosine triphosphatase-activating protein (GAP). Like the SH2 domain found in Src, Crk, and Abl, the SH2 domain of tensin binds specifically to a number of phosphotyrosine-containing proteins from v-src-transformed cells. Tensin is also found to be phosphorylated on tyrosine residues. These findings suggest that by possessing both actin-binding and phosphotyrosine-binding activities and being itself a target for tyrosine kinases, tensin may link signal transduction pathways with the cytoskeleton (Davis, 1991).

A 7.1-kilobase cDNA encoding chick cardiac muscle tensin has been cloned. It contains an open reading frame of 1,744 amino acid residues. Sequence analysis reveals that, in addition to the previously noted SH2 domain, tensin contains virtually all of the known sequence (362 aa) of insertin, an actin-capping protein that allows actin monomer to be 'inserted'. Moreover, tensin shares partial homology with actin (46.7% identity in 30 aa), beta-spectrin's actin-binding consensus (40% identity in 26 aa), BCR (40% identity in 25 aa), catenin alpha (35% identity in 45 aa), synapsin Ia (25.6% identity in 156 aa), IL-3 receptor (20.2% identity in 384 aa), and IL-2/EPO receptors (14% identity in 20 aa). Recombinant full-length tensin, tagged with an influenza-derived epitope, was over-expressed by a baculovirus system and purified to apparent homogeneity. It migrates as a 200-kDa protein in SDS-polyacrylamide gel electrophoresis, similar to the native tensin. The structure of the tensin molecule has been characterized by light scattering, electron microscopy, and gel filtration. Nine monoclonal antibodies recognizing different regions of tensin have been prepared and characterized. The epitope-tagged recombinant tensin gene was subcloned into a pRcCMV vector and transfected into NIH 3T3 cells. Immunofluorescence stainings with monoclonal antibodies specific for chick tensin (not cross-reactive with mouse tensin) show that the expressed protein is indeed localized at focal contacts, as is that of native tensin (Lo, 1994b).

Tensin, an actin filament capping protein, and auxilin (see Drosophila auxilin), a component of receptor-mediated endocytosis, are known to have 350 residue regions of significant sequence similarity near their N-termini. These regions are homologous, not only to each other, but also to the catalytic domain of a putative protein tyrosine phosphatase (PTP) from Saccharomyces cerevisiae and to other PTPs. It is proposed that the PTP-like portion of the homology region of tensin and auxilin represents a distinct domain. A detailed sequence comparison indicates that the PTP-like domain in tensin is unlikely to exhibit phosphatase activity, whereas in auxilin it may possess a different phosphatase specificity from tyrosine phosphatases. It is probable that the PTP-like domains in tensin and auxilin mediate binding interactions with phosphorylated polypeptides; they may therefore represent members of a distinct class of phosphopeptide recognition domain (Haynie, 1996).

Tensin is a focal-adhesion molecule that binds to actin filaments and interacts with phosphotyrosine-containing proteins. To analyse tensin's function in mammals, tensin cDNAs were cloned from human and cow. The isolated ~7.7-kb human cDNA contains an open reading frame encoding 1735 amino acid residues. The amino acid sequence of human tensin shares 60% identity with chicken tensin, and contains all the structural features described previously in chicken tensin. This includes the actin-binding domains, the Src homology domain 2, and the region similar to a tumor suppressor, PTEN. Two major differences between human and chicken tensin are (1) the lack of the first 54 residues present in chicken tensin, and (2) the addition of 34- and 38-residue inserts in human and bovine tensin. In addition, interspecies sequencing data have uncovered the presence of a glutamine/CAG repeat that appears to have expanded in the course of evolution. Northern-blot analysis reveals a 10-kb message in most of the human tissues examined. An additional 9-kb message is detected in heart and skeletal muscles. The molecular mass predicted from the human cDNA is 185 kDa, although both endogenous and recombinant human tensin migrate as 220-kDa proteins on SDS/PAGE. The discrepancy is due to the unusually low electrophoretic mobility of the central region of the tensin polypeptide (residues 306-981). A survey of human prostate and breast cancer cell lines by Western-blot analysis shows a lack of tensin expression in most cancer cell lines, whereas these lines express considerable amounts of focal-adhesion molecules such as talin and focal-adhesion kinase. Finally, tensin is rapidly cleaved by a focal-adhesion protease, calpain II. Incubation of cells with a calpain inhibitor, MDL, prevents tensin cleavage and induces morphological change in these cells, suggesting that cleavage of tensin and other focal-adhesion constituents by calpain disrupts maintenance of normal cell shape (Chen, 2000).

A previously undocumented tensin family member, tensin2/KIAA 1075, has been cloned and characterized. Human tensin2 cDNA encodes a 1,285-aa sequence that shares extensive homology with tensin1 at its amino- and carboxyl-terminal ends; this homology includes the actin-binding domain, the Src homology 2 (SH2) domain, and the phosphotyrosine binding (PTB) domain. Analysis of the genomic structures of tensin1 and tensin2 further confirm that they represent a single gene family. Examination of tensin2 mRNA distribution has revealed that heart, kidney, skeletal muscle, and liver are tissues of high expression. The endogenous and recombinant tensin2 were expressed as a 170-kDa protein in NIH 3T3 cells. The subcellular localization of tensin2 was determined by transfection of green fluorescence protein (GFP)-tensin2 fusion construct. The results indicated that tensin2 is also localized to focal adhesions. Finally, functional analysis of tensin genes has demonstrated that expression of tensin genes is able to promote cell migration on fibronectin, indicating that the tensin family plays a role in regulating cell motility (Chen, 2002).

A new tensin family member, tensin3, functions in the epidermal growth factor (EGF) signaling pathway. Human tensin3 cDNA encodes a 1445 amino acid sequence that shares extensive homology with tensin1, tensin2, and COOH-terminal tensin-like protein. Tensin3 is expressed in various tissues and in different cell types such as endothelia, epithelia, and fibroblasts. The potential role of tensin3 in EGF-induced signaling pathway is explored. EGF induces tyrosine phosphorylation of tensin3 in MDA-MB-468 cells in a time- and dose-dependent manner, but it is independent of an intact actin cytoskeleton or phosphatidylinositol 3-kinase. Activation of EGF receptor is necessary but not sufficient for tyrosine phosphorylation of tensin3. It also requires Src family kinase activities. Furthermore, tensin3 forms a complex with focal adhesion kinase and p130Cas (see CAS/CSE1 segregation protein) in MDA-MB-468 cells. Addition of EGF to the cells induces dephosphorylation of these two molecules, leads to disassociation of the tensin3-focal adhesion kinase-p130Cas complex, and enhances the interaction between tensin3 and EGF receptor. The results demonstrate that tensin3 may function as a platform for the disassembly of EGF-related signaling complexes at focal adhesions (Cui, 2003).

Tyrosine phosphorylation of tensin

A small number of proteins becomes tyrosine-phosphorylated in response to integrin-mediated cell adhesion to extracellular matrix proteins. Two of these tyrosine-phosphorylated proteins have been identified as the focal adhesion kinase and paxillin. A third focal adhesion protein, tensin, has been identified that becomes tyrosine-phosphorylated during cell adhesion to extracellular matrix proteins. The tyrosine phosphorylation of tensin does not occur when cells adhere to plastic or polylysine and is blocked when microfilament assembly and cell spreading are inhibited with cytochalasin D. In addition, other focal adhesion proteins such as talin and vinculin do not become tyrosine-phosphorylated under the same conditions of cell spreading on extracellular matrix proteins (Bockholt, 1993).

Tensin interaction with integrin

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, an antibody-chasing technique was developed for simultaneous tracking of two integrin populations by different antibodies. Whereas the vitronectin receptor alpha(v)beta(3) remains within focal contacts, the fibronectin receptor alpha(5)beta(1) translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 +/- 0.7 microm/h and is independent of cell migration. It is induced by ligation of alpha(5)beta(1) integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied alpha(5)beta(1) integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocks ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. It is proposed that translocating alpha(5)beta(1) integrins induces initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly (Pankov, 2000).

The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrin biological functions by binding to cytoplasmic proteins. Most integrin beta tails contain one or two NPXYF motifs that can form beta turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-alpha bind to integrin beta tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction the binding of a series of recombinant PTB domains to a panel of short integrin beta tails was examined. In addition to the known integrin-binding proteins, Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domains bind to integrin tails. Furthermore, Dok-1 physically associates with integrin alpha IIb beta 3. Mutations of the integrin beta tails confirm that these interactions are canonical PTB domain-ligand interactions: (1) the interactions were blocked by mutation of an NPXY motif in the integrin tail; (2) integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. This specificity, and a molecular model of an integrin beta tail-PTB domain interaction, was used to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in beta 7 and beta 3 tails. These data establish that short integrin beta tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism (Calderwood, 2003).

Tensin interaction with actin

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Three distinct actin-binding domains have been identified within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463 cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts (Lo, 1994a).

Tensin, an actin filament capping protein first purified from chicken gizzard, is localized to various types of adherens junctions in muscle and nonmuscle cells. Chicken tensin cDNA has been isolated and characterized from a chicken cardiac library. The 6.3-kb chicken cardiac tensin cDNA encodes an open reading frame of 1,792 amino acids. Mammalian cells transfected with the chicken tensin cDNA expressed a polypeptide of approximately 200 kD recognizable by antibodies to chicken gizzard tensin. The express protein was incorporated into focal adhesions and other actin-containing structures in the transfected cells. To map the domain associated with tensin's high affinity, barbed-end F-actin-capping activity, bacterially expressed recombinant fusion proteins containing various segments of tensin were prepared and assayed for activity. The results of these experiments show that the high affinity capping domain (kD = 1.3 nM) lies within amino acid residues R1037-V1169. Additional studies on a shorter construct, S1061-H1145, show that these 85 residues are sufficient for producing complete inhibition of actin polymerization and depolymerization. The data showing complete inhibition of polymerization and shift in critical concentration are consistent with a simple barbed-end capping mechanism (Chuang, 1995).

Tensin interaction with PI 3-kinase

Tensin is an SH2 domain-containing cytoskeletal protein that binds to and caps actin filaments. Investigation of signal transduction mechanisms associated with tensin has revealed the presence of phosphoinositide 3-kinase (PI 3-kinase) activity in tensin immunoprecipitates from platelet-derived growth factor-treated cells. Association of PI 3-kinase activity with tensin is transitory, and the amount of activity is approximately 1% of the total PI 3-kinase activity found in anti-phosphotyrosine (anti-pY) immunoprecipitates. In vitro, PI 3-kinase activity associates with the SH2 domain of tensin in a platelet-derived growth factor-dependent manner. The optimal phosphopeptide binding specificity of the SH2 domain of tensin was determined to be phospho-Y (E or D), N, (I, V, or F). Synthetic phosphopeptides containing the sequence YENI can specifically block the association of PI 3-kinase activity with tensin in a dose-dependent manner. These results suggest that PI 3-kinase interacts with the cytoskeleton via the SH2 domain of tensin and may play an important role in platelet-derived growth factor-induced cytoskeletal reorganization that is concomitant with cell migration and proliferation (Auger, 1996).

Signaling downstream of tensin

Cells organize diverse types of specialized adhesion sites upon attachment to extracellular matrix (ECM) components. One of the physiological roles of such cell-ECM interactions is to initiate and regulate adhesion-mediated signal transduction responses. The association of cells with fibronectin fibrils has been shown to regulate the JNK and p38 signaling pathways. Whether tensin, a cytoskeletal component localized to both focal contacts and fibronectin-associated fibrillar adhesions, can induce these signaling pathways was tested. Tensin overexpression results in activation of both the c-Jun amino-terminal kinase (JNK) and p38 pathways. Tensin-mediated JNK activation is independent of the activities of the small GTP binding proteins Rac and Cdc42, but does depend on SEK, a kinase involved in the JNK pathway. It is suggested that tensin may directly activate the JNK and p38 pathways, acting downstream or independent of the activities of the small GTP binding proteins Rac and Cdc42 (Katz, 2000).

Effects of tensin mutation

Tensin is a focal adhesion phosphoprotein that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions of tensin, the mouse tensin gene was cloned, its program of expression was determined, and gene targeting was used to generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis, tensin null mice developed normally and appeared healthy postnatally for at least several months. Over time, -/- mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of tensin. Mice with overt signs of weakness exhibited signs of renal failure and possessed multiple large cysts in the proximal kidney tubules, but even in tensin null mice with normal blood analysis, cysts were prevalent. Ultrastructurally, noncystic areas showed typical cell-matrix junctions that readily labeled with antibodies against other focal adhesion molecules. In abnormal regions, cell-matrix junctions were disrupted and tubule cells lacked polarity. Taken together, these data imply that, in the kidney, loss of tensin leads to a weakening, rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be compensated for by other focal adhesion proteins (Lo, 1997).

Regeneration of skeletal muscle requires the activation, proliferation, differentiation and fusion of satellite cells to generate new muscle fibers. This study was designed to determine the role of tensin in this process. Cardiotoxin was used to induce regeneration in the anterior tibial muscles of tensin-knockout and wild-type mice. From histological analysis, it was found that the regeneration process lasted longer in knockout than in wild-type mice. To investigate the mechanism involved in this delay, each regeneration step was examined in animals and cultured primary cells. Fewer proliferating myogenic cells identified by bromodeoxyuridine and desmin double labelling were found in knockout mice on the first 2 days after injury. Expression of myosin, paxillin, dystrophin and dystrophin-associated proteins were delayed in knockout mice. Withdrawal from the cell cycle was less efficient in isolated knockout myoblasts, and the fusion capacity was reduced in these cells as well. These defects in regeneration most likely contributed to the 9-fold increase of centrally nucleated fibers occurring in the non-injected knockout mice. These results demonstrated clearly that tensin plays a role in skeletal-muscle regeneration (Ishii, 2001).

Tensin1 is an actin- and phosphotyrosine-binding protein that localizes to focal adhesions. Both tensin1 and tensin2 promote cell migration. Since localization of proteins to particular intracellular compartments often regulates their functions, and Src homology domain 2 may mediate signals related to cell migration, it was hypothesized that tensin-mediated cell migration is regulated by the focal adhesion localization and the Src homology domain 2 of tensin. To test this hypothesis, the effects of a series of tensin1 mutants on cell migration were analyzed. The results have shown that (1) tensin1 contains two focal adhesion-binding sites, (2) the wild-type tensin1 significantly promotes cell migration, (3) mutants with one focal adhesion-binding site do not promote cell migration, (4) the non-focal adhesion localized mutant suppresses cell migration and (5) the mutant that is not able to bind to phosphotyrosine-containing proteins has no effect on cell migration. These results have indicated that focal adhesion localization of tensin1 and the phosphotyrosine-binding activity are two critical factors in regulating tensin-mediated cell migration (Chen, 2003).

blistery: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.