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Biomedical subjects

K Burridge

Publications and source records attributed to K Burridge.

At least 37 records · Page 2Linked to original sources
PubMed1997

Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing.

Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton- matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction.

Actins↗
PubMed1997

Integrin-mediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation. Studies with activated and inhibitory beta3 cytoplasmic domain mutants.

Integrin alphaIIbbeta3 functions as the fibrinogen receptor on platelets and mediates platelet aggregation and clot retraction. Among the events that occur during either "inside-out" or "outside-in" signaling through alphaIIbbeta3 is the phosphorylation of focal adhesion kinase (pp125(FAK)) and the association of pp125(FAK) with cytoskeletal components. To examine the role of pp125(FAK) in these integrin-mediated events, pp125(FAK) phosphorylation and association with the cytoskeleton was determined in cells expressing two mutant forms of alphaIIbbeta3: alphaIIbbeta3(D723A/E726A), a constitutively active integrin in which the putative binding site for pp125(FAK) is altered, and alphaIIbbeta3(F727A/K729E/F730A), in which the putative binding site for alpha-actinin is altered. Both mutants were expressed on the cell surface and were able to bind ligand, either spontaneously or upon activation. Whereas cells expressing alphaIIbbeta3(D723A/E726A) were able to form focal adhesions and stress fibers upon adherence to fibrinogen, cells expressing alphaIIbbeta3(F727A/K729E/F730A) adhere to fibrinogen, but had reduced focal adhesions and stress fibers. pp125(FAK) is recruited to focal adhesions in adherent cells expressing alphaIIbbeta3(D723A/E726A) and is phosphorylated in adherent cells or in cells in suspension in the presence of fibrinogen. In adherent cells expressing alphaIIbbeta3(F727A/K729E/F730A), pp125(FAK) was phosphorylated despite reduced formation of focal adhesions and stress fibers. We conclude that activation of pp125(FAK) can be dissociated from two important events in integrin signaling, the assembly of focal adhesions in adherent cells and integrin activation following ligand occupation.

Amino Acid Sequence↗
PubMed1997

Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells.

Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell-cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.

ADP Ribose Transferases↗
PubMed1997

E-cadherin engagement stimulates tyrosine phosphorylation.

Cadherins are cell adhesion molecules concentrated at intercellular adherens junctions, where they form a multiprotein complex with cytoplasmic catenins. Although cell-cell interactions affect many aspects of cell behavior, little is known about signaling pathways triggered by cadherin engagement. We show here that E-cadherin-mediated cell-cell adhesion leads to a rapid increase in tyrosine phosphorylation at sites of cell-cell contact and that this stimulation of tyrosine phosphorylation can be mimicked by aggregation of E-cadherin with antibodies. The proteins that become phosphorylated are distinct from those previously shown to be tyrosine phosphorylated in response to integrin-mediated adhesion and include ras-GAP. We also find that E-cadherin-mediated tyrosine phosphorylation is not required for the assembly of adherens-type junctions.

Cadherins↗
PubMed1996

Regulation of vinculin binding to talin and actin by phosphatidyl-inositol-4-5-bisphosphate.

Vinculin, a prominent cytoskeletal protein at cell-substrate adhesions (focal adhesions) and cell-cell adhesions (adherens junctions), interacts with other cytoskeletal proteins, including talin and actin. An intramolecular interaction between the head and tail domains of vinculin masks the binding sites for both proteins. The exposure of cryptic binding sites may be important for promoting focal adhesion assembly. Several agents that induce the formation of focal adhesions act through the GTP-binding protein Rho, which elevates phosphatidylinositol-4,5-bisphosphate (PtdInsP2) levels by activating phosphatidyl-inositol-4-phosphate-5-OH kinase (PtdIns-5-OH kinase). PtdInsP2 regulates several actin-binding proteins, including profilin, gelsolin and alpha-actinin, and interacts with vinculin. Here we report that PtdInsP2 dissociates vinculin's head-tail interaction, unmasking its talin- and actin-binding sites. Microinjection of antibodies against PtdInsP2 inhibit assembly of stress fibres and focal adhesions.

3T3 Cells↗
PubMed1996

Overexpression of the Ras-related TC21/R-Ras2 protein may contribute to the development of human breast cancers.

Although experimental studies suggest that aberrant Ras function can promote the malignant progression of human breast epithelial cells, the occurrence of mutated ras genes in breast tumors is infrequent. One possible explanation for this apparent paradox is that aberrant function of the Ras-related protein TC21/R-Ras2, which causes malignant transformation of NIH 3T3 cells via upregulation of the Ras signal transduction pathway, may contribute to breast tumor development in the absence of Ras mutations. To address this possibility, we utilized two complementary approaches. First, we determined that aberrant TC21 function caused transformation of the MCF-10A human breast epithelial cell line. TC21-transformed MCF-10A cells exhibited altered cellular morphology associated with a disruption of cell-cell adherens junctions, formed colonies in soft agar, and showed enhanced motility in vitro. These alterations were similar to, but more dramatic than, those observed with oncogenic Ras-transformed MCF-10A cells. Furthermore, overexpression of normal TC21, but not Ras, also caused transformation of these cells. Second, we observed that TC21 protein expression was greatly elevated in 7 of 9 breast tumor lines when compared to untransformed MCF-10A cells. Taken together, these results support the possibility that overexpression of TC21 may contribute to aberrant growth properties of breast carcinoma cells.

Breast Neoplasms↗
PubMed1996

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

Amino Acid Sequence↗
PubMed1996

Rho-stimulated contractility drives the formation of stress fibers and focal adhesions.

Activated rhoA, a ras-related GTP-binding protein, stimulates the appearance of stress fibers, focal adhesions, and tyrosine phosphorylation in quiescent cells (Ridley, A.J., and A. Hall, 1992. Cell. 70:389-399). The pathway by which rho triggers these events has not been elucidated. Many of the agents that activate rho (e.g., vasopressin, endothelin, lysophosphatidic acid) stimulate the contractility of smooth muscle and other cells. We have investigated whether rho's induction of stress fibers, focal adhesions, and tyrosine phosphorylation is the result of its stimulation of contractility. We demonstrate that stimulation of fibroblasts with lysophosphatidic acid, which activates rho, induces myosin light chain phosphorylation. This precedes the formation of stress fibers and focal adhesions and is accompanied by increased contractility. Inhibition of contractility by several different mechanisms leads to inhibition of rho-induced stress fibers, focal adhesions, and tyrosine phosphorylation. In addition, when contractility is inhibited, integrins disperse from focal adhesions as stress fibers and focal adhesions disassemble. Conversely, upon stimulation of contractility, diffusely distributed integrins are aggregated into focal adhesions. These results suggest that activated rho stimulates contractility, driving the formation of stress fibers and focal adhesions and elevating tyrosine phosphorylation. A model is proposed to account for how contractility could promote these events.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Focal adhesions, contractility, and signaling.

Focal adhesions are sites of tight adhesion to the underlying extracellular matrix developed by cells in culture. They provided a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction that relate to growth control. The assembly of focal adhesions is regulated by the GTP-binding protein Rho. Rho stimulates contractility which, in cells that are tightly adherent to the substrate, generates isometric tension. In turn, this leads to the bundling of actin filaments and the aggregation of integrins (extracellular matrix receptors) in the plane of the membrane. The aggregation of integrins activates the focal adhesion kinase and leads to the assembly of a multicomponent signaling complex.

Animals↗
PubMed1995

An in-gel assay for protein tyrosine phosphatase activity: detection of widespread distribution in cells and tissues.

A method is described for the detection of protein tyrosine phosphatase activity in sodium dodecyl sulfate-polyacrylamide gels. A radiolabeled substrate, 32P-labeled poly(glutamic acid-tyrosine) (random copolymer) is incorporated into gels prior to polymerization. Following electrophoresis, the sodium dodecyl sulfate is removed; the proteins are fully denatured by soaking gels in 6 M guanidine hydrochloride and then renatured by incubation in buffers containing 0.04% Tween 40 and high concentrations of reducing agents. Protein tyrosine phosphatase activity is detected in autoradiographs of dried gels as regions from which the 32P has been selectively removed. Electrophoresis of known cytoplasmic protein tyrosine phosphatases indicates activity as the predicted molecular weights. As little as 10 pg of some cytoplasmic phosphatases is detectable. However, transmembrane tyrosine phosphatases, such as CD45, are detected only at very high protein loadings in this assay. Electrophoresis of whole cell lysates indicates multiple bands of tyrosine phosphatase activity, some of which comigrate with known cytoplasmic protein tyrosine phosphatases. The activity is inhibited by sodium orthovanadate or the omission of reducing agents during the renaturation process. The assay has been used to analyze embryonic and adult tissues, as well as whole cell lysates. A similar profile of bands of tyrosine phosphatase activity is seen with many different cells and tissues. However, some that are highly differentiated, such as adult skeletal muscle, erythrocytes, or sperm, reveal either a reduced level of tyrosine phosphatase activity or a simplified profile of bands.

Adult↗
PubMed1995

Association of aciculin with dystrophin and utrophin.

Aciculin is a recently identified 60-kDa cytoskeletal protein, highly homologous to the glycolytic enzyme phosphoglucomutase type 1, (Belkin, A. M., Klimanskaya, I. V., Lukashev, M. E., Lilley, K., Critchley, D., and Koteliansky, V. E. (1994) J. Cell Sci. 107, 159-173). Aciculin expression in skeletal muscle is developmentally regulated, and this protein is particularly enriched at cell-matrix adherens junctions of muscle cells (Belkin, A. M., and Burridge, K. (1994) J. Cell Sci. 107, 1993-2003). The purpose of our study was to identify cytoskeletal protein(s) interacting with aciculin in various cell types. Using immunoprecipitation from cell lysates of metabolically labeled differentiating C2C12 muscle cells with anti-aciculin-specific antibodies, we detected a high molecular weight band (M(r) approximately 400,000), consistently coprecipitating with aciculin. We showed that this 400 kDa band comigrated with dystrophin and immunoblotted with anti-dystrophin antibodies. The association between aciculin and dystrophin in C2C12 cells was shown to resist Triton X-100 extraction and the majority of the complex could be extracted only in the presence of ionic detergents. In the reverse immunoprecipitation experiments, aciculin was detected in the precipitates with different anti-dystrophin antibodies. Immunodepletion experiments with lysates of metabolically labeled C2C12 myotubes showed that aciculin is a major dystrophin-associated protein in cultured skeletal muscle cells. Double immunostaining of differentiating and mature C2C12 myotubes with antibodies against aciculin and dystrophin revealed precise colocalization of these two cytoskeletal proteins throughout the process of myodifferentiation in culture. In skeletal muscle tissue, both proteins are concentrated at the sarcolemma and at myotendinous junctions. In contrast, utrophin, an autosomal homologue of dystrophin, was not codistributed with aciculin in muscle cell cultures and in skeletal muscle tissues. Analytical gel filtration experiments with purified aciculin and dystrophin showed interaction of these proteins in vitro, indicating that their association in skeletal muscle is due to direct binding. Whereas dystrophin was shown to be a major aciculin-associated protein in skeletal muscle, immunoblotting of anti-aciculin immunoprecipitates with antibodies against utrophin showed that aciculin is associated with utrophin in cultured A7r5 smooth muscle cells and REF52 fibroblasts. Immunodepletion experiments performed with lysates of metabolically labeled A7r5 cells demonstrated that aciculin is a major utrophin-binding protein in this cell type. Taken together, our data show that aciculin is a novel dystrophin- and utrophin-binding protein. Association of aciculin with dystrophin (utrophin) in various cell types might provide an additional cytoskeletal-matrix transmembrane link at sites where actin filaments terminate at the plasma membrane.

Animals↗
PubMed1995

Immunodetection of alpha-actinin in focal adhesions is limited by antibody inaccessibility.

In this study we demonstrate that alpha-actinin is a prominent component of the focal adhesions of nonmuscle cells but that the alpha-actinin in focal adhesions is largely inaccessible to staining with antibodies against alpha-actinin. Our results explain a controversy that has existed in the literature. Investigators who microinject alpha-actinin into nonmuscle cells have routinely observed significant incorporation of alpha-actinin into focal adhesions as well as stress fibers. Immunofluorescence and immunoelectron microscopy have, however, indicated that alpha-actinin is located farther from the membrane than either talin or vinculin. Immunofluorescence studies of smooth muscle dense plaques and myotendinous junctions have also yielded conflicting results regarding the presence or absence of alpha-actinin at these sites. Here, we confirm that alpha-actinin immunofluorescence of fibroblasts yields weak or absent staining of focal adhesions. We also demonstrate that microinjected alpha-actinin readily incorporates into focal adhesions. However, various antisera against either the cell's endogenous alpha-actinin or against the microinjected chicken gizzard alpha-actinin fail to stain focal adhesions despite the presence of microinjected alpha-actinin at these sites. Furthermore, disassembly of stress fibers induced by dibutyrl cAMP demonstrates that alpha-actinin persists in focal adhesions in the absence of associated stress fibers, suggesting that alpha-actinin's association with focal adhesions is independent of stress fibers.

Actinin↗
PubMed1995

Localization of utrophin and aciculin at sites of cell-matrix and cell-cell adhesion in cultured cells.

Aciculin is a phosphoglucomutase-related cytoskeletal protein associated with dystrophin and/or utrophin in various tissues and cell types. Comparison of expression patterns for aciculin, dystrophin, and utrophin in cultured cells demonstrated that aciculin is coexpressed with utrophin, but not with dystrophin, in cultures of A7r5 smooth muscle cells and REF52 fibroblasts. Some other nonmuscle cells synthesized only trace levels of or no aciculin and utrophin. Aciculin was detected by immunoblotting in antiutrophin immunoprecipitates from A7r5 and REF52 cultured cells, indicating an association between these two proteins. The aciculin-utrophin complex in fibroblasts and smooth muscle cells was mostly resistant to Triton X-100 extraction and was detected predominantly in the Triton-insoluble fraction, enriched in actin and actin-associated proteins. By immunofluorescence both aciculin and utrophin were identified in a similar dot-like or streak-like pattern in A7r5 and REF52 cultured cells. Immunolocalization of utrophin in cultured fibroblasts and smooth muscle cells in combination with interference reflection microscopy demonstrated that utrophin staining was mostly codistributed, but not exclusively confined to the areas of focal adhesions, sites of closest cell attachment to the substrate. Double immunostaining of A7r5 and REF52 cells for aciculin and utrophin revealed a precise colocalization of both cytoskeletal proteins at focal adhesions and along microfilaments. Costaining of cultured fibroblasts and smooth muscle cells with antibodies against utrophin and major focal adhesion components, vinculin and talin, showed that utrophin is concentrated in focal adhesions both at initial stages of cell spreading and in well spread cells of nearly confluent monolayers. In MCF10 breast epithelial cells both utrophin and aciculin were localized at cell-cell adherens-type junctions. Our data show that utrophin is a cytoskeletal component of cell-matrix and cell-cell adhesions in various cultured cells. In certain cell types the aciculin-utrophin complexes may contribute to the linking actin filaments to the plasma membrane.

Actin Cytoskeleton↗
PubMed1995

Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia.

Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens-type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta-catenin is found in association with E-cadherin, p120 Cas is not. In the ras-transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine-phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras-transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.

Breast↗
PubMed1995

Adhesion-induced tyrosine phosphorylation of the p130 src substrate.

Adhesion of cells to the extracellular matrix leads to an increase in the tyrosine phosphorylation of a specific set of proteins, three of which have now been identified as the focal adhesion proteins pp125FAK, paxillin and tensin. In addition, we have previously noted the adhesion-induced tyrosine phosphorylation of a fourth protein, with an apparent molecular mass of 130. As in the case of FAK, paxillin and tensin, a 130 kDa protein is also found to be highly tyrosine phosphorylated in Rous sarcoma virus (RSV)-transformed cells. This protein forms a stable complex with pp60src and is directly phosphorylated by activated forms of c-src. Using a monoclonal antibody (mAb 4F4) specific for the src-associated p130 we show that p130 is also phosphorylated in response to cell adhesion. Immunoprecipitation of p130 followed by an anti-phosphotyrosine immunoblot revealed that adhesion of rat embryo fibroblasts (REF52) to fibronectin (FN) led to a significant increase in the phosphotyrosine content of p130. Furthermore, a comparison of cell lysates before and after immunoprecipitation confirmed the absence of tyrosine phosphorylated p130 from lysates immunoprecipitated with mAb 4F4. Immunofluorescence staining of REF52s revealed that p130 is found in focal adhesions as well as along stress fibers in a pattern reminiscent of that exhibited by alpha-actinin. In addition, in many cells, we found significant staining in the nucleus, but evidence is presented that the nuclear staining is not due to tyrosine phosphorylated p130. Finally, unlike pp125FAK, p130 does not appear to be itself a kinase as evidence by immune-complex kinase assays carried out in the presence or absence of exogenous substrates.

Animals↗