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Biomedical subjects

C E Turner

Publications and source records attributed to C E Turner.

At least 55 records · Page 3Linked to original sources
PubMed1993

Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells.

Treatment of Swiss 3T3 cells with bombesin caused a striking increase (21-fold) in the tyrosine phosphorylation of the cytoskeleton-associated protein paxillin, as judged by anti-phosphotyrosine Western blots of anti-paxillin immunoprecipitates. Vasopressin and endothelin also stimulated paxillin tyrosine phosphorylation. Bombesin-stimulated tyrosine phosphorylation of paxillin was detectable within 1 min and was concentration-dependent (half-maximum effect at 0.09 nM). Bombesin stimulation of paxillin tyrosine phosphorylation could be dissociated from both protein kinase C (PKC) activation and the mobilization of Ca2+ from intracellular stores. Activation of PKC in quiescent Swiss 3T3 cells using the tumor promoter phorbol 12,13-dibutyrate (PDB) increased the tyrosine phosphorylation of paxillin in a time-dependent manner but was less effective than bombesin and stimulated detectable phosphorylation only within 5 min, considerably slower than bombesin-induced tyrosine phosphorylation of paxillin. Furthermore, the selective PKC inhibitor, GF109203X, or down-regulation of PKC using prolonged treatment with PDB markedly inhibited the stimulation of paxillin tyrosine phosphorylation by PDB but had little effect on the response to bombesin. In contrast, cytochalasin D, an agent that selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced paxillin tyrosine phosphorylation. This is the first report to identify paxillin as a substrate for neuropeptide-stimulated tyrosine phosphorylation.

3T3 Cells↗
PubMed1993

Detection of Src homology 3-binding proteins, including paxillin, in normal and v-Src-transformed Balb/c 3T3 cells.

The Src homology 3 (SH3) domain, located in the amino-terminal, noncatalytic half of pp60src, is highly conserved among members of the Src family of tyrosine kinases. SH3 domains have also been identified in a variety of proteins otherwise unrelated to protein-tyrosine kinases. The presence of SH3 domains in proteins with diverse functions suggests this domain may be important for directing protein-protein interactions necessary for protein function or cellular localization. To explore possible interactions between the SH3 domain and cellular proteins, we have established conditions for the isolation of proteins that bind in solution to the Src SH3 domain. A 67-amino acid fragment of c-Src containing either the entire glutathione S-transferase-SH3 domain (GST-SH3) or the SH3 domain from the neuronal form of c-Src (GST-SH3+) was expressed as a glutathione S-transferase fusion protein. The GST fusion proteins were incubated with lysates from [35S]methionine-labeled Balb/c 3T3 cells or v-Src-transformed Balb/c 3T3 cells. We found that GST-SH3, but not wild-type GST, specifically interacted with multiple cellular proteins, whereas GST-SH3+ only weakly associated with a small subset of these proteins. The majority of the SH3-binding proteins were found in particulate and detergent-insoluble cell fractions. Anti-phosphotyrosine immunoblots of the SH3-binding proteins revealed that several of the SH3-binding proteins are phosphorylated on tyrosine in v-Src-transformed cells. In addition, a number of the SH3-binding proteins were phosphorylated on serine and/or threonine in in vitro kinase assays, suggesting that one or more of the SH3-binding proteins has kinase activity. We identified paxillin, a vinculin-binding protein, as one of the Src SH3-binding proteins. This finding strongly supports the hypothesis that SH3 domains may be involved in subcellular localization of proteins to cytoskeleton and/or cellular membranes.

3T3 Cells↗
PubMed1993

Tyrosine phosphorylation of the focal adhesion kinase pp125FAK during development: relation to paxillin.

Significant changes in the level of protein tyrosine phosphorylation accompany avian embryonic development. A comparison of different tissues reveals that a similar and remarkably restricted complement of proteins is modified in this manner. In each case the major proteins detected using anti-phosphotyrosine antibodies have molecular masses of approximately 170, 150, 125, 70 and 50 kDa. As a first step in determining the function of this protein modification in embryogenesis we have initiated a study to identify these phosphoproteins. We have previously reported that the 70 kDa band is paxillin, a component of actin-membrane attachment sites associated with regions of cell adhesion (Turner, C. E. (1991) J. Cell Biol. 115, 201-207). We report here that the 125 kDa phosphotyrosine-containing protein is the tyrosine kinase pp125FAK, a protein that co-localizes with paxillin at sites of adhesion (Schaller et al. (1992) Proc. Nat. Acad. Sci. USA 89, 5192-5196). Tyrosine phosphorylation of both pp125FAK and paxillin was detected at low levels as early as embryonic day 3 and increased steadily during the first half of development, reached a maximum between embryonic days eight and twelve, and declined to background levels prior to hatching. Paxillin protein expression also increased during the first half of embryogenesis, suggesting little change in the overall phosphorylation of this protein through embryonic day 8. In contrast, pp125FAK, following an initial increase, is expressed at a constant high level during these early embryonic stages, implying an increase in its overall phosphotyrosine content.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Treatment of khat addiction.

The authors present two cases of khat addiction that were successfully treated with bromocriptine. Khat is a bush cultivated in the Mid East because of its highly stimulant effects. Its leaves contain a variety of sympathomimetics. While khat is rarely found in the U.S., American soldiers stationed in the Arabian peninsula may be exposed to it. Because of an alcohol interdiction during the current Persian Gulf crisis, these troops may be tempted to use this plant as an alternative recreational drug.

Adult↗
PubMed1992

Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.

Cells in culture reveal high levels of protein tyrosine phosphorylation in their focal adhesions, the regions where cells adhere to the underlying substratum. We have examined the tyrosine phosphorylation of proteins in response to plating cells on extracellular matrix substrata. Rat embryo fibroblasts, mouse Balb/c 3T3, and NIH 3T3 cells plated on fibronectin-coated surfaces revealed elevated phosphotyrosine levels in a cluster of proteins between 115 and 130 kD. This increase in tyrosine phosphorylation was also seen when rat embryo fibroblasts were plated on laminin or vitronectin, but not on polylysine or on uncoated plastic. Integrin mediation of this effect was suggested by finding the same pattern of elevated tyrosine phosphorylation in cells plated on the cell-binding fragment of fibronectin and in cells plated on a synthetic polymer containing multiple RGD sequences. We have identified one of the proteins of the 115-130-kD cluster as pp125FAK, a tyrosine kinase recently localized in focal adhesions (Schaller, M. D., C. A. Borgman, B. S. Cobb, R. R. Vines, A. B. Reynolds, and J. T. Parsons. 1992. Proc. Natl. Acad. Sci. USA. 89:5192). A second protein that becomes tyrosine phosphorylated in response to extracellular matrix adhesion is identified as paxillin, a 70-kD protein previously localized to focal adhesions. Treatment of cells with the tyrosine kinase inhibitor herbimycin A diminished the adhesion-induced tyrosine phosphorylation of these proteins and inhibited the formation of focal adhesions and stress fibers. These results suggest a role for integrin-mediated tyrosine phosphorylation in the organization of the cytoskeleton as cells adhere to the extracellular matrix.

3T3 Cells↗
PubMed1991

Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins.

The integrin family of cell adhesion receptors mediates many of the interactions between cells and the extracellular matrix. Because the extracellular matrix has profound influences on cell behavior, it seems likely that integrins transduce biochemical signals across the cell membrane. The nature of these putative signals has, thus far, remained elusive. Antibody-mediated clustering of integrin receptors was used to mimic the integrin clustering process that occurs during formation of adhesive contacts. Human epidermal carcinoma (KB) cells were incubated with an anti-beta 1 integrin monoclonal antibody for 30 min on ice followed by incubation at 37 degrees C with anti-rat IgG. This treatment, which induced integrin clustering, stimulated the phosphorylation on tyrosine residues of a 115- to 130-kDa complex of proteins termed pp130. When integrins were clustered in the presence of the phosphatase inhibitor sodium orthovanadate, pp130 showed a substantial increase in phosphorylation compared to the case in which integrins were clustered in the absence of vanadate. Maximal pp130 phosphorylation was observed 10-20 min after initiation of integrin clustering in the absence of vanadate or after 5-10 min in its presence. These time courses roughly parallel the formation of integrin clusters on the cell surface as observed by fluorescence microscopy. pp130 phosphorylation depended on the amount of anti-integrin antibody present. Additionally, the tyrosine phosphorylation of pp130 showed specificity since it was stimulated by antibodies to the integrin alpha 3 and beta 1 subunits but not by antibodies to other integrin alpha subunits or to nonintegrin cell surface proteins. Immunoprecipitation experiments clearly demonstrated that pp130 is not itself a beta 1 integrin. It is postulated, therefore, that the integrin-stimulated tyrosine phosphorylation of pp130 may reflect part of an important signal transduction process between the extracellular matrix and the cell interior.

Antigen-Antibody Complex↗
PubMed1991

Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle.

In this report we have demonstrated that paxillin, a cytoskeletal protein which is present in focal adhesions, localizes in vivo to regions of cell-extracellular matrix interaction which are believed to be analogous to focal adhesions. Specifically, it is enriched in the dense plaques of chicken gizzard smooth muscle tissue and in the myotendinous junctions formed in Xenopus laevis tadpole tail skeletal muscle. In addition, paxillin was identified at the rat diaphragm neuromuscular junction. The distribution of paxillin is thus comparable to that of other focal adhesion proteins, for example, talin and vinculin, in these structures.

Animals↗
PubMed1991

Revaccination with pneumococcal vaccine of elderly persons 6 years after primary vaccination.

We vaccinated 15 persons, age 56 to 79 years, with 14-valent pneumococcal polysaccharide vaccine and revaccinated them with 23-valent vaccine 6 years later to assess adverse reactions and booster responses of anticapsular antibodies. No systemic reactions occurred after administration of either vaccine. After booster vaccination, five vaccinees developed only mild soreness or tenderness at the site of injection. The (arithmetic) mean antibody level to 12 capsular types measured by radioimmunoassay increased 3.1-fold 1 month after the first vaccine; the range was 1427-5124 ng antibody nitrogen per ml (N ml-1). Six years later, mean antibody levels waned to about half these levels. One month after administration of the second dose of vaccine, the mean antibody level increased 1.5-fold; the range was 1011-3954 ng antibody N ml-1. Revaccination with pneumococcal vaccine of elderly persons can be carried out safely; the levels of anticapsular antibody achieved after revaccination are about half the levels after primary vaccination.

Aged↗
PubMed1991

Transmembrane molecular assemblies in cell-extracellular matrix interactions.

Several new interactions have been identified and proteins characterized in focal adhesions. Together they suggest alternative or parallel linkages between actin filaments and members of the integrin family of extracellular receptors. Transformed cells continue to serve as models for studying the assembly and disassembly of these adhesions.

Actins↗
PubMed1991

Paxillin is a major phosphotyrosine-containing protein during embryonic development.

Phosphotyrosine-containing proteins were immunoprecipitated from embryonic chicken tissue extracts using anti-phosphotyrosine antibody coupled to agarose beads. Major phosphotyrosine-containing proteins of 110, 70, and 50 kD were observed following blotting with anti-phosphotyrosine antibody. The 70-kD band was selectively removed from the samples by precipitation with antibodies to the focal adhesion protein paxillin, therefore identifying paxillin as one of the major tyrosine kinase substrates during chick embryonic organogenesis. The tyrosine phosphorylation of paxillin is regulated developmentally: during embryogenesis, a marked decrease in its phosphotyrosine content was observed, although the total level of paxillin remained essentially constant. Approximately 20% of the paxillin was phosphorylated on tyrosine in the early embryo. In contrast, tyrosine phosphorylation of paxillin was undetectable in the adult. A similar profile of phosphotyrosine-containing proteins was identified in rat embryos. Paxillin was also found to be a major phosphotyrosine-containing protein in the rat embryo. These data suggest that the regulated phosphorylation of tyrosine residues on paxillin may perform a critical role in controlling cell and tissue cytoarchitecture rearrangement during vertebrate development.

Animals↗
PubMed1990

Functional studies of the domains of talin.

The protein talin has two domains of approximately 200 and 47 kD, which can be cleaved apart by a variety of proteases. To examine the function of these two structural domains of talin, we have digested purified talin with a calcium-dependent protease and separated the resulting fragments chromatographically. Both fragments were radioiodinated and used to probe Western blots of whole fibroblasts and chicken gizzard extracts. The large talin fragment bound to vinculin and metavinculin. The small fragment did not demonstrate any binding in this assay. The fragments were labeled fluorescently and microinjected into fibroblasts in tissue culture. The large talin fragment incorporated quickly into focal adhesions where it remained stable for at least 14 h. The small fragment associated with focal adhesions of fibroblasts but was also distributed diffusely in the cytoplasm and the nucleus. These experiments suggest that talin has at least two sites that contribute to its localization in focal adhesions. Intact talin microinjected into Madin-Darby bovine kidney epithelial cells localized to the focal adhesions but was excluded from the zonulae adherentes, despite the localization of vinculin to both of these sites. In contrast, the large talin fragment, when microinjected into these epithelial cells, incorporated into both focal adhesions and zonulae adherentes. The difference in localization between the large talin fragment and intact talin seems to be due to the removal of the small domain. This difference in localization suggests that talin binding sites in zonulae adherentes have limited accessibility.

Actins↗
PubMed1990

Paxillin: a new vinculin-binding protein present in focal adhesions.

The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pIs ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of approximately 85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60src in Rous sarcoma virus-transformed cells (Glenney, J. R., and L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this interaction with vinculin suggests paxillin may be a key component in the control of focal adhesion organization.

Animals↗
PubMed1990

Beta-endorphin decline in late luteal phase dysphoric disorder.

The beta-endorphin hypothesis of late luteal phase dysphoric disorder (premenstrual syndrome or L2D2) was tested. Twenty-two PMS patients were compared to twenty-two controls. Levels of beta-endorphin, ACTH, FSH, LH, cortisol, prolactin and TRH were measured on the first and twentieth days after menses. PMS subjects exhibited a significantly greater drop in the opiate, beta-endorphin, (p less than .001) than controls. No relationship or significant e was seen with the other hormones/transmitters tested. The symptoms of PMS may be due to noradrenergic rebound following beta-endorphin decline. Symptomatic and pharmacological morphine withdrawal and manic phase of bipolar disorder are discussed as possible models for L2D2.

Adult↗
PubMed1989

The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity.

Chemical agents which activate specific kinases were employed to disrupt the stress fiber and focal adhesion organization of cells spread on a substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted a rapid loss of stress fibers and focal adhesions from African green monkey kidney (BSC-1) cells. This was paralleled by an increase in the level of talin phosphorylation suggesting that this may play a role in the removal of talin from focal adhesions. Similar morphological changes were produced in the rat embryo fibroblast line (REF 52) by dibutyryl-cAMP, which stimulates protein kinase A. In contrast, however, the phosphorylation of talin was reduced in REF 52 cells when treated with dibutyryl cAMP. In untreated cells we found that the levels of vinculin phosphorylation were very low relative to the levels of talin phosphorylation and did not change following drug treatment in either cell line. Although limited proteolytic cleavage of cytoskeletal proteins represents a potential mechanism for focal adhesion disruption, we observed no proteolysis of talin or vinculin in response to either drug treatment.

Animals↗
PubMed1989

The relationship between talin and acetylcholine receptor clusters in Xenopus muscle cells.

Talin is involved in mediating the cytoskeleton-extracellular matrix interaction at focal contacts in cultured fibroblasts. Recently this protein has been localized at both the myotendinous junction (MTJ) and the neuromuscular junction (NMJ) in skeletal muscle. At the MTJ, talin may mediate the insertion of myofibrils into the plasma membrane, thus serving a function similar to that seen at focal contacts. However, the function of talin at the NMJ is unknown. In this study, we examined its distribution at both mature and developing acetylcholine receptor (AChR) clusters in Xenopus muscle cells both in vivo and in vitro with immunofluorescence. At the NMJs of both myotomal and submaxillaris muscles, talin was absent from the AChR clusters. In cultured myotomal muscle cells, it was absent from 40% of both the nerve-associated AChR patches and the spontaneously formed AChR clusters located on the top surface of the cells. We therefore conclude that this protein is not essential for maintenance of AChR clusters at the NMJ. In addition to MTJs, talin was invariably associated with AChR clusters induced by polyornithine-coated beads, and, to a large extent, with spontaneously formed clusters on the ventral side of cultured cells. A common feature of these talin-positive domains is the deep membrane infoldings, where bundles of actin filaments are inserted into the membrane. Thus, talin may be involved in the formation and maintenance of these structures. The deep membrane infoldings, though prominent at most NMJs, are absent from the two muscles under study in vivo. Our work thus suggests that the postjunctional membrane at the NMJ is heterogeneous, consisting of an AChR cluster domain and, often but not always, a domain for proteins involved in cytoskeleton-membrane linkage as exemplified by talin.

Animals↗
PubMed1989

Detection of metavinculin in human platelets using a modified talin overlay assay.

Talin was purified from human platelets and proteolytically cleaved by the calcium-dependent protease (CDP II) to two stable fragments of 200 and 47 kDa. The 200 kDa fragment was radiolabeled and used in Western blot overlay assays of fractionated platelet proteins. This procedure revealed vinculin to be the major talin binding protein. However, in addition, a less abundant protein of approximately 150 kDa also interacted strongly with the talin fragment. Using conventional immunoblot analysis we have confirmed that this protein is metavinculin, a protein previously believed to be confined to cardiac and smooth muscle tissue.

Blood Platelets↗
PubMed1989

Effects of capping on the non-ionic detergent solubility of rat thymocyte glycoproteins.

The non-ionic detergent solubility of surface antigens during capping, following their cross-linking by specific antibodies, was investigated for three rat thymocyte glycoproteins, the leucocyte-common antigen (L-CA), the leucocyte sialoglycoprotein (LSGP) and Thy-1, using a combination of immunofluorescence microscopy, covalent surface radiolabeling and quantitative analysis using a radiolabeled antibody. Prior to the addition of cross-linking antibody, both L-CA and LSGP were soluble in the non-ionic detergent Triton X-100, while Thy-1 was largely insoluble. Addition of sufficient antibody to induce capping led to a significant reduction in the solubility of L-CA and LSGP, even prior to warming to induce capping of the antigen under investigation. Subsequent capping did not increase the amount of insoluble antigen, suggesting that ligand binding, rather than the process of capping itself, is sufficient to cause this partial Triton insolubility. These results indicate the formation of an association between these liganded glycoproteins and the cell's detergent-insoluble cytoskeleton. In contrast, the Thy-1 antigen became progressively more extractable by Triton X-100 as the antigen was capped, suggesting that the insertion of the Thy-1 lipid tail into the plasma membrane was progressively more easily perturbed by the non-ionic detergent during the capping process. The reciprocal maintenance of solubility of either LSGP or L-CA during the capping of the other shows that the majority of the 180 kDa thymocyte L-CA molecules play no role in the mechanism of capping of LSGP. Furthermore, immunoprecipitates of solubilized L-CA contained no detectable amounts of the cytoskeletal protein fodrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗