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Biomedical subjects

C E Turner

Publications and source records attributed to C E Turner.

At least 37 records · Page 2Linked to original sources
PubMed1998

Association of Bovine Papillomavirus Type 1 E6 oncoprotein with the focal adhesion protein paxillin through a conserved protein interaction motif.

We have found that the E6 oncoprotein of Bovine Papillomavirus Type 1 (BE6) as well as the E6 protein of the cancer associated HPV-16 (16E6) interact with the focal adhesion protein paxillin. Mutational analysis of paxillin revealed that BE6 binds paxillin through small protein interaction motifs called LD motifs that have been previously identified as important in regulating association of paxillin with vinculin and focal adhesion kinase (FAK), and that BE6 can interact with at least two separate binding sites on paxillin. The LD motifs of paxillin that bind BE6 share homology with the E6 binding site of E6-AP, a ubiquitin ligase that together with 16E6 targets the degradation of the p53 tumor suppressor. Paxillin binding to BE6 excludes simultaneous binding to E6-AP. Mutational analysis of BE6 can distinguish the interaction of BE6 with E6-AP compared to paxillin and revealed that the interaction of BE6 with paxillin may be necessary for the induction of anchorage independent growth of cells by BE6.

Animals↗
PubMed1998

Paxillin.

Paxillin is a 68 kDa cytoplasmic protein that localizes to discrete sites of cell attachment to the extracellular matrix called focal adhesions. It is a multi-domain adapter protein capable of interacting with several structural and signaling proteins including vinculin, FAK, PYK2, Src and Crk. Phosphorylation of paxillin in response to integrin-mediated cell adhesion and growth factor stimulation regulates some of these interactions. Thus, paxillin functions as a scaffold for the recruitment of molecules into a signal transduction complex that is closely apposed to the plasma membrane. This is likely to facilitate the efficient processing of external stimuli that modulate important cellular events including cell adhesion, cell motility and growth control. Since paxillin interacts with several proteins known to cause cell transformation, the binding sites for these proteins on paxillin represent potential targets for therapeutic agents.

Animals↗
PubMed1998

Human monoclonal antibodies encoded by the V4-34 gene segment show cold agglutinin activity and variable multireactivity which correlates with the predicted charge of the heavy-chain variable region.

We have characterized the reactivities of a panel of V4-34-encoded human IgM monoclonal antibodies (mAb) which bind the erythrocyte Rh D antigen, derived from an immunized individual. These were compared with the specificities of V4-34-encoded autoantibodies with I/i reactivity produced from patients with cold agglutinin disease (CAD), and other V4-34-encoded autoantibodies. The antibodies were evaluated for cold agglutinin activity using haemagglutination tests, immunofluorescence microscopy for reactivity with tissue components, and in solid phase radiobinding assays with purified antigens. We found that (i) cold agglutinin activity was a property of all the V4-34-encoded mAb (ii) the cold agglutinins from CAD patients were generally monospecific for I/i whereas most of the anti-D and the other V4-34-encoded mAb displayed multireactive properties, frequently binding to strongly acidic antigens (iii) computation of the net charge of the heavy-chain V regions showed that the multireactive mAb were generally more positively charged than the monospecific cold agglutinins, which could contribute to their multireactive phenotype. The involvement of charge interactions was further indicated by the effects of pH and ionic strength on the immunofluorescence staining patterns.

Adult↗
PubMed1998

Serine and threonine phosphorylation of the paxillin LIM domains regulates paxillin focal adhesion localization and cell adhesion to fibronectin.

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to "inside-out" integrin-mediated signal transduction.

Amino Acid Sequence↗
PubMed1997

Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin.

Tyrosine phosphorylation of paxillin by the focal adhesion kinase (FAK) has been implicated as a signal transduction mechanism associated with cell adhesion and cytoskeletal reorganization. The potential role of serine phosphorylation of paxillin in these events has not been well characterized. In this study we have examined the phosphorylation profile of paxillin both in vitro and in vivo. By using glutathione S-transferase-paxillin fusion proteins in precipitation-kinase assays in vitro we observed that a fusion protein spanning amino acid residues 54-313 of paxillin, and containing a FAK-binding site, precipitated substantial serine kinase activity as well as FAK activity from a smooth-muscle lysate. Together these kinases phosphorylated paxillin on tyrosine residue 118, a site that has been identified previously as a target for FAK phosphorylation, and on serine residues 188 and/or 190. The binding site for the serine kinase, the identity of which is currently unknown, was further mapped to residues 168-191 of paxillin. To assess the physiological relevance of these sites phosphorylated in vitro, the profile of paxillin phosphorylation in vivo stimulated by seeding fibroblasts on fibronectin was characterized. As expected, plating cells on fibronectin enhanced the tyrosine phosphorylation of paxillin. However, 96% of the phosphorylation of paxillin occurred on serine residues. Comparison by two-dimensional phosphopeptide analyses indicated that the major sites of tyrosine and serine phosphorylation detected in the assays in vitro co-migrate with phosphopeptides derived from paxillin phosphorylated in vivo in response to plating cells on fibronectin. These findings support a role for both tyrosine and serine kinases in the signal transduction pathway linking integrin activation to paxillin phosphorylation.

Animals↗
PubMed1996

Thrombin receptor activation elicits rapid protein tyrosine phosphorylation and stimulation of the raf-1/MAP kinase pathway preceding delayed mitogenesis in cultured rat aortic smooth muscle cells: evidence for an obligate autocrine mechanism promoting cell proliferation induced by G-protein-coupled receptor agonist.

Treatment of quiescent rat aortic smooth muscle cells with either alpha-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) resulted in pronounced increases in [3H]thymidine incorporation that were concentration dependent and reached a maximum of approximately 15-fold above serum-starved controls. However, in contrast to FBS, PDGF-BB, or basic fibroblast growth factor (bFGF), that initiated DNA synthesis promptly after 16-19 h, thymidine incorporation in response to thrombin was delayed by an additional 3-6 h. Delayed mitogenesis correlated with the appearance of a potent mitogenic activity in conditioned media samples obtained from thrombin-stimulated rat aortic smooth muscle cells, as assayed using Swiss 3T3 fibroblasts. This activity was not inhibited by neutralizing antibodies directed against PDGF or bFGF. Furthermore, in the Swiss 3T3 cells, simple addition of either alpha-thrombin or SFLLRNP failed to elicit a significant mitogenic response. In signal transduction studies, both thrombin and SFLLRNP treatment led to rapid tyrosine phosphorylation of proteins with apparent molecular masses of 42, 44, 75, 120, and 190 kD, respectively, as assessed by antiphosphotyrosine immunoblotting. The overall pattern of protein tyrosine phosphorylation was distinct from that observed after PDGF-BB addition. Activation of Raf-1 and the mitogen-activated protein (MAP) kinases p44mapk and p42mapk was also observed. However, the time course and duration of Raf-1/MAP kinase activation after thrombin stimulation were similar to those elicited by PDGF-BB. Taken together, our results indicate that thrombin-stimulated vascular smooth muscle proliferation is delayed and requires the de novo expression of one or more autocrine mitogens. In addition, the rapid induction of discrete intracellular signaling mechanisms by thrombin, including the Raf-1/MAP kinase pathway, appears to be insufficient alone to promote vascular smooth muscle cell mitogenesis.

Animals↗
PubMed1996

Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding.

Paxillin is a 68-kD focal adhesion phosphoprotein that interacts with several proteins including members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal proteins vinculin and the tyrosine kinase, focal adhesion kinase (FAK). This suggests a function for paxillin as a molecular adaptor, responsible for the recruitment of structural and signaling molecules to focal adhesions. The current study defines the vinculin- and FAK-interaction domains on paxillin and identifies the principal paxillin focal adhesion targeting motif. Using truncation and deletion mutagenesis, we have localized the vinculin-binding site on paxillin to a contiguous stretch of 21 amino acids spanning residues 143-164. In contrast, maximal binding of FAK to paxillin requires, in addition to the region of paxillin spanning amino acids 143-164, a carboxyl-terminal domain encompassing residues 265-313. These data demonstrate the presence of a single binding site for vinculin, and at least two binding sites for FAK that are separated by an intervening stretch of 100 amino acids. Vinculin- and FAK-binding activities within amino acids 143-164 were separable since mutation of amino acid 151 from a negatively charged glutamic acid to the uncharged polar residue glutamine (E151Q) reduced binding of vinculin to paxillin by >90%, with no reduction in the binding capacity for FAK. The requirement for focal adhesion targeting of the vinculin- and FAK-binding regions within paxillin was determined by transfection into CHO.K1 fibroblasts. Significantly and surprisingly, paxillin constructs containing both deletion and point mutations that abrogate binding of FAK and/or vinculin were found to target effectively to focal adhesions. Additionally, expression of the amino-terminal 313 amino acids of paxillin containing intact vinculin- and FAK-binding domains failed to target to focal adhesions. This indicated other regions of paxillin were functioning as focal adhesion localization motifs. The carboxyl-terminal half of paxillin (amino acids 313-559) contains four contiguous double zinc finger LIM domains. Transfection analyses of sequential carboxyl-terminal truncations of the four individual LIM motifs and site-directed mutagenesis of LIM domains 1, 2, and 3, as well as deletion mutagenesis, revealed that the principal mechanism of targeting paxillin to focal adhesions is through LIM3. These data demonstrate that paxillin localizes to focal adhesions independent of interactions with vinculin and/or FAK, and represents the first definitive demonstration of LIM domains functioning as a primary determinant of protein subcellular localization to focal adhesions.

Animals↗
PubMed1995

The alpha 6A beta 1 and alpha 6B beta 1 integrin variants signal differences in the tyrosine phosphorylation of paxillin and other proteins.

Integrin receptors can mediate transmembrane signaling in response to ligand binding. To further examine the role of the integrin alpha subunit in these signaling functions, we assessed the contribution of the alpha 6 cytoplasmic domain variants to the signaling properties of the alpha 6 beta 1 integrin using P388D1 cells that had been transfected with either the alpha 6A or the alpha 6B cDNA. The alpha 6A beta 1 and alpha 6B beta 1 receptors induced marked quantitative differences in the tyrosine phosphorylation of several proteins after binding to laminin. Specifically, the alpha 6A cytoplasmic domain was more effective than the alpha 6B cytoplasmic domain in inducing the tyrosine phosphorylation of three major proteins (molecular mass, 120, 110, and 76 kDa). In addition to these proteins, we also observed that the tyrosine phosphorylation of the cytoskeletal protein paxillin was increased significantly more by alpha 6A beta 1 integrin-mediated adhesion to laminin than by that of alpha 6B beta 1. This differential pattern of tyrosine phosphorylation induction does not appear to be a secondary event initiated by cell shape changes. Also, differences in tyrosine phosphorylation in the alpha 6 transfectants were not evident in response to attachment to other substrates. These findings provide biochemical evidence for functional differences between alpha subunit cytoplasmic domain variants of the same integrin.

Animals↗
PubMed1995

Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase.

The concomitant tyrosine phosphorylation of the focal adhesion protein, paxillin, and the tyrosine kinase, focal adhesion kinase (FAK), in response to multiple stimuli including integrin-mediated cell adhesion suggests that paxillin phosphorylation is closely coupled to FAK activity. In the present study, we have identified a specific tyrosine residue within paxillin, tyrosine 118 (Tyr-118), that represents the principle site of phosphorylation by FAK in vitro. The identification of this site as a target for FAK phosphorylation was accomplished by immunoprecipitating FAK and performing in vitro kinase assays, using as substrate either glutathione S-transferase (GST)-paxillin fusion proteins containing truncations in paxillin sequence or fusion proteins with phenylalanine substitutions for tyrosine residues. GST-paxillin containing a phenylalanine substitution at Tyr-118 (Y118F) was not phosphorylated by FAK immunoprecipitates; however, this mutant was shown to bind FAK equally as well as the wild type fusion protein. As a first step toward assessing the function of paxillin phosphorylation on Tyr-118, a Y118F paxillin cDNA construct was transiently transfected into NIH 3T3 cells. Similar to wild type paxillin, mutated paxillin localized to focal adhesions, indicating that the phosphorylation of paxillin on Tyr-118 is not essential for the recruitment of paxillin to sites of cell adhesion.

3T3 Cells↗
PubMed1995

Nerve growth factor triggers microfilament assembly and paxillin phosphorylation in human B lymphocytes.

Increasing evidence suggests that the nervous system is involved in allergic inflammation. One of the potential regulatory molecules of the neuroimmune system is nerve growth factor (NGF). Recent studies from our group demonstrated the presence of a functional NGF receptor (NGFR) on human B lymphocytes. Moreover, we showed that gp140trk tyrosine kinase, which serves as an NGFR, was involved in transduction of early signaling events in human B lymphocytes. The mechanisms by which NGF initiates the signaling cascade and the link between the neuroimmune systems are unknown. We have focused on the role of the cytoskeleton as a possible mediator for transduction of signals induced by NGF. Polymerized actin (F-actin) content was determined by fluorescent staining and immunoblotting with antiactin antibody. Addition of NGF caused a time- and concentration-dependent increase in F-actin content, and maximum effects were noted after 1 min. These increases in F-actin content and NGF-induced thymidine incorporation could be blocked by incubating the cells with cytochalasin D and botulinum C2 toxin before the addition of NGF. Incubation of human B lymphocytes with 10 nM K252a, an inhibitor of Trk kinase, decreased NGF-induced microfilament assembly by 75%. In immunoprecipitation experiments, addition of NGF to B cells induced a rapid increase in the tyrosine phosphorylation of paxillin, one of a group of focal adhesion proteins involved in linking actin filaments to the plasma membrane. Coimmunoprecipitation studies demonstrated the association between gp140trk kinase and paxillin. Together, these observations suggest that actin assembly is involved in NGF signaling in human B cells, and that paxillin may be essential in this pathway after phosphorylation by gp140trk kinase.

Actin Cytoskeleton↗
PubMed1995

Angiotensin II stimulation of rapid paxillin tyrosine phosphorylation correlates with the formation of focal adhesions in rat aortic smooth muscle cells.

Angiotensin II is a potent vasoconstrictor that has been also implicated in vascular hyperproliferative diseases, including atherosclerosis and restenosis following angioplasty. Treatment of cultured, serum-starved rat aortic smooth muscle cells with angiotensin II causes rapid protein tyrosine phosphorylation that precedes cell mitogenesis. We have identified two of the phosphoproteins as paxillin (75 kilodaltons) and the tyrosine kinase pp125Fak, both components of actin-associated focal adhesion sites. Angiotensin II stimulated a 5-fold increase in the tyrosine phosphorylation of paxillin and a smaller (1.5-fold) increase in pp125Fak tyrosine phosphorylation. Paxillin tyrosine phosphorylation was evident within 1 minute, and was maximal after 10 minutes. Similar elevated protein tyrosine phosphorylation levels of paxillin were obtained with exposure of the rat aortic smooth muscle cells to peptides endothelin-1 and alpha-thrombin that function, as angiotensin II, through binding to members of the seven transmembrane domain G protein coupled receptors. Angiotensin II treatment also stimulated the production of a well-ordered actin-containing stress fiber network and prominent paxillin-containing focal adhesions. The focal adhesions stained intensely with anti-phosphotyrosine antibody suggesting the tyrosine phosphorylation of paxillin and cytoskeletal reorganization were tightly coupled. Angiotensin II receptor occupancy has been shown previously to lead to protein kinase C activation. However, compared to angiotensin II stimulation, a smaller, delayed increase in paxillin tyrosine phosphorylation was observed following direct protein kinase C activation by the phorbol ester phorbol 12-myristate-13-acetate. Paxillin tyrosine phosphorylation was selective for certain agonists since no increase in tyrosine phosphorylation of this protein was observed following exposure to the potent mitogen PDGF. Thus, actin-based cytoskeletal changes involving sites of cell adhesion to the extracellular matrix may play an important role in normal and pathophysiologic smooth muscle cell growth regulation in response to certain angiotensin II-type vasoactive agonists.

Angiotensin II↗
PubMed1994

Paxillin: a cytoskeletal target for tyrosine kinases.

Paxillin is a recently identified member of the complex of cytoskeletal proteins that is found concentrated in cultured cells and in vivo at the cytoplasmic face of regions of cell attachment to the extracellular matrix. These sites, in view of their close proximity to the extracellular matrix, are well positioned to act as signal-transducing centers to 'report on' changes in the cells, immediate environment. Recent findings indicate that such signals are in part mediated through the activation of tyrosine kinases concentrated at the sites of adhesion. Changes in the phosphotyrosine content of paxillin accompanying this elevation in kinase activity suggest that paxillin may be an important intermediary in these pathways.

Animals↗
PubMed1994

Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.

Catechols↗
PubMed1994

Localization of the achondroplasia gene to the distal 2.5 Mb of human chromosome 4p.

Achondroplasia has been mapped to 4p16.3 using 18 multigenerational families with achondroplasia and 10 short tandem repeat polymorphic markers from this region. No evidence of genetic heterogeneity was found. Analysis of a recombinant family localizes the achondroplasia locus to the 2.5 Mb region between D4S43 and the telomere. Multipoint linkage analysis favors placement telomeric of D4S412. The establishment of closely linked markers will facilitate positional cloning of the achondroplasia gene and permit prenatal diagnosis of homozygous achondroplasia for at risk couples.

Achondroplasia↗
PubMed1994

Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region.

Paxillin is a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix. Extensive tyrosine phosphorylation of this protein occurs during integrin-mediated cell adhesion, embryonic development, fibroblast transformation and following stimulation of cells by mitogens that operate through the family of seven membrane-spanning G-protein-coupled receptors. Paxillin binds in vitro to the focal adhesion protein vinculin as well as to the SH3 domain of c-src and, when tyrosine phosphorylated, to the SH2 domain of v-crk. Here, we report the complementary DNA, and derived amino acid sequence, that codes for approximately 90% of the paxillin protein. We have identified a region in the amino-terminal half of the protein that supports the binding of both vinculin and the focal adhesion tyrosine kinase, pp125Fak. Although there is no significant overall homology with other identified proteins, the carboxyl third of paxillin contains one LIM domain and three LIM-like sequences. The LIM motif is common to a number of transcription factors and to two other focal adhesion proteins, zyxin and cysteine-rich protein. In addition to several potential tyrosine phosphorylation sites there are five tyrosine-containing sequences that conform to SH2-binding motifs. The protein also contains a short proline-rich region indicative of a SH3-binding domain. Taken together, these data suggest that paxillin is a unique cytoskeletal protein capable of interaction with a variety of intracellular signalling, and structural, molecules important in growth control and the regulation of cytoskeletal organization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin.

Paxillin and vinculin are cytoskeletal proteins that colocalise to focal adhesions, specialised regions of the cell involved in attachment to the extracellular matrix. These two molecules form part of a complex of proteins that link the actin network to the plasma membrane. Paxillin has been shown to bind directly in vitro to the C-terminal region of vinculin (Turner et al. (1990). J. Cell Biol. 111, 1059-1068), which also contains a focal adhesion targeting sequence (Bendori et al. (1989). J. Cell Biol. 108, 2383-2393). In the present study, we have used a series of vinculin deletion mutants to map more precisely the sites in vinculin responsible for paxillin binding and focal adhesion localisation. A glutathione-S-transferase fusion protein spanning vinculin residues 881-1000 was sufficient to support 125I-paxillin binding in a gel-blot assay while no detectable binding was observed to a fusion protein spanning residues 881-978. Transfection experiments using cDNAs encoding chick vinculin residues 398-1066 and 398-1028 demonstrated that amino acids C-terminal to residue 1028 were not necessary for targeting to focal adhesions. In contrast, a vinculin polypeptide expressed from a cDNA encoding residues 398-1000 failed to localise to focal adhesions in stably transfected NIH3T3 cells. We have therefore identified a region of 50 amino acids (residues 979-1028) within the C-terminal region of vinculin that contains both the paxillin-binding site and the focal adhesion targeting sequence. This region is highly conserved in human and chicken vinculin and is likely to be important in regulation of the assembly of focal adhesions.

3T3 Cells↗