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A B Reynolds

Publications and source records attributed to A B Reynolds.

At least 37 records · Page 2Linked to original sources
PubMed1996

The novel catenin p120cas binds classical cadherins and induces an unusual morphological phenotype in NIH3T3 fibroblasts.

p120cas (CAS) is a tyrosine kinase substrate whose phosphorylation has been implicated in cell transformation by Src and in ligand-induced signaling through the EGF, PDGF, and CSF-1 receptors. More recently, CAS has been shown to associate with E-cadherin and its cofactors (catenins), molecules that are involved in cell adhesion. Although both CAS and beta-catenin contain armadillo repeat domains (Arm domains), the amino acid identity between these proteins in this region is only 22%, and it is not yet clear whether CAS will emulate other catenins by associating with other members of the cadherin family. Here we report that in addition to binding E-cadherin, wild-type CAS associated with N-cadherin and P-cadherin. Transient transfection of cloned CAS isoforms into MDCK epithelial cells indicated that CAS1 and CAS2 isoforms are equally capable of binding to E-cadherin even though these cells preferentially express CAS2 isoforms. In addition, CAS colocalized with N-cadherin in NIH3T3 cells and analysis of CAS mutants in vivo indicated that the CAS-N-cadherin interaction requires an intact CAS Arm domain. The data suggest that CAS-cadherin interactions in general are dictated by the conserved armadillo repeats and are not heavily influenced by sequences added outside the Arm domain by alternative splicing. Interestingly, overexpression of CAS in NIH3T3 cells induced a striking morphological phenotype characterized by the presence of long dendrite-like processes. This branching phenotype was specific for CAS, since (i) overexpression of the structurally similar beta-catenin had little effect on cell morphology, and (ii) the branching was abolished by deletions in the CAS Arm domain. Our data indicate that, like other catenins, CAS is a cofactor for multiple members of the cadherin family. However, the dramatically distinct phenotype exhibited by fibroblasts overexpressing CAS, versus beta-catenin, support recent data suggesting that these catenins have fundamentally different and possibly opposing roles in cadherin complexes.

3T3 Cells↗
PubMed1996

Identification of murine p120 isoforms and heterogeneous expression of p120cas isoforms in human tumor cell lines.

p120cas (CAS) is a protein tyrosine kinase substrate that associates directly with the cytoplasmic tail of the cell-cell adhesion molecule E-cadherin. CAS is thus part of a multimolecular complex that, along with other cadherin-binding proteins (catenins), mediates interactions between E-cadherin and the actin cytoskeleton. Down-regulation of E-cadherin expression and defects in catenin function have been implicated in tumor metastasis, but the role of CAS in these processes has not been addressed. Recently, the study of CAS was complicated when new anti-CAS antibodies revealed the presence of at least four putative CAS isoforms that appeared to vary in abundance between cell types. Here, we identify the four major isoforms expressed in murine fibroblasts, and we show that they are products of alternative splicing. Analysis of CAS isoforms in a variety of murine cell lines indicates that motile cells like fibroblasts and macrophages preferentially express CAS1 (i.e., CAS1A and CAS1B isoforms), and epithelial cells preferentially express CAS2 (i.e., CAS2A and CAS2B isoforms), whereas nonadherent cells (e.g., B cells, T cells, and myeloid cells) do not express detectable levels of CAS. Interestingly, CAS1 expression is dramatically up-regulated in a Src-transformed Madin-Darby canine kidney cell line, indicating that the pattern of isoform expression can be altered by cell transformation. Analysis of a variety of differentiated and metastatic human tumor cell lines reveals that CAS isoform expression in these cells is quite heterogeneous. Furthermore, several poorly differentiated cell lines fail to express particular isoforms that are typically observed in well-differentiated cell lines. These data raise the possibility that unbalanced expression of CAS isoforms in human carcinomas may influence cadherin function and contribute to malignant or metastatic cell phenotypes.

3T3 Cells↗
PubMed1996

The gene encoding p120cas, a novel catenin, localizes on human chromosome 11q11 (CTNND) and mouse chromosome 2 (Catns).

The p120cas gene encodes a protein tyrosine kinase substrate that associates with the cell-cell adhesion protein complex containing E-cadherin and its cytoplasmic cofactors alpha-catenin, beta-catenin, and plakoglobin. Like other components of the cadherin/catenin complex, defects in p120cas may contribute to cell malignancy. We have determined the chromosomal location of the p120cas gene in human and mouse using fluorescence in situ hybridization and interspecific backcross analysis, respectively. The human p120cas gene (CTNND) is localized immediately adjacent to the centromere on the long arm of chromosome 11 in band 11q11. The murine p120cas gene (Catns) was assigned to the middle of chromosome 2. Neither locus is currently known to be associated with disease or malignancy.

Animals↗
PubMed1995

Use of monoclonal antibody 1H1, anticortactin, to distinguish normal and neoplastic smooth muscle cells: comparison with anti-alpha-smooth muscle actin and antimuscle-specific actin.

In preliminary experiments, we found that 1H1, a monoclonal antibody directed against the v-src substrate cortactin, reacts with smooth muscle, myoepithelium, myofibroblasts, and macrophages in formaldehyde-fixed human tissues. To evaluate the use of this antibody as a diagnostic reagent, we tested the immunohistochemical distribution of cortactin in 61 mesenchymal neoplasms, 11 neuroectodermal neoplasms, and eight embryonal epithelial neoplasms. The results were compared with those obtained using antibodies against alpha-smooth muscle actin and muscle-specific actin on a similar set of tissues. With the exception of positive staining in rhabdomyosarcoma, in this series only tumors with smooth muscle differentiation appeared to contain cortactin (16 of 19 leiomyosarcomas, one infantile fibrosarcoma, one malignant fibrous histiocytoma). Immunoelectron microscopy localized cortactin to the actin-associated dense bodies of the microfilament network. We conclude that cortactin may be a useful adjunct to alpha-smooth muscle actin and muscle-specific actin as a marker for the study and diagnosis of smooth muscle neoplasms and related lesions.

Actins↗
PubMed1995

The tyrosine kinase substrate p120cas binds directly to E-cadherin but not to the adenomatous polyposis coli protein or alpha-catenin.

The tyrosine kinase substrate p120cas (CAS), which is structurally similar to the cell adhesion proteins beta-catenin and plakoglobin, was recently shown to associate with the E-cadherin-catenin cell adhesion complex. beta-catenin, plakoglobin, and CAS all have an Arm domain that consists of 10 to 13 repeats of a 42-amino-acid motif originally described in the Drosophila Armadillo protein. To determine if the association of CAS with the cadherin cell adhesion machinery is similar to that of beta-catenin and plakoglobin, we examined the CAS-cadherin-catenin interactions in a number of cell lines and in the yeast two-hybrid system. In the prostate carcinoma cell line PC3, CAS associated normally with cadherin complexes despite the specific absence of alpha-catenin in these cells. However, in the colon carcinoma cell line SW480, which has negligible E-cadherin expression, CAS did not associate with beta-catenin, plakoglobin, or alpha-catenin, suggesting that E-cadherin is the protein which bridges CAS to the rest of the complex. In addition, CAS did not associate with the adenomatous polyposis coli (APC) tumor suppressor protein in any of the cell lines analyzed. Interestingly, expression of the various CAS isoforms was quite heterogeneous in these tumor cell lines, and in the colon carcinoma cell line HCT116, which expresses normal levels of E-cadherin and the catenins, the CAS1 isoforms were completely absent. By using the yeast two-hybrid system, we confirmed the direct interaction between CAS and E-cadherin and determined that CAS Arm repeats 1 to 10 are necessary and sufficient for this interaction. Hence, like beta-catenin and plakoglobin, CAS interacts directly with E-cadherin in vivo; however, unlike beta-catenin and plakoglobin, CAS does not interact with APC or alpha-catenin.

Adenomatous Polyposis Coli Protein↗
PubMed1994

Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes.

p120cas is a tyrosine kinase substrate implicated in ligand-induced receptor signaling through the epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor receptors and in cell transformation by Src. Here we report that p120 associates with a complex containing E-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Furthermore, p120 precisely colocalizes with E-cadherin and catenins in vivo in both normal and Src-transformed MDCK cells. Unlike beta-catenin and plakoglobin, p120 has at least four isoforms which are differentially expressed in a variety of cell types, suggesting novel means of modulating cadherin activities in cells. In Src-transformed MDCK cells, p120, beta-catenin, and plakoglobin were heavily phosphorylated on tyrosine, but the physical associations between these proteins were not disrupted. Association of p120 with the cadherin machinery indicates that both Src and receptor tyrosine kinases cross talk with proteins important for cadherin-mediated cell adhesion. These results also strongly suggest a role for p120 in cell adhesion.

3T3 Cells↗
PubMed1993

Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate.

Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.

Actins↗
PubMed1992

pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions.

Expression of the Rous sarcoma virus-encoded oncoprotein, pp60v-src, subverts the normal regulation of cell growth, which results in oncogenic transformation. This process requires the intrinsic protein-tyrosine kinase activity of pp60v-src and is associated with an increase in tyrosine phosphorylation of a number of cellular proteins, candidate substrates for pp60v-src. We report here the isolation of a cDNA encoding a protein, pp125, that is a major phosphotyrosine-containing protein in untransformed chicken embryo cells and exhibits an increase in phosphotyrosine in pp60v-src-transformed chicken embryo cells. This cDNA encodes a cytoplasmic protein-tyrosine kinase which, based upon its predicted amino acid sequence and structure, is the prototype for an additional family of protein-tyrosine kinases. Immunofluorescence localization experiments show that pp125 is localized to focal adhesions; hence, we suggest the name focal adhesion kinase.

Amino Acid Sequence↗
PubMed1992

Platelet activation leads to increased c-src kinase activity and association of c-src with an 85-kDa tyrosine phosphoprotein.

We have used platelets as a model system to study the function of c-src in signal transduction and cell adhesion. Numerous proteins were found to be phosphorylated on tyrosine in response to thrombin-induced platelet activation and aggregation. Two phases of phosphorylation were observed, with the second phase, but not the first, being inhibited by blocking platelet aggregation with an Arg-Gly-Asp-Ser tetrapeptide. As a first step towards identifying those proteins phosphorylated on tyrosine and to determine the specific role of p60src during platelet activation, we looked for changes in p60src kinase activity and for associations of p60src with other tyrosine phosphoproteins. The data presented here demonstrate an increase in p60src kinase activity within 1 min of thrombin-induced activation. Furthermore, p60src transiently associates with a tyrosine phosphoprotein during platelet activation and aggregation. This tyrosine phosphoprotein, p80/85, is a previously characterized cytoskeletal substrate for v-src in transformed cells. The data presented here suggest a model in which p60src functions in platelets to link upstream events, such as cell-surface adhesive interactions, with changes in platelet shape and cytoskeletal organization.

Amino Acid Sequence↗
PubMed1992

p120, a novel substrate of protein tyrosine kinase receptors and of p60v-src, is related to cadherin-binding factors beta-catenin, plakoglobin and armadillo.

A novel protein tyrosine kinase (PTK) substrate, p120, has been previously implicated in ligand-induced signaling through the epidermal growth factor, platelet-derived growth factor and colony-stimulating factor 1 receptors, and in cell transformation by p60v-src. We have isolated a near full-length cDNA encoding murine p120. The encoded protein lacks significant homology with any reported protein, but it contains four copies of an imperfect 42 amino acid repeat that occurs 12.5 times in the protein encoded by Drosophila armadillo (arm), and its direct homologs, human plakoglobin (plak) and Xenopus laevis beta-catenin (beta-cat). The presence of this motif implies that p120 may share at least one aspect of its function with the arm protein and its homologs.

3T3 Cells↗
PubMed1991

The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110.

Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine-containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.

Animals↗
PubMed1991

Identification and characterization of a novel cytoskeleton-associated pp60src substrate.

Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies revealed a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 revealed an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure.

Amino Acid Sequence↗
PubMed1991

Regulation of the oncogenic activity of the cellular src protein requires the correct spacing between the kinase domain and the C-terminal phosphorylated tyrosine (Tyr-527).

Repression of the tyrosine kinase activity of the cellular src protein (pp60c-src) depends on the phosphorylation of a tyrosine residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids surrounding Tyr-527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report, we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine. Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed.

Amino Acid Sequence↗
PubMed1991

Tyrosine phosphorylation of a 120-kilodalton pp60src substrate upon epidermal growth factor and platelet-derived growth factor receptor stimulation and in polyomavirus middle-T-antigen-transformed cells.

The monoclonal antibody 2B12 is directed toward p120, a 120-kDa cellular protein originally identified as a protein tyrosine kinase substrate in cells expressing membrane-associated oncogenic variants of pp60src. In this report, we show that p120 was tyrosine phosphorylated in avian cells expressing membrane-associated, enzymatically activated variants of c-src, including variants having structural alterations in the src homology regions 2 and 3. In contrast, p120 was not tyrosine phosphorylated in cells expressing enzymatically activated, nonmyristylated pp60src. Furthermore, p120 was tyrosine phosphorylated in avian cells expressing middle T antigen, the transforming protein of polyomavirus, as well as in rodent cells stimulated with either epidermal growth factor (EGF) or platelet-derived growth factor. Analysis of the time course of p120 tyrosine phosphorylation in EGF-stimulated cells revealed a rapid onset of tyrosine phosphorylation. In addition, both the extent and duration of p120 phosphorylation increased when cells overexpressing the EGF receptor were stimulated with EGF. Biochemical analysis showed that p120 (in both normal and src-transformed cells) was membrane associated, was myristylated, and was phosphorylated on serine and threonine residues. Hence, p120 appears to be a substrate of both nonreceptor- and ligand-activated transmembrane receptor tyrosine kinases and of serine/threonine kinases and is perhaps a component of both mitogen-stimulated and tyrosine kinase oncogene-induced signaling pathways.

Animals↗
PubMed1991

PDGF, CSF-1, and EGF induce tyrosine phosphorylation of p120, a pp60src transformation-associated substrate.

Transformation by activated pp60c-src has been correlated by genetic analysis with the tyrosine phosphorylation of a 120 kilodalton (kDa) protein, p120. We now demonstrate tyrosine phosphorylation of p120 following stimulation of cells by growth factors whose receptors have intrinsic tyrosine-specific protein kinase activity. Stimulation of quiescent NIH3T3 cells with platelet-derived growth factor (PDGF) resulted in the tyrosine phosphorylation of p120 that was maximal by 5 min and returned to background levels by 30 min. p120 was also phosphorylated on tyrosine after addition of colony-stimulating factor 1 (CSF-1) or epidermal growth factor (EGF) to NIH3T3 cells engineered to express high levels of their respective receptors. Two additional src substrates, p110 and p85, were analysed under identical assay conditions. PDGF, CSF-1, and EGF induced only a minimal increase in the tyrosine phosphorylation of p85 and no change in the phosphorylation of p110. Thus, the marked ligand-induced tyrosine phosphorylation of p120 was a property not shared by the other src substrates examined. Immunoblotting with antibodies to p120 and the ras GTPase activating protein, GAP, suggests that p120 and GAP are unrelated. In addition, the amino acid sequences of four cyanogen bromide peptides derived from p120 showed no homology to GAP or to sequences in either the PIR or Swiss-Prot databases. These data suggest that tyrosine phosphorylation of p120 may contribute to both signal transduction through growth factor receptors and pp60src induced transformation.

Amino Acid Sequence↗
PubMed1990

Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases.

Cellular transformation by oncogenic retroviruses encoding protein tyrosine kinases coincides with the tyrosine-specific phosphorylation of multiple protein substrates. Previous studies have shown that tyrosine phosphorylation of a protein of 120 kDa, p120, correlated with src transformation in chicken embryo fibroblasts. Additionally, we previously identified two phosphotyrosine-containing cellular proteins, p130 and p110, that formed stable complexes with activated variants of pp60src, the src-encoded tyrosine kinase. To study transformation-relevant tyrosine kinase substrates, we have generated monoclonal antibodies to individual tyrosine phosphoproteins, including p130, p120, p110, and five additional phosphoproteins (p210, p125, p118, p85, and p185/p64). These antibodies detected several of the same tyrosine phosphoproteins in chicken embryo fibroblasts transformed by avian retroviruses Y73 and CT10, encoding the yes and crk oncogenes, respectively. Protein substrates in mouse, rat, hamster, and human cells overexpressing activated variants of chicken pp60src were also detected by several of the monoclonal antibodies.

Animals↗
PubMed1990

Glycoprotein tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryo fibroblasts.

The level of tyrosine phosphorylation of cellular glycoproteins isolated by wheat germ agglutinin chromatography in cells infected with a variety of kinase-positive/transformation-defective src mutants was examined in an effort to identify cellular membrane proteins whose phosphorylation correlates with phenotypic transformation. We have identified two glycoproteins, with molecular masses of 95 and 135 kilodaltons, whose phosphorylation correlates with morphological transformation, growth in soft agar, and an increase in the rate of 2-deoxyglucose uptake. The strong correlation obtained between transformation and phosphorylation of these proteins suggests that they may be substrates for pp60src which are important in the process of transformation.

Animals↗