ABSTRACT
Zyxin is a component of adhesion plaques that has been suggested to perform regulatory functions at these specialized regions of the plasma membrane. Here we describe the isolation and characterization of cDNAs encoding human and mouse zyxin. Both the human and mouse zyxin proteins display a collection of proline-rich sequences as well as three copies of the LIM domain, a zinc finger domain found in many signaling molecules. The human zyxin protein is closely related in sequence to proteins implicated in benign tumorigenesis and steroid receptor binding. Antibodies raised against human zyxin recognize an 84-kDa protein by Western immunoblot analysis. The protein is localized at focal contacts in adherent erythroleukemia cells. By Northern analysis, we show that zyxin is widely expressed in human tissues. The zyxin gene maps to human chromosome 7q32-q36.
Subject(s)
Cell Adhesion , Metalloproteins/chemistry , Zinc Fingers , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Chromosome Mapping , Cytoskeletal Proteins , DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , Glycoproteins , Humans , Mice , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/metabolism , ZyxinABSTRACT
As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59fyn, pp60v-src, and activated pp60c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60c-src, p59fyn, or pp62c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyn-, src- and yes- fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59fyn and pp62c-yes, and particularly in those lacking pp60c-src. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells.
Subject(s)
Phosphotyrosine/metabolism , Protein-Tyrosine Kinases/deficiency , Proto-Oncogene Proteins pp60(c-src)/deficiency , Proto-Oncogene Proteins/deficiency , src-Family Kinases , Animals , Animals, Newborn , Cell Adhesion , Fibroblasts/enzymology , Fibronectins , Immunoblotting , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation , Phosphotyrosine/analysis , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-fyn , Proto-Oncogene Proteins c-yes , Proto-Oncogene Proteins pp60(c-src)/metabolismABSTRACT
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.
Subject(s)
Cell Adhesion , Phosphoproteins/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Animals , Antibodies, Monoclonal , Avian Sarcoma Viruses , Cell Adhesion Molecules/analysis , Cell Adhesion Molecules/isolation & purification , Cell Adhesion Molecules/metabolism , Cell Line , Cell Transformation, Neoplastic , Embryo, Mammalian , Fibroblasts , Fibronectins , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Genes, src , Microscopy, Fluorescence , Molecular Weight , Phosphoproteins/isolation & purification , Phosphorylation , Phosphotyrosine , Protein-Tyrosine Kinases/analysis , Protein-Tyrosine Kinases/isolation & purification , Protein-Tyrosine Kinases/metabolism , Rats , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
A small number of proteins becomes tyrosine-phosphorylated in response to integrin-mediated cell adhesion to extracellular matrix proteins. Previous work has identified two of these tyrosine-phosphorylated proteins as the focal adhesion kinase and paxillin. Here we identify a third focal adhesion protein, tensin, that becomes tyrosine-phosphorylated during cell adhesion to extracellular matrix proteins. The tyrosine phosphorylation of tensin does not occur when cells adhere to plastic or polylysine and is blocked when microfilament assembly and cell spreading are inhibited with cytochalasin D. In addition, we show that other focal adhesion proteins such as talin and vinculin do not become tyrosine-phosphorylated under the same conditions of cell spreading on extracellular matrix proteins.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Movement , Fibronectins/metabolism , Laminin/metabolism , Microfilament Proteins/metabolism , Tyrosine/metabolism , Animals , Cell Movement/drug effects , Cells, Cultured , Cytochalasin D/pharmacology , Phosphorylation , Rats , TensinsABSTRACT
Tyrosine phosphorylation of cytoskeletal proteins at adhesive junctions has been speculated to play a role in the regulation of cell signaling at these sites. Previously, monoclonal antibodies were generated against phosphotyrosine-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts, resulting in two antibodies which recognized antigens of 76 and 215 kDa that localized to focal contacts. We have now localized the 215-kDa antigen to a number of adhesive junctions in vivo, including the zonula adherens, intercalated discs, and myotendinous and neuromuscular junctions. In sections of skeletal muscle and in isolated myofibrils, the 215-kDa protein was localized to the I-band. By immunoprecipitation and immunoblot analysis, we determined that the 215-kDa antigen cross-reacts with a polyclonal anti-tensin antibody.
