Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus

Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.     

Histone kinase activities in normal and transformed mouse cells

Crude extracts from replicating normal and transformed cells were assayed for protein kinase activities specific for different sites in purified Hl histone in vitro. Extracts from normal cells favored the NH2-terminal region while extracts from transformed cells favored the COOH-terminal region. Analysis of phosphopeptides demonstrated that histone kinases from both normal and transformed cells catalyzed the phosphorylation of a number of sites in common, and these were typical of sites phosphorylated in replicating cells. The preference for the NH2-terminal region by extracts from normal cells was due to the extensive phosphorylation of a site previously shown to be phosphorylated by cyclic AMP-dependent protein kinase. This activity was very low in transformed cells.     

Normal rat kidney cells secrete both phosphorylated and nonphosphorylated forms of osteopontin showing different physiological properties

We have reported previously that the 69-kDa major phosphoprotein, secreted by normal rat kidney (NRK) cells, is osteopontin, a glycosylated bone matrix protein. Here we show that this 69-kDa osteopontin is secreted by NRK cells in both phosphorylated (pp69) and nonphosphorylated (np69) forms, with estimated isoelectric points of 3.8 and 4.5, respectively. Electrophoretic analysis of radioiodinated cell surface proteins immunoprecipitated with an anti-69-kDa osteopontin serum, demonstrates that the 69-kDa osteopontin is also present on the cell surface, but only its phosphorylated form (pp69) shows such cell surface association. Because osteopontin mediates cell adhesion and spreading, and contains an Arg-Gly-Asp-Ser cell-binding sequence, our observations strongly suggest that the cell surface localization of pp69 osteopontin is receptor-mediated, and the modification by phosphorylation may be crucial for its receptor binding activity. We also report that antisera directed against either fibronectin or 69-kDa osteopontin co-immunoprecipitate both np69 osteopontin and fibronectin as a heat-dissociable complex. In contrast, pp69 osteopontin does not co-precipitate with fibronectin. These observations demonstrate an interactive relationship between np69 and soluble fibronectin. Furthermore, compared to NRK cells, vanadyl sulfate-treated NRK cells which acquire a reversible transformed phenotype, including anchorage-independent growth, show increased levels of pp69 on the cell surface, concomitant with significantly decreased levels of pp69 and elevated levels of np69 in the conditioned media. The data presented here establish transformation sensitivity of NRK cell-secreted osteopontin with respect to its secretion and cell surface localization, and demonstrate that phosphorylated and nonphosphorylated forms of osteopontin have different physiological properties, which may regulate the functional roles of this extracellular matrix protein.     

Phosphorylation of the ribosomal protein S6 is elevated in cells transformed by a variety of tumor viruses.

We have analyzed the phosphorylation of the ribosomal protein S6 in several cultures of normal cells and cells transformed with RNA or DNA tumor viruses. Serum deprivation of confluent, normal cells results in low levels of S6 phosphorylation. In contrast, under identical conditions, this protein is highly phosphorylated in virally transformed cells. No differences in the phosphopeptides generated by one-dimensional limited proteolysis were detected in S6 prepared from serum-stimulated or transformed cells. These results suggest that a common event in oncogenic transformation is the phosphorylation of S6.     

Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein.

We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Stimulation of Ca2+ uptake and protein phosphorylation in tumor cells by fibronectin

Suspensions derived from attached HeLa cells transported 45Ca2+ considerably faster than those derived from spinner cultures grown in liquid medium. Incubation of spinner cells with fibronectin or cold-insoluble globulin in the presence of 5% calf serum at 37 degrees C for 1 to 2 h greatly increased the rate of Ca2+ flux into the cells. Suspensions of cells transformed by Rous sarcoma virus transported Ca2+ much more slowly than cell suspensions of the parent strain of normal rat kidney. Incubation of the transformed cells or Ehrlich ascites tumor cells with fibronectin increased the rate of Ca2+ uptake, while no effect was seen on Ca2+ transport by this treatment of normal kidney cells grown in tissue cultures. A 45,500-dalton protein was found to interact firmly with Ca2+ that entered into attached HeLa cells or fibronectin-treated spinner cells. This Ca2+-associated protein was detected by lithium dodecyl sulfate gel electrophoresis at 0 degrees C after 30 s of exposure to radioactive Ca2+. In tumor cells without fibronectin treatment, the radioactive band was not seen under the same conditions, even after 10 min incubation with 45Ca2+. In fibronectin-treated tumor cells, addition of Ca2+ to buffered solutions resulted in increased phosphorylation of a protein in the 45,000-dalton region. The phosphorylated protein band which appears to be associated with the cytoskeleton can be resolved by isoelectric focusing into four polypeptide chains. The relation of these observations to the cascade of protein kinases involved in the phosphorylation of the beta-subunit of the (Na+-K+)-ATPase is discussed.     

Similarities and differences between the fibronectins of normal and transformed hamster cells

Fibronectins from normal and virally transformed hamster cells were compared by several criteria. The fibronectin from transformed cells was similar to that from normal cells in being an intact dimeric glycoprotein with the ability to bind to gelatin, activated thiol-Sepharose, and cells. No evidence was found for proteolytic cleavages or abnormalities in disulfide bonding of transformed cell fibronectin. This fibronectin was also shown to be active in promoting cell attachment, elongation, and alignment. Therefore, the fibronectin produced by transformed cells is not defective. However, it was shown that the transformed cells were partially deficient in their capacity to bind fibronectins from either normal or transformed cells. This deficiency has implications for the significance of the loss of fibronectin on oncogenic transformation. Partial proteolysis of the fibronectins from normal and transformed cells gave rise to the same fragments. However, the glycosylated fragments from transformed cell fibronectin appeared somewhat larger than those from normal cell fibronectin. Analysis of fibronectin glycopeptides showed that transformation leads both to more branches per core and to a higher sialylation of the asparagine-linked complex carbohydrate side chains.     

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.     

Phosphorylation of Serine 402 Regulates RacGAP Protein Activity of FilGAP Protein

FilGAP is a Rho GTPase-activating protein (GAP) that specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at Ser-402 is localized to the cytoplasm but not at the cytoskeleton. Although Ser-402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of Ser-402 is accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin, whereas cells transfected with FilGAP S402A mutant were not affected by calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP, and phosphorylation of Ser-402 may play a role in the regulation of cell spreading on fibronectin.     

Phosphorylated 350 kD protein in the nucleus as it is associated with cell transformation

Protein kinases are thought to play a key role in signal transduction and oncogenesis, but little is known about the intranuclear phosphorylation events associated with transformation. Here we report on cell cycle-dependent phosphorylation of cytoskeleton-associated 350 kD protein and the regular interchange in its location between the nucleus and cytoplasm of normal cells. Persistent intranuclear location of the phosphorylated 350 kD protein was also found throughout the cell cycle in transformed cells, as detected by immunoprecipitation of 32P-phosphorylated 350 kD protein from isolated nuclei and immunofluorescent staining with a monoclonal antibody that recognized phosphorylated site of 350 kD protein. A conditional transformed phenotype induced by a temperature-sensitive (ts) viral oncogene or a transforming growth factor was also associated with the intranuclear presence of the phosphorylated 350 kD protein. Thus the 350 kD protein seems to be a target molecule of protein kinases that are stimulated directly or indirectly by growth factors or by oncogene products in the nucleus, and appears to be a new transformation-related nuclear antigen.     

The migratory behavior of avian embryonic cells does not require phosphorylation of the fibronectin-receptor complex

When locomotory embryonic cells become stationary, they acquire new substratum-adhesion properties. In particular, the distribution of fibronectin receptors shifts from diffuse and highly mobile on the cell membrane to immobilized in close association with fibronectin molecules and cytoskeletal elements in focal contacts. Receptor phosphorylation has been proposed as a possible regulator of the interaction between the receptor and its intracellular and extracellular ligands. In the present study, we have compared the phosphorylation state of the fibronectin receptor in motile neural crest and somitic cells, in stationary somitic cells, and in Rous-sarcoma virus transformed-chick embryo fibroblasts, using immunoprecipitation following metabolic labeling. While no receptor phosphorylation was detected in motile embryonic cells, the beta subunit of the receptor was phosphorylated in stationary cells. This subunit was also highly phosphorylated in Rous-sarcoma virus-transformed chicken cells. These results suggest that phosphorylation of the fibronectin receptor cannot account for its distribution in the cell membrane and for the nature of the interactions between this receptor and its ligands in embryonic cells.     

Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants.

We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology.     

Transformation by Rous sarcoma virus induces clathrin heavy chain phosphorylation

We have shown that the heavy chain of clathrin is phosphorylated in chicken embryo fibroblast cells transformed by Rous sarcoma virus, but not in normal cells. Approximately 1 mol of phosphate is bound for every 5 mol of heavy chain in the maximally phosphorylated transformed cells. Two-thirds of the phosphate is on serine and one-third on tyrosine residues. Clathrin heavy chain is a substrate for pp60v-src in vitro. Cleveland analysis of the in vivo and in vitro clathrin heavy chain phosphopeptides, generated by protease V8 digestion, show labeled proteolytic fragments of similar molecular weight, suggesting that pp60v-src could be directly responsible for the in vivo phosphorylation of clathrin. Phosphate is equally incorporated into clathrin in both the unassembled and the assembled clathrin pools, whereas [35S]methionine is preferentially incorporated into the assembled pool. In normal cells, clathrin visualized by immunofluorescent staining appears in a punctate pattern along the membrane surface and concentrated around the nucleus; in transformed cells the perinuclear staining is completely absent. The phosphorylation of clathrin heavy chain in transformed cells may be linked to previously observed transformation-dependent alterations in receptor-mediated endocytosis of ligands such as EGF and thrombin.     

Constitutive phosphorylation of the receptor for insulinlike growth factor I in cells transformed by the src oncogene.

Many oncogene products have been shown to bear strong homology to or to interact with components of normal cellular signal transduction. We have previously shown that a glycoprotein band of 95 kilodaltons (kDa) becomes tyrosine phosphorylated in chick cells transformed by Rous sarcoma virus and that tyrosine phosphorylation of this protein band correlates tightly with phenotypic transformation in cells infected with a large and diverse panel of src mutants (L. M. Kozma, A. B. Reynolds, and M. J. Weber, Mol. Cell. Biol. 10:837-841, 1990). In this communication, we report that a component of the 95-kDa glycoprotein band is related or identical to the 95-kDa beta subunit of the receptor for insulinlike growth factor I (IGF-I). We found that the beta subunit of the IGF-I receptor comigrated on polyacrylamide gels with a component of the 95-kDa glycoprotein region from src-transformed cells under both reducing and nonreducing gel conditions and had a very similar partial phosphopeptide map. To further test the hypothesis that the beta subunit of the IGF-I receptor becomes tyrosine phosphorylated in cells transformed by pp60src, a human cell line that expressed the IGF-I receptor was transformed by src. Comparison of IGF-I receptors immunoprecipitated from normal and transformed cells revealed that the beta subunit of the IGF-I receptor became constitutively tyrosine phosphorylated in src-transformed cells. Moreover, IGF-I receptor phosphorylation induced by src was synergistic with that induced by the hormone: IGF-I-stimulated autophosphorylation of the receptor was much greater in src-transformed cells than in untransformed HOS cells even at maximal concentrations of IGF-I. This increased responsiveness to IGF-I was not due to increases in receptor number, time course of phosphorylation, or affinity for hormone. Finally, no IGF-I-like activity could be detected in culture supernatants collected from the src-transformed cells, suggesting that the increased receptor phosphorylation observed in the src-transformed cells may be mediated by an intracellular mechanism rather than an external autocrine stimulation. Our data demonstrate that the IGF-I receptor becomes constitutively tyrosine phosphorylated in src-transformed cells. This finding raises the possibility that pp60v-src alters growth regulation at least in part by phosphorylating and activating this growth factor receptor.     

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.     

Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.     

The characteristics of actin cytoskeleton structure and its rearrangements by extracellular matrix proteins in normal, immortalized and transformed rat fibroblasts

Comparative analysis of actin cytoskeleton structure in rat embryonic fibroblasts, E1A-immortalized and E1A + cHa-ras-transformed cells has been carried out. A decrease in adhesiveness and the rate of changes in actin cytoskeleton structures was shown to correlate with the level of morphological transformation of cells. E1A + cHa-ras-transformants show the lowest adhesiveness and complete disorganization of actin structures. Cultivation on serum-free media promoted disassembling of actin cytoskeleton structures in a small part of normal fibroblast population, only in a few immortalized cells, but exerted no influence on transformed cells. The influence of immobilized extracellular matrix proteins fibronectin, laminin and collagens type I and III on actin cytoskeleton structure in normal, immortalized and transformed fibroblasts was studied. Transformed cells spread on fibronectin completely restored highly organized actin structures, displayed a lot of stress fibers and focal contacts. The use of laminin revealed differences in locomotion between normal and transformed cells. Normal, immortalized and transformed fibroblasts spread on fibronectin and laminin demonstrate some peculiarities in actin cytoskeleton structures as a result of specificity of ligand-receptor interaction. Cells spread on fibronectin have polygonal shapes, many stress fibers and focal contacts, whereas cells spread on laminin are highly polarized and develop broad lamellae filled with actin microfilament meshwork. Collagens type I and III can affect adhesive properties and actin cytoskeleton structure in all cell lines studied only slightly, in comparison with fibronectin and laminin.     

Sequential enhanced tyrosine phosphorylation during progressive malachite green induced malignant transformation of Syrian hamster embryo cells in culture is associated with no change in the activity levels of tyrosine phosphatases.

Malachite green (MG) consisting green crystals with a metallic lustre is extremely soluble in water and is highly cytotoxic to mammalian cells and also acts as liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported that MG induces malignant transformation in Syrian hamster embryo (SHE) cells. Since tyrosine phosphorylation and dephosphorylation reactions are known to play critical roles during normal and abnormal cellular proliferation, in this study we have studied the tyrosine phosphorylation, tyrosine phosphorylated proteins and protein tyrosine phosphatases in malignantly transformed cells and during sequential development of cellular transformation by MG compared to control cells. The present investigation shows that enhanced tyrosine phosphorylation and tyrosine phosphorylated proteins associated with the static levels of tyrosine protein phosphatases may probably contribute to the abnormal cellular proliferation during malignant transformation of SHE cells by MG.     

Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape

The major cell surface glycoprotein of chick embryo fibroblasts, cellular fibronectin (formerly known as CSP or LETS protein), was purified and used to produce monospecific antisera. After affinity purification, the anti-fibronectin was used to investigate fibronectin's localization, its transfer from intracellular to extracellular pools, its antibody-induced redistribution on the cell surface, and its role in cell shape. Anti-fibronectin localizes to extracellular fibrils located under and between sparse cells, and to a dense matrix that surrounds confluent cells. Cellular fibronectin is also present in granular intracytoplasmic structures containing newly synthesized fibronectin before secretion. This intracellular staining disappears 2 h after treatment with cycloheximide or puromycin, and returns after removal of these protein synthesis inhibitors. In pulse-chase experiments using cycloheximide, fibronectin was sequentially transferred from the intracellular to the fibrillar extracellular forms. Transformation of chick fibroblasts results in decreases in both extracellular and intracellular fibronectin, and in altered cell shape. Treatment of untransformed chick fibroblasts with anti-fibronectin results in rapid (30 min) alteration to a rounder cell shape resembling that of many transformed cells. These rapid shape changes are followed by a slow, antibody-induced redistribution of fibronectin to supranuclear caplike structures. This "capping" is inhibited by metabolic inhibitors. Reconstitution of cell surface fibronectin onto transformed cells restores a more normal fibroblastic phenotype. The reconstituted fibronectin on these cells organizes into fibrillar patterns similar to those of untransformed cells. As with untransformed cells, treatment of these reconstituted cells with anti-fibronectin also results in cell rounding and "capping" of fibronectin.