Cell-substratum attachment and cell surface hyaluronate of Rous sarcoma virus-transformed chondrocytes

Hyaluronate is associated with the cell surface of cultured Rous sarcoma virus-transformed chondrocytes. Detachment of these cells from their substratum by a variety of reagents is accompanied by release of 75-100% of this hyaluronate into solution. Treatment of the cells with 200 U/ml protease-free Streptomyces hyaluronidase at 37 degrees C cause release of greater than 90% of the cell surface hyaluronate and complete cell detachment. Treatment with a lower concentration of Streptomyces hyaluronidase (30 U/ml) at 25 degrees C or a corresponding activity of testicular hyaluronidase gives similar results, but only in the presence of mM EGTA. Treatment with the lower activities of either hyaluronidase or with 1 mM EGTA alone release only approximately 45% of the cell surface hyaluronate and does not cause significant cell detachment. It is concluded that there are two populations of cell surface hyaluronate differing in their accessibility or their resistance to dissociation from other components of the cell surface. It is proposed that the less readily released fraction is located between the transformed chondrocyte surface and substratum and is necessary for their interaction.     

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus- transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.     

The synthesis of surface coat materials in normal and transformed cells

The rate of synthesis and thickness of the surface coat material in a range of virus-transformed and chemically-transformed cell lines were measured by ellipsometry. Cell lines transformed by polyoma virus, SV 40 virus, Rous sarcoma virus and murine sarcoma virus had a significantly thicker coat than the normal parent cells. An increase in the thickness of the cell coat was not a consistent feature of the transformed cell state since this change was not detected in cell lines transformed by methylcholanthrene. The rate of synthesis of the surface coat was significantly faster in transformed cells than in normal cells. Coat synthesis in normal and transformed cells was inhibited rapidly by treatment with cycloheximide. Inhibition of cellular RNA synthesis by actinomycin D produced rapid inhibition of coat synthesis in normal and chemically-transformed cell, but in certain virus-transformed cell lines coat synthesis continued for up to h. The significance of these changes in the pattern of coat synthesis in transformed cells in relation to their altered surface properties is discussed.     

Changes in the pericellular matrix during differentiation of limb bud mesoderm

Mesodermal cells in the developing chick embryo limb bud appear morphologically homogeneous until stage 21. At stage 22 the prechondrogenic and premyogenic areas begin to condense, culminating in the appearance of cartilage and muscle by stage 25-26. We have examined changes in the hyaluronate-dependent pericellular matrices elaborated by mesodermal cells of the limb bud from different developmental stages and the corresponding changes in production of cell surface-associated and secreted glycosaminoglycans. When placed in culture, most early mesodermal cells (stage 17 lateral plate and stage 19 limb bud) exhibited pericellular coats as visualized by the exclusion of particles. These coats were removed by treatment of the cultures with Streptomyces hyaluronidase. Cells from stage 20-21 limb buds (precondensation) had smaller coats, whereas cells derived from stage 22, 24, and 26 limb buds (condensed chondrogenic and myogenic regions) lacked coats. However, coats were reformed during subsequent cytodifferentiation of chondrocytes; chondrocytes from stage 28 and 30 limb buds, and more mature chondrocytes from stage 38 tibiae, had pericellular coats. Thus, cytodifferentiation of cartilage is accompanied by extensive intercellular matrix accumulation in vivo and reacquisition of pericellular coats in vitro. Although their structure was still dependent on hyaluronate, chondrocyte coats were associated with increased proteoglycan content compared to the coats of early mesodermal cells. The amount of incorporation of [3H]acetate into cell surface hyaluronate remained relatively constant from stages 17 to 38, whereas in the medium compartment, incorporation into hyaluronate was more than 4-fold greater by stage 17 and 19 mesodermal cells than by cells from stages between 20 and 38. However, there was a progressive increase in incorporation into cell surface and medium chondroitin sulfate throughout these developmental stages. Thus, at the time of cellular condensation in the limb bud in vivo, we have observed a reduction in size of hyaluronate-dependent pericellular coats and a dramatic change in the relative proportion of hyaluronate and chondroitin sulfate produced by the mesodermal cells in vitro.     

Transformation of chick embryo fibroblasts by rous sarcoma virus does not inhibit the assembly of fibronectin into reduction-sensitive dimer and high molecular weight complex

Chick embryo fibroblasts (CEFs) transformed by Rous sarcoma virus synthesize reduced amounts of fibronectin and also shed this protein into the medium more rapidly than do uninfected cells. We wanted to know whether or not the increased fibronectin shedding rate observed in RSV-transformed CEFs could be explained by an inability of fibronectin to form dimers and/or HMW complex. Our studies on normal and RSV-transformed fibroblasts labelled either metabolically or by lactoperoxidase-catalysed iodination indicate that RSV-induced transformation does not alter the subunit structure of either cell-bound or shed fibronectin, nor does it appear to alter the kinetics of conversion of dimeric fibronectin into HMW complex. We conclude that transformation of CEFs by Rous sarcoma virus does not prevent the assembly of fibronectin into dimeric and HMW complex forms and we offer alternative hypotheses for the more rapid shedding of fibronectin protein by these cells.     

Effects of vanadate on tyrosine phosphorylation and the pattern of glycosaminoglycan synthesis in rabbit chondrocytes in culture

The present study examined the effects of high doses of vanadate on glycosaminoglycan (GAG) synthesis and tyrosine phosphorylation in rabbit chondrocytes in confluent cultures. Although 6 microM vanadate increased the incorporation of [3H]glucosamine into chondroitin sulfate proteoglycans twofold, 40-60 microM vanadate suppressed this incorporation fourfold. Although 6 microM vanadate had little effect on [3H]glucosamine incorporation into hyaluronate, 40-60 microM vanadate increased this incorporation threefold. Chemical analyses confirmed that the increase in [3H]glucosamine incorporation into hyaluronate and the decrease in the incorporation into chondroitin sulfate proteoglycan correlated with increased hyaluronate content and decreased chondroitin sulfate content in the cell layers of vanadate-transformed cells. Chondrocytes exposed to 40-60 microM vanadate became typically transformed spindlelike cells. Furthermore, vanadate, at 6 and 60 microM, increased the overall level of phosphotyrosine by 8- and 31-fold, respectively, and 60 microM vanadate enhanced phosphorylation of many phosphotyrosine-containing proteins. These observations suggest that vanadate induces transformation-associated changes in the pattern of GAG synthesis when it induces excess phosphorylation on tyrosine in chondrocyte proteins.     

Production of macrophage migration inhibitory factor(s) (MIF) by virus-transformed cells

Rodent cell lines transformed by SV40, polyoma virus and Rous sarcoma virus cultured in vitro release material into the culture medium which inhibits the migration of guinea pig macrophages. Similar macrophage migration inhibitory factors (MIF) were not detected in cell-free supernatants harvested from untransformed cell cultures. Comparison of the MIF produced by virus-transformed cells with MIF derived from peripheral lymphocytes stimulated in vitro by phytohemagglutinin (PHA) revealed that they had similar molecular weights (25 000), heat stability and were both inhibited by α-fucose and lotus agglutinin. Incubation of MIF-containing cell-free supernatants from transformed cells with pancreatic trypsin inhibitor, soybean trypsin inhibitor and diisopropylfluorophosphate eliminated the MIF activity. The possible identity of the MIF released by transformed cells as a protease is discussed with reference to a potential role in modifying the surface properties of transformed cells.     

Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes

Rous sarcoma virus-transformed BHK cells (RSV/B4-BHK) adhere to a fibronectin-coated substratum primarily at specific dot-shaped sites. Such sites contain actin and vinculin and represent close contacts with the substratum as revealed by interference reflection microscopy. Only a few adhesion plaques and actin filament bundles can be detected in these cells as compared to untransformed parental fibroblasts. In thin sections examined with transmission electron microscopy (TEM) these adhesion sites correspond to short protrusions of the ventral cell surface that contact the substratum at their apical portion. These structures, which may represent cellular feet, are therefore called podosomes. By screening a number of different transformed fibroblasts plated on a fibronectin-coated substratum we find that podosomes are common to mammalian and avian cell lines transformed either by Rous sarcoma virus (RSV) or by Fujinami avian sarcoma virus (FSV), whose oncogenes encode specific tyrosine kinases. Using antibodies reacting with phosphotyrosine in immunofluorescence experiments, we show that phosphotyrosine-containing molecules are concentrated in podosomes. Podosomes are not detected in fibroblasts transformed by other retroviruses (Snyder-Theilen sarcoma virus, Abelson leukemia virus and Kirsten sarcoma virus) or by DNA tumor viruses (polyoma, SV40), indicating that podosome-mediated adhesion in transformed fibroblasts is related to the peculiar properties of some oncoproteins and possibly to their tropism for adhesion systems. Podosomes and adhesion plaques, although similar in cytoskeletal protein composition, have different mechanisms and kinetics of formation. Assembly of podosomes, in fact (i) does not require fetal calf serum (FCS) in the adhesion medium, that is necessary for the organization of adhesion plaques; (ii) does not require protein synthesis; and (iii) is insensitive to the ionophore monensin, that prevents adhesion plaque formation. Moreover, during attachment to fibronectin-coated dishes, podosomes appear in the initial phase (60 min) of attachment, while adhesion plaques require a minimum of 180 min. In conclusion podosomes of RSV- and FSV-transformed fibroblasts represent a phenotypic variant of adhesion structures.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells.

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polyoma virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Materials released into culture medium by normal and oncogenic virus-transformed mammalian cells

Materials released into culture medium by transformed and untransformed baby hamster kidney cells labelled with glucosamine, sulfate, fucose or leucine were characterized. Some of the components could also be labelled by iodination of intact cells, indicating their surface origin. Analysis on gradient polyacrylamide sodium lauryl sulfate gels demonstrated that a group of high apparent molecular weight glucosamine-labelled components were more abundant in materials released from Rous sarcoma virus-transformed baby hamster kidney cells than from baby hamster kidney cells or polymo virus-transformed baby hamster kidney cells. The relative rates of release of glucosamine-labelled components from transformed and untransformed cells were similar except that the transformed baby hamster kidney cells released some large molecular weight components slightly more rapidly than baby hamster kidney cells. Treatment of labelled medium materials with testicular hyaluronidase removed much glucosamine label from the materials but did not affect the amounts of other labels. After treatment with hyaluronidase, the patterns of labelled conditioned media from both transformed and untransformed baby hamster kidney cells were qualitatively and quantitatively very similar, suggesting that the differences seen in untreated labelled conditioned media were due to the presence of hyaluronidase-sensitive materials associated with medium materials rather than to actual differences in glycoproteins.     

Influence of cytochalasin d-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes

There is growing evidence that cell shape regulates both proliferation and differentiated gene expression in a variety of cell types. We have explored the relationship between the morphology of articular chondrocytes in culture and the amount and type of proteoglycan they synthesize, using cytochalasin D to induce reversible cell rounding. When chondrocytes were prevented from spreading or when spread cells were induced to round up, 35SO4 incorporation into proteoglycan was stimulated. Incorporation into the cell layer was stimulated more than into the medium. When the cells were allowed to respread by removing cytochalasin D, proteoglycan synthesis returned to control levels. Cytochalasin D-induced stimulation of 35SO4 incorporation reflected an increase in core protein synthesis rather than lengthening of glycosaminoglycan chains, because [3H]serine incorporation into core protein was also stimulated. The observed stimulation of proteoglycan synthesis was not due to an overall stimulation of protein synthesis, to inhibition of DNA synthesis, or to accumulation of cells in one phase of the cell cycle. Cytochalasin D-treatment of cells in suspension caused no further stimulation of 35SO4 incorporation, suggesting that the observed effects were due to cell rounding rather than exposure to cytochalasin D per se; nevertheless, we cannot completely rule out other, nonspecific, effects of the drug. Fibroblasts and chondrocytes that had been passaged to stimulate dedifferentiation did not incorporate more 35SO4 when treated with cytochalasin D, suggesting that increased proteoglycan synthesis in response to rounding may itself be a differentiated property of chondrocytes.     

Immunochemical purification of probe-labeled plasma membrane proteins: An approach to the molecular anatomy of the cell surface

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labelling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80–100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37°C but not at 41°C, the expression of this component is related to the expression of the transformed phenotype.     

Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk- supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.     

Demonstration of T antigens on the surface of cells transformed with primate polyoma viruses

Primate polyoma virus-transformed hamster, mouse, and rat cell lines were examined by indirect immunofluorescence staining for cell surface-associated T antigens, by using a rabbit antiserum prepared against sodium dodecyl sulfate-denatured large T antigen of simian virus 40 (anti-SV40-SDS-T serum). Positive surface staining was shown not only on SV40-transformed cells, but also on BK and JC virus-transformed cells. In contrast, normal cells and cells transformed with mouse polyoma-, human adeno-, and murine sarcoma viruses were negative. The data on SV40-transformed cells confirmed the reports of others demonstrating the cell surface location of SV40 large T antigen, and the data on BK and JC virus-transformed cells proved that these cells have cell-surface T antigens that cross-react with anti-SV40-SDS-T serum.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. Transformation by rous sarcoma virus induces a switch in the collagen type synthesis and enhances fibronectin expression

Epithelial-like chondrocytes obtained from chick embryo were transformed with Rous sarcoma virus. Cellular transformation was monitored looking at the morphology change, the cell growth, and the expression of plasminogen activator. Analysis on polyacrylamide gel of intracellular and secreted proteins showed: 1) a disappearance of the specific products of differentiated chondrocytes; 2) a switch in the collagen synthesis from the type II, the chondrocyte-specific type, to the type I, characteristic of fibroblasts and other cells of mesenchymal origin; 3) an enhancement of fibronectin synthesis. Analysis of the proteins from chondrocytes infected with Rous-associated virus 1, a virus unable to induce cell transformation in vitro, indicated that the altered expression of the differentiated proteins in Rous sarcoma virus-infected chondrocytes depended upon the action of src gene product.     

Hyaluronate-protein complex of rous sarcoma virus-transformed chick embryo fibroblasts

Involvement of covalently linked protein or peptide in the structure or synthesis of hyaluronate has not previously been convincingly demonstrated. We have developed conditions for double-labeling with [3H]leucine and [14C]acetate, then isolating and characterizing the cell-associated and secreted hyaluronate-protein complexes of Rous sarcoma virus-transformed chick embryo fibroblasts. The preparations were purified by Bio-Gel A-15m gel filtration and CsCl density gradient ultracentrifugation under dissociative conditions, followed by acid agarose gel electrophoresis in the presence of 0.1% Nonidet P-40. The purified hyaluronate preparations did not change their 3H:14C ratios after further sodium dodecyl sulfate or alkali treatment. The cell-derived hyaluronate-protein was resistant to pronase but susceptible to proteinase K in the presence of sodium dodecyl sulfate. After chondroitinase ABC digestion, the cell-derived 3H-labeled protein was separated from the 14C-labeled hyaluronate disaccharides, then shown to give a broad band corresponding to Mr approximately 12,000 on sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and to be susceptible to both pronase and proteinase K. The corresponding 3H-labeled peptide was prepared in the same manner from the medium hyaluronate and the [3H]leucine shown to be present in material smaller in amount and size than that from the cell. We propose from these and other published data that the cell-associated hyaluronate-protein may be bound to the cell surface and that the hyaluronate in the medium may be derived from it as a result of proteolytic scission.     

The response of chicken embryo dermal fibroblasts to cytochalasin B is altered by Rous sarcoma virus-induced cell transformation.

The drug cytochalasin B (CB), which disrupts the cellular microfilament network, allows the identification of as yet unclassified structural differences between normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. When exposed to CB, normal chick fibroblasts attain an arborized or dendritic morphology. This results as the cytoplasm collapses upon the remaining structural and adhesive components of the cell. Rous sarcoma virus-transformed cells did not form or maintain these dendritic-like processes in the presence of CB and, as a result, rounded up but still remained attached to the substrate. With a temperature-sensitive mutant of Rous sarcoma virus, LA24A, it was possible to show that these effects are completely reversible and dependent on the expression of pp60src. The cytoskeleton in these CB-treated cells was examined by both immunofluorescence and electron microscopy. After exposure to CB, the microfilaments were found to be disrupted similarly throughout both the transformed and the nontransformed cells. In the nontransformed cells arborized by exposure to CB, the extended processes were found to contain intermediate filaments in an unusually high concentration and degree of organization. The distribution of these filaments in the central body of the arborized cells was random. This lower concentration and random distribution was similar to that seen throughout the transformed cells rounded up by exposure to CB. The failure of these transformed cells to arborize in CB indicates that the structural component(s) which is necessary for the formation or maintenance or both of the arborized state is altered by the expression of pp60src.     

Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture.

The fibrinolytic activity of cells in culture varied with the type of serum employed in the growth medium. Degradation of iodinated fibrin occurred slowly when Rous sarcoma virus-transformed chick embryo fibroblasts were grown in medium containing fetal bovine serum (FBS), and rapidly when chicken serum was employed. This difference reflected the low plasminogen and high inhibitor content of FBS. The inhibitors were found to be serum macromolecules that were precipitated with ammonium sulfate or polyethylene glycol, and were inactivated by boiling or upon exposure to acidic conditions. No inhibitor activity was detected in fetuin, one of the major proteins present in FBS. Acidified FBS was similar to chicken serum in that both supported high rates of cell-mediated fibrinolytic activity. Although virally transformed hamster, mouse and chicken cells grew well in acid-treated FBS, their normal counterparts did not. Apparently, acifification resulted in the formation of materials that were toxic to normal cells. These agents rapidly blocked cellular DNA synthesis.     

Repression of quiescence-specific polypeptides in chicken heart mesenchymal cells transformed by Rous sarcoma virus.

Chicken heart mesenchymal cells do not proliferate in medium of physiological composition containing plasma (S. Balk, Proc. Natl. Acad. Sci. USA 77:6606-6610, 1980). To understand the molecular events involved in cell quiescence and in the initiation of cell division under physiological conditions, we examined the differences in the patterns of protein synthesis of quiescent, hormone-stimulated, and Rous sarcoma virus-transformed chicken heart mesenchymal cells. We describe the expression of a 20,000-kilodalton (kDa) polypeptide actively synthesized by quiescent cells but not by their transformed counterparts. Normal chicken heart mesenchymal cells stimulated with epidermal growth factor and insulin also repressed the synthesis of the 20,000-kDa polypeptide while actively growing but synthesized increasing amounts of the protein at high cell density (confluence). The synthesis of the 20,000-kDa protein is not restricted to chicken heart mesenchymal cells, since confluent, density-arrested chicken embryo fibroblasts also expressed high levels of the protein. Transformed chicken heart mesenchymal cells and embryo fibroblasts did not synthesize the protein even at high cell density. The 20,000-kDa polypeptide accumulated in the culture medium.     

Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies.

The protein substrates for the tyrosine protein kinases in cells transformed by avian sarcoma viruses were analyzed by gel electrophoresis in combination with immunoblotting or immunoprecipitation by antibodies against phosphotyrosine. We found that greater than 90% of phosphotyrosine-containing cellular proteins can be immunoprecipitated by these antibodies. The level of phosphotyrosine-containing cellular proteins detectable by this method markedly increased upon transformation with Rous sarcoma virus, and more than 20 distinct bands of such proteins were found in lysates of Rous sarcoma virus-transformed cells. Most of these phosphotyrosine-containing proteins had not been identified by other methods, and their presence appeared to correlate with morphological transformation in cells infected with various Rous sarcoma virus mutants and Y73, PRCII, and Fujinami sarcoma viruses. However, considerably different patterns were obtained with cells infected with nontransforming Rous sarcoma virus mutants that encode nonmyristylated src kinases, indicating that most substrates that correlate with transformation can only be recognized by p60v-src associated with the plasma membrane.