Rous Sarcoma Virus-transformed Cells Research Articles

Identification of Amino Acid Residues of the Matrix Metalloproteinase-2 Essential for Its Selective Inhibition by β-Amyloid Precursor Protein-derived Inhibitor

The extracellular domain of beta-amyloid precursor protein (APP) contains an inhibitor against matrix metalloproteinase-2 (MMP-2, gelatinase A). Our previous study ( Higashi, S. and Miyazaki, K. (2003) J Biol Chem 278, 14020-14028 ) demonstrated that the inhibitor is localized within the ISYGN-DALMP sequence of APP, and a synthetic decapeptide containing this sequence (named APP-derived inhibitory peptide, APP-IP) selectively inhibits the activity of MMP-2. To determine the region of interaction that correlates with the selective inhibition, we constructed various MMP-2 mutants. An MMP-2 mutant, which had the hemopexin-like domain and three fibronectin-like type II domains of MMP-2 deleted, and native MMP-2 showed similar affinities for APP-IP, suggesting that only the catalytic domain of MMP-2 is essential for the interaction. Studies of chimeric proteases, consisting of various parts of the MMP-2 catalytic domain and those of MMP-7 (matrilysin) or MMP-9 (gelatinase B), further revealed that Ala(88) and Gly(94) in the non-prime side and Tyr(145) and Thr(146) in the prime side of the substrate-binding cleft of MMP-2 contribute separately to the selective inhibition. Replacement of the amino acid residue at position 94 of a chimeric MMP mutant affected its interaction with the C-terminal Pro(10) of APP-IP, whereas that of residues 145-148 affected the interaction with Tyr(3) of the inhibitor, suggesting that the N to C direction of APP-IP relative to the substrate-binding cleft of MMP is analogous to that of propeptide in proMMP, and opposite to that of substrate. When the APP-IP sequence was added to the N terminus of the catalytic domain of MMP-2, the activity of the protease was intramolecularly inhibited. We speculate that the direction of interaction makes the active site-bound APP-IP resistant to cleavage, thereby supporting the inhibitory action of the peptide inhibitor.

Changes in the Balance between Caldesmon Regulated by p21-activated Kinases and the Arp2/3 Complex Govern Podosome Formation

Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.

Palmitoylation of Caveolin-1 at a Single Site (Cys-156) Controls Its Coupling to the c-Src Tyrosine Kinase: TARGETING OF DUALLY ACYLATED MOLECULES (GPI-LINKED, TRANSMEMBRANE, OR CYTOPLASMIC) TO CAVEOLAE EFFECTIVELY UNCOUPLES c-Src AND CAVEOLIN-1 (TYR-14)

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma virus-transformed cells. Previous studies have shown that caveolin-1 is phosphorylated on tyrosine 14 by c-Src and that lipid modification of c-Src is required for this phosphorylation event to occur in vivo. Phosphocaveolin-1 (Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain largely unknown. Here, we show that palmitoylation of caveolin-1 at a single site (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kinase. Furthermore, upon evaluating a battery of nonreceptor and receptor tyrosine kinases, we demonstrate that the tyrosine phosphorylation of caveolin-1 by c-Src is a highly selective event. We show that Src-induced tyrosine phosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dually acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH(2)-terminal domain of Galpha(i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, when these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptide derived from the extreme N terminus of c-Src, the tyrosine phosphorylation of caveolin-1 is abrogated; the same peptide lacking myristoylation has no inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-14) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactions as important regulatory factors for coupling caveolin-1 to the c-Src tyrosine kinase in vivo.

A Novel Group IIA Phospholipase A2Interacts with v-Src Oncoprotein from RSV-transformed Hamster Cells

We have isolated a novel isoform of phospholipase A(2). This enzyme was designated srPLA(2) because it was discovered while analyzing the proteins interacting with different forms of the v-Src oncoproteins isolated from Rous sarcoma virus-transformed hamster cells. It contains all the functional regions of the PLA(2) group IIA proteins but differs at its C-terminal end where there is an additional stretch of 8 amino acids. The SrPLA(2) isoform was detected as a 17-kDa precursor in cells and as a mature 14-kDa form secreted in culture medium. A direct interaction of the 17-kDa precursor with the Src protein was observed in lysates of transformed cells. Both the 17- and 14-kDa forms were found to be phosphorylated on tyrosine. To our knowledge, this is the first report of a PLA(2) group II protein that is tyrosine phosphorylated. We surmise that srPLA(2) interacts with the Src protein at the cell membrane during the process of its maturation.

A product of DAN, a novel candidate tumour suppressor gene, is secreted into culture medium and suppresses DNA synthesis.

Our previous studies have shown that the DAN gene product possesses an ability to revert phenotypes of transformed rat fibroblasts and represents a candidate tumour suppressor gene for neuroblastoma. In the present study, characterisation of DAN was carried out using rat fibroblast 3Y1 cells and their DAN-overexpressor counterparts (S-9). The N-terminal region of DAN (amino acids 1-24) was highly hydrophobic and DAN protein was found to be secreted into the culture medium. When DAN was treated with PNGase F, a enzyme that cleaves most N-linked carbohydrate residues, the mobility of both cytoplasmic and secreted DAN was increased in SDS-polyacrylamide gel electrophoresis, suggesting DAN is N-glycosylated, irrespective of its localisation. When partially purified, DAN was able, when added to the culture, to suppress DNA synthesis of Rous sarcoma virus-transformed 3Y1 cells, which lack the expression of DAN.

The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes.

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.

The SH3 domain of p56lck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes.

Many of the Src-like tyrosine kinases are thought to participate in multiprotein complexes that modulate transmembrane signalling through tyrosine phosphorylation. We have used in vitro binding studies employing bacterially expressed glutathione S-transferase-p56lck fusion proteins and cell extracts to map regions on p56lck that are involved in binding to phosphatidylinositol 3'-kinase (PI3K). Deletions within the SH3 domain of p56lck abolished binding of PI3K activity from T-cell lysates, whereas deletion of the SH2 domain caused only a slight reduction in the level of PI3K activity bound to p56lck sequences. The binding of PI3K from T-cell extracts to p56lck was not blocked by antiphosphotyrosine antibodies, but p56lck-bound PI3K activity was sensitive to phosphatase treatment. The SH3 domain of p56lck also bound the majority of PI3K activity from uninfected chicken embryo fibroblasts. However, a drastically different binding specificity was observed with use of extracts of Rous sarcoma virus v-src-transformed cells, in which the majority of PI3K activity bound to the SH2 domain of p56lck in a phosphotyrosine-dependent manner. These results suggest that are two modes of PI3K binding to p56lck, and presumably to other Src-like tyrosine kinases. In one mode, PI3K from T cells or uninfected chicken embryo fibroblasts binds predominantly to the SH3 domain of p56lck. In the other mode, involving PI3K from Rous sarcoma virus-transformed cells, binding is largely phosphotyrosine dependent and requires the SH2 domain of p56lck.

Rous sarcoma virus-transformed cells develop peculiar adhesive structures along the cell periphery.

Alteration of the cell/substratum adhesive structures of rat fibroblasts (3Y1 cells) upon transformation by Rous sarcoma virus (RSV) was investigated by immunofluorescence microscopy. In serum-containing culture medium, 3Y1 cells developed focal adhesions as their main adhesive structures, while BY1 cells expressed peculiar close contacts along the cell periphery with the vitronectin receptor integrin, in addition to podosomes. These peripheral close contacts are referred to as the peripheral adhesions. The peripheral adhesions were observed as a darker region than podosomes by interference reflection microscopy. They were more easily destroyed by incubating the cells with RGD-containing peptide than were the focal adhesions. In contrast to focal adhesions and podosomes, actin bundles were not detected within the peripheral adhesions, where pp60v-src and tyrosine-phosphorylated proteins accumulated. Expression of the integrin was determined by the substratum composition when BY1 cells were cultured in serum-free culture medium. Under such conditions, BY1 cells expressed the peripheral adhesions within 3 hours on adhesion molecule-coated glass. On the other hand, in serum-containing medium, they first developed focal adhesions transiently at their early stage of adhesion, and then the peripheral adhesions were predominantly expressed within 12 hours. Podosomes were formed in a time course similar to that of the peripheral adhesions. These findings suggest that the peripheral adhesion is a class of stable adhesive structure distinct from the focal adhesion or podosome of BY1 cells. Similar close contact-type peripheral adhesions with the integrin were also observed in a variety of cultured cells such as normal fibroblasts at their logarithmic growth phase, phorbol ester-treated fibroblasts, and several malignant tumor cells, with poorly organized focal adhesions and stress fibers. These findings further suggest that the peripheral adhesions may be widely involved in the adhesion of cells that inadequately develop stress fibers and focal adhesions.

Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases.

We have used myelin basic protein immobilized in sodium dodecyl sulfate-polyacrylamide gels to identify protein kinases after gel electrophoresis, followed by protein kinase reactions. This technique has permitted us to detect three protein kinases in serum-deprived cells transformed by p60src. On induction of cellular transformation by a temperature-sensitive v-src, a p87 protein kinase is activated within 30 min and remains activated in fully transformed cells. The p63 protein kinase is not fully activated until 24 h but remains activated in transformed cells. The commonly studied p42MBPK is rapidly activated within 30 min, and its kinase activity decreases significantly by 24 h, when the p63 enzyme is fully activated. The p42MBPK, as well as the p63 and p87 enzymes, are stimulated by transforming p60c-src mutants but not normal c-src or nonmyristylated p60c-src. In addition, the kinase activity of p63 enzyme, but not of p42MBPK, can be induced in okadaic acid-treated chicken embryo fibroblasts, indicating that phosphatase 2A and/or phosphatase 1 may be involved in the regulation of its activity. Additional data indicate that either p42MBPK or p63 activity correlates with the stimulation of the protein kinase p90RSK. Thus, there may be two independent pathways leading to the activation of the RSK gene product.

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with 3H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.

Induction of hsp 72/73 by herbimycin A, an inhibitor of transformation by tyrosine kinase oncogenes

Herbimycin A, which has been known to inactivate and degrade p60 v-src tyrosine kinase, induced an elevated synthesis of a protein with a molecular size of 70 kDa in A431 human epidermoid carcinoma cells. This protein showed the same migration distance on SDS—polyacrylamide gel electrophoresis as that of the protein induced in the cells by heat shock treatment, and this 70-kDa protein was identified as a member of the heat shock protein 70 family (hsp70) through immunoprecipitation with anti-hsp72/73 antibody and partial digestion with V8 protease. The induced level of the 70-kDa protein was dependent on the length of period and the concentration of herbimycin A treatment. Cellular fractionation and indirect immunofluorescence analyses revealed that the 70-kDa protein induced by herbimycin A was localized in the cytoplasm, in contrast to the nuclear distribution of hsp70 induced by heat treatment. Induction of hsp70 by herbimycin A was also observed in several other cells, including HeLa S3 cells, chicken embryo fibroblasts, NIH3T3 cells, and Rous sarcoma virus-transformed NIH3T3 cells.

Cellular invasion into matrix beads: Localization of fa integrins and fibronectin to the invadopodia

We have examined the contribution of adhesion mechanisms to cell invasiveness by growing chicken embryo fibroblasts (CEF) or Rous sarcoma virus-transformed cells (RSVCEF) on fibronectin-coated crosslinked gelatin beads (FN-beads). RSVCEF attached more readily and spread more rapidly on FN-beads than CEF, suggesting an increase in the adhesion-related motility of the transformed cells. In addition, RSVCEF invaded the FN-beads, but CEF did not, by extending specialized cell surface protrusions called invadopodia at sites of cell invasion. FN removal by RSVCEF cultured on prelabeled fluorescent FN-beads (FL-FN) was evident at sites of invadopodia, and internalized FL-FN occurred in vacuoles near the ventral membrane of cells at sites of FN removal. The precise distribution of FN and integrins in cells invading FN-beads was determined by immunofluorescence and immunoelectron microscopy of frozen thin-sections. In both CEF and RSVCEF, beta 1 integrins and FN occupied separate intracellular compartments during the early stage of spreading on FN-beads. Later, beta 1 integrins were largely localized at the ventral cell surface of both CEF and RSVCEF. Polyclonal anti-integrin antibody recognizing beta 1 and several alpha chains, however, labeled both ventral and dorsal cell surfaces. During invasion by RSVCEF, beta 1 integrins were concentrated at extended invadopodia and also colocalized with internalized FL-FN material in phagocytic vesicles. Furthermore, secreted FN was deposited by RSVCEF at the base of invadopodia colocalizing with beta 1 integrin. Both FL-FN matrix removal and formation of the invadopodia were found to be resistant to treatment with GRGDS at concentrations that inhibit the interaction between cells and FN-beads. Thus, the localization of beta 1 integrins to the plasma membrane contacting immobilized FN results in an extremely tight cellular adherence to the matrix bead, that stabilizes invadopodia and also mediates endocytic clearance of degraded FN-matrix material.

Induction of morphological change by tyrosine kinase inhibitors in Rous sarcoma virus-transformed rat kidney cells

Erbstatin and methyl 2,5-dihydroxycinnamate, related tyrosine kinase inhibitors, induced a morphological change in temperature-sensitive Rous sarcoma virus-transformed rat kidney (RSV 15·NRK) that brought the cells close to the morphology of their normal counterpart. Erbstatin did not change the morphology of normal or Kirsten sarcoma virus-transformed rat kidney cells. Erbstatin also inhibited morphological transformation of RSV 15·NRK cells induced by a shifting in temperature. Actin stress fibres were observed only in normal cells and not in transformed cells. Erbstatin induced stress fibre organization in transformed cells. Erbstatin and methyl 2.5-dihydroxycinnamate increased fibronectin gene expression in RSV-transformed cells. Thus, tyrosine kinase inhibitors induced normal phenotypes specifically in v-sre-expressing cells.

Purification and Properties of Extracellular Matrix-Degrading Metallo-Proteinase Overproduced by Rous Sarcoma Virus-Transformed Rat Liver Cell Line, and Its Identification as Transin1

Rous sarcoma virus-transformed rat liver cell line RSV-BRL secreted a neutral proteinase in a latent precursor form with a molecular weight (Mr) of 57,000 (57k) as a major secreted protein. This enzyme was a calcium-dependent metallo-proteinase. The proenzyme was purified from the serum-free conditioned medium of the transformed cells by affinity chromatographies on a zinc chelate Sepharose column and a reactive red agarose column. When activated by treatment with trypsin or p-aminophenylmercuric acetate (APMA) in the presence of Ca2+, the purified enzyme effectively hydrolyzed casein, fibronectin, and laminin. Type IV collagen was hydrolyzed at 37 degrees C but not at 30 degrees C by the enzyme, whereas type I and type III collagens were hardly hydrolyzed even at 37 degrees C. The treatment with trypsin or AMPA in the presence of Ca2+ converted this 57k proenzyme to an active and stable enzyme with Mr 42k. In the absence of Ca2+, however, APMA converted the proenzyme to an intermediate form with Mr 45k, while trypsin digested it to an inactive peptide with Mr 30k. These results demonstrate that calcium ion is essential for the activation, activity expression, and stabilization of this metallo-proteinase. Analysis of its partial amino acid sequence and amino acid composition showed that the 57k proenzyme was identical or closely related to the putative protein transin, a rat homologue of stromelysin.

Paxillin: a new vinculin-binding protein present in focal adhesions.

The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pIs ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of approximately 85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60src in Rous sarcoma virus-transformed cells (Glenney, J. R., and L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this interaction with vinculin suggests paxillin may be a key component in the control of focal adhesion organization.

Myristoylated [formula omitted]-oncogene products are potently detected by the monoclonal antibody to the NH 2-terminal myristoyl-Gly-Ser-Ser-Lys [formula omitted]-peptide

Monoclonal antibodies were raised against a synthetic NH 2-terminal myristoyl tetrapeptide ( N -myristoyl-Gly-Ser-Ser-Lys) which is characteristic of an NH 2-terminal portion of pp60 src , the transforming protein of src -oncogene. The antibody reacted with the albumin conjugated with both the N -myristoyl and N -lauroyl-tetrapeptides, but concentrations at which 50% of the immunoreaction was inhibited were 5 pmol for the N -myristoyl and 830 pmol for N -lauroyl tetrapeptidyl albumin. On the other hand, N -palmitoyl tetrapeptidyl and underivatized albumin, and Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys octapeptide had no effects. These results suggest a high affinity of the antibody for an N -myristoyl-Gly-Ser-Ser-Lys moiety. src -Oncogene products in Rous sarcoma virus-transformed cells and human colon carcinoma tumor cells were selectively identified as myristoylated pp60 src by immunoprecipitation analyses with the antibody.

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.

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.

Processing of 9E3 mRNA and regulation of its stability in normal and Rous sarcoma virus-transformed cells.

We studied the expression of 9E3 mRNA, which is known to be induced in chicken embryo fibroblasts by p60v-src activity and by serum. In addition to full-length 9E3 mRNA, we identified several smaller RNAs that hybridized with 9E3 cDNA. One of these RNAs hybridized with a 5' 9E3 cDNA probe but not with a 3' cDNA probe. The other hybridized with a 3' cDNA probe but lacked 5' sequences, including the entire 9E3 coding region. Only the latter RNA was polyadenylylated, as determined by RNase H digestion in the presence of oligo(dT). The level of the small RNAs increased after treatment with cycloheximide and actinomycin D, indicating that the small RNAs were produced by processing of preexisting transcripts. The derivation of the small RNAs from 9E3 mRNA rather than from a related gene was confirmed by S1 nuclease analysis. The 3' terminus of the 5' RNA and the 5' terminus of the 3' RNA mapped to the same position, which suggested that the small RNAs were formed by endonucleolytic cleavage of 9E3 mRNA at a specific site in the 3' noncoding region. We also found that the stability of 9E3 mRNA was increased after serum stimulation and was greater in Rous sarcoma virus-transformed than in uninfected cells. The relative amount of the small RNAs as compared with the full-length transcript was greatest under conditions in which the full-length transcript was least stable. These data suggest that site-specific endonucleolytic cleavage regulates the stability of 9E3 mRNA.