Allan R. Goldberg

Increased protease levels in transformed cells: A casein overlay assay for the detection of plasminogen activator production

Abstract Using a casein-agarose overlay assay, Rous sarcoma virus-transformed chick embryo fibroblasts show a markedly higher level of plasminogen activator activity than do normal, untransformed chick embryo fibroblasts. Infection by avian sarcoma viruses of subgroups A, B, and C, but not avian leukosis viruses of groups A and B, elevates the level of plasminogen activators produced by the fibroblasts. The activators produced by transformed chick embryo fibroblasts can convert plasminogen from calf, fetal calf, as well as chicken serum, to plasmin. Soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, and fetuin inhibit plasmin mediated caseinolysis. The active-site alkylating agents TLCK, TPCK, APB, GPB, ZPBK, ZLCK, ZPCK also are inhibitory. Activation is specific for plasminogen; the zymogens trypsinogen, chymotrypsinogen, and pepsinogen cannot be activated.

Proteolytic cleavage by plasmin of the HA polypeptide of influenza virus: Host cell activation of serum plasminogen☆

Abstract Cleavage of the HA polypeptide, the largest glycoprotein of the WSN strain of influenza virus, to polypeptides HA1 plus HA2 occurs when influenza virus is grown in MDBK cells in medium containing 2% calf serum, but does not occur in serum-free medium. Addition of highly purified plasminogen in concentrations as low as 1.5 μg/ml to serum-free medium results in complete cleavage. The release of plasminogen activators by the cell is essential for the activation of the zymogen and cleavage of the HA polypeptide. Either chick or calf plasminogen can be converted to the active enzyme, plasmin, by activators produced by either infected or uninfected MDBK cells. Cleavage of the HA polypeptide by plasmin can be prevented by trypsin inhibitors from bovine pancreas (Kunitz) or soybean. Cleavage does not occur in the presence of 2% calf serum from which plasminogen has been removed specifically by affinity chromatography, indicating that plasminogen is the only serum component involved in the conversion of the HA polypeptide to HA1 plus HA2. The results obtained indicate that in the MDBK cell-WSN strain system, cleavage of the HA polypeptide can be explained by the activation of serum plasminogen by cellular activators, and that cleavage of the influenza virus hemagglutinin polypeptide is a sensitive indicator of the production of plasminogen activators by cells.

Evidence that the src gene product of rous sarcoma virus is membrane associated

Abstract We have investigated the intracellular localization of the src phosphotransferase and of pp60src by cellular fractionation. Fractionation of cell lysates by differential centrifugation showed that both the phosphotransferase activity, measured using an immune complex assay, and pp60src cosedimented with particulate fractions enriched in cellular membranes. Upon further fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients the src phosphotransferase and pp60src fractionated in parallel with plasma membranes. The phosphotransferase activity remained associated with cellular membranes under conditions of high ionic strength or of high concentrations of divalent cation chelators further supporting a direct membrane association. The src phosphotransferase activity is higher in buffer containing only Triton X-100 detergent as compared to a more complicated detergent mixture, but under these assay conditions the pp60scr appears to undergo proteolysis to a 52K-dalton polypeptide (pp52src) which is active as a phosphotransferase. Pyrimidine triphosphates were shown to act as donors in the immune complex assay of src phosphotransferase activity. [γ-32P]CTP was about one-third as efficient as [γ-32P]ATP in serving as a donor. Both CTP and UTP were effective inhibitors of src phosphotransferase activity when either radiolabeled ATP or GTP were used as substrates. The ability of src to interact efficiently with both purine and pyrimidine triphosphates contrasts markedly with most well-studied protein kinases which have been shown to use almost exclusively purine triphosphates.

Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity.

We have suggested previously that the amino-terminal 8 kilodaltons of pp60src may serve as a structural hydrophobic domain through which pp60src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, ion contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60src, and suggest that the amino terminus of pp60src may have a critical role in the promotion of in vivo tumorigenicity.

Subcellular localization of pp60src in RSV-transformed cells.

In 1911 Rous demonstrated that a retrovirus, subsequently named Rous sarcoma virus (RSV), could cause a tumor in a chicken. Nearly 60 years elapsed before it was shown that a specific virus function encoded by the src gene of RSV was required for the maintenance of the transformed state. Martin (1970) isolated a temperature-sensitive RSV mutant whose properties indicated that the viral transforming gene product is required for transformation, but not for viral replication. Seven years later, Martin’s genetic experiment was provided with a biochemical foundation when Brugge and Erikson (1977) showed that the transformation gene src encoded a 60 000–dalton (60-kd) protein present in RSV-transformed cells. Since 1977, many genes capable of inducing oncogenic transformation, and hence given the generic name onc, have been recognized, and many of the oncogenes’ protein products have been identified. The molecular biology of retroviruses is reviewed extensively and in great detail by Weiss et al. (1982), and the reader is referred to this volume and numerous reviews referenced therein on specific aspects of retrovirology.

Effects of the src gene product on microfilament and microtubule organization in avian and mammalian cells infected with the same temperature-sensitive mutant of Rous sarcoma virus.

Abstract We have studied cytoskeletal organization in both mammalian (rat kidney cells) and avian cells (chick embryo fibroblasts) that have been transformed with temperature-sensitive src gene mutants of Rous sarcoma virus. The functioning of the src gene in rat cells affected the organization of actin-containing microfilament bundles, but not microtubules at the permissive temperature for transformation. These cells formed colonies in soft agar at permissive temperature, but not at nonpermissive temperature. In contrast, the src gene product affected both microtubule and microfilament organization in chick embryo fibroblasts at permissive temperature.

Novel localization of pp60src in rous sarcoma virus-transformed rat and goat cells and in chicken cells transformed by viruses rescued from these mammalian cells

Abstract We have investigated by indirect immunofluorescence and subcellular fractionation the intracellular location of pp60 src in RSV-transformed mammalian cells and in CEF cells transformed by virus rescued from these cells. Two independently derived cell lines were examined: RR1022 cells isolated from an in vivo sarcoma induced in an Amsterdam rat by infection with SR-RSV-D; and Pcl, cells isolated from a soft agar colony of normal goat skin fibroblasts transformed in vitro by SR-RSV-D. Transforming viruses (RSV-RR and RSV-Pcl) were rescued from RR1022 and Pcl cells by fusion with CEF cells. Immunofluorescence microscopy showed association of pp60 src with the nuclear envelope and the juxtanuclear reticular membranes in the transformed mammalian cells and in CEF cells transformed by the rescued viruses, in contrast to the plasma membrane localization of pp60 src seen in SR-RSV-transformed CEF cells. Results of subcellular fractionation by differential centrifugation and fractionation of particulate fractions by equilibrium centrifugation in discontinuous sucrose gradients were in agreement with the differences in pp60 src distribution observed by immunofluorescence microscopy. Although the mammalian cell lines were independently derived, pp60 src s isolated from RR1022 and Pcl cells both lacked amino-terminal 21- and 18-kilodalton [ 35 S]methionine S. aureus V8 protease peptides found in SR-RSV-D pp60 src . Proteolytic peptides identical to those of pp60 src from the mammalian cells were obtained from pp60 src proteins isolated from rescued virus-transformed CEF cells, suggesting that the alteration in the amino-terminal half of the src protein represents a stable change, and that an alteration in the primary structure of pp60 src is responsible for the altered intracellular membrane localization of pp60 src in these cells.

INVOLVEMENT OF CELL AND SERUM PROTEASES IN EXPOSING TUMOR‐SPECIFIC AGGLUTININ SITES*

Animal cells transformed by DNA or RNA containing tumor viruses are more sensitive to agglutination by certain lectins than comparable normal cells.lr These lectins, especially the wheat germ agglutinin isolated from wheat germ3 and concanavalin A4 isolated from jack bean meal, appear to react with carbohydrate determinants on the cell surface. Presumably the enhanced agglutinability of cells transformed by oncogenic viruses results from an alteration of the cell surface, which may involve an increase in the number of agglutinin-binding sites at the cell surface, an alteration in their distribution, or other as yet undefined alterations in the molecular structure of the membrane. Changes in agglutinability, on the transformation or reversion of transformed cell lines to a greater degree of contact inhibition, are usually accompanied by changes in growth properties. It has been suggested by Burger that the surface alteration detected through the change in agglutinability is necessary for the alteration in growth properties that follows viral transformation. In fact, mild and brief proteolytic treatment of normal cells, which have been contact-arrested both in growth and movement, causes these cells to overgrow and to become agg1utinable.l. * It therefore appeared relevant to ask the following questions: (1) Can inhibitors of proteases prevent the agglutinability of transformed cells? (2) Do transformed cells elaborate substances that cause cells both to undergo multiplication and to become agglutinable? Several protease inhibitors were tested for their ability to affect the agglutinability of several different transformed cell lines (TABLE 1). Agglutination was scored on a serological scale, from 0 to (++++). Under the conditions of this experiment all transformed cells, including polyomaand SV4O-transformed 3T3 cells, Schmidt-Ruppin-Rous sarcoma virus-transformed chick embryo fibroblasts, and L1210 cells, a mouse lymphocytic leukemia, agglutinated in the presence of a homogeneous preparation of wheat germ agglutinin (WGA) that had been purified by affinity chromatography on ovomucoidSepharose.6 Normal 3T3 fibroblasts and normal chick embryo fibroblasts do not agglutinate at a WGA concentration of 20 pg/ml, which causes tumor cells to agglutinate. Inhibitors were incubated with cells for 6 hours and agglutination was scored 18 hours later. Ovomucoid, a macromolecular inhibitor of trypsin, blocked agglutination of all transformed cells when used at 50 pglml. Several inhibitors capable of active-center alkylation of proteases were tested. The chloromethyl ketone (TLCK) prevented agglutination of SV40 and L1210 cells but had no effect on the agglutinability of polyoma-transformed 3T3 cells.

Temperature-sensitive membrane association of pp60src in tsNY68-infected cells correlates with increased tyrosine phosphorylation of membrane-associated proteins

Incubation of membrane vesicles from normal and Rous sarcoma virus-transformed chick embryo fibroblasts (CEF) with [gamma-32P]ATP resulted in the phosphorylation of a large number of proteins. The major differences observed between the membrane vesicles of untransformed and transformed cells were: (1) a 5- to 10-fold increase in the proportion of labeled phosphotyrosine in transformed vesicles and (2) the phosphorylation of pp60src in vesicles from transformed cells. Of the many proteins labeled in vitro, only pp60src was immunoprecipitated by TBR serum. Phosphorylation of the immunoprecipitated pp60src occurred on tyrosine in the 26-kDa carboxy-terminal Staphylococcus aureus V8 protease fragment. pp60src was not phosphorylated in vitro in membrane vesicles prepared from tsNY68-infected cells grown at the nonpermissive temperature. The proportion of labeled phosphotyrosine in membrane proteins from tsNY68-infected cells grown at the nonpermissive temperature was only slightly increased relative to that observed in membranes prepared from normal cells. Subcellular fractionation indicated that while pp60src was membrane associated in tsNY68-infected cells grown at the permissive temperature, pp60src was chiefly soluble in tsNY68-infected cells grown at the nonpermissive temperature. Temperature-sensitive membrane association of pp60src in tsNY68-infected cells was also observed by indirect immunofluorescence microscopy. When membranes were prepared from tsNY68-infected cells that had been downshifted from the nonpermissive to the permissive temperature, the reappearance of in vitro phosphorylated pp60src and the increase in the proportion of labeled phosphotyrosine in membrane vesicles correlated with the kinetics of src immune complex kinase reactivation and membrane association of pp60src.

Properties and mechanism of action of viral and cellular tyrosyl protein kinases

We have demonstrated the usefulness of several angiotensin analogs as substrates for a number of tyrosyl protein kinases of viral or cellular origin. Results of initial rate studies with pp60src at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 microM and 0.24 microM respectively and Vmax was 1.0 nmol/min/mg. The end-product ADP and the ATP analog, AMP-PNP, were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [delta Phe4]-angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. The available evidence allowed us to conclude that while pp60src contained essential histidine and lysine residues in its active site, the kinase reaction does not involve a phosphoryl enzyme intermediate. Phosphorylation of the angiotensin peptides in vitro also has allowed us to demonstrate the presence of at least two tyrosyl protein kinases in the cytoplasm of normal rat liver cells. These kinases appear to be novel in that they are present in normal cells and are not stimulated by growth factors. Also, results of preliminary experiments indicate that these kinases are not immunologically related to the transforming gene products of Rous and Fujinami sarcoma viruses (unpublished observations). The identification of these new kinases represents another application of the angiotensin peptides as substrates for tyrosyl kinases (13). The results obtained do not exclude the possibility that there exist in rat liver other tyrosyl kinases that do not phosphorylate these particular peptide substrates. No attempt has been made to characterize tyrosyl kinases associated with the plasma membrane fraction. Although they represent only a small fraction of the total activity of liver cells, the plasma membrane kinases have a relatively high specific activity. These kinases may be identical with growth factor receptor kinases previously identified in liver cell membranes (5). The most abundant tyrosyl kinase in rat liver cytoplasm has a molecular weight of 75,000 daltons and was found in cytosol and microsomal salt-wash fraction. The observation that the purified 75 Kd enzyme phosphorylates a 75 Kd protein on tyrosine residues suggests that the enzyme may possess autophosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)