E A Garber

A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation.

We have constructed mutants by using linker insertion followed by deletion in the region of cloned Rous sarcoma virus DNA coding for the N-terminal 9 kilodaltons of the src protein. Previous work implicated this region in the membrane association of the protein. The mutations had little effect on src tyrosine kinase activity. Substitution of a tri- or tetrapeptide for amino acids 15 to 27, 15 to 49, or 15 to 81 had little effect on the in vitro transforming capacity of the virus. Like wild-type p60src, the src proteins of these mutants associated with plasma membranes and were labeled with [3H]myristic acid. In contrast, a mutant whose src protein had the dipeptide Asp-Leu substituted for amino acids 2 to 81 and a mutant with the tripeptide Asp-Leu-Gly substituted for amino acids 2 to 15 were transformation defective, and the mutant proteins did not associate with membranes and were not labeled with [3H]myristic acid. These results suggest that amino acids 2 to 15 serve as an attachment site for myristic acid and as a membrane anchor. Since deletions including this region prevent transformation, and since tyrosine kinase activity is not diminished by the deletions, these results imply that target recognition is impaired by mutations altering the very N terminus, perhaps through their effect on membrane association.

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.

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.

Processing of p60v-src to its myristylated membrane-bound form

p60src of wild-type Rous sarcoma virus is myristylated at its N-terminal glycine residue. We have shown previously that this myristylation is necessary for p60src membrane association and for cell transformation by using src mutants with alterations within the N-terminal 30 kilodaltons of p60src. In this study we analyzed the process of p60src myristylation in wild type- and mutant-infected cells. All myristylated src proteins examined lack the initiator methionine, but two mutant src proteins lacking the initiator methionine are not myristylated, indicating that removal of the initiator methionine and myristylation are not obligatorily coupled. Analysis of the kinetics of myristylation and the association of p60src with cellular proteins p50 and p90 indicated that myristylation occurs before p60src becomes membrane associated and that transient association with p50 and p90 occurs regardless of myristylation. Myristylation is required for stable association of p60src with the plasma membrane but is not sufficient for membrane association. A mutant with an src deletion of amino acids 169 through 264 has an src protein that is myristylated but not membrane bound, remaining stably associated with p50 and p90. This mutant is transformation defective. Several N-terminal deletion mutants possessing tyrosine kinase activity have myristylated and membrane-bound src proteins but are not fully active in cell transformation, suggesting that additional N-terminal functional domains exist.

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein.

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.

Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.

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.

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.