Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins.

We have isolated Swiss 3T3 subclones that are resistant to the mitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen-activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v-Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Rac-mediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.

Perturbed hemopoiesis and the generation of multipotential stem cell clones in src-infected bone marrow cultures is an indirect or transient effect of the oncogene.

Multipotential stem cell lines, derived specifically from long-term bone marrow cultures infected with a recombinant retrovirus carrying v-src, lack v-src. Stable consequences thus result from transient actions or indirect effects of v-src on other cells, with the latter possibility being favored by its mosaic expression in marrow cultures.

Expression of the v-Src oncoprotein in fibroblasts disrupts normal regulation of the CDK inhibitor p27 and inhibits quiescence.

To determine how an oncogenic tyrosine kinase disturbs cell cycle control we examined expression of cell cycle proteins and growth of fibroblasts reversibly transformed by a temperature sensitive mutant of v-Src (ts LA 29). ts LA 29 Rat-1 cells and normal Rat-1 cells had similar growth rates but the transformed cells traversed the G1 phase of the cell cycle more rapidly and failed to exit cycle efficiently in response to serum starvation and cell confluence. Cyclin D1 and cyclin E levels were not elevated in growing ts LA 29 Rat-1 cells and the abbreviated G1 was further accelerated by overexpression of cyclin E. A fall in cyclin E and cyclin A dependent kinase activities in Rat-1 cells in response to inhibitory growth conditions was abrogated in ts LA 29 Rat-1 cells and correlated with lack of p27 accumulation or cyclin A down regulation, the latter due to sustained cyclin A promoter activity. The expression of p27 mRNA was lower in ts LA 29 Rat-1 cells than Rat-1 cells and was elevated following v-Src inactivation concurrent with an increase in p27 promoter activity and temporary cell cycle exit. The suppression of mRNA or transcription is a novel way an oncoprotein can induce down regulation of p27 and contributes to the G1 shortening and perturbed cell cycle regulation of the v-Src transformed cells.

Mitogenesis of quiescent chick fibroblasts by v-Src: dependence on events at the membrane leading to early changes in AP-1.

Activation of rapidly reversible temperature-sensitive (ts) v-Src in quiescent chicken embryo fibroblasts (CEFs) results in both morphological transformation and exit from G0 to G1, resulting in mitosis. This phenomenon permits examination of cellular responses very soon after activating the oncoprotein, and we have used this to study changes in endogenous AP-1, and the regulation of its major components, in the first few hours after activating v-Src. This approach contrasts with a number of studies that have demonstrated enhanced activity of exogenously added AP-1 components in cells transformed by v-Src. Reactivation of a membrane-associated tyrosine kinase (tsRCAN-29) results in a several-fold increase in AP-1 DNA binding and a similar increase in the activity of an AP-1-responsive reporter soon after temperature shift. c-Jun and c-Fos are regulated at a number of levels in response to both stimuli. In quiescent RCAN-29-infected CEFs stimulated into cycle by shift to permissive temperature, c-fos transcripts are elevated by 15 min and remain above basal level for at least 4 h. Serum induces much greater elevation of c-fos transcripts, although this response is transient. Despite the difference in magnitude of the transcript responses, the stimulation of nuclear c-Fos protein is similar in both serum and v-Src-stimulated cultures. No elevation in c-jun transcripts or nuclear c-Jun protein level is evident in v-Src-stimulated quiescent CEFs. However, there is an early change in the tryptic phosphopeptide map of p39 c-Jun in response to both v-Src and serum. Upon stimulation we observed a novel redistribution of phosphate in the carboxy-terminal tryptic phosphopeptide that may be responsible in part for the increase in AP-1 DNA binding. Phosphorylation of amino-terminal serines 63 and 73 on peptides Y and X, believed to be responsible for regulation of the transactivation function of c-Jun, is constitutively high in resting CEF cultures; stimulation with serum or v-Src results in only a modest increase in phosphorylation at these sites. Significantly, reactivation of a non-myristylated, transformation-defective version of the tsRCAN-29 v-Src protein (RCAN-29A2) is unable to induce resting CEFs to re-enter cycle. In addition, this mutant fails to induce early increases in AP-1 activity, implying that these nuclear changes require crucial signalling events at the cell periphery, and that these events correlate with the biological consequences of expression of v-Src.

The mechanism of cell cycle regulation by v-Src.

The tyrosine kinase oncoprotein v-Src can overcome the requirements for serum growth factors and anchorage which restrain normal cell growth. Here we investigated the biochemical mechanisms whereby v-Src induces quiescent cells to enter S phase in the absence of serum mitogens. Activating a temperature sensitive v-Src in quiescent cells sequentially induced cyclins D1, E and A and also down regulated p27. We addressed whether p27 down regulation was required to activate cyclin D1/CDK4/6 or cyclin E/CDK2 by engineering cells with inducible p27. Both S phase entry and activation of cyclin/CDKs were inhibited by over expression of p27. Using cells engineered with inducible p16 we showed that Cyclin D/CDK4/6 activity was required for v-Src to increase expression of cyclin A but not cyclin E. To determine which downstream kinases mediated these effects of v-Src we added pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3-K), LY294002 or mitogen activated protein kinase kinase (MEK), U0126. PI3-K was required for v-Src to activate MEK and MEK was required for v-Src to increase expression of cyclins D1 and E. However, the MEK inhibitor prevented p27 protein down regulation whereas the PI3-K inhibitor did not. This was because reduced PI3-K activity lead to proteolytic degradation of p27.

Mitogenesis induced by pp60v-src is not accompanied by increased expression of immediate early response genes.

Rat-1 cells infected with a temperature sensitive mutant of RSV (ts LA 29 Rat-1) can be rendered quiescent by serum deprivation at restrictive temperature. Shift to permissive conditions activates the v-src protein tyrosine kinase within 10 minutes and either this stimulus, or serum addition at restrictive temperature, leads to progression of the cell from G0 to G1, S-phase and mitosis. The effects of serum and temperature shift are not synergistic, suggesting that they may operate by convergent mechanisms. However, the characteristic serum-stimulated transient increases in transcripts of three immediate early response genes, c-fos, c-jun and c-myc are absent or much reduced when mitogenesis in ts LA 29 Rat-1 is induced by pp60v-src. Nonetheless, upon activating the pp60v-src protein kinase there is a marked and rapid increase in the ability of ts LA 29 Rat-1 nuclear extracts to retard the gel migration of oligonucleotides containing the AP-1 binding site, indicating that pp60v-src activity leads to an enhanced functioning of Fos and Jun related proteins that may, in turn, affect their transcriptional activation. Furthermore, these findings, and comparison with those of other laboratories, suggest that the mitogenic and transforming activities of pp60v-src have different effects on the transcription of immediate early response genes.

v-Src-induced degradation of focal adhesion kinase during morphological transformation of chicken embryo fibroblasts.

Morphological transformation of cells by the v-Src tyrosine kinase is incompletely understood. However, it is independent of nuclear functions and probably involves phosphorylation of targets associated with the cytoskeleton and focal adhesions, structures which tether the cytoskeleton to the points of cell attachment. v-Src activity both stimulates tyrosine phosphorylation of a tyrosine kinase present in focal adhesions (focal adhesion kinase or pp125FAK) and disrupts focal adhesions, leading to cell rounding and detachment. However, pp125FAK is also phosphorylated on tyrosine as a result of integrin stimulation which induces quite different biological consequences including the organisation of focal adhesions when cells spread on fibronectin (reviewed in Schaller and Parsons, 1993). To address this paradox, we examined changes in pp125FAK during activation and shut-off of temperature sensitive mutant v-Src proteins that induce varying degrees of transformation in chick embryo fibroblasts. An efficiently transforming v-Src mutant initially stimulated pp125FAK tyrosine phosphorylation, but induced subsequent pp125FAK degradation prior to the onset of cell rounding and detachment. v-Src mutants which are impaired in their ability to induce morphological transformation were much less efficient at inducing degradation of pp125FAK. Moreover, cell spreading during restitution of normal morphology did not require detectable tyrosine phosphorylation of pp125FAK, or its potential substrate paxillin, suggesting that pp125FAK may function more in the turnover of focal adhesions than in their assembly.

Transcription of Rous sarcoma proviruses in rat cells is determined by chromosomal position effects that fluctuate and can operate over long distances.

Most Rous sarcoma proviruses integrated in rat DNA are inactive. The active minority are not necessarily a consequence of long terminal repeat mutations and their transcription can fluctuate in parallel with previously demonstrated transitions in chromatin configuration. Transcriptional alternations cannot be attributed to variations in positive or negative factors that operate independently of the site of provirus integration. Moreover, distinct proviruses in the same cell can be differentially expressed and flanking cellular elements can act over several kilobases to inhibit provirus transcription. We postulate that position-dependent fluctuations in proviral repression can be mediated by trans-acting factors, and DNA transfections indicate that initiation and maintenance of this phenomenon are separable entities. Provirus activity presumably reflects otherwise inapparent cell controls that affect chromatin structure over long distances.

Effects of inhibitors of lipid synthesis on the replication of Rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins.

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholestetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1(-14C)]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [35S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examinatin of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.

Cell surface and metabolic labelling of the proteins of normal and transformed chicken cells.

We have studied the surface proteins of normal and transformed chick cells using four-labelling techniques with different specificities, (a) lactoperoxidase catalysed iodination (b) galactose oxidase/B3H4 (c) pyridoxal phosphate/B3H4 and (d) periodate/B3H4. All methods labelled a large external transformation-sensitive (LETS) protein, in agreement with previous studies. In addition, using galactose oxidase and periodate labelling techniques, we present evidence which suggests that the transformed cell surface glycoproteins are more sialylated. The LETS protein was also labelled with (14C) glucosamine and after trypsinization a small band of identical molecular weight to LETS remained, possibly representing an internal pool of the protein. In contrast LETS protein labelled with (3H) fucose was completely removed by trypsin, suggesting that the internal pool of the protein is incompletely glycosylated. Evidence is also presented to show that although the level of the protein is drastically reduced at the transformed cell surface, it is still synthesised and shed into the medium.

A single point mutation has pleiotropic effects on pp60v-src function.

The Rous sarcoma virus mutant tsLA29 encodes a pp60v-src molecule that is temperature sensitive for both tyrosine kinase activity and its ability to locate at the cell periphery. The defect in localization appears to be due to a perturbation in events following complex dissociation, since the mutant enzyme shows a rapidly reversible association with the cytoskeleton when shifted between permissive and restrictive temperatures. Although tsLA29 pp60v-src differs from the wild type at three amino acid residues, studies with chimeric proteins show that only one of the mutations, an alanine-for-proline substitution at residue 507, accounts for all the temperature-sensitive characteristics. Moreover, a single second site mutation, at residue 427, can restore the wild phenotype. Cells infected with a chimeric virus encoding only the alanine substitution at position 507 have a conspicuously fusiform morphology, suggesting that this mutation also has subtle effects on pp60v-src function that are apparently compensated for by the other mutations in native tsLA29.

Integrated proviruses as probes for chromosomal position effects in mammalian cells and their hybrids.

Proviruses are susceptible to transcriptional regulation by cellular influences operating at their integration sites. Two experimental systems have been exploited to analyse such chromosomal position effects but have identified contrasting molecular mechanisms. We propose a reconciliation of these apparently paradoxical results and suggest ways in which proviruses may serve as sensors for the further analysis of normal mammalian gene regulatory mechanisms.

The genome of RNA tumor viruses: a functional requirement for a polyploid structure?

This paper points out certain theoretical problems in DNA synthesis associated with antiprimer transcription and with circularization that could oblige RNA tumor viruses to rely on a polyploid genome. It is suggested that each completed act of reverse transcription may be coupled with an act of genetic recombination aimed at recovering the antiprimer information from an adjacent genome subunit in a polyploid train. A partially double-stranded DNA transcript could then be formed with sufficient terminal redundancy to permit circularization. The model provides satisfactory explanations for observed genetic interactions (particularly recombination and heterozygote formation), for inactivation data and for selective subunit transcription.