Pp60src Kinase Activity Research Articles

Specific signaling cascades involved in cell spreading during healing of micro‐wounded gastric epithelial monolayers

Mechanisms that specifically modulate cell spreading and/or cell migration following epithelial wounding are poorly understood. Using micro-wounded human gastric epithelial monolayers, we show herein that EGF and TGFalpha maximally increase spreading of epithelial sheets under a cell proliferation-independent mechanism. Treatment of confluent HGE-17 cells with the phosphatidylinositol 3-kinase inhibitor, LY294002, and the epidermal growth factor receptor inhibitor, PD153035, strongly reduced basal and TGFalpha-stimulated cell spreading. While pharmacological inhibition of pp60src-kinase activity also attenuated basal epithelial spreading, addition of the mTOR/p70S6K inhibitor rapamycin or a specific siRNA targeting ILK sequence did not affect the kinetic rates of wound closure. Epithelial wound healing was initiated by actin purse-string contraction followed by lamellae formation. Conversely, disruption of actin and tubulin stability with cytochalasin D and nocodazole, respectively, inhibited epithelial sheet spreading. Finally, antibodies directed against the alpha3 integrin subunit, but not against the alpha6 or alpha2 subunits, attenuated epithelial sheet spreading as well as lamellae formation. In conclusion, the current investigation establishes that EGF/TGFalpha and the alpha3beta1 integrin, pp60c-src, EGFR and PI3K pathways are mainly associated with the cell spreading of the restitution process during healing of human gastric epithelial wounds.

The Src signaling pathway regulates osteoclast lysosomal enzyme secretion and is rapidly modulated by estrogen.

To investigate the role of the pp60src signaling pathway in osteoclast activity, we have used dominant negative pp60src, c-ras, and c-raf expression vectors to individually disrupt their functions in osteoclasts. Osteoclasts were transiently transfected and secretions of cathepsin B/K and tartrate-resistant acid phosphatase (TRAP) were monitored. Expression of these constructs increased secretion of lysosomal enzymes. In contrast, constitutively active pp60src expression caused decreased lysosomal enzyme secretion. Osteoclasts respond to 17-beta estradiol (17betaE2) treatment with decreased lysosomal enzyme secretion. Therefore, we investigated the effects of E2 on pp60src kinase activity and observed an E2 time- and dose-dependent decrease in cytoskeletal membrane-associated pp60src tyrosine kinase activity. We have shown that estrogen decreases lysosomal enzyme gene expression and secretion; so we have examined the effects of the expression constructs on estrogen regulation of enzyme secretion. Constitutively active pp60src blocked E2 effects on secretion whereas expression of dominant negative pp60src, c-Ras, or c-Raf enhanced E2 effects. These data support that the kinase domain of cytoskeletal-associated pp60src is likely to be involved in the regulation of lysosomal enzyme secretion.

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells.

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.

Early Activation of Endogenous pp60src Kinase Activity during Neuronal Differentiation of Cultured Human Neuroblastoma Cells

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.

Altered pp60 src kinase activity in regressing tumors induced by Rous sarcoma virus

Tumors which are induced in chickens by Rous sarcoma virus (RSV) usually grow progressively for several weeks and then regress. pp60 src kinase activity was found to be reduced in cultured tumor cells which derived from regressing sarcomas by 60–75% as compared to progressively growing neoplasms. We further found that a cellular pp89 which co-precipitated with pp60 src was largely diminished in tumor cells derived from regressing as compared with progressively growing sarcomas. In contrast, immunoprecipitation analyses from membrane or cytosol fractions did not reveal any significant differences between these 2 types of tumor cells in distribution of pp60 src. Moreover, partial proteolysis or isoelectric focussing of labelled immunoprecipitated pp60 src did not reveal major differences between the 2 tumor cell types. These data indicate that the lack of pp89 cell binding to pp60 src in regressing sarcoma cells correlates well with the diminution of the pp60 src kinase activity in these cells, but does not affect the transport of pp60 src to the plasma membrane.

Expression of avian sarcoma virus genes in tumor cells derived from different periods of tumor growth

Avian sarcoma virus (ASV)-induced tumor cells that are derived from regressing neoplasms contain about 75% less pp60 src kinase activity than do progressively-growing tumor cells. Paradoxically, the polyadenylated RNAs extracted from ‘regressor’ cells found in regressing neoplasms are much more productive than those from progressively-growing tumors in a reticulocyte lysate in vitro translation system, as determined both by time course and concentration curve studies. Electrophoretic analysis of polypeptides corresponding to known ASV proteins further suggests that the translation product from the mRNAs of regressing tumor cells contains much more viral protein than does that derived from progressively-growing sarcoma cells. The identity of two of these viral proteins was confirmed by immunoprecipitation of the respective translation products, using a tumor-bearing rabbit serum with specificity for pp60 src and Pr76 gag precursor polypeptides, and showed that much more of pp60 src and Pr76 gag were produced by the mRNAs from regressor than from progressor cells. We further found that ASV genes including genomic and subgenomic species (8.6, 5.0 and 3.2 kb) were present in greater amounts in regressing as opposed to progressively-growing tumor cells. These results support the notion that viral RNAs are accumulated in ‘regressor’ tumor cells.

Pp60 src-Dependent protein phosphorylation in membranes from Rous sarcoma virus-transformed chicken embryo fibroblasts

The Rous sarcoma virus (RSV)-transforming protein, pp60 src , is a plasma membrane-associated tyrosine-specific protein kinase. A 36,000-Da cellular polypeptide (p36) which is phosphorylated at tyrosine in RSV-transformed chicken embryo fibroblasts (RSV-CEF) is also plasma membrane associated. To determine if p36 is directly phosphorylated by pp60 src , and to examine the effects of mutations within src on pp60 src phosphorylation and kinase activity in situ in the plasma membrane, src-dependent protein phosphorylation in membranes isolated from RSV-CEF has been characterized. These membrane preparations contained high ATPase and phosphoprotein phosphatase activities; but when sufficient concentrations of [γ- 32P]ATP were used, the phosphorylation of pp60 src and the phosphorylation of p36 were linear for 1 min or more, and the initial rates of phosphorylation could therefore be determined. In membranes from RSV-CEF pp60 src and p36 became phosphorylated predominantly at tyrosine, while in membranes from uninfected cells p36 was phosphorylated at low levels at serine. When membranes from RSV-CEF were preincubated with tumor-bearing rabbit (TBR) serum, the IgG became phosphorylated while the phosphorylation of p36 was inhibited, suggesting that p36 is directly phosphorylated by pp60 src . Phosphorylation of pp60 src , p36, and TBR-IgG was dependent on growth temperature in membranes from cells infected by a temperature-sensitive mutant, tsNY68, although some dependence on growth temperature was observed even with membranes from wild-type RSV-infected cells. However, at the nonpermissive temperature, tsNY68 pp60 src retained 20–40% of its kinase activity, providing supporting for the proposal (B. M. Sefton, T. Hunter, and K. Beemon (1980, J. Virol, 33, 220–229) that transformation may result from a small quantitative change in pp60 src activity. The phosphorylation of pp60 src and its kinase activity were not coordinately affected by growth temperature or mutations within src, indicating that different factors affect the phosphoacceptor capacity and kinase activity of the protein.

Immunological responsiveness against tumors induced by avian sarcoma virus: reduced expression of pp60src kinase activity in regressing tumors.

Tumors which are induced in chickens by avian sarcoma virus frequently grow progressively for several weeks and then regress. We showed that tumor cells which are derived from the progressively growing phase of tumor growth produce large quantities of progeny-transforming virus, are reactive with antiviral antibody, and are susceptible to lysis in cell-mediated cytotoxicity assays by splenic lymphocytes of sensitized hosts. In contrast, tumor cells derived from regressing sarcomas are poor producers of progeny virus and are relatively unreactive with both antiviral antibody and sensitized lymphocytes. We further found that pp60src kinase activity was reduced by about 75% in regressing compared with progressively growing tumor cells. The half-lives of directly precipitable pp60src in tumor cells derived from progressively growing and regressing neoplasms were 6 and 1.5 h, respectively. Studies on each of three other cellular enzymes did not reveal any regression-associated decreases in enzyme activity. These data support the notion that expression of adequate levels of long-lived pp60src kinase activity is essential to progressive tumor growth.

Intercellular communication and the control of growth: XI. Alteration of junctional permeability by the src gene in a revertant cell with normal cytoskeleton.

To learn whether the reduction of cell-to-cell communication in transformation is a possible primary effect of pp60src phosphorylation or secondary to a cytoskeletal alteration, we examined the junctional permeability in transformed cells with normal cytoskeleton. The permeability to fluorescent-labelled mono- and diglutamate was compared in clones of Faras' vole cells--clones transformed by Rous sarcoma virus and reverted from that transformation. One revertant clone (partial revertant), had the high level of pp60src kinase activity and tumorigenicity of the fully transformed parent clone, but had lost the cytoskeletal alterations of that clone. Another revertant clone (full revertant) had lost the tumorigenicity and most of the pp60src kinase activity, in addition (J.F. Nawrocki et al., 1984, Mol. Cell Biol. 4:212). The junctional permeability of the partial revertant with normal cytoskeleton was similar to that of the fully transformed parent clone with abnormal cytoskeleton. The permeabilities of both were lower than those of the full revertant and the normal uninfected cell, demonstrating that the junctional change by the src gene is independent of the cytoskeletal one.

Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts

Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 °C) instead of the restrictive temperature (41.5 °C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 °C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.

Differentiation and transformation of neural plate cells

The developmental potential of presumptive neural plate cells of prestreak chick embryos (stage 1) and neural plate cells from definitive streak chick embryos (stage 4) has been examined in cell culture using specific markers that identify the major cell types in the vertebrate central nervous system. The prestreak presumptive neural plate (PSPNP) cells, stage 1, assume an epithelial appearance in vitro and synthesize cellular fibronectin, but do not express markers for the neuronal, astrocytic, melanocytic, or oligodendrocytic lineages. Conversely, definitive-streak neural plate (DSNP) cells contain precursors which express cell-type-specific markers for terminally differentiated neurons, astrocytes, and melanocytes, and synthesize an extracellular matrix of cellular fibronectin. Differentiation of DSNP cells in vitro can be prevented by infection with a temperature-sensitive mutant of Rous sarcoma virus (RSV), tsNY68. Differentiation of DSNP cell transformants can be resumed by a temperature shift to the nonpermissive temperature. The morphological and biochemical changes associated with tsNY68 transformation are accompanied by alterations in pp60 src kinase activity in the transformed cells.

Cyclic AMP treatment of Rous sarcoma virus-transformed Chinese hamster ovary cells increases phosphorylation of pp60src and increases pp60src kinase activity.

Treatment of growing Rous sarcoma virus-transformed Chinese hamster ovary cells with the cyclic AMP analog 8-bromo-cyclic adenosine 3',5'-monophosphate (8-bromo-cyclic AMP) stimulates the incorporation of 32Pi into the viral transforming protein pp60src. Based on one-dimensional and two-dimensional peptide analysis and phosphoamino acid analysis, the increase is on a single phosphoserine residue at the NH2 terminus of the protein. The phosphate incorporation increases during the first 4 h of treatment. The pp60src kinase activity in extracts of cells treated with 8-bromo-cyclic AMP was stimulated about 2- to 3-fold. This stimulation of kinase activity increased during the first 3 h of treatment with 1 mM 8-bromo-cAMP and the activity was increased in both the soluble and particulate fraction of the cells. These results suggest that cyclic AMP can modulate the activity of pp60src in transformed cells.

Monoclonal antibody against the carboxy terminal peptide of pp60src of Rous sarcoma virus reacts with native pp60src.

A monoclonal mouse antibody has been prepared against a synthetic peptide corresponding to the six carboxy-terminal amino acids (C' peptide) of the src gene product pp60v -src of Rous sarcoma virus (RSV). The antibody was able to precipitate pp60v -src and to bind pp60v -src kinase activity in a competition test, indicating that this peptide can serve as an antibody-binding site (epitope). Furthermore, the finding that three out of 28 pp60src-specific tumor-bearing rabbit (TBR) sera contained antibody against the C' peptide argues for an in vivo role for the carboxy terminus of pp60src. C' peptide-specific IgG was purified from one TBR serum using affinity chromatography, and was shown to precipitate significant amounts of pp60src, and bind most of the pp60src kinase activity from SRA, PrA, and B77-C strains of avian sarcoma virus (ASV), but not endogenous pp60c -src, a cellular homologue to the viral pp60v -src. Similar results were obtained with IgG isolated from a C' peptide immune rabbit serum. None of the three C' peptide-specific IgGs could serve as a phosphate acceptor in an immune complex protein kinase reaction.

Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.

Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src.

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.

F-actin aggregates in transformed cells.

Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait.

Association of the src-gene product of rous sarcoma virus with a pyruvate-kinase inactivating factor

A casein-kmase preparation was isolated from extracts of mouse tumors induced by Rous sarcoma virus with the aid of affinity chromatography. The preparation was several hundredfold enriched with respect to casein-inase as well as pp60 src-kinase activity and contained a pyruvate-kinase inactivating factor. The pp60 src- kinase is expressed by a phosphoprotein ( pp60 src) which is the product of the transforming src gene of the Rous sarcoma virus (RSV). The pp60 src- kinase phosphorylates IgG from sera of rabbits bearing the RSV tumor (TBR sera) in free solution exclusively in tyrosine residues. The activity of pp60 src- kinase, as well as casein phosphorylation, was inhibited by μmolar concentrations of quercetin. The protein-kinase preparation caused MgATP-dependent inactivation of co-eluting pyruvate kinase. The activation of pyruvate kinase was blocked by phosphoribosyl pyrophosphate, fructose 1,6-diphosphate and P 1, p 5-di (adenosine-5')-pentaphosphate. These compounds also inhibited tyrosine phosphorylation of TBR-IgG and the casein-kinase activity. Immunoadsorption of the protein-kinase preparation by TBR-IgG was accompanied by a loss of the protein-kinase preparation's ability to cause MgATP-dependent inactivation of pyruvate kinase. Essentially the same results were obtained when tumor extracts were first passed over Blue Sepharose and subsequently over the casein-affinity column. Inactivation of purified chicken-liver pyruvate kinase, type M 2, was observed with an 800-fold purified pp60 src-kinase preparation obtained from RSV-transformed chicken-embryo fibroblasts. Incubation of the pyruvate kinase with pp60 srcin the presence of MgATP resulted in increased tyrosine phosphorylation of proteins with sub-unit molecular weights of 52 000 and 56 000 dalton. The latter corresponded to the sub-unit molecular weight of chicken-liver pyruvate kinase. The results show that the pyruvate-kinase inactivating factor is closely associated with pp60 src-kinase. The findings support the hypothesis that substrates of pp60 src, essential for cell proliferation, are among the enzymes that control glycolysis.

Loss of tumorigenicity correlates with a reduction in pp60 src kinase activity in a revertant subclone of avian sarcoma virus-infected field vole cells

We have recently isolated an interesting revertant subclone (revertant 866-4) of ESV-infected field vole cells that is indistinguishable from uninfected vole cells with respect to its lack of transformed cell properties. These revertants are not only normal morphologically, but they do not grow in soft agar and are nontumorigenic in athymic nude mice. Despite this lack of transformed cell properties, we have found that this cell line still contains pp60 src at concentrations (0.30 μg/mg cell protein) similar to those (0.13–0.42 μg/mg cell protein) found in transformed and morphologically reverted, but tumorigenic vole cells (partial revertants). However, the most interesting aspect of this newly isolated subclone is the marked reduction in its pp60 src kinase activity (2–3%) when compared with the specific activity of pp60 src immunoprecipitated from transformed and partially revertant vole cell lines. Since the reduction in pp60 src kinase activity strongly correlates with the loss of tumorigenicity in this particular revertant cell line, these data support the contention that this enzymatic activity is a crucial factor in the tumorigenic conversion of cells by avian sarcoma virus. Proteolytic peptide analysis of the structure of pp60 src from revertant 866–4 indicates that it is similar to pp60 src obtained from avian sarcoma virus-transformed chick embryo fibroblasts. Moreover, the reduction in kinase activity does not appear to be due to a lack of phosphorylation of the tyrosine residue in pp60 src. Thus neither an obvious structural alteration nor a reduction in phosphorylation of pp60 src appears responsible for the reduced kinase activity observed, suggesting that some as of yet undetermined feature of pp60 src can influence the pp60 src phosphorylating event.

Lack of correlation between pp60src kinase activity and transformation parameters in cells infected with temperature-conditional mutants of Rous sarcoma virus.

The pp60src kinase activity of cells infected with temperature-conditional mutants of Rous sarcoma virus (RSV), which induce only a partial transformation, was compared to the pattern of transformation parameters induced by these mutants. The tsGI251-infected cells were thermosensitive for hexose transport, fibrinolysis, morphological alterations and pp60src kinase activity, but cold-sensitive for loss of growth control. The tsGI253-infected cells were similar to tsGI251-infected cells except that they were only slightly cold-sensitive for loss of growth control and had a very low pp60src kinase activity which was temperature-insensitive. Thus, the pp60src kinase activity measured in vitro with IgG as a substrate did not correlate in a consistent way with any of the measured parameters of transformation.

Biological properties of "partial" transformation mutants of Rous sarcoma virus and characterization of their pp60src kinase.

We have isolated mutants of Rous sarcoma virus from an unmutagenized stock of the Schmidt-Ruppin strain of Rous sarcoma virus. These mutants induce only a "partial" transformation, and the transformation properties induced show unusual properties or combinations. Cells infected with mutant CU2 have a unique "blebby" morphology, have lost surface fibronectin, form very small colonies in soft agar, and are nearly normal with respect to adhesiveness and hexose transport. Cells infected with mutant tsCU11 have a nearly normal morphology, but grow well in soft agar. Cells infected with mutant CU12 have a fusiform morphology, intermediate levels of hexose transport and fibronectin, and form very large colonies in soft agar. Because the appearance of the different parameters of transformation is dissociated in these mutant-infected cells, these data are interpreted as supporting a model in which the transforming protein pp60src interacts with more than one primary target in generating the transformed phenotype. All of the mutants display levels of pp60src kinase activity less than that of the wild type. In the case of mutant CU12, the lower kinase activity is in part a consequence of a lower steady-state amount of pp60src inside the cell.