Pp60src Kinase Research Articles

The submembranous location of p11 and its interaction with the p36 substrate of pp60 src kinase in situ

p36, a major cytoplasmic substrate of pp60 src kinase, is present beneath the plasma membrane. It can be isolated either as a monomer or as a heterotetramer (protein I) containing two copies each of p36 and a unique p11 polypeptide. To compare the expression rules of p36 and p11 as well as their cellular distributions, monoclonal antibodies to the two porcine proteins were isolated. In tissue culture cells p11-specific antibodies decorated the same submembranous compartment previously seen with antibodies to p36 and fodrin or spectrin and followed the p36 images under all fixation/extraction conditions tested. Immunofluorescence microscopy on tissue sections showed coincident expression patterns of both proteins confirming and extending previous results with p36 antibodies. Antibodies with limited cross-species reaction have been used to trace the fate of porcine p11 and p36 injected into cultured cells. Both proteins are incorporated in the submembranous compartment, where they remain in Triton cytoskeletons prepared in the presence but not in the absence of Ca 2+. The incorporation of p36 in vivo conforms with its Ca 2+-dependent binding to actin, fodrin, and certain phospholipids in vitro. In contrast, the incorporation of p11 seems to depend on an in situ interaction with p36 or an exchange with endogenous p11 present on p36. The combined results indicate a strong coupling of p11 and p36 in cellular compartmentalization and tissue differentiation.

Isolation of a calcium-sensitive, 35,000-dalton microfilament- and liposome-binding protein from ascites tumor cell microvilli: identification as monomeric calpactin.

Microvilli isolated from the MAT-C1 ascites subline of the 13762 rat mammary adenocarcinoma contain a major calcium-sensitive microfilament-binding protein, AMV-p35 (ascites microvillar p35). Association of AMV-p35 with microfilament cores during Triton X-100 extraction of the microvilli is half-maximal at 0.1-0.2 mM calcium. The protein, which comprises 6% of the total microvillar protein, can be isolated from microfilament cores prepared in the presence of calcium by extraction with EGTA and purification by ion-exchange chromatography. Alternatively, the protein can be isolated from Triton extracts of microvilli prepared in the absence of calcium by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on an ion-exchange column. AMV-p35 binds to phosphatidylserine liposomes and F-actin with half-maximal calcium concentrations of about 10 microM and 0.2 mM, respectively. Treatment of AMV-p35 with chymotrypsin yields a 33,000-dalton fragment, behavior similar to the tyrosine kinase substrates calpactins I and II and lipocortins I and II. Immunoblot analyses using antibodies directed against calpactin I, lipocortin I, and lipocortin II showed strong reactivity of AMV-p35 with anti-calpactin I and anti-lipocortin II, but little reactivity toward anti-lipocortin I. The close relationship between AMV-p35 and calpactin I was verified by amino acid sequence analyses of peptides isolated from cyanogen bromide digests of AMV-p35. By gel filtration and velocity sedimentation analyses purified AMV-p35 is a 35,000-dalton monomer. Moreover, AMV-p35 extracted directly from microvilli in Triton/EGTA also behaves as a 35,000-dalton menomer. These findings indicate that AMV-p35 is closely related to the pp60src kinase substrate calpactin I (p36). However, AMV-p35 occurs in the microvilli as a monomer rather than as the heterotetrameric calpactin found in several other cell types.

Calcium-dependent isolation of the 36 kD substrate of pp60src kinase: Fractionation of the phosphorylated and unphosphorylated species

Calcium-dependent isolation of the 36 kD substrate of pp60src kinase: Fractionation of the phosphorylated and unphosphorylated species

Amiloride inhibits the protein tyrosine kinases associated with the cellular and the transforming src-gene products

Amiloride inhibits a protein tyrosine kinase from rat brain extracts. The kinase activity is characterized by an anti-serum (TBR-serum) which immunoprecipitates pp60 c-src, the cellular counterpart of the transforming protein pp60 v-src of Rous sarcoma virus. In immunocomplexes, TBR-IgG serves as an artificial but specific phosphate acceptor. The phosphate incorporation into TBR-IgG is a time- and temperature-dependent process. In the presence of amiloride the TBR-IgG phosphorylation is reduced. The drug does not influence the immunocomplexes formed by TBR-IgG and pp60 src and no amiloride-activated protein tyrosine phosphatase can be detected in the immunocomplex system. Half-maximal inhibition of the tyrosine kinase occurs at 300 μM amiloride and is competitive with respect to ATP. Viral pp60 src kinase of transformed cells is more sensitive to amiloride ( ic 50: 50–100 μM). Furthermore, normal cellular tyrosine kinases are to a lesser extent inhibited by amiloride as compared to the transforming viral pp60 src kinase. These results may indicate different amiloride-sensitive forms of cellular pp60 src kinases.

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.

Detection of a tyrosine kinase in human sera and blood cells by pp60 src antiserum

Using sera of Rous sarcoma virus-tumor bearing rabbits (TBR-sera) as a tool to detect pp60 src kinase in immunoprecipitates, we report here that about 10% of our TBR-sera revealed tyrosine kinase activity in human serum, plasma and in soluble extracts of human blood cells. The activity found in serum represents 5–12% of the total kinase activity in blood. Most of the enzyme activity was detected in lymphocytes and polymorphnuclear cells rather than in platelets and erythrocytes. We also demonstrate that the tyrosine kinase in serum is inhibited by quercetin, a potent inhibitor of the viral pp60 src protein kinase.

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.

Cells transformed with a ts viral src mutant are temperature sensitive for in vivo growth.

Studies on ts mutants of avian sarcoma viruses have previously implicated the src gene product (pp60src) kinase function in in vitro transformation. The role of src in vivo, however, has not been clearly defined. Using a sensitive and quantitative assay that was developed in chicken embryos (Chambers et al., Cancer Res. 42:4018-4025, 1982), we tested the in vivo tumorigenic properties of cells transformed with LA23, an avian sarcoma virus that is temperature sensitive for in vitro transformation. We found that the in vivo growth ability of these cells was temperature sensitive and that this in vivo behavior correlated with the in vitro transformation behavior (growth in soft agar and saturation density).

Cells transformed with a ts viral src mutant are temperature sensitive for in vivo growth.

Studies on ts mutants of avian sarcoma viruses have previously implicated the src gene product (pp60src) kinase function in in vitro transformation. The role of src in vivo, however, has not been clearly defined. Using a sensitive and quantitative assay that was developed in chicken embryos (Chambers et al., Cancer Res. 42:4018-4025, 1982), we tested the in vivo tumorigenic properties of cells transformed with LA23, an avian sarcoma virus that is temperature sensitive for in vitro transformation. We found that the in vivo growth ability of these cells was temperature sensitive and that this in vivo behavior correlated with the in vitro transformation behavior (growth in soft agar and saturation density).

Identification of calcium-binding proteins associated with the lymphocyte plasma membrane

The Nonidet P40 insoluble fraction of lymphocyte plasma membrane contains three polypeptides of about 68,000-, 33,000- and 28,000-Mr ∗ ∗ Abbreviations: Mr, molecular weight; SDS/PAGE, sodium dodecylsulfate/ polyacrylamide gel electrophoresis; 2D, two dimensional which are solubilised by Ca 2+-chelators.As judged by various criteria the 33,000-Mr polypeptide is homologous to the 36,000-Mr pp60 src kinase substrate of chicken fibroblasts and the 68,000-Mr polypeptide is related to the 67,000-Mr “calelectrin” of bovine liver. The 28,000-Mr polypeptide may also be related to calelectrin.

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.

Transformation of erythroid cells by rous sarcoma virus (RSV)

RSV transforms several nonhematopoietic cell types and as reported here also has the capacity to transform hematopoietic cells of the erythroid lineage. In vitro, the three RSV isolates tested induced erythroblast-like colonies in infected bone marrow cells that were distinguishable by size and cell arrangement from those induced by avian erythroblastosis virus (AEV). Also in contrast to AEV-transformed erythroblast cultures, isolated cell colonies induced by RSV required complex growth conditions in liquid medium similar to the in vitro conditions necessary for erythroblasts transformed by the acute leukemia virus E26. Temperature-shift experiments using temperature-sensitive ( ts) NY68 RSV revealed that when grown at the nonpermissive temperature (42°), mutant-infected cells became benzidine positive and partially differentiated into erythrocytes. Wild-type ( wt) RSV-transformed cells did not undergo similar changes. However, both wt RSV-, and to a greater extent, ts RSV-transformed cultures at the permissive temperature (37°) did contain populations of spontaneously differentiating erythroid cells signifying that the transforming activity of the virus did not fully arrest erythroid maturation. In addition, the RSV-transformed cells did express tyrosine kinase activity. When injected intravenously into birds, RSV induced an erythroblastosis-like disease similar to AEV but also caused fibrosarcomas and leg paralysis. These results show that RSV can alter the pattern of erythroid differentiation in a manner similar to, but distinct from, AEV and indicate that the tyrosine-specific pp60 src kinase is involved in erythroid cell transformation. Since the arc and erb B proteins share a significant amino acid homology, these data suggest that both may also share a common functional homology.

Visualization of phosphotyrosine containing molecules within the detergent insoluble cell matrix of v-src transformed fibroblasts

The RSV oncogene v-src is known to transform host cells through the action of a single gene protein product (pp60src) endowed with tyrosine specific kinase activity. The nature and cellular localization of substrates of pp60src kinase are largely unknown. Detergent insoluble cell matrix was prepared by treating RSV-transformed mouse fibroblasts with the non-ionic detergent NP-40. These preparations, highly enriched in cytoskeletal proteins, when observed in the E M consisted mainly of a meshwork of filaments; fragments of plasma membrane and nuclear “ghosts” were also present. Antibodies against phosphotyrosine -previously shown to be reactive with protein phosphorylated at tyrosine residues- were prepared and affinity purified using a synthetic hapten (azobenzyl phosphonic acid, ABP). By means of the immunogold techniques applied to electron microscopy, phosphotyrosine containing molecules were found to be present in RSV transformed, but not in control fibroblasts. Gold particles were mostly bound to electron dense granular material associated with the filaments. These results are consistent with the idea that pp60src itself and some tyrosine phosphorylated proteins may be found among detergent-insoluble cell structures.

Inhibition by quercetin of the release of density dependent-inhibition of cell growth in RSV-transformed chicken cells

The expression of src gene in dense cultures of chick embryo fibroblasts (CEF) infected by a thermosensitive mutant (NY68) of RSV released densitydependent inhibition of growth and induced in these cells a large increase in DNA, RNA and protein synthesis. This stimulation of cellular metabolism was abolished in the presence of quercetin. Furthermore, quercetin added to the culture medium also inhibited the stimulation of pp60src kinase due to the expression of transformation.

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.

The use of synthetic peptides for defining the specificity of typrosine protein kinases

The tyrosine protein kinases are a large family of enzymes that may be involved in regulating cell growth and differentiation. An important property of these enzymes is their substrate specificity. Defining the specificity of these enzymes will contribute to a greater understanding of their biological functions. Synthetic peptides provide a useful means for studying the specificities of protein kinases. The utility of synthetic peptides for specificity studies is exemplified by the results obtained with the cyclic nucleotide-dependent protein kinases. A large number of synthetic peptides have been tested as substrates for these enzymes. There are three important conclusions from this work. First, in terms of primary sequence, the two cyclic nucleotide-dependent protein kinases both require the presence of a pair of basic residues on the N-terminal side of the phosphorylatable residue. Second, recognition of a specific secondary structure of these enzymes. Third, the specificities of the two cyclic nucleotide-dependent protein kinases are remarkably similar in terms of both primary and secondary structure recognition. Sequences at the sites of tyrosine phosphorylation often show the presence of numerous acidic residues on the N-terminal side of the tyrosine. This result led to the suggestion that these acidic residues might be important for the recognition of these sites by the tyrosine protein kinases. Specificity studies using synthetic peptidese have provided some experimental verification of this concept. In the case of four tyrosine protein kinases, LSTRA cell tyrosine protein kinase, epidermal growth factor receptor kinase, insulin receptor kinase and pp60 gag-yes tyrosine protein kinase, the presence of acidic residues on the N-terminal side of the tyrosine in a synthetic peptide was a favorable determinant. In the case of a fifth member of the tyrosine protein kinase family, namely pp60 src kinase, the data are less clear that the presence of acidic residues is involved in substrate recognition. This enzyme readily phosphorylated several peptides that did not have acidic residues on the N-terminal side of the tyrosine. Although there is some indication that the presence of acidic residues on the N-terminal side of the tyrosine is a factor in substrate recognition by several of the tyrosine protein kinases, the changes in kinetic parameters with the various peptide substrates were rather small. In addition, some sites phosphorylated in proteins do not have any acidic residues on the N-terminal side of the phosphorylated tyrosine residue. The presence of acidic residues on the primary sequence near the tyrosine is not an absolute determinant for the recognition of sites of phosphorylation by the tyrosine protein kinases. As in the case of the cyclic nucleotide-dependent protein kinases, secondary structure is probably also important for the recognition of sites of phosphorylation by the tyrosine protein kinases.

Differential inhibition of cellular and viral pp60src kinase by P1,P4-di(adenosine-5')tetraphosphate.

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.