The expression in eukaryotes of a tyrosine kinase which is reactive with pp60v-src antibodies

All specimens of Eumetazoa and Parazoa, ranging from mammals, birds, teleosts, sharks, lampreys, amphioxus, insects, down to sponges showed the pp60c-src associated kinase activity, indicating that c-src, which is the cellular homologue of the oncogene v-src of Rous sarcoma virus (RSV) is probably present in all multicellular animals. Protozoa and plants did not show pp60c-src kinase activity. The degree of c-src expression depends on the taxonomic rank of the Eumetazoa tested, and is organ-specific with nervous tissues displaying the highest kinase activities. In the central nervous system of mammals and birds we found a high c-src expression, and in that of the lampreys, amphioxus, and insects the lowest. Unexpectedly, total extracts of sponges showed an amount of pp60c-src kinase activity similar to that of brain cell extracts of mammals and birds. These findings suggest that pp60c-src is a phylogenetic old protein that might have evolved together with the multicellular organisation of Metazoa, and that might be of importance in proliferation and differentiation of nontransformed cells.     

The Expression in Eukaryotes of a Tyrosine Kinase Which is Reactive with pp60v-src Antibodies

Abstract. All specimens of Eumetazoa and Parazoa, ranging from mammals, birds, teleosts, sharks, lampreys, amphioxus, insects, down to sponges showed the pp60 ^c-src associated kinase activity, indicating that c-src , which is the cellular homologue of the oncogene v-src of Rous sarcoma virus (RSV) is probably present in all multicellular animals. Protozoa and plants did not show pp60 ^c-src kinase activity.
The degree of c-src expression depends on the taxonomic rank of the Eumetazoa tested, and is organ-specific with nervous tissues displaying the highest kinase activities. In the central nervous system of mammals and birds we found a high c-src expression, and in that of the lampreys, amphioxus, and insects the lowest. Unexpectedly, total extracts of sponges showed an amount of pp60 ^c-src kinase activity similar to that of brain cell extracts of mammals and birds. These findings suggest that pp60 ^c-src is a phylogenetic old protein that might have evolved together with the multi-cellular organisation of Metazoa, and that might be of importance in proliferation and differentiation of nontransformed cells.     

ATP- and polyphosphate-mediated stimulation of pp60c-src kinase activity in extracts from vascular smooth muscle

Recently, we reported that pp60c-src kinase activity was present in adult bovine coronary arterial smooth muscle and showed that the activity of the enzyme in in vitro immunoprecipitation assays was stimulated 20-60-fold by ATP (Di Salvo, J., Gifford, D., and Kokkinakis, A. (1988) Biochem. Biophys. Res. Commun. 153, 388-394). In the present study, ATP-mediated stimulation of activity was also demonstrated in extracts from aortic vascular smooth muscle. In contrast, no stimulation was apparent in extracts from brain. Stimulation of activity in vascular preparations was also induced with beta,gamma-imidoadenosine 5'-triphosphate (AMP.PNP), a nonmetabolizable analog of ATP, and with several other polyphosphates including ADP and sodium pyrophosphate. No stimulation occurred in response to monophosphates such as AMP or KH2PO4. As expected, the specific activity of pp60c-src in brain extracts did not change when the amount of extracted protein included in immunoprecipitation mixtures was increased. Unexpectedly, however, the specific activity of the vascular enzyme decreased markedly as the amount of extracted protein subjected to immunoprecipitation was increased. Following stimulation of pp60c-src in vascular extracts with ATP, the enzyme behaved in a fashion similar to pp60c-src extracted from brain. That is, the enhanced specific activity of the stimulated vascular enzyme did not decrease with increasing amounts of extracted protein. Moreover, mixing experiments in which vascular smooth muscle extracts were added to brain extracts showed that the muscle extracts contained a factor which inhibited pp60c-src kinase activity. This inhibition was blocked when the mixed extracts were immunoprecipitated in the presence of ATP, or when inhibitory extract was treated with trypsin. Taken together, these data suggest that pp60c-src kinase activity in vascular tissue may be subject to a novel regulatory mechanism involving an inhibitory protein factor which can be nullified by polyphosphates.     

Expression of v-src and chicken c-src in rat cells demonstrates qualitative differences between pp60v-src and pp60c-src

The retroviral oncogene v-src arose by transduction of the cellular gene c-src. The similarity between these genes raised the possibility that c-src might be able to elicit neoplastic growth. We explored this by constructing a chimeric plasmid that allows the expression of chicken c-src. A rat cell line containing ten times the normal intracellular level of pp60c -src was isolated after transfecting rat-2 cells with the chimeric DNA. These cells produce the protein encoded by c-src ( pp60c -src) in quantities at least three times greater than required to achieve transformation by the product of v-src ( pp60v -src). The cells remain phenotypically normal, contain actin cables, and do not grow in soft agar. However, transfection of the cell line containing elevated cells of pp60c -src or Rat-2 cells with a molecular clone of v-src produces cells that exhibit properties of biologically transformed cells: round morphology, disrupted actin cables, and ability to grow in soft agar.     

v-src mutations outside the carboxyl-coding region are not sufficient to fully activate transformation by pp60c-src in NIH 3T3 cells.

Previous studies have shown that carboxyl-terminal mutation of pp60c-src can activate its transforming ability. Conflicting results have been reported for the transforming ability of pp60c-src mutants having only mutations outside its carboxyl-terminal region. To clarify the effects of such mutations, we tested the activities of chimeric v(amino)- and c(carboxyl)-src (v/c-src) proteins at different dosages in NIH 3T3 cells. The focus-forming activity of Rous sarcoma virus long terminal repeat (LTR)-src expression plasmids was significantly reduced when the v-src 3' coding region was replaced with the corresponding c-src region. This difference was masked when the Rous sarcoma virus LTR was replaced with the Moloney murine leukemia virus LTR, which induced approximately 20-fold more protein expression, but even focus-selected lines expressing v/c-src proteins were unable to form large colonies in soft agarose or tumors in NFS mice. This suggests that pp60c-src is not equally sensitive to mutations in its different domains and that there are at least two distinguishable levels of regulation, the dominant one being associated with its carboxyl terminus. v/c-src chimeric proteins expressed with either LTR had high in vitro specific kinase activity equal to that of pp60v-src but, in contrast, were phosphorylated at both Tyr-527 and Tyr-416. Total cell protein phosphotyrosine was enhanced in cells incompletely transformed by v/c-src proteins to the same extent as in v-src-transformed cells, suggesting that the carboxyl-terminal region may affect substrate specificity in a manner that is important for transformation.     

pp60c-src kinase activity in bovine coronary extracts is stimulated by ATP

pp60c-src kinase is believed to participate in regulating key cellular mechanisms including signal transduction and differentiation of smooth muscle during early embryogenesis. In this study, pp60c-src kinase activity was demonstrated in extracts from adult bovine coronary arterial smooth muscle. Activity, reflected by autophosphorylation of pp60c-src, phosphorylation of exogenous substrates, and phosphorylation of several endogenous substrates, was enhanced about 2 fold when added Mg2+ was replaced by Mn2+. Unexpectedly, activity was dramatically stimulated 20-50 fold by prior incubation with ATP. Such stimulation appears to be mediated through a novel mechanism which is independent of ATP-induced phosphorylation of reaction components. These new observations strongly suggest that a unique mechanism exists for regulation of coronary arterial pp60c-src kinase activity. Conceivably, this mechanism may serve important roles in modulating signal transduction and contractility of vascular smooth muscle.     

Activation of the pp60c-src kinase during differentiation of monomyelocytic cells in vitro.

The proto-oncogene c-src, the cellular homolog of the Rous sarcoma virus (RSV) transforming gene v-src, is expressed in a tissue-specific and age-dependent manner. Its physiological function, although still unknown, appears to be more closely related to differentiation processes than to proliferation processes. To obtain more information about the physiological role of the c-src gene in cells, we have studied differentiation-dependent alterations using the human HL-60 leukaemia cell line as a model system. Induction of monocytic and granulocytic differentiation of HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and dimethylsulfoxide (DMSO) is associated with an activation of the pp60c-src tyrosine kinase, but not with increased c-src gene expression. Control experiments exclude an interaction of TPA and DMSO themselves with the pp60c-src kinase.     

pp60c-src in the developing cerebellum.

pp60c-src was localized in the cerebellum of developing chicken embryos by immunoperoxidase staining with antisera raised against bacterially expressed pp60v-src. Immunoreactivity (IR) appeared in the cerebellum of the chicken embryos at the time of neuronal differentiation. pp60c-src IR was detected in regions of the developing cerebellum where processes of developing neurons and glia are located. In the early embryo (stage 17), pp60c-src IR was localized in the marginal zone of the cerebellar plate. By stage 40, pp60c-src IR was localized in the process-rich molecular layer of the cerebellum and between the cells of the developing internal granular layer. Cell bodies of cerebellar neurons did not show pp60c-src IR at any stage of development. Mitotically active neuroepithelial cells of the metencephalon did not express pp60c-src before the onset of differentiation in the early embryo, nor did proliferating cells of the external granular layer express pp60c-src at later stages. Although it is not possible to ascertain whether pp60c-src is localized in developing neurons or glia at the light microscope level, the time of its appearance and pattern of distribution in the molecular layer is suggestive of a localization within the developing neuronal processes which compose the bulk of this layer. Biochemical analyses of pp60c-src in the developing cerebellum by the immune complex protein kinase activity and sensitivity of the kinase to inhibition by P1,P4-di(adenosine-5')tetraphosphate confirmed that the expression of pp60c-src coincided with the time of neuronal differentiation. We conclude from these results that in the central nervous systems, pp60c-src may be more important in an aspect of cell differentiation or a mature neuronal function than in the proliferation of neuronal or glial precursors.     

Analysis of pp60c-src protein kinase activity in human tumor cell lines and tissues

We have evaluated the level of pp60c-src protein kinase activity in a variety of human tumor tissues and human tumor cell lines, and have estimated the abundance of the c-src protein in several of these tissues and cell lines. All cell lines derived from tumors of neuroectodermal origin that express a neural phenotype were found to possess c-src molecules with high levels of tyrosine-specific protein kinase activity. In contrast, cell lines derived from tumors of neuroectodermal origin that do not express neural characteristics, such as glioblastomas and melanomas, were found to have pp60c-src molecules with low levels of protein kinase activity. A similar pattern was observed when we analyzed the activity of c-src molecules extracted directly from corresponding tumor tissues. Analysis of human tumor cell lines derived from tissues other than those of neuroectodermal origin revealed that pp60c-src protein kinase activity was low in most cases. Exceptions to this observation were all rhabdomyosarcoma, osteogenic sarcoma, Ewing's sarcoma, and colon carcinoma lines tested. Comparison of pp60c-src kinase activity in normal skeletal muscle and rhabdomyosarcoma tissue and in normal breast tissue and breast adenocarcinoma tissue revealed that pp60c-src kinase activity was specifically elevated in the tumor tissues in both cases. However, the amount of pp60c-src protein in both normal and tumor tissues was found to be similar. These observations suggest that increases in the specific activity of the pp60c-src phosphotransferase in some rhabdomyosarcomas and breast carcinomas may be a characteristic acquired during the malignant transformation of the cells that is retained in cell lines established from these tumors.     

pp60c-src在血管平滑肌细胞内丝裂原活化蛋白激酶激活中的作用

观察ｐｐ６０ｃ－ｓｒｃ在血管紧张素Ⅱ（ＡｎｇⅡ）诱导血管平滑肌细胞（ＶＳＭＣｓ）内丝裂原活化蛋白激酶（ＭＡＰＫ）激活中的作用,以了解ＡｎｇⅡ促ＶＳＭＣｓ增殖的信号转导过程。将合成的反义ｃ－ｓｒｃ寡脱氧核苷酸（ｏｌｉｇｏｄｅｏｘｙｎｕｃｌｅ－ｏｔｉｄｅｓ,ＯＤＮｓ）以脂质体包裹转染培养的大鼠ＶＳＭＣｓ,用Ｗｅｓｔｅｒｎ印迹测得细胞裂解液中ｐｐ６０ｃ－ｓｒｃ含量明显下降,免疫沉淀方法测得ｐｐ６０ｃ－ｓ     

Polyoma virus middle T antigen-pp60c-src complex associates with purified phosphatidylinositol 3-kinase in vitro.

Reconstitution of the polyoma virus middle T antigen (mT)-pp60-src complex and phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been accomplished in vitro with immunopurified baculovirus-expressed mT-pp60c-src and PtdIns 3-kinase purified from rat liver. Both the 110- and 85-kDa subunits of the PtdIns 3-kinase associated with the mT-pp60c-src complex. The association of PtdIns 3-kinase with the mT-pp60c-src complex was dependent on the protein-tyrosine kinase activity of pp60c-src as a kinase-inactive mutant (pp60(295c-src)) still complexed with mT, but the mT-pp60(295c-src)) complex was unable to bind PtdIns 3-kinase. The mT-pp60c-src complex phosphorylated both subunits of PtdIns 3-kinase on tyrosine residues. The immunopurified mT-pp60c-src complex also associated with PtdIns 3-kinase activity from whole cell lysates, and this association was dependent upon the protein-tyrosine kinase activity of pp60c-src. Comparison of 35S-labeled proteins from whole cell lysates which associated with immunopurified mT-pp60c-src and mT-pp60(295c-src) revealed proteins of 110 and 85 kDa as the major peptides dependent on protein-tyrosine kinase activity for association with the complex. In addition, a synthetic phosphopeptide (13-mer) containing sequences conserved between the major tyrosine phosphorylation site of murine polyoma virus mT, hamster polyoma virus mT, and the insulin receptor substrate (IRS-1) specifically blocked the association of the 85- and 110-kDa polypeptides with the mT-pp60c-src complex. The ability to block the association was dependent on the tyrosine phosphorylation of the peptide. Association of PtdIns 3-kinase activity was blocked concurrently. This is the first demonstration that the 110-kDa subunit of PtdIns 3-kinase can associate with mT-pp60c-src. This association in vitro is a step toward understanding protein-protein interactions important in the signal transduction pathway of oncogenic proteins.     

pp60c-src Kinase is in chick and human embryonic tissues

The normal cellular protein pp60c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60c-src distributed throughout the neural tube and heart. The distribution of pp60c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60c-src kinase. The results of our studies suggest that pp60c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.     

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.     

Osteoclasts express high levels of pp60c-src in association with intracellular membranes

Deletion of the c-src gene in transgenic mice by homologous recombination leads to osteopetrosis, a skeletal defect characterized by markedly deficient bone resorption (Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Cell. 64:693-702), demonstrating a critical functional role of pp60c-src in osteoclast activity. Since decreased bone resorption could result from a defect either within the osteoclast or within other cells present in its environment, indirectly affecting osteoclast functions, we determined which cell(s) in bone expressed high levels of pp60c-src Measuring pp60c-src protein and kinase activities in osteoclasts and immunolocalizing pp60c-src in bone, we find that expression of pp60c-src is nearly as high in osteoclasts as in brain and platelets. In contrast, other bone cells contain only very low levels of the protein. In addition, expression of the c-src gene product increases when bone marrow cells are induced to express an osteoclast-like phenotype by 1,25-dihydroxy-vitamin D3, further suggesting that high expression of pp60c-src is part of the osteoclast phenotype. Three other src-like kinases, c-fyn, c-yes, and c-lyn, are also expressed in osteoclasts at ratios to pp60c-src similar to what is found in platelets. These src-related proteins do not, however, compensate for the absence of pp60c-src in the src- mice, thereby suggesting that pp60c-src may have a specific function in osteoclasts. Although further work is necessary to elucidate what the critical role of pp60c-src in osteoclasts is, our observation that the protein is associated mostly with the membranes of intracellular organelles suggests the possibility that this role might be at least in part related to the targeting or fusion of membrane vesicles.     

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.     

Expression of c-src in cultured human neuroblastoma and small-cell lung carcinoma cell lines correlates with neurocrine differentiation.

Human cell lines with neuronal and neuroendocrine features were examined for their expression of pp60c-src, the cellular homolog of the transforming gene product pp60v-src of Rous sarcoma virus. Four neuroblastoma (LA-N-5, SH-SY5Y, Paju, and SK-N-MC) and three small-cell lung carcinoma (U-2020, U-1690, and U-1285) cell lines were selected on the basis of their stage of neurocrine differentiation, as determined by the expression of neuron-specific enolase. In an immune complex protein kinase assay, all seven cell lines displayed c-src kinase activity which was considerably higher than that found in nonneurocrine cells (human diploid fibroblasts, glioma, and non-small cell lung carcinoma cell lines). Furthermore, the c-src kinase activity, as determined by autophosphorylation or phosphorylation of an exogenous substrate, enolase, correlated with the stage of neurocrine differentiation. There was an approximately 30-fold difference in c-src kinase autophosphorylation activity between the cell lines representing the highest and lowest stages of neurocrine differentiation. A similar variation was found in the steady-state levels of the c-src protein of these cell lines. Highly differentiated neuroblastoma cells expressed two forms of the src protein. Digestion by Staphylococcus aureus V8 protease did reveal structural diversity in the amino-terminal ends of these c-src molecules. In summary, we found a clear correlation between c-src kinase activity and the stage of neuronal and neuroendocrine differentiation. Thus, the phenotypic similarity between neurons and neuroendocrine cells includes high c-src expression.     

Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation.

The transforming protein of polyoma virus, middle T antigen, associates with the protein tyrosine kinase pp60c-src, and analysis of mutants of middle T suggests that this complex plays an important role in transformation by polyoma. It has recently been reported that pp60c-src from polyoma virus-transformed cells has enhanced tyrosine kinase activity in vitro. The data presented here confirm these findings and show that the enhanced kinase activity of pp60c-src is due to an increase in the Vmax of the enzyme. Sucrose density gradient analysis demonstrates that only the form of pp60c-src which is bound to middle T antigen is activated. The difference in enzyme activity between pp60c-src from normal and middle T-transformed cells is more marked when the enzyme is prepared from lysates containing the phosphotyrosine protein phosphatase inhibitor, sodium orthovanadate. pp60c-src from middle T transformed cells is unaffected, but pp60c-src from normal cells has reduced kinase activity if dephosphorylation is prevented. The kinase activity of pp60c-src thus appears to be regulated by its degree of phosphorylation at tyrosine, and data are presented which support this hypothesis. pp60c-src is the first example of a protein tyrosine kinase whose activity is inhibited by phosphorylation at tyrosine. Middle T antigen may increase the kinase activity of pp60c-src by preventing phosphorylation at this regulatory site.