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K Radke

Publications and source records attributed to K Radke.

31 records · Page 2Linked to original sources
PubMed1984

The role of protein phosphorylation at tyrosine in transformation and mitogenesis.

In cells transformed by avian sarcoma viruses or stimulated by growth factors, certain polypeptides become phosphorylated at tyrosine residues. It is not known if these cellular polypeptides are phosphorylated directly by the tyrosine-kinase activities which are associated with the viral transforming proteins and with growth factor receptors. It is also not clear if phosphorylation of these polypeptides is required for viral transformation or the response to growth factors. We describe here some observations which bear on these questions and discuss possible future approaches.

Alpharetrovirus↗
PubMed1984

Cellular localization of the transforming protein of wild-type and temperature-sensitive Fujinami sarcoma virus.

Fujinami sarcoma virus (FSV) encodes a 140,000-dalton transforming protein, P140, which contains gag- and fps-specific sequences. The cellular localization of this protein was examined by fractionation of [35S]methionine-labeled, FSV-infected chicken embryo fibroblasts. In homogenates of cells infected by wild-type, temperature-resistant FSV prepared in either hypotonic or isotonic buffer, 60 to 80% of the P140 was particulate. Isopycnic separation on discontinuous sucrose gradients indicated that the majority of the particulate P140 was present in a light membrane fraction enriched for plasma membranes. Much of the particulate P140 could be solubilized by the addition of 0.6 M salt to a postnuclear supernatant, suggesting that P140 is not an integral membrane protein. Particulate P140 may be associated with membranes either directly as a peripheral membrane protein or indirectly via cytoskeletal elements. In cells infected by mutants of FSV temperature sensitive for cellular transformation, most of the P140 is particulate at the permissive temperature, whereas most is soluble at the nonpermissive temperature; this change in distribution is not a secondary consequence of the change in cellular phenotype, since it also occurs in nonconditionally transformed cells doubly infected with temperature-sensitive FSV and wild-type Rous sarcoma virus. The movement of P140 from the particulate to the soluble fraction occurs rapidly when cells infected by temperature-sensitive FSV are shifted from the permissive to the nonpermissive temperature. Furthermore, P140 moves from the soluble to the particulate fraction, although somewhat more slowly, when cells are shifted from the nonpermissive to the permissive temperature. These observations suggest that the association of P140 with plasma membranes or the cytoskeleton may play a role in transformation by FSV.

Animals↗
PubMed1983

Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses.

A cellular protein of 36,000 daltons becomes phosphorylated at tyrosine in chicken embryo fibroblasts transformed with avian sarcoma viruses. We have used cellular fractionation and immunofluorescence to locate the 36-kdalton protein in virus-transformed and uninfected chicken fibroblasts. The 36-kdalton protein in transformed cells fractionated mainly with high-speed particulate material, and in density gradient separations, the 36-kdalton protein was found in association with light density membranes together with most of the plasma membrane marker. Increasing the concentration of salt or adding ion chelators solubilized some of the 36-kdalton protein that otherwise was pelletable with high g forces. Based on these data, we conclude that this protein is peripherally or indirectly attached to light density membranes, including plasma membranes. Indirect immunofluorescent staining of the 36-kdalton protein in fixed cells revealed that it was located inside the cell in an extensive reticulum apposed to surface membranes. The same pattern of staining was found in both uninfected and virus-transformed cells. Pretreatment of cells with nonionic detergents before fixation altered or abolished 36-kdalton staining. The 36-kdalton protein appeared to be excluded from regions of the cells where actin cables were present. The pattern of staining observed with the anti-36-kdalton antibody was similar, but not identical, to that observed with antiserum against nonerythroid spectrin. Thus, the data obtained by biochemical fractionation and by immunofluorescent staining indicate that the 36-kdalton protein is found in a reticulum at the inner surface of the plasma membrane, possibly in association with cytoskeletal proteins.

Animals↗
PubMed1982

Transformation of both erythroid and myeloid cells by E26, an avian leukemia virus that contains the myb gene.

E26 and avian myeloblastosis virus are replication-defective avian retroviruses that contain the myb oncogene and cause leukemia in chickens with short periods of latency. Animals infected with E26 develop erythroleukemia and also contain low numbers of transformed myeloid cells, while avian myeloblastosis virus induces a purely myeloid leukemia. In both cases the type of leukemia induced is independent of the subgroup of the helper virus used. E26-transformed erythroid and myeloid cells can each be propagated selectively from explanted leukemic cells with media supplemented with factors that promote the growth either of normal chicken erythroid precursors or of myeloid progenitor cells. E26 also induces the outgrowth of transformed cells from bone marrow cells infected in vitro. These cells are also either erythroid or myeloid, depending on the culture conditions employed. Most of the erythroid cells transformed by E26 are erythroblast-like, but a significant number are more mature, including erythrocyte-like cells as well as some cells that appear to be aberrant in differentiation. Both erythroid and myeloid E26-transformed cells produce infectious virus and express P135 E26, the putative (gag-myb-x) transforming protein of the virus. Thus E26 is a virus that is capable of generating factor-dependent transformed cells in two different hematopoietic lineages.

Animals↗
PubMed1982

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.

Animals↗
PubMed1981

Tumor promoters alter gene expression and protein phosphorylation in avian cells in culture.

We have investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the synthesis and modification of polypeptides in normal avian cells and cells infected by wild-type and temperature-sensitive Rous sarcoma virus (RSV). Using two-dimensional gel electrophoresis, we have detected alterations in both the abundance of cellular polypeptides and in their phosphorylation that seem unique to TPA treatment. However, the state of phosphorylation of the major putative substrate for the action of the src gene-associated protein kinase, the 34- to 36-kilodalton protein, was not altered. Moreover, examination of the phosphorylated amino acid content of total cellular phosphoproteins revealed that the response to TPA was not associated with detectable increases in their phosphotyrosine content. These results make it unlikely that TPA acts by the activation of the phosphorylating activity of the cellular proto-src gene or by the activation of other cellular phosphotyrosine-specific kinases. We have shown previously that temperature-sensitive RSV-infected cells at nonpermissive temperature demonstrate an increased sensitivity to TPA treatment [Bissell, M. J., Hatie, C. & Calvin, M. (1979) Proc. Natl. Acad. Sci. USA 76, 348-352]. Our present results indicate that this is not due to reactivation of the phosphorylating activity of the defective src gene product or to its leakiness, and they lend support to the notion of multistep viral carcinogenesis.

Animals↗
PubMed1980

Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine.

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.

Animals↗
PubMed1980

A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation.

Cells infected by one strain of Fujinami sarcoma virus (FSV) are transformed at 38 degrees C but are phenotypically normal at 41.5 degrees C. FSV encodes a 140,000 molecular weight protein (P140) with gag gene-related and FSV-specific peptide sequences. At 41.5 degrees C, P140 is weakly phosphorylated at serine residues, and is inactive in the immune complex protein kinase assay. At 38 degrees C, P140 is highly phosphorylated, contains phosphotyrosine in addition to phosphoserine, and in the immune complex kinase assay becomes phosphorylated at three tyrosine residues. Phosphorylation of cellular polypeptides at tyrosine residues in FSV-infected cells is also temperature-sensitive. These observations indicate that P140 is the transforming protein of FSV and that protein phosphorylation at tyrosine residues is involved in transformation by this virus.

Alpharetrovirus↗
PubMed1979

Transformation by Rous sarcoma virus: effects of src gene expression on the synthesis and phosphorylation of cellular polypeptides.

Infection of chicken embryo fibroblasts by Rous sarcoma virus induces a variety of alterations in cellular growth and morphology. We have used two-dimensional polyacrylamide gel electrophoresis to examine the effects of viral transformation on the pattern of synthesis and phosphorylation of cellular polypeptides. Infection by Rous sarcoma virus does not appear to induce the de novo synthesis, or the complete suppression, of any of the [35S]methionine-labeled cellular polypeptides that can be resolved with this technique; however, there are quantitative changes in a minor fraction (approximately 4%) of the [35S]methionine-labeled polypeptides. When cells labeled with [32P]orthophosphate were examined, a phosphorylated polypeptide, Mr 36,000, was detected in transformed cells; this polypeptide appears within 20 min when cells infected by a temperature-sensitive mutant of Rous sarcoma virus are shifted from the nonpermissive to the permissive temperature. Phosphorylation of the 36,000 Mr polypeptide thus represents an early event in the process of transformation, and it is possible that this polypeptide is a target for the kinase activity associated with pp60src.

Animals↗
PubMedDate not supplied

Administrative preparation of clinical nurse specialists.

A study is reported in which the authors examined the administrative preparation needs of a sample of 46 graduates of a clinical nurse specialist program who were surveyed 3 to 5 years after graduation. The study focused on three areas: current administrative responsibilities, administrative content in the graduate curriculum, and respondents' future goals for involvement in administration. Findings showed that the respondents' highest levels of current participation were in planning and policy functions (51 per cent) with a low overall involvement in other administrative functions. Eighty-five per cent of graduates felt that the master's degree nursing program should have allowed for more content in administration. A majority (54 per cent) had enhanced their skills in administration on the job or through continuing education. Twenty-five per cent planned a career focus on administration within 5 years. Study implications concerning future curricular priorities and pathways are discussed.

Administrative Personnel↗