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H Hanafusa

Publications and source records attributed to H Hanafusa.

At least 181 records · Page 10Linked to original sources
PubMed1985

Preferential expression of the c-fps protein in chicken macrophages and granulocytic cells.

We have studied the expression of the protein kinase activity of NCP98, the c-fps gene product, in several hemopoietic tissues of chickens as a function of the developmental stage of these organs. We found that in bone marrow, spleen, and bursa, maximum NCP98 kinase activity on a per-cell basis correlates with the peak of granulopoiesis in these organs. Furthermore, in a bovine serum albumin density gradient fractionation of bone marrow cells, granulocytic cells appeared to account for most of the NCP98 kinase activity. No correlation was found between the distribution of erythrocytic, lymphocytic, or thrombocytic cells and the distribution of the expression of NCP98 kinase activity. However, NCP98 protein and kinase activity were 10-fold higher in macrophages than in bone marrow. In addition, depletion by complement-mediated lysis of erythrocytic cells in bone marrow did not significantly reduce the total recovery of NCP98 kinase activity. These results argue for the specific expression of the c-fps gene product in granulocytic cells and macrophages.

Animals↗
PubMed1985

Phosphatidylinositol kinase activity in virus-transformed and nontransformed cells.

We assayed phosphatidylinositol (PI) kinase (EC 2.7.1.67) activity in detergent extracts of nontransformed or virus-transformed cells. Nontransformed chicken embryo fibroblasts (CEF) contain PI kinase activity with an apparent specific activity of 20 pmol/min per mg of protein. This activity sedimented as a single peak with a molecular weight of approximately 60,000 in a glycerol gradient, although immunoprecipitation with anti-p60src sera showed that the PI kinase activity is distinct from p60c-src. Extracts from CEF transformed by Rous sarcoma virus, Fujinami sarcoma virus, or avian sarcoma virus UR2 showed no elevation of PI kinase activity over nontransformed CEF. Removal of the oncogene products from extracts by immunoprecipitation did not change the level of PI kinase activity in extracts, suggesting that putative virus-coded PI kinases do not make a significant contribution to overall levels of PI kinase activity in transformed cells. Additionally, P140gag-fps was separated from cellular PI kinase by phosphocellulose chromatography. This partially purified fraction contained low PI kinase activity distinct from P140gag-fps, indicating that P140gag-fps has no detectable PI kinase activity.

1-Phosphatidylinositol 4-Kinase↗
PubMed1984

Rous sarcoma virus variants that carry the cellular src gene instead of the viral src gene cannot transform chicken embryo fibroblasts.

The transforming activity of the cellular src (c-src) gene as well as of hybrid genes between viral and cellular src was tested by constructing derivatives of Rous sarcoma virus DNA in which all or part of the viral src gene (v-src) was replaced by the corresponding portion of the c-src gene. After these derivatives were introduced into chicken embryo fibroblasts by transfection, replication-competent virus was recovered, which induced the expression of p60src at a level equivalent to p60v-src expression in cells infected with Rous sarcoma virus wild type. Replacement of the portion of the v-src gene, either upstream or downstream of the Bgl I site, with the homologous portion of the c-src gene resulted in fully transforming viruses. On the other hand, the virus stock obtained from cells transfected with Rous sarcoma virus DNA containing the entire c-src gene had a very low titer of focus-forming virus, while it contained a high titer of infectious virus. We present evidence that the rare small foci are formed by mutant viruses generated from the original c-src-containing virus. These results indicate that overproduction of the c-src gene product does not cause cell transformation, and that this proto-oncogene is subject to a relatively high rate of mutation when incorporated in a retrovirus genome, resulting in the acquisition of transforming capacity.

Animals↗
PubMed1984

DNA sequence of the Bryan high-titer strain of Rous sarcoma virus: extent of env deletion and possible genealogical relationship with other viral strains.

The genetic structure of the Bryan high-titer strain of Rous sarcoma virus (BH-RSV) was analyzed by using a molecular clone obtained from proviral DNA. DNA sequencing of the pol-src junction of BH-RSV revealed that the env sequence was almost entirely absent; only six base pairs following the pol termination codon remained. Beginning at nucleotide 7 (relative to the end of pol), a 91-base pair sequence identical to the 91 base pairs immediately upstream from src in other strains of RSV was found. The helper virus-related sequence of about 100 base pairs, which is present as a direct repeat in the 5' and 3' regions flanking src in other RSVs, was present only on the 3' side of src in BH-RSV. The 3' end of BH-RSV, from the last 16 base pairs of src through the U3 region, was virtually identical to a region downstream of env through U3 in the nontransforming helper virus Rous-associated virus-2, suggesting that BH-RSV may have been derived by recombination between Rous-associated virus-2 and cellular src DNA. The possibility that the original RSV may have been a defective transforming virus and a precursor of the nondefective RSV strains is discussed.

Amino Acid Sequence↗
PubMed1984

Identification of the viral sequence required for the generation of recovered avian sarcoma viruses and characterization of a series of replication-defective recovered avian sarcoma viruses.

The ability of transformation-defective deletion mutants of Schmidt-Ruppin Rous sarcoma virus to induce tumors and generate recovered sarcoma viruses (rASVs) was correlated with the partial src sequences retained in the transformation-defective viral genomes. Since all the transformation-defective viruses that were capable of generating rASVs retained a portion of the 3' src sequence, regardless of the extent of the 5' src deletion, and those lacking the 3' src were unable to generate rASVs, it appears that the 3', but most likely not the 5', src sequence retained in the transformation-defective viral genome is essential for rASV formation. However, rASVs derived from a particular mutant, td109, which retained a portion of the 3' src sequence, but lacked most (if not all) of the 5' src sequence, were all found to be defective in replication. Analyses of the genomic sequences of 13 isolates of td109-derived rASVs revealed that they contained various deletions in viral envelope (env), polymerase (pol), and structural protein (gag) genes. Ten isolates of rASVs contained env deletions. One isolate (rASV3812) contained a deletion of env and the 3' half of pol, and one isolate (rASV398) contained a deletion of env and pol. The one with the most extensive deletion (rASV374) had a deletion from the p12-coding sequence through pol and env. In addition, the 5' src region of td109-derived rASVs were heterogeneous. Among the 7 isolates analyzed in detail, one isolate of rASV had a small deletion of the 5' src sequence, whereas three other isolates contained extra new sequences upstream from src. Both env- and env- pol- rASVs were capable of directing the synthesis of precursor and mature gag proteins in the infected nonproducer cells. We attribute the deletions in the replication-defective rASVs to the possibility that the 5' recombination site between the td109 and c-src sequence, involved regions of only partial homology due to lack of sufficient 5' src sequence in the td109 genome for homologous recombination. A model of recombination between the viral genome and the c-src sequence is proposed to account for the requirement of the 3' src sequence and the basis for the generation of deletions in td109-derived rASVs.

Animals↗
PubMed1984

Revertants and partial transformants of rat fibroblasts infected with Fujinami sarcoma virus.

Fifteen revertants were isolated from three independent clones of rat fibroblasts transformed by Fujinami sarcoma virus (FSV). Three revertant clones resulted from the deletion of the one copy of the FSV provirus, and one encoded an enzymatically inactive, transformation-defective protein. The remaining revertant clones were characterized by a transcriptional block of the provirus. Digestion of chromosomal DNA with MspI and HpaII revealed that the FSV provirus was hypermethylated in these revertants, whereas proviral DNA of their spontaneous retransformants was hypomethylated. Furthermore, the revertants had lost DNase I-hypersensitive sites in and around the FSV provirus. The effect of transcriptional regulation of the FSV provirus was further analyzed in clones showing various degrees of phenotypic transformation. We quantitated v-fps mRNA levels in these cells by liquid hybridization and found that increasing levels of viral RNA correlated with a more pronounced transformed phenotype. These results suggest that transcription of FSV proviral DNA is under both viral and cellular control and that transformation by FSV is a function of the dosage of v-fps mRNA.

Animals↗
PubMed1984

Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.

Animals↗
PubMed1984

A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation.

We have constructed mutants by using linker insertion followed by deletion in the region of cloned Rous sarcoma virus DNA coding for the N-terminal 9 kilodaltons of the src protein. Previous work implicated this region in the membrane association of the protein. The mutations had little effect on src tyrosine kinase activity. Substitution of a tri- or tetrapeptide for amino acids 15 to 27, 15 to 49, or 15 to 81 had little effect on the in vitro transforming capacity of the virus. Like wild-type p60src, the src proteins of these mutants associated with plasma membranes and were labeled with [3H]myristic acid. In contrast, a mutant whose src protein had the dipeptide Asp-Leu substituted for amino acids 2 to 81 and a mutant with the tripeptide Asp-Leu-Gly substituted for amino acids 2 to 15 were transformation defective, and the mutant proteins did not associate with membranes and were not labeled with [3H]myristic acid. These results suggest that amino acids 2 to 15 serve as an attachment site for myristic acid and as a membrane anchor. Since deletions including this region prevent transformation, and since tyrosine kinase activity is not diminished by the deletions, these results imply that target recognition is impaired by mutations altering the very N terminus, perhaps through their effect on membrane association.

Amino Acid Sequence↗
PubMed1983

Temperature-sensitive membrane association of pp60src in tsNY68-infected cells correlates with increased tyrosine phosphorylation of membrane-associated proteins.

Incubation of membrane vesicles from normal and Rous sarcoma virus-transformed chick embryo fibroblasts (CEF) with [gamma-32P]ATP resulted in the phosphorylation of a large number of proteins. The major differences observed between the membrane vesicles of untransformed and transformed cells were: (1) a 5- to 10-fold increase in the proportion of labeled phosphotyrosine in transformed vesicles and (2) the phosphorylation of pp60src in vesicles from transformed cells. Of the many proteins labeled in vitro, only pp60src was immunoprecipitated by TBR serum. Phosphorylation of the immunoprecipitated pp60src occurred on tyrosine in the 26-kDa carboxy-terminal Staphylococcus aureus V8 protease fragment. pp60src was not phosphorylated in vitro in membrane vesicles prepared from tsNY68-infected cells grown at the nonpermissive temperature. The proportion of labeled phosphotyrosine in membrane proteins from tsNY68-infected cells grown at the nonpermissive temperature was only slightly increased relative to that observed in membranes prepared from normal cells. Subcellular fractionation indicated that while pp60src was membrane associated in tsNY68-infected cells grown at the permissive temperature, pp60src was chiefly soluble in tsNY68-infected cells grown at the nonpermissive temperature. Temperature-sensitive membrane association of pp60src in tsNY68-infected cells was also observed by indirect immunofluorescence microscopy. When membranes were prepared from tsNY68-infected cells that had been downshifted from the nonpermissive to the permissive temperature, the reappearance of in vitro phosphorylated pp60src and the increase in the proportion of labeled phosphotyrosine in membrane vesicles correlated with the kinetics of src immune complex kinase reactivation and membrane association of pp60src.

Animals↗
PubMed1983

Transforming genes of avian (v-fps) and mammalian (v-fes) retroviruses correspond to a common cellular locus.

The Gardner (GA) and Snyder-Theilen (ST) isolates of feline sarcoma virus (FeSV) represent genetic recombinants between feline leukemia virus (FeLV) and transformation-specific sequences (v-fes gene) of cat cellular origin. A related transforming gene (v-fps), common to the Fujinami, PRC II, and UR 1 strains of avian sarcoma virus has also been described. Translational products of each of these recombinant virus isolates are expressed in the form of polyproteins exhibiting protein kinase activities with specificity for tyrosine residues. In the present study, v-fes and v-fps homologous sequences of GA-FeSV, ST-FeSV, and Fujinami sarcoma virus (FSV) are defined and these independently derived transforming genes are shown to correspond to a common cellular genetic locus which has remained highly conserved throughout vertebrate evolution.

Animals↗
PubMed1983

Structure and sequence of the cellular gene homologous to the RSV src gene and the mechanism for generating the transforming virus.

We determined the nucleotide sequences of all coding regions and a significant part of the flanking regions of the chicken c-src gene, which is a cellular homolog of the v-src gene of Rous sarcoma virus. The c-src gene consists of 12 exons; the boundaries of the exons were determined by assuming that the amino acid sequence of its product, pp60c-src, is basically the same as that of pp60v-src. The deduced amino acid sequence of pp60c-src was very similar to that of pp60v-src, but the last 19 carboxy-terminal amino acids of pp60c-src were replaced by a new set of 12 amino acids of pp60v-src. The sequence encoding the carboxy-terminal sequence of pp60v-src was found 900 bp downstream from the termination codon of the c-src gene. We suggest that the c-src sequence was captured by a virus through recombination at both sides of the c-src gene, and that the recombinations occurred at the level of proviral DNA.

Animals↗
PubMed1983

Local mutagenesis of Rous sarcoma virus: the major sites of tyrosine and serine phosphorylation of pp60src are dispensable for transformation.

We have constructed mutants of Rous sarcoma virus expressing p60srcs that are underphosphorylated on serine or tyrosine, by linker insertion or insertion/deletion into cloned Rous sarcoma virus DNA, and recovery of mutant virus by transfection of chicken embryo fibroblasts. Cells infected with mutants whose p60srcs lack the major site of either serine or tyrosine phosphorylation were morphologically transformed and formed colonies in soft agar. The tyrosine kinase activities of the mutant p60srcs measured in vivo and in vitro were close to the wild type activity. Peptide mapping showed that phosphorylation on tyrosine and serine of p60src is independent: the major phosphorylated tyrosine and the major phosphorylated serine can each be phosphorylated in the absence of phosphorylation of the other.

Animals↗
PubMed1983

Cytoplasmic localization of the transforming protein of Fujinami sarcoma virus: salt-sensitive association with subcellular components.

Fujinami sarcoma virus (FSV) encodes a transforming protein of 130,000 daltons (P130) which is associated with a tyrosine-specific protein kinase activity. To elucidate mechanisms involved in cell transformation by FSV, we have studied the intracellular location of P130 in rat cells nonproductively infected with FSV. Immunofluorescent staining of several FSV-transformed rat cell lines with a tumor regressor antiserum specific against the fps sequences of P130 showed that the major staining was localized in the cytoplasm. Staining was also seen in cell ruffles and in some cases at areas of cell contact. The cytoplasmic location of P130 staining in cells infected with temperature-sensitive mutants of FSV was unchanged when they were grown at permissive or nonpermissive temperature. Cell fractionation of FSV-transformed cells under various conditions showed that the ionic strength used during cell fractionation had a striking effect on the distribution of P130. At 10 mM NaCl, 70% of P130 sedimented in the large granule fraction, whereas at 500 mM NaCl 70 to 90% of P130 was recovered in the cytosol fraction. Furthermore, a combination of ionic and nonionic detergents that effectively solubilized subcellular membranes was insufficient to solubilize P130 unless the salt concentration was raised. We conclude that the majority of P130 and its associated protein kinase activity are localized in the cytoplasm and that P130 is not an integral membrane protein.

Animals↗
PubMed1983

A fps gene without gag gene sequences transforms cells in culture and induces tumors in chickens.

From molecularly cloned DNAs of Fujinami sarcoma virus (FSV) and the Schmidt-Ruppin-A strain of Rous sarcoma virus (SRA), viral DNA was constructed in which fps-specific sequences encoded in FSV replaced the src gene of SRA. A 3' fragment of FSV DNA, from an ATG methionine coding sequence 148 base pairs downstream from the gag-fps junction through the long terminal repeat, was joined to cloned SRA DNA at the translation start site for the src gene. The resultant DNA clone contained the splice acceptor site for src mRNA processing in SRA, but contained no src coding sequences from SRA nor any gag sequences from FSV. All genes for the replication of SRA were retained. Transfection of this cloned viral DNA genome into chicken embryo fibroblasts induced morphological transformation of the cells in culture. However, the morphology of the transformed cells was distinct from that observed in cells infected with wild-type FSV. The transformed cells produced a nondefective transforming virus called F36 which contained a hybrid FSV-SRA long terminal repeat. F36-infected cells produced a protein with the expected molecular weight of 91,000, which had an associated protein kinase activity and was immunoprecipitated by antibodies raised against fps gene determinants but not by antibodies raised against gag or src proteins. Injection of F36 virus into 8-day-old chicks produced tumors at the site of inoculation, detectable within 7 days. These results demonstrated that the gag portion of the gag-fps fusion protein of FSV is not required for transformation or tumorigenesis.

Animals↗
PubMed1982

Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity.

We have suggested previously that the amino-terminal 8 kilodaltons of pp60src may serve as a structural hydrophobic domain through which pp60src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, ion contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60src, and suggest that the amino terminus of pp60src may have a critical role in the promotion of in vivo tumorigenicity.

Amino Acid Sequence↗
PubMed1982

A cellular protein is immunologically crossreactive with and functionally homologous to the Fujinami sarcoma virus transforming protein.

We obtained a regressing-tumor antiserum specific for the unique sequence of the transforming protein P140 of Fujinami sarcoma virus by injecting Fischer rats with syngeneic embryo cells transformed with Fujinami sarcoma virus. This serum is capable of immunoprecipitating a protein of 98,000 daltons from cell extracts of normal, uninfected chicken bone marrow cells. This normal cellular protein (NCP98) was shown to be structurally related to P140, sharing the majority of 35S-methionine-labeled tryptic peptides with the viral gene product P140. NCP98 is a phosphoprotein in vivo, with an associated in vitro protein kinase activity, capable of phosphorylating specifically at tyrosine residues of NCP98 itself and alpha-casein, an externally added substrate. This kinase activity is biochemically indistinguishable from the kinase activity associated with P140 by all criteria tested. Moreover, in vitro-phosphorylated NCP98 and P140 shared the same phosphopeptides. The expression of NCP98 is tissue-specific. It is readily detectable in bone marrow cells and detectable to a lesser extent in liver and lung cells from 6--18 day old chickens.

Animals↗