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H Hanafusa

Publications and source records attributed to H Hanafusa.

At least 127 records · Page 7Linked to original sources
PubMed1990

A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src.

A potential substrate of p60v-src in Rous sarcoma virus-transformed cells was found to be a 130-kilodalton (kDa) glycoprotein which binds to lectin-Sepharose and can be immunoprecipitated by an anti-phosphotyrosine antibody. This glycoprotein was shown to be distinct from the fibronectin receptor and a cellular protein phosphorylated in p60v-src immune complexes. The protein was a transmembrane protein localized in the plasma membrane and resistant to extraction with Triton X-100. The 130-kDa protein was also highly phosphorylated in cells transformed by Fujinami sarcoma virus or Y73 but not in cells infected with Rous sarcoma virus mutants that encode p60v-src lacking myristoylated N termini. Phosphorylation of this glycoprotein was temperature dependent in cells infected with temperature-sensitive mutants. The good correlation between its phosphorylation and morphological transformation, together with its relative abundance among phosphorylated proteins and its subcellular localization, suggests that phosphorylation of the 130-kDa glycoprotein is one of the primary events important for cell transformation by p60v-src and related oncogene products.

Animals↗
PubMed1990

Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain.

To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. Expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus.

Amino Acid Sequence↗
PubMed1990

Expression of 9E3 mRNA is associated with mitogenicity, phosphorylation, and morphological alteration in chicken embryo fibroblasts.

Transformation of chicken embryo fibroblasts (CEF) with viruses encoding src, ros, yes, and fps as well as ras, mos, middle T, erbA and erbB, myc, and crk stimulated 9E3 mRNA expression. Treatment of CEF with agents that modulate cell shape or attachment to the substratum caused an increase in 9E3 mRNA without an increase in tyrosine phosphorylation. 9E3 mRNA was also increased in CEF in response to several agents which modulate phosphorylation, including phorbol myristic acetate, vanadate, and okadaic acid, which suggests that the rapid induction of 9E3 mRNA expression in CEF by the src protein occurs downstream of morphological or phosphorylation events.

Animals↗
PubMed1989

Event-related potentials (P300) in myotonic dystrophy.

The P300 component of the auditory event-related potential in 8 patients with myotonic dystrophy was studied and compared with that of 13 healthy controls. Abnormalities of P300 (prolongation of the latency and/or decrease of the amplitude) were observed in 6. These observations imply that the function of cognitive and information processing are impaired in myotonic dystrophy.

Acoustic Stimulation↗
PubMed1989

Suppression of Rous sarcoma virus-induced tumor formation by preinfection with viruses encoding src protein with novel N termini.

Two recovered avian sarcoma viruses (rASVs), rASV157 and rASV1702, encode src products which contain novel, nonmyristoylated N-terminal amino acids. These viruses transform chicken embryo fibroblasts and cause tumors in chicks. However, the tumors rASVs induce are small and regress within 2 weeks. To determine whether this regression results from weak tumorigenicity or from the active immunity of the host, we injected 1-week-old chicks with rASV and several days later injected the chicks with challenge virus of a different subgroup. Of the rASV1702-preinfected chicks challenged 5 days later with Rous sarcoma virus (RSV), 40% showed no subsequent tumor formation and 60% formed tumors which regressed within 1 week. The potency of this protective effect depended on the dosage of preinfection virus used and increased as the interval between preinfection and challenge infection was lengthened (when the interval was 9 days, none of the challenged chicks formed tumors). rASV157-preinfected chicks challenged with RSV after 9 days showed only partial protection: 42% formed tumors which regressed, whereas 58% formed tumors which continued to grow. Challenging rASV-preinfected chicks with Fujinami sarcoma virus or a RSV vector encoding the v-fps oncogene or polyomavirus middle T resulted in no suppression of tumor formation. Preinfection with src mutants or a RSV vector encoding polyomavirus middle T antigen, both of which induce slow-growing tumors, failed to elicit the protective effect. Finally, a novel N-terminal domain encoded by rASV1702 src was shown to be involved in but not sufficient for full protection. These data indicate that determinants on or induced by rASV157 and rASV1702 can elicit a potent protection against the tumorigenic potential of RSV-encoded p60v-src.

Animals↗
PubMed1989

Modulation of arachidonic acid metabolism by Rous sarcoma virus.

Arachidonic acid (C20:4) metabolites were released constitutively from wild-type Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF). 3H-labeled C20:4 and its metabolites were released from unstimulated and uninfected CEF only in response to stimuli such as serum, phorbol ester, or the calcium ionophore A23187. High-pressure liquid chromatography analysis showed that the radioactivity released from [3H]arachidonate-labeled transformed cells was contained in free arachidonate and in the cyclooxygenase products prostaglandin E2 and prostaglandin F2 alpha; no lipoxygenase products were identified. The release of C20:4 and its metabolites from CEF infected with pp60src deletion mutants was correlated with serum-independent DNA synthesis and with the expression of the mRNA for 9E3, a gene expressed in Rous sarcoma virus-transformed cells which has homology with several mitogenic and inflammatory peptides. 3H-labeled C20:4 release was not correlated with p36 phosphorylation, which argues against a role for this protein as a phospholipase A2 inhibitor. CEF infected with other oncogenic viruses encoding a tyrosine kinase also released C20:4, as did CEF infected with viruses that contained mos and ras; however, infection with a crk-containing virus did not result in stimulation of 3H-labeled C20:4 release, suggesting that utilization of this signaling pathway is specific for particular transformation stimuli.

Animals↗
PubMed1989

Processing of 9E3 mRNA and regulation of its stability in normal and Rous sarcoma virus-transformed cells.

We studied the expression of 9E3 mRNA, which is known to be induced in chicken embryo fibroblasts by p60v-src activity and by serum. In addition to full-length 9E3 mRNA, we identified several smaller RNAs that hybridized with 9E3 cDNA. One of these RNAs hybridized with a 5' 9E3 cDNA probe but not with a 3' cDNA probe. The other hybridized with a 3' cDNA probe but lacked 5' sequences, including the entire 9E3 coding region. Only the latter RNA was polyadenylylated, as determined by RNase H digestion in the presence of oligo(dT). The level of the small RNAs increased after treatment with cycloheximide and actinomycin D, indicating that the small RNAs were produced by processing of preexisting transcripts. The derivation of the small RNAs from 9E3 mRNA rather than from a related gene was confirmed by S1 nuclease analysis. The 3' terminus of the 5' RNA and the 5' terminus of the 3' RNA mapped to the same position, which suggested that the small RNAs were formed by endonucleolytic cleavage of 9E3 mRNA at a specific site in the 3' noncoding region. We also found that the stability of 9E3 mRNA was increased after serum stimulation and was greater in Rous sarcoma virus-transformed than in uninfected cells. The relative amount of the small RNAs as compared with the full-length transcript was greatest under conditions in which the full-length transcript was least stable. These data suggest that site-specific endonucleolytic cleavage regulates the stability of 9E3 mRNA.

Animals↗
PubMed1989

Phosphatidylinositol kinase activity associates with viral p60src protein.

Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.

1-Phosphatidylinositol 4-Kinase↗
PubMed1989

In vivo phosphorylation states and kinase activities of transforming p60c-src mutants.

To further investigate regulation of p60src by tyrosine phosphorylation, the in vivo phosphorylation states and kinase activities of transforming mutants derived from p60c-src were examined. One set of six in vitro recombinants encoded chimeric proteins containing the carboxyl terminus of chicken p60c-src (including Tyr 527) in combination with various isolated amino acid substitutions present in p60v-src. Another set of seven viral isolates were randomly selected from soft-agar colonies infected with replicating retroviruses that originally encoded normal p60c-src. Results show that mutations at multiple sites throughout amino- and carboxy-terminal domains are sufficient to alter the in vivo phosphorylation state and kinase activity of p60c-src. Moreover, all of the transforming p60c-src mutants exhibited elevated in vivo kinase activities, as assayed by immune blot analysis of cellular proteins using antibodies to phosphotyrosine, which correlated well with enhanced auto-phosphorylation at Tyr 416. By contrast, Tyr 527 phosphorylation in the transforming mutants varied from low to high levels that were correlated to a limited extent with levels of in vivo kinase activities. Taken together, results suggest that, in addition to complete dephosphorylation of Tyr 527, other mechanisms contribute to p60c-src activation.

Animals↗
PubMed1989

Localization of major potential substrates of p60v-src kinase in the plasma membrane matrix fraction.

Subcellular localization of potential substrates of a tyrosine-protein kinase, p60v-src, was analyzed by cell fractionation in combination with immunoblotting with antiphosphotyrosine antibody. In cells transformed by wild type Rous sarcoma virus, most phosphotyrosine-containing proteins were found both in plasma membranes and in a cytoplasmic matrix structure associated with plasma membranes and resistant to nonionic detergent extraction (plasma membrane matrix). A similar localization of phosphotyrosine-containing proteins was obtained in cells transformed by PRCII, Y73, or Fujinami sarcoma virus. On the other hand, in cells infected with Rous sarcoma virus mutants that encode nonmyristylated p60v-src, tyrosine phosphorylation was found mostly in proteins which were different from those identified in wild type-infected cells and were distributed to both plasma membrane and cytosolic fractions. These results suggest that most cellular substrate proteins, phosphorylation of some of which may be critical for the initiation of transformation, are present primarily in the plasma membrane-matrix structure.

Animals↗
PubMed1989

Phosphatidylinositol kinase type I activity associates with various oncogene products.

We have assayed immunoprecipitates of several oncogene products from retrovirally infected chicken embryo fibroblasts (CEF) for phosphatidylinositol (PI) kinase activity. Immunoprecipitates of P68gag-ros, P130gag-tps, P47gag-crk, polyoma middle T (mT)-p60c-src complex, and mT-p62c-yrs complex exhibited PI kinase activity when assayed without detergents. This activity was sensitive to the nonionic detergent, Triton X-100, and the product was indistinguishable from phosphatidylinositol-3-phosphate, the product of kinase type I. Immunoprecipitates of p21Ha-ros protein did not contain any PI kinase type I activity. It has been suggested that an 81 kD protein phosphorylated in in vitro kinase assays of immunoprecipitates from mT-transformed rodent cells is responsible for the PI kinase type I activity seen in these immunoprecipitates. We have detected a chicken homologue of this 81 kD protein in immunoprecipitates of lysates from mT-transformed CEF. However, the chicken 81 kD protein sedimented more quickly than the PI kinase activity in sucrose gradients. In addition, the 81 kD protein was not detectable in protein kinase assays of immunoprecipitates of P68gag-ros or P130gag-fps. These results suggest that the 81 kD protein may not be the PI kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

A novel viral oncogene with structural similarity to phospholipase C.

Numerous oncogenes have been isolated from acutely transforming retroviruses. To date, the products of these viral oncogenes have been protein kinases, nuclear proteins, growth factors, or GTP-binding proteins. We have cloned the previously uncharacterized avian sarcoma virus CT10 and sequenced its genome. This virus encodes a protein, p47gag-crk, that has blocks of sequence similarity to the amino-terminal, non-catalytic region of the non-receptor class of tyrosine kinases. In addition, the structure of p47gag-crk has striking similarity to a 180-amino acid region of bovine brain phospholipase C. Biochemical data suggest that p47gag-crk activates one or several endogenous tyrosine kinases.

Amino Acid Sequence↗
PubMed1988

Avian sarcoma viruses.

Twelve independent isolates of avian sarcoma viruses (ASVs) can be divided into four groups according to the transforming genes harbored in the viral genomes. The first group is represented by viruses containing the transforming sequence, src, inserted in the viral genome as an independent gene; the other three groups of viruses contain transforming genes fps, yes or ros fused to various length of the truncated structural gene gag. These transforming sequences have been obtained by avian retroviruses from chicken cellular DNA by recombination. The src-containing viruses code for an independent polypeptide, p60src; and the representative fps, yes and ros-containing ASVs code for P140/130gag-fps, P90gag-yes and P68gag-ros fusion polypeptides respectively. All of these transforming proteins are associated with the tyrosine-specific protein kinase activity capable of autophosphorylation and phosphorylating certain foreign substrates. p60src and P68gag-ros are integral cellular membrane proteins and P140/130gag-fps and P90gag-yes are only loosely associated with the plasma membrane. Cells transformed by ASVs contain many newly phosphorylated proteins and in most cases have an elevated level of total phosphotyrosine. However, no definitive correlation between phosphorylation of a particular substrate and transformation has been established except that a marked increase of the tyrosine phosphorylation of a 34,000 to 37,000 dalton protein is observed in most ASV transformed cells. The kinase activity of ASV transforming proteins appears to be essential, but not sufficient for transformation. The N-terminal domain of p60src required for myristylation and membrane binding is also crucial for transformation. By contrast, the gag portion of the FSV P130gag-fps is dispensable for in vitro transformation and removal of it has only an attenuating effect on in vivo tumorigenicity. The products of cellular src, fps and yes proto-oncogenes have been identified and shown to also have tyrosine-specific protein kinase activity. The transforming potential of c-src and c-fps has been studied and shown that certain structural changes are necessary to convert them into transforming genes. Among the cellular proto-oncogenes related to the four ASV transforming genes, c-ros most likely codes for a growth factor receptor-like molecule. It is possible that the oncogene products of ASVs act through certain membrane receptor(s) or enzyme(s), such as protein kinase C, in the process of cell transformation.

Animals↗
PubMed1988

p60c-src is complexed with a cellular protein in subcellular compartments involved in exocytosis.

We found high levels of the c-src gene product in neuroendocrine tissues from adult animals. To understand the role of this proto-oncogene product, the subcellular localization of p60c-src was studied in neuroendocrine tissue from adrenal medulla. The results indicate that p60c-src was highly enriched in chromaffin granule membranes, in stable association with a protein of 38 kD. The complex with the 38-kD protein was also detected in brain, a tissue known to carry high levels of p60c-src. The 38-kD protein is not calpactin I, II, or synaptophysin. Comparison of its peptide map showed a high degree of conservation among the different species and tissues examined. The interaction between p60c-src and the 38-kD protein involves disulphide bonds that are stable even when the cell fractionation is performed in the presence of a reducing agent. Since the presence of disulphide bonds among cytoplasmic proteins is very unlikely, the possibility of a noncovalent association between p60c-src and the 38-kD protein in vivo is discussed. The 38-kD protein may be involved in a function of p60c-src related to secretory organelles.

Adrenal Medulla↗
PubMed1988

The putative trans-activator in the MAgag region of Rous sarcoma virus is not required for cell transformation.

A stop codon created by oligonucleotide-directed mutagenesis in the proposed transcriptional trans-activator of Rous sarcoma virus (S. Broome and W. Gilbert, Cell 40:537-546, 1985) which truncated the trans-activator by half did not alter the transforming activity or the replication of the virus in primary chicken embryo fibroblasts. This result proves that this trans-activator is not essential for transformation of primary cells by Rous sarcoma virus.

Amino Acid Sequence↗