Biomedical subjects

H Hanafusa

Publications and source records attributed to H Hanafusa.

At least 163 records · Page 9Linked to original sources

PubMed1986

Isolation of chicken cellular DNA sequences with homology to the region of viral oncogenes that encodes the tyrosine kinase domain.

A library of chicken genomic DNA was screened for sequences that could hybridize to a cloned DNA fragment containing the transforming gene (v-fps) of Fujinami sarcoma virus. In addition to c-fps, two unique chicken cellular DNA sequences were isolated that hybridized weakly to v-fps. These sequences hybridized with many other viral oncogenes encoding tyrosine kinases. Sequence analysis of the region where homology was detected revealed a region that is highly conserved among the tyrosine kinases both at the nucleotide and amino acid levels. Although we were unable to detect expression of either chicken cellular DNA sequence in a variety of avian tissues, the data suggest the existence of additional members of the tyrosine kinase gene family. Screening genomic libraries for sequences that hybridize weakly to functional regions of other genes may prove useful for the isolation and characterization of additional members of other gene families.

Animals↗

PubMed1986

Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

Amino Acid Sequence↗

PubMed1986

Antipeptide antiserum identifies a widely distributed cellular tyrosine kinase related to but distinct from the c-fps/fes-encoded protein.

We raised antibodies directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of the transforming protein of Fujinami sarcoma virus (FSV) (P140). The antiserum obtained specifically recognized FSV-P140 and its cellular homolog and in addition, it recognized a new cellular protein of 94,000 daltons (NCP94) in avian and mammalian cells. NCP94 was found to be associated with a cyclic nucleotide-independent protein kinase activity that was specific for tyrosine residues. Although NCP94 and FSV-P140 share antigenic determinants, NCP94 is not a cellular homolog of FSV-P140: NCP94 and the previously identified c-fps/fes product were different in their tryptic fingerprints and in their tissue specificities. Thus, the function of NCP94 in normal cells is probably different than that of the c-fps/fes product. NCP94 was expressed in every tissue and cell line that was examined. In chickens, NCP94 levels were highest during embryonic development and NCP94 expression was high in gizzard, brain, and spleen throughout embryonic and adult life. The universal expression of NCP94 suggests that this protein may be involved in an essential function of normal cells. NCP94 may be a new cellular tyrosine kinase of the src gene family.

Amino Acid Sequence↗

PubMed1986

Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector.

The middle T antigen of polyomavirus transformed primary chicken embryo fibroblasts when expressed from a replication-competent avian retrovirus. This in vitro-constructed retrovirus, SRMT1, is a variant of Rous sarcoma virus that encodes the middle T antigen in place of v-src. Inoculation of SRMT1 into 1-week-old chickens rapidly induced hemangiomas and hemangiosarcomas. As shown with mammalian cells infected with polyomavirus, polyomavirus middle T antigen appears to be associated with p60c-src in chicken cells infected with SRMT1. When lysates of SRMT1-infected cells immunoprecipitated with either a monoclonal antibody against p60src or anti-T serum were assayed in an in vitro kinase reaction, the middle T antigen was heavily phosphorylated. To see whether an excess of p60c-src could alter the extent of phosphorylation of the middle T protein or the process of cell transformation by middle T, cells were doubly infected with SRMT1 and NY501, a virus which overexpresses p60c-src. Doubly infected chicken embryo fibroblasts transformed with the same kinetics and were morphologically indistinguishable from chicken embryo fibroblasts infected with SRMT1 alone. Phosphorylation of the middle T antigen was elevated two- to fivefold relative to cells infected only with SRMT1.

Animals↗

PubMed1986

Cellular proteins homologous to the viral yes gene product.

We raised antibodies in rabbits against the amino-terminal portion of the viral yes protein produced in bacteria with the use of an expression vector based on the lac operon. The anti-yes serum thus obtained precipitated P90gag-yes from Yamaguchi 73 virus-transformed chicken embryo fibroblasts, and this immunoprecipitation was blocked by the purified antigen. The anti-yes serum did not recognize viral src, fps, or fgr proteins. Affinity-purified anti-yes immunoglobulin G (IgG) precipitated two proteins of 59 and 62 kilodaltons from lysates of normal chicken embryo fibroblasts. Two-dimensional tryptic peptide mapping showed that these proteins are closely related to P90gag-yes and that they are different from pp60c-src. Similar to P90gag-yes, the 59- and 62-kilodalton proteins were phosphorylated exclusively on tyrosine in an in vitro kinase reaction, whereas in vivo they were phosphorylated on serine and, to a lesser extent, on tyrosine as well. Expression of the 59- and 62-kilodalton proteins, determined by the immune complex kinase assay, was relatively high in brain, retina, kidney, and liver. The presence in normal chicken embryo fibroblasts and in chicken kidney of two transcripts, 3.7 and 3.9 kilobases in length, that hybridize with a yes-specific DNA probe, as well as the two proteins recognized by anti-yes IgG, suggests either differential splicing of cellular yes gene transcripts or the existence of another yes-related gene.

Animals↗

PubMed1986

Transformation by p60src with altered N-terminal sequences.

The transforming activity of the src gene product of Rous sarcoma virus, p60src, depends on both tyrosine specific protein kinase activity and N-terminal myristylation that is required for the plasma membrane association of this protein. The src proteins of two recovered avian sarcoma viruses, rASV157 and rASV1702, are exceptional in that they are not myristylated and yet are active in transformation. These viruses also induce tumors that regress rapidly. We found that their src proteins have unusual N-terminal structures: 30-45 amino acids of the env signal peptide are attached to internal (6th and 76th) amino acids in the src sequences. These altered N-terminal structures seem to be responsible for many abnormal properties of these mutant src proteins, including the early regression of tumors they induce.

Amino Acid Sequence↗

PubMed1985

Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein.

The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.

Acylation↗

PubMed1985

Activation of the transformation potential of the cellular fps gene.

Chicken cellular-fps (c-fps) sequences were substituted for viral-fps (v-fps) sequences in two retroviral genome structures, one that expressed a c-fps gene product that was indistinguishable from the normal c-fps gene product expressed in chicken bone marrow cells, and another that expressed a gag-fps fusion protein. When c-fps gene sequences (without linked gag gene sequences) were expressed at high levels in a viral vector, no transformation of fibroblasts was detected. It was previously demonstrated that the corresponding v-fps sequences could transform fibroblasts. When the same c-fps sequences were expressed in a form linked to gag gene sequences, transformation of fibroblasts and induction of tumors were observed. The data suggest that the c-fps gene product lacks transformation potential by itself even when overexpressed and that the transformation potential of the c-fps gene can be activated by either mutation (or mutations) in the fps coding region or by fusion with viral gag gene sequences.

Animals↗

PubMed1985

Fine structural mapping of a critical NH2-terminal region of p60src.

We have recently demonstrated that an NH2-terminal sequence required for myristylation and membrane association of the Rous sarcoma virus transforming protein, p60src, is contained within amino acids 2-14 [Cross, F.R., Garber, E. A., Pellman, D. & Hanafusa, H. (1984) Mol. Cell. Biol. 4, 1834-1842]. This sequence is also required for cell transformation. We have now constructed five mutants of Rous sarcoma virus that contain alterations in the src sequence coding for these 14 amino acids. Mutants encoding src proteins with a peptide insertion between amino acids 1 and 2, or peptide substitutions for amino acids 2-4, 3-4, or 7-15, were transformation-defective. The src proteins of these mutants differed from the wild-type protein in that they were not myristylated and did not fractionate with the plasma membrane of infected cells. The fifth mutant encoded a src protein with a short peptide substituted for amino acids 11-15. This protein was myristylated and plasma membrane associated, and the virus transformed cells. We therefore conclude that a sequence required for myristylation and membrane association of p60src is located within the first 7-10 amino acids of the src protein, and that p60src myristylation and membrane association are required for cell transformation. Consistent with this idea, we have isolated four transforming revertants from one of the transformation-defective mutants. The src proteins of all four revertants were found to be myristylated and membrane associated.

Amino Acid Sequence↗

PubMed1985

Specific expression of the human cellular fps/fes-encoded protein NCP92 in normal and leukemic myeloid cells.

We have found that both an antibody directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of transforming protein P140 of Fujinami sarcoma virus and a regressing tumor antiserum recognized the products of the c-fps/fes genes of both avian and mammalian cells. The anti-peptide antibody also recognized a 94-kilodalton protein that was related to but distinct from the c-fps/fes product in structure and in tissue distribution. A 92-kilodalton protein, NCP92, was found to be the mammalian counterpart of the previously identified avian c-fps/fes protein NCP98 by its structural similarity to NCP98, its associated tyrosine kinase activity, and its similar tissue distribution. The highest levels of NCP92 were found in tissue macrophages and in bone marrow. In bone marrow NCP92 expression was restricted to cells of the monocyte/macrophage and granulocyte lineages. That the expression of NCP92 is limited to these cell types was confirmed by the analysis of murine and human hematopoietic tumors representing different cell lineages: NCP92 was positive in leukemic cells of granulocytic and monocytic origin but not in B-lymphocytic, T-lymphocytic, or erythroid tumor cells. The expression of NCP92 seems to be related to the capacity of myeloid cells to differentiate and to respond to certain colony-stimulating factors.

Animals↗

PubMed1985

Partial nucleotide sequence of Rous sarcoma virus-29 provides evidence that the original Rous sarcoma virus was replication defective.

Rous sarcoma virus-29 (RSV-29) is the strain of RSV that has the least number of passages beyond its isolation from chicken tumor no. 1 among all current strains of RSV. Biological characterization indicated that it was replication defective. RNA analysis of nonproducer clones of RSV-29-infected chicken embryonic fibroblasts showed the presence of a subgenomic message of 2.6 kilobases containing src and a genomic RNA of 7.7 kilobases that contains gag, pol, and src, but not env. The src-containing EcoRI fragment of RSV-29 proviral DNA was molecularly cloned. Sequence analysis of the regions flanking src revealed that the env gene was completely deleted in RSV-29 and that the sequence across the deletion was exactly the same as the Bryan high-titer strain of RSV. The sequence immediately 3' to src in RSV-29 was closely related to that of the Prague strain of RSV. The fact that the strain of RSV which has the minimal number of passages beyond its isolation is replication defective supports the hypothesis of Lerner and Hanafusa (J. Virol. 49:549-556, 1984) that the original RSV is a defective transforming virus. This defective transforming virus is postulated to be the precursor to other defective RSVs like the Bryan high-titer strain and to nondefective RSVs like the Prague strain. The particular clone of RSV-29 that we studied also had a short stretch of sequence duplication at the 3' end of the pol gene, which was presumably created by an error of reverse transcription.

Animals↗

PubMed1985

Two independent mutations are required for temperature-sensitive cell transformation by a Rous sarcoma virus temperature-sensitive mutant.

We molecularly cloned the src coding region of tsNY68, a mutant of Rous sarcoma virus temperature sensitive (ts) for transformation, and constructed a series of ts wild-type recombinant src genes. DNA containing the hybrid genes was transfected into chicken cells together with viral vector DNA and helper viral DNA, and infectious transforming viruses were recovered. Characterization of these recombinant viruses indicated that at least two mutations are present in the 3' half of the mutant src gene, both of which are required for ts. Nucleotide sequence analysis revealed three differences in the deduced amino acid sequence compared with the parental virus. Two of these changes, a deletion of amino acids 352 to 354 and an amino acid substitution at position 461, are responsible for the ts phenotype.

Animals↗

PubMed1985

Membrane association of the transforming protein of avian sarcoma virus UR2 and mutants temperature sensitive for cellular transformation and protein kinase activity.

The localization of the transforming protein P68gag-ros of avian sarcoma virus UR2, which has a hydrophobic region at the N terminus of its ros-specific tyrosine kinase-encoding sequence, was examined by subcellular fractionation. P68 behaved as an integral membrane protein associated with the plasma membrane of transformed cells. P68 became membrane associated very rapidly in its biogenesis. Three temperature-sensitive mutants of UR2 were isolated and characterized. Cells infected with the mutants were temperature sensitive for morphological alteration and colony formation. The mutant P68 proteins were membrane associated in mutant-infected cells regardless of the temperature but were active as protein kinases only at the permissive temperature. The results suggest that P68 is a membrane-associated protein whose kinase activity plays a crucial role in UR2-mediated cell transformation.

Animals↗

PubMed1985

Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses.

The level of phosphotyrosine in vinculin was determined in chicken embryo fibroblasts transformed by various strains of avian sarcoma virus. As previously reported (Sefton et al., Cell 24:165-174, 1981), vinculin was phosphorylated at tyrosine residues in most cultures examined, but the level varied greatly and no detectable change was found in cultures infected with Fujinami sarcoma virus or UR2 sarcoma virus. Regardless of the level of vinculin phosphorylation, the number of organized microfilament bundles was found to be decreased in all transformed cells. These results strongly suggest that tyrosine phosphorylation of vinculin is not an obligatory step in cell transformation by this class of oncogenes, nor is it correlated with the associated cytoskeletal disarray.

Animals↗

PubMed1985

Processing of p60v-src to its myristylated membrane-bound form.

p60src of wild-type Rous sarcoma virus is myristylated at its N-terminal glycine residue. We have shown previously that this myristylation is necessary for p60src membrane association and for cell transformation by using src mutants with alterations within the N-terminal 30 kilodaltons of p60src. In this study we analyzed the process of p60src myristylation in wild type- and mutant-infected cells. All myristylated src proteins examined lack the initiator methionine, but two mutant src proteins lacking the initiator methionine are not myristylated, indicating that removal of the initiator methionine and myristylation are not obligatorily coupled. Analysis of the kinetics of myristylation and the association of p60src with cellular proteins p50 and p90 indicated that myristylation occurs before p60src becomes membrane associated and that transient association with p50 and p90 occurs regardless of myristylation. Myristylation is required for stable association of p60src with the plasma membrane but is not sufficient for membrane association. A mutant with an src deletion of amino acids 169 through 264 has an src protein that is myristylated but not membrane bound, remaining stably associated with p50 and p90. This mutant is transformation defective. Several N-terminal deletion mutants possessing tyrosine kinase activity have myristylated and membrane-bound src proteins but are not fully active in cell transformation, suggesting that additional N-terminal functional domains exist.

Acylation↗

PubMed1985

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein.

Animals↗

PubMed1985

Lack of induction of neuroretinal cell proliferation by Rous sarcoma virus variants that carry the c-src gene.

Expression of p60v-src of Rous sarcoma virus in cultured chicken embryo neuroretinal cells was previously shown to result in the transformation and sustained proliferation of normally quiescent cell populations. We show here that Rous sarcoma virus variants that encode p60c-src, the cellular homolog of p60v-src, lack the ability to induce morphological transformation and cell proliferation of cultured neuroretinal cells. Neuroretinal cells infected with c-src-containing viruses, however, possess no less p60 protein kinase activity assayed in the immune complex than those infected with the transformation-defective Rous sarcoma virus mutants PA101 or PA104, which do stimulate the growth of these cells.

Animals↗

PubMed1985

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.

Animals↗