Nucleotide sequence and topography of chicken c-fps. Genesis of a retroviral oncogene encoding a tyrosine-specific protein kinase

We isolated molecular clones of chicken DNA that carry portions of the cellular proto-oncogene c-fps and then determined the nucleotide sequence of all regions of the gene that are related to the retroviral oncogene v-fps. The homology of v-fps within c-fps resides on at least 19 interspersed segments, 17 of which represent complete exons and two of which may represent only portions of exons. Fusion of these segments reconstructs a facsimile of v-fps. The arrangement of introns and exons within c-fps differs from that of the related proto-oncogene c-src in the domains of the two genes that encode tyrosine-specific protein kinase activity. It therefore appears likely that the introns arose subsequent to the gene duplication that engendered c-src and c-fps. The data also reveal potential junctions between viral and cellular domains in the genomes of two independently isolated avian sarcoma viruses (the PRCII and Fujinami strains). The lefthand junctions can be well defined: they occur at the same position in c-fps but at different positions in the viral gene gag. The righthand junctions cannot be defined as precisely because they include a sequence of 10 to 15 nucleotides whose origin is not known. In the genome of PRCII virus, the composition of this sequence suggests that it arose from the polyadenylated 3' terminus of the c-fps messenger RNA. If this deduction proves to be correct, the data will provide direct evidence that the righthand recombination during transduction by retroviruses occurs between RNA intermediates. Irrespective of these ambiguities, both junctions are located within exons of c-fps, and both may have been formed by non-homologous recombination (although the evidence for the latter statement is not decisive). A sequence of 1020 nucleotides has been deleted from the transduced version of c-fps in the genome of PRCII virus, apparently by homologous recombination between sequences repeated within c-fps. Fujinami virus may contain the entire coding domain of c-fps, but mutations have created 26 amino acid substitutions in the viral version of the gene. By contrast, the partially deleted version of c-fps in PRCII virus contains no mutations that would alter the amino acid sequence.     

Nucleotide sequence of v-fps in the PRCII strain of avian sarcoma virus.

PRCII is an avian retrovirus whose oncogene (v-fps) induces fibrosarcomas in birds. The viral gene v-fps arose by transduction of an undetermined portion of a cellular gene known as c-fps. PRCII is weakly oncogenic when compared with Fujinami sarcoma virus, another transforming virus containing v-fps. As a first step in the elucidation of the molecular basis for the decreased virulence of PRCII, we have determined the entire nucleotide sequence of v-fps in the PRCII genome. The v-fps domain in PRCII encodes a polypeptide with a molecular weight of ca. 60,500 fused to a portion of the polyprotein encoded by the viral structural gene gag. The hybrid gag-fps polyprotein of PRCII would have a molecular weight of ca. 98,100, in accord with results of previous studies of the protein encoded by the PRCII genome. The leftward junctions between fps and gag in Fujinami sarcoma virus and PRCII are located at the same position in fps, but at different positions in gag. A sequence of 1,020 nucleotides, bounded by direct repeats of 6 nucleotides, is present in v-fps of Fujinami sarcoma virus but absent from PRCII. Our data should permit further explorations of the relationship between structure and function in the transforming protein encoded by v-fps.     

Cellular localization of c-fps gene product NCP98.

We compared the intracellular location of the product of the c-fps proto-oncogene, NCP98, with that of its viral homolog P140, the transforming protein of Fujinami sarcoma virus. Using the technique of biochemical subcellular fractionation, we determined that 60 to 90% of NCP98 and its associated kinase activity are in the soluble fraction of a chicken myeloblast cell line. This fractionation behavior differs from that of P140, which is found predominantly in the particulate fraction, both in Fujinami sarcoma virus-infected chicken embryo fibroblasts and in Fujinami sarcoma virus-infected myeloblasts. The fractionation behavior of NCP98 is, however, similar to that of the P140 encoded by a temperature-sensitive strain of Fujinami sarcoma virus in infected cells grown at the nonpermissive temperature. The absence of gag sequences from NCP98 is not responsible for the difference in fractionation behavior: the v-fps transforming protein of strain F36, P91, which lacks gag sequences, is also predominantly particulate. These results indicate that association with cellular structural components correlates with the transforming activity of proteins containing fps sequences.     

The human c-fps/fes gene product expressed ectopically in rat fibroblasts is nontransforming and has restrained protein-tyrosine kinase activity.

A 13-kilobase EcoRI genomic restriction fragment containing the human c-fps/fes proto-oncogene locus was expressed transiently in Cos-1 monkey cells and stably in Rat-2 fibroblasts. In both cases, human c-fps/fes directed synthesis of a 92-kilodalton protein-tyrosine kinase (p92c-fes) indistinguishable from a tyrosine kinase previously identified with anti-fps antiserum which is specifically expressed in human myeloid cells. Transfected Rat-2 cells containing approximately 50-fold more human p92c-fes than is found in human leukemic cells remained morphologically normal and failed to grow in soft agar. Synthesis of p92c-fes in this phenotypically normal line exceeded that of the P130gag-fps oncoprotein in a v-fps-transformed Rat-2 line. Despite this elevated expression, human p92c-fes induced no substantial increase in cellular phosphotyrosine and was not itself phosphorylated on tyrosine. In contrast, p92c-fes immunoprecipitated from these Rat-2 cells or expressed as an enzymatically active fragment in Escherichia coli from a c-fps/fes cDNA catalyzed tyrosine phosphorylation with an activity similar to that of v-fps/fes polypeptides. Thus, p92c-fes is not transforming when ectopically overexpressed in Rat-2 fibroblasts. This lack of transforming activity correlates with a restriction imposed on the kinase activity of the normal c-fps/fes product in vivo which is apparently lifted for v-fps/fes oncoproteins, suggesting that regulatory interactions within the host cell modify fps/fes protein function and normally restrain its oncogenic potential.     

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.     

Chicken myeloid stem cells infected by retroviruses carrying the v-fps oncogene do not require exogenous growth factors to differentiate in vitro

To determine the function of c-fps in chicken macrophages and granulocytic cells we have infected chicken bone marrow cells with retroviruses containing the v-fps oncogene. Normal chicken macrophage progenitors, M-CFCs, give rise to macrophage colonies in semisolid cultures when macrophage colony stimulating factor (M-CSF) is added into the culture medium. Upon infection with v-fps bearing retroviruses, we observed that M-CFCs were induced to develop macrophage colonies in vitro without exogenous M-CSF. This activation results from a direct effect of v-fps on the M-CFCs. No leukemic transformation was observed in the infected colonies. By comparing the effects of several retroviruses, we showed that the induction of M-CFC development is specific to v-fps containing viruses and mediated by the v-fps protein. These observations support the hypothesis that the c-fps gene is involved in the control of proliferation and/or differentiation of myeloid cells.     

Efficient transformation by Prague A Rous sarcoma virus plasmid DNA requires the presence of cis-acting regions within the gag gene.

A region in addition to and outside the long terminal repeats (LTRs) in the gag gene of the Prague A strain of Rous sarcoma virus was found to be essential in cis for efficient cell transformation by cloned viral DNA. Transformation in chicken embryo fibroblasts, which requires infectious virus production and reinfection, was facilitated in cis by sequences between nucleotides 630 and 1659. Efficient transformation of NIH 3T3 cells in which secondary spread of virus is not necessary (as it is in chicken embryo fibroblasts) required sequences between nucleotides 630 and 1149. A src cDNA clone which also lacks this region demonstrated low transformation efficiency, indicating that the role of the cis element cannot be attributed to interference with RNA splicing. The gag gene segment required in cis for transformation, between nucleotides 630 and 1149, could substitute for the simian virus 40 enhancer in either orientation, and cells transfected with Rous sarcoma virus LTR-driven plasmids containing the gag cis element had a two- to threefold increase in steady-state viral RNA levels compared with plasmids lacking this region. Thus, additional cis-acting regulatory elements located outside the viral LTRs may modulate viral gene expression and contribute to the efficiency of cell transformation.     

Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus.

The single protein encoded by Abelson murine leukemia virus is a fusion of sequence from the retroviral gag genes with the v-abl sequence. Deletion of most of the gag region from the transforming protein results in a virus capable of transforming fibroblasts but no longer capable of transforming lymphoid cells. Smaller deletions in gag reveal that p15 gag sequences are responsible for this effect, whereas deletion of p12 sequences had no effect on lymphoid transformation. In transformed fibroblasts, p15-deleted and normal proteins had similar activities and subcellular localization. When the p15-deleted genome was introduced into previously transformed lymphoid lines, its protein product exhibited a marked instability. The tyrosine-specific autophosphorylation activity per cell was less than 1/20th that of the nondeleted protein. Although pulse-Ia-beling showed that the p15-deleted protein was synthesized efficiently, immunoblotting demonstrated that its steady-state level was less than 1/10th that of the nondeleted Abelson protein. The specific instability of the p15-deleted protein in lymphoid cells explains the requirement of these sequences for lymphoid but not fibroblast transformation.     

The structure of the human c-fes/fps proto-oncogene.

We have determined the complete nucleotide sequence of a human DNA fragment of approximately 13 kbp, which was shown by Southern blot analysis to contain the entire v-fes/fps cellular homolog. The v-fes/fps homologous sequences were dispersed over 11 kbp in 18 interspersed segments which were flanked by splice junctions. Fusion of these segments created a DNA fragment in which coding regions similar to those observed in the viral oncogenes v-fes of the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus and v-fps found in Fujinami sarcoma virus could be identified. A potential initiation site in the first exon was found. About 200 nucleotides downstream of a translational stop codon in the v-fes/fps homologous region, a poly(A) addition signal was identified. The deduced amino acid sequence has a molecular weight of 93 390 dalton resembling NCP92, the recently described human c-fes/fps product. The topography of human c-fes/fps appeared to resemble that of chicken c-fps.     

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.     

The amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences.

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180gag-fms encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180gag-fms) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120v-fms, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. Both constructs were biologically active when transfected into NIH 3T3 cells and produced morphologically transformed foci at equivalent efficiencies. When transfected into a cell line (psi 2) expressing complementary viral gene functions, G418-resistant (Neor) cells containing either of these vector DNAs produced high titers of transforming viruses. Analysis of proteins produced in cells containing the vector lacking gag gene sequences showed that gP180gag-fms was not synthesized, whereas normal levels of both immature gp120v-fms and mature gp140v-fms were detected. The glycoprotein was efficiently transported to the cell surface, and it retained wild-type tyrosine kinase activity. We conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180gag-fms is mediated by signal peptidase and that the amino termini of gp140v-fms and the c-fms gene product are identical.     

A highly efficient retroviral vector allows detection of the transforming activity of the human c-fps/fes proto-oncogene.

We have constructed an efficient new retroviral vector containing strong promoting elements derived from the Friend murine leukemia virus (F-MuLV) long terminal repeat (LTR) and have used the vector to demonstrate that overexpression of human c-fps/fes can transform established mouse cells. When a c-fps/fes cDNA was cloned into the vector, this viral DNA and the recovered virus induced very high levels of the c-fps/fes product NCP92 and tumorigenic transformation of NIH 3T3 cells. Compared with an isogenic vector under control of a Moloney MuLV-derived LTR, the vector driven by the F-MuLV LTR induced 3- to 10-times-higher levels of expression of c-fps/fes, a higher level of phosphotyrosine in cellular proteins, and a virus whose transforming activity was 2 orders of magnitude greater. We conclude (i) that normal c-fps/fes can induce morphologic transformation and that its transforming activity is a function of the level of expression of NCP92 and (ii) that the vector based on the F-MuLV LTR is more efficient than the vector driven by a Moloney MuLV LTR in inducing high levels of expression and measurable biological activity.     

Cellular transformation by subgenomic feline sarcoma virus DNA

The genome of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) is a 4.3-kilobase-pair (kbp) RNA molecule that contains a 1.5-kbp cellular insertion (fes gene) flanked by feline leukemia virus sequences at its 5' end (1.6 kbp) and 3' end (1.2 kbp) (Sherr et al., J. Virol. 34:200-212, 1980). DNA transfection techniques have been utilized to determine the regions of the ST-FeSV genome involved in malignant transformation. I have found that the 3.7-kbp 5'-end fragment of the ST-FeSV provirus (which corresponds to the 3.4-kbp 5'-end fragment of the viral genome) is sufficient to transform NIH/3T3 fibroblasts. Enzymes that cleave the ST-FeSV provirus DNA within the feline leukemia virus gag gene sequences or within the fes gene abolished the transforming activity. Preservation of the proviral large terminal repeats was also required for transformation. Transformed NIH/3T3 cells obtained by transfection of total or subgenomic ST-FeSV DNA expressed normal levels of the ST-FeSV gene product ST P85 and of its associated protein kinase activity. Furthermore, these cells contained high levels of phosphotyrosine residues, a biochemical marker associated with cellular transformation induced by certain retroviruses including ST-FeSV. These results, taken together, strongly support the concept that only those ST-FeSV proviral sequences necessary for ST P85 expression are involved in malignant transformation.     

Construction and biological analysis of deletion mutants of Fujinami sarcoma virus: 5''-fps sequence has a role in the transforming activity.

Fujinami sarcoma virus (FSV) genome codes for the gag-fps fusion protein FSV-P130. The amino acid sequence of the 3' one-third portion in v-fps is partially homologous to the 3' half of pp60src, or the kinase domain, but the sequence of the 5' portion is unique to v-fps. To identify a possible domain structure in the v-fps sequence responsible for cell transformation, we constructed various deletion mutants of FSV with molecularly cloned viral DNA. Their transforming activities were assayed by measuring focus formation on chicken embryo fibroblasts and rat 3Y1 cells and tumor formation in chickens. The mutants carrying a deletion at the 3' portion in v-fps, the kinase domain, lost transforming activity. The mutants carrying an approximately 1-kilobase deletion within the 5' portion of the v-fps sequence retained focus-forming activity and tumorigenicity in the chicken system, but the efficiency of focus formation was about 10 times lower than that of the wild type. The morphology of these transformed cells was distinct from that observed in cells infected with wild-type FSV. Furthermore, these mutants could not transform rat 3Y1 cells, although wild-type FSV DNA transformed rat 3Y1 cells at a high frequency. The mutants carrying a larger deletion in the 5' portion of fps completely lacked the transforming activity. These results suggest that the 3' portion of the v-fps sequence is necessary but not sufficient for cell transformation and that the 5' portion of v-fps has a role in the transforming activity.     

A major site of tyrosine phosphorylation within the SH2 domain of Fujinami sarcoma virus P130gag-fps is not required for protein-tyrosine kinase activity or transforming potential.

Phosphorylation of the major autophosphorylation site (Tyr-1073) within Fujinami sarcoma virus P130gag-fps activates both the intrinsic protein-tyrosine kinase activity and transforming potential of the protein. In this report, a second site of autophosphorylation Tyr-836 was identified. This tyrosine residue is found within a noncatalytic domain (SH2) of P130gag-fps that is required for full protein-kinase activity in both rat and chicken cells. Autophosphorylation of this tyrosine residue implies that the SH2 region lies near the active site in the catalytic domain in the native protein and thus possibly regulates its enzymatic activity. Four mutations have occurred within the SH2 domain between the c-fps and v-fps proteins. Tyr-836 is one of these changes, being a Cys in c-fps. Site-directed mutagenesis was used to investigate the function of this autophosphorylation site. Substitution of Tyr-836 with a Phe had no apparent effect on the transforming ability or protein-tyrosine kinase activity of P130gag-fps in rat-2 cells. Mutagenesis of both autophosphorylation sites (Tyr-1073 and Tyr-836) did not reveal any cooperation between these two phosphorylation sites. The implications of the changes within the SH2 region for v-fps function and activation of the c-fps oncogenic potential are discussed.     

Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome.

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus.     

Monoclonal antibodies to the transforming protein of Fujinami avian sarcoma virus discriminate between different fps-encoded proteins

Two monoclonal antibodies have been obtained that recognize antigenic determinants within the C-terminal fps-encoded region of P140gag-fps, the transforming protein of Fujinami avian sarcoma virus (FSV). The hybridomas which secrete these antibodies (termed 88AG and p26C) were isolated after the fusion of NS-1 mouse myeloma cells with B lymphocytes from Fischer rats that had been immunized with FSV-transformed rat-1 cells. FSV P140gag-fps immunoprecipitated by either antibody is active as a tyrosine-specific kinase and is able to autophosphorylate and to phosphorylate enolase in vitro. The fps-encoded proteins of all FSV variants, including the gag- p91fps protein of F36 virus, are recognized by both monoclonal antibodies. However, the product of the avian cellular c-fps gene. NCP98, and the transforming proteins of the recently isolated fps-containing avian sarcoma viruses 16L and UR1 are recognized only by the p26C antibody. The 88AG antibody therefore defines an epitope specific for FSV fps, whereas the epitope for p26C is conserved between cellular and viral fps proteins. The P105gag-fps protein of the PRCII virus is not precipitated by p26C (nor by 88AG), presumably as a consequence of the deletion of N-terminal fps sequences. These data indicate that the fps-encoded peptide sequences of 16L P142gag-fps and UR1 P150gag-fps are more closely related to NCP98 than that of FSV P140gag-fps. This supports the view that 16L and UR1 viruses represent recent retroviral acquisitions of the c-fps oncogene. The P85gag-fes transforming protein of Snyder-Theilen feline sarcoma virus is not precipitated by either monoclonal antibody but is recognized by some antisera from FSV tumor-bearing rats, demonstrating that fps-specific antigenic determinants are conserved in fes-encoded proteins.