Transformation of avian lymphoid cells by reticuloendotheliosis virus

Avian reticuloendotheliosis virus (REV-T) is the most virulent of all retroviruses, inducing an invariably fatal leukemia in chickens with a latent period of 7-10 days. Unlike avian cells transformed by other acutely transforming viruses, lymphoid cells transformed by REV-T are immortalized. Furthermore, in vitro derived, REV-T transformed cells which do not produce virus are tumorigenic and induce lethal reticuloendotheliosis when injected into histocompatible birds. Thus REV-T transforms its target cell both in vitro and in vivo. In addition this transformation is independent of any helper virus functions. Like other acute leukemia viruses, REV-T is replication-defective and must co-replicate with a reticuloendotheliosis associated virus (REV-A). During evolution, a substantial portion of its genome has been deleted and replaced with a host-derived genetic sequence, designated v-rel. Presumably, the v-rel oncogene was transduced from a normal turkey DNA locus, c-rel. There are 9 regions of homology between c-rel and v-rel, however, several differences exist between these genes, suggesting that transformation by REV-T results from the production of an altered v-rel protein. The v-rel sequence is distinct from other known oncogenes and encodes a 57-kDa phosphoprotein. In REV-T transformed cells, this pp57v-rel protein is localized in the cytoplasm. The product of the v-rel oncogene is present at a low level, representing only about 0.003% of total methionine-labelled protein. In addition, pp57v-rel is relatively stable, having an estimated half-life of 4-10 h. The v-rel protein when purified close to homogeneity is complexed with a 40-kDa cellular phosphoprotein in transformed lymphoid cells and possesses serine kinase activity. This review discusses the molecular aspects of transformation by REV-T in the context of other oncogene-encoded proteins.     

Avian reticuloendotheliosis virus-transformed lymphoid cells contain multiple pp59v-rel complexes.

The v-rel oncogene of avian reticuloendotheliosis virus type T (REV-T) encodes a 59-kilodalton (kDa) phosphoprotein located principally in the cytosol of transformed lymphoid cells. All of the detectable pp59v-rel was present in high-molecular-weight complexes containing at least five cellular proteins (p124, p115, p75c-rel, p70hsc, and pp40). Antiserum was developed against the 40-kDa protein, the most abundant cellular protein associated with the complex. The 40-kDa phosphoprotein was complexed with pp59v-rel in REV-T-transformed lymphoid cell lines arrested at different stages of B-cell development as well as in lymphoid tumor cells and in fibrosarcomas. The half-life (8 h) of pp40 in REV-T-transformed lymphoid cells was the same as that of pp59v-rel. Antiserum against pp40 permitted the identification of two pp59v-rel complexes. The most abundant cytoplasmic complex contained approximately 75% of the pp59v-rel and all of the detectable pp40 in REV-T-transformed lymphoid cells. Twenty-five percent of the pp59v-rel was present in a minor complex that contained the majority of p75c-rel along with p115 and p124. In nuclear extracts of REV-T-transformed lymphoid cells, pp59v-rel was complexed with pp40. The two high-molecular-weight proteins (p115 and p124) and p75c-rel were not detected in the nuclear complex. In the cytosolic complexes, pp40 was heavily phosphorylated, whereas the nuclear form was much less extensively phosphorylated.     

Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location.     

v-rel oncoproteins in the nucleus and in the cytoplasm transform chicken spleen cells.

The transforming protein encoded by the v-rel oncogene of the highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (Rev-T) is a 59,000-dalton protein, p59v-rel. The mechanism by which p59v-rel induces transformation of early lymphoid cells is unknown. As a step towards understanding the mechanism of v-rel-induced transformation, we sought to establish the subcellular site of action of p59v-rel. In this report, we show that p59v-rel contains sequences that are necessary for its efficient localization in the nucleus of infected chicken embryo fibroblasts. These v-rel sequences when added to the normally cytoplasmic protein, beta-galactosidase, directed that protein to the nucleus. A mutation in the v-rel nuclear-localizing sequence did not affect the transforming function, although it did alter the nuclear-localizing function. The addition of a supplemental nuclear-localizing sequence from simian virus 40 large T-antigen to v-rel resulted in the expression of a transforming rel protein which was located exclusively in the nucleus of transformed spleen cells, in contrast to wild-type p59v-rel, which was largely cytoplasmic in transformed spleen cells. Our results support the hypothesis that v-rel encodes a protein which can act either in the nucleus or in the cytoplasm to transform spleen cells.     

p59v-rel, the transforming protein of reticuloendotheliosis virus, is complexed with at least four other proteins in transformed chicken lymphoid cells

Previous studies have identified the protein product of v-rel, the oncogene carried by reticuloendotheliosis virus (REV), as a 59,000-dalton phosphoprotein located predominantly in the cytosol of transformed chicken lymphoid cells. In immune precipitates of p59v-rel, there is a closely associated protein kinase activity. In chicken lymphoid cells that do not contain REV, p68c-rel is found free in the cytosol not associated with other proteins and not detectably phosphorylated. In this study, we found that immune precipitates of 59v-rel from REV-transformed cells contain at least four other proteins, of approximate molecular weights 124, 115, 68, and 36 kilodaltons (kDa). The 124-, 115-, and 36-kDa proteins are apparently unrelated to p59v-rel in sequence, and their coprecipitation suggests that they are complexed with p59v-rel. The coprecipitating 68-kDa protein was found to be p68c-rel, which, like the other three proteins, precipitates by virtue of its association with p59v-rel. Glycerol gradient analysis suggested the presence of more than one type of complex: one containing p115, p68c-rel, p59v-rel, and p36, and another containing p124, p115, p59v-rel, and possibly p68c-rel. In vitro kinase activity was found in all size classes, coinciding with the distribution of p115 and p59v-rel. The complex(es) was stable under a variety of conditions, including a wide range of ionic strengths, chelators, and detergents, and through multiple cycles of immune precipitation and elution. This suggests a specific and functionally significant interaction among the members that may be of direct relevance to the mechanism of REV-induced transformation.     

Localization of the v-rel protein in reticuloendotheliosis virus strain T-transformed lymphoid cells.

The protein (p59rel) encoded by the transforming gene of reticuloendotheliosis virus strain T (REV-T) has been identified in REV-T-transformed avian lymphoid cells by using antisera raised against synthetic peptides whose sequences were derived from three nonoverlapping regions of v-rel (N. R. Rice, T. D. Copeland, S. Simek, S. Oroszlan, and R. V. Gilden, Virology 149:217-229, 1986). To obtain polyclonal antibodies directed against a larger number of p59rel epitopes, a 262-amino acid segment was expressed in bacteria. Antisera raised against this fusion protein (v-delta-rel) precipitated p59rel from lysates of [35S]methionine-labeled REV-T-transformed cells, thus confirming previous results obtained with the peptide antisera. We used this new antiserum to localize p59rel in REV-T-transformed cells by subcellular fractionation using differential centrifugation and by indirect immune fluorescent staining. After fractionation and immune precipitation, the majority of p59rel was found in the cytosolic fraction. Indirect immunofluorescence experiments also gave results consistent with the cytoplasmic localization of the v-rel protein in transformed lymphoid cells. In previous studies (Rice et al., Virology 149:217-229, 1986) it was shown that immune precipitates formed with one of the three p59rel peptide antisera possessed in vitro protein kinase activity. Immune precipitates formed with the fusion protein antiserum also showed kinase activity in the in vitro assay. Most of this activity was found in the soluble cytoplasmic fraction, indicating that the kinase may be p59rel or a protein closely associated with it.     

A mutant v-rel with increased ability to transform B lymphocytes.

We observed that two strains of REV-T differ in the ability to transform bursal cells in vitro. REV-TW, with v-rel derived from a well-characterized clone and considered the prototype of the wild type, fails to generate colonies in soft agar. In contrast, REV-S2A3, derived from the S2A3 cell line, readily transforms bursal cells. With PCR, a 1,591-bp fragment containing v-rel from the REV-S2A3 provirus was cloned into plasmid pREV-0. Except for the absence of v-rel, pREV-0 is identical to pREV-TW. Five clones of pREV-PCR, each produced by an independent amplification, were obtained. The REV-PCR viruses displayed the strong transforming phenotype of REV-S2A3. Two mutations were identified in the 5' region of v-rel from REV-PCR1 to REV-PCR5: a silent mutation and a G-to-T transversion, changing the alanine at position 40 to serine. To confirm the relevance of this amino acid substitution, a 478-bp fragment containing the mutations was exchanged between REV-TW and REV-PCR1. Only the mutant viruses were able to form large colonies of bursal cells in liquid culture and to generate bursal cell colonies in soft agar. When tested on splenocytes, the wild-type viruses induced predominantly non-B-cell colonies while the mutant viruses gave origin mainly to B-cell colonies. The above results indicate that the substitution of serine for alanine at position 40 of v-Rel enhances the ability of REV-T to transform B lymphocytes in vitro. This mutation is close to the DNA-binding region, and the variant v-Rel oncoprotein shows increased kappa B-binding activity, thus confirming the relevance of this property for transformation.     

Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation.     

Specific phosphorylation by calcium-EGTA complex of a 75 kDa human tumor plasma membrane protein (pp75)

1. The major functional role played by phosphorylation of plasma membrane proteins in the biological properties of tumor cells suggests that identification of protein kinases and their substrates will contribute to our understanding of the molecular basis of the malignant process and of the aberrant behavior of tumor cells. 2. The present study has investigated the phosphorylation of surface proteins of human tumor cells. Incubation of plasma membranes isolated from cultured human melanoma cells with [gamma-32P]ATP in the presence of Ca2+ and ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) resulted in specific phosphorylation of serine and threonine residues on a 75kDa protein (pp75). 3. Neither Ca2+ or EGTA alone, nor any other divalent metal ion tested could induce phosphorylation of pp75. 4. The phosphorylation of pp75 was directly dependent upon the presence of non-ionic detergents, and was influenced by length of incubation and concentration ratio of Ca2+ and EGTA. 5. Incubation of isolated plasma membranes with [gamma-32P]ATP in the presence of Ca2+ and EGTA and immunochemical analysis by Western blotting with an anti pp75 xenoantiserum detected the pp75 in human melanoma, neuroblastoma, ovarian carcinoma and lymphoid T cells and fibroblasts but not in B-lymphoid cells, renal carcinoma cells, peripheral blood lymphocytes and splenocytes. 6. These results suggest the presence of a new class of plasma membrane bound protein kinases activated by chelated calcium and differentially expressed in normal and transformed human cells.     

Avian reticuloendotheliosis virus: identification of the hematopoietic target cell for transformation

Non-virus-producing hematopoietic cells transformed in vitro by reticuloendotheliosis virus (REV-T) induce lethal "reticuloendotheliosis" when inoculated into histocompatible chickens. This is the first direct demonstration that an in vivo target cell of an avian acute leukemia virus can be transformed in vitro. The tumorigenic, REV-T-transformed non-virus-producing cells fail to express helper-virus-coded proteins. REV-T transformed tumorigenic cells therefore do not require helper-virus functions. Cells transformed in vivo or in vitro by REV-T have lymphoblastoid morphology and express low levels of terminal-deoxynucleotidyl-transferase activity and bursal-cell determinants. One clone synthesized Ig mu. The preferred target cells for REV-T transformation are therefore immature lymphoid cells that express B-cell determinants. We propose that the unique transforming sequence of REV-T be designated rel (lymphoid).     

Effects of phosphorylation of avian retrovirus nucleocapsid protein pp12 on binding of viral RNA

The major nucleocapsid protein of avian retroviruses, pp12, preferentially binds to the single-stranded regions of 60 S viral RNA with a apparent binding constant (Kapp) of 1.2 X 10(11) M-1. If the phosphate associated with serine residues of pp12 is hydrolyzed by either alkali treatment or with partially purified phosphoprotein phosphatase activities isolated from virions, the Kapp for binding to 60 S RNA decreases 100-fold. The high affinity binding of pp12 to viral RNA can be restored by phosphorylation of the protein with a protein kinase, protease-activated kinase I. The same serine residues phosphorylated in vivo are phosphorylated by protease kinase I in vitro. These residues have been identified as serine residues 40 and either 76 or 77. The protein purified from virions is phosphorylated primarily at serine residue 40 (greater than 90%). This suggests that phosphoserine residue 40 is responsible for modulating the binding of the protein to RNA. Thus, the phosphorylation state of pp12 can be reversibly altered in vitro resulting in the interconversion of the protein between a state of high and low affinity for single-stranded viral RNA.     

Expression, purification, and characterization of the cytoplasmic domain of the human IGF-1 receptor using a baculovirus expression system.

The cytoplasmatic domain of the beta-subunit of the human IGF-1 receptor (residues 929-1337) has been overexpressed in insect cells using the baculovirus expression system. Synthesis of the soluble protein (IGFK, M(r) 46 kDa) in Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation was achieved 40-48 h postinfection. Rapid purification to near homogeneity (>/=95% pure protein) was accomplished by sequential chromatography on Resource-Q and phenyl-Sepharose with a specific activity of 142 nmol/min/mg using poly[Glu:Tyr] as substrate. The purified IGFK showed a preference for Mn(2+) ions and a linear incorporation of (32)P from [gamma-(32)P]ATP over a 20-fold dilution of the protein and was stimulated 20-fold by the polycation poly-L-lysine. Interestingly, the kinase autophosphorylated on tyrosine and serine residues. In contrast, a kinase-negative mutant, IGFK-K1003A, did not undergo phosphorylation on tyrosine or serine residues, respectively, suggesting that IGF-1 receptor kinase is a dual specific kinase.     

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.     

Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with 32Pi. The 120 kDa 32P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the 32Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.