BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias.

The c-abl proto-oncogene encodes a cytoplasmic tyrosine kinase which is homologous to the src gene product in its kinase domain and in the upstream kinase regulatory domains SH2 (src homology region 2) and SH3 (src homology region 3). The murine v-abl oncogene product has lost the SH3 domain as a consequence of N-terminal fusion of gag sequences. Deletion of the SH3 domain is sufficient to render the murine c-abl proto-oncogene product transforming when myristylated N-terminal membrane localization sequences are also present. In contrast, the human BCR/ABL oncogene of the Philadelphia chromosome translocation has an intact SH3 domain and its product is not myristylated at the N terminus. To analyze the contribution of BCR-encoded sequences to BCR/ABL-mediated transformation, the effects of a series of deletions and substitutions were assessed in fibroblast and hematopoietic-cell transformation assays. BCR first-exon sequences specifically potentiate transformation and tyrosine kinase activation when they are fused to the second exon of otherwise intact c-ABL. This suggests that BCR-encoded sequences specifically interfere with negative regulation of the ABL-encoded tyrosine kinase, which would represent a novel mechanism for the activation of nonreceptor tyrosine kinase-encoding proto-oncogenes.     

Identification and characterization of p59fyn (a src-like protein tyrosine kinase) in normal and polyoma virus transformed cells.

fyn is a member of the growing family of protein tyrosine kinase genes whose sequences are highly related to that of c-src. We have generated antibodies to peptides corresponding to two different amino-terminal sequences encoded by this gene. Antisera to both peptides recognized a 59 kd protein from human and mouse fibroblasts. p59fyn was phosphorylated in vivo on serine and tyrosine residues and was also myristylated. Furthermore, immune precipitates of p59fyn had tyrosine kinase activity in vitro, as measured by autophosphorylation and by phosphorylation of substrates such as enolase. This kinase activity was shown to be negatively regulated by tyrosine phosphorylation. We have also established that, like pp60c-src and p62c-yes, p59fyn was complexed with middle T antigen, the transforming protein of polyoma virus. However, the tyrosine kinase activity of p59fyn was not elevated in middle T transformed cells. Possible explanations for this are discussed.     

Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus.

The c-fyn proto-oncogene is a member of a family of closely related genes of which c-src is the prototype. Using peptide antibodies which had been raised against sequences predicted to be specific for the human c-fyn gene product, the c-fyn protein was identified. It is a tyrosine kinase with apparent mol. wt of 59 kd that is also phosphorylated and myristylated. Like pp60c-src and pp62c-yes, pp59c-fyn is able to form a stable complex with middle-T antigen, the transforming protein of polyomavirus. The transformation-defective middle-T mutant NG59, which is unable to associate stably with pp60c-src does not associate with pp59c-fyn. In contrast to pp60c-src, complex formation with middle-T antigen does not lead to a significant increase in the tyrosine kinase activity of pp59c-fyn. These findings lead us to suggest that middle-T mediated transformation may be a consequence of the deregulation of several members of the src-family of protein tyrosine kinases.     

N-terminal deletion in the src gene of Rous sarcoma virus results in synthesis of a 45,000-Mr protein with mitogenic activity.

Expression of the v-src gene of Rous sarcoma virus in avian embryo neuroretina cells results in transformation and sustained proliferation of these normally resting cells. Transformed neuroretina cells are also tumorigenic upon inoculation into immunodeficient hosts. We have previously described conditional mutants of Rous sarcoma virus encoding p60v-src proteins which induce proliferation of neuroretina cells in the absence of transformation and tumorigenicity. These results suggest that p60v-src is composed of functionally distinct domains which may interact with multiple cellular targets. In this study, we describe a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion of 278 base pairs in the 5' portion of the v-src gene but which has retained the ability to induce proliferation of quail neuroretina cells. The deleted v-src gene encodes a 45,000-molecular-weight phosphoprotein which contains both phosphoserine and phosphotyrosine, is myristylated, and possesses tyrosine kinase activity indistinguishable from that of wild-type p60v-src. Molecular cloning and sequence analysis of the mutant v-src gene have shown that this deletion extends from amino acid 33 to 126 of the wild-type p60v-src. Therefore, this portion of the v-src protein is dispensable for the mitogenic activity of Rous sarcoma virus in neuroretina cells.     

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.     

Biochemical properties of p60v-src mutants that induce different cell transformation parameters.

PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation.     

Myristylation is involved in intracellular retention of hepatitis B virus envelope proteins.

The envelope of hepatitis B virus contains three related proteins, one of which is myristylated. The nonmyristylated small and middle protein are assembled into empty envelope particles which are secreted from cells, whereas the myristylated large envelope protein is mainly found in complete virions and is not secreted in the absence of the nucleocapsid. The block to secretion can be partially overcome by mutation or deletion of the myristylation site. Creation of a myristyl attachment site in the small protein impairs the secretion of empty envelope particles but not their intracellular assembly. Myristylation may therefore play a crucial role in hepatitis B virus replication by channeling the envelope proteins into complete viral particles.     

A novel oncogene, v-ryk, encoding a truncated receptor tyrosine kinase is transduced into the RPL30 virus without loss of viral sequences.

The RPL viruses are acute oncogenic avian retroviruses isolated from chicken tumors. We carried out a genetic analysis of three of the viruses, RPL25, RPL28, and RPL30. While RPL25 and RPL28 were shown to contain the erbB oncogene, RPL30 appeared to contain a novel protein tyrosine kinase oncogene. This gene, v-ryk, was cloned and sequenced. The v-ryk oncogene contains a 1.39-kb nonretroviral sequence that includes a tyrosine kinase domain which was inserted into the viral envelope protein gp37-coding region and fused in frame with upstream gp37 to generate a P69gp37-ryk fusion oncoprotein. Unlike that of other acutely transforming retroviruses, transduction of the v-ryk gene into RPL30 did not result in deletion of viral sequences. Sequence analysis suggested that v-Ryk is more homologous to receptor-type tyrosine kinases than to nonreceptor-type kinases. By reconstitution of a virus from its cDNA, the v-ryk oncogene has been shown to be fully responsible for the transforming activity of the RPL30 virus. Antibodies specific to v-Ryk immunoprecipitated the v-Ryk oncoprotein from cells transformed by the RPL30 virus. The v-Ryk protein was shown to be first synthesized as a 150-kDa precursor and then cleaved into the mature 69-kDa gp37-Ryk fusion protein, both parts of which were found to be localized to the membrane fraction. As expected from the sequence of v-Ryk, immunoprecipitates of v-Ryk from RPL30-transformed cells were found to display a protein tyrosine kinase activity in vitro, and the levels of tyrosine-phosphorylated proteins are elevated in v-ryk-transformed cells.     

Characterization of early stages in vaccinia virus membrane biogenesis: implications of the 21-kilodalton protein and a newly identified 15-kilodalton envelope protein.

Vaccinia virus (VV) membrane biogenesis is a poorly understood process. It has been proposed that cellular membranes derived from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) are incorporated in the early stages of virion assembly. We have recently shown that the VV 21-kDa (A17L gene) envelope protein is essential for the formation of viral membranes. In the present work, we identify a 15-kDa VV membrane protein encoded by the A14L gene. This protein is phosphorylated and myristylated during infection and is incorporated into the virion envelope. Both the 21- and 15-kDa proteins are found associated with cellular tubulovesicular elements related to the ERGIC, suggesting that these proteins are transported in these membranes to the nascent viral factories. When synthesis of the 21-kDa protein is repressed, organized membranes are not formed but numerous ERGIC-derived tubulovesicular structures containing the 15-kDa protein accumulate in the boundaries of the precursors of the viral factories. These data suggest that the 21-kDa protein is involved in organizing the recruited viral membranes, while the 15-kDa protein appears to be one of the viral elements participating in the membrane recruitment process from the ERGIC, to initiate virus formation.     

Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA.

UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a large precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and alpha-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tyrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations.     

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.     

Activation of the c-abl oncogene by viral transduction or chromosomal translocation generates altered c-abl proteins with similar in vitro kinase properties.

The v-abl protein of Abelson murine leukemia virus is a tyrosine-specific kinase. Its normal cellular homolog, murine c-abl, does not possess detectable tyrosine kinase activity in vitro. Previously, we have detected tyrosine kinase activity in vitro for an altered c-abl gene product (c-abl P210) in the K562 human chronic myelogenous leukemia cell line. The expression of this variant c-abl gene product correlates with chromosomal translocation and amplification of the c-abl gene in K562 cells. Like v-abl, c-abl P210 is a fusion protein containing non-abl sequences near the amino terminus of c-abl. We compared the in vitro tyrosine kinase activity of c-abl P210 with that of wild-type murine v-abl. The remarkable similarities of these two proteins with respect to cis-acting autophosphorylation, trans-acting phosphorylation of exogenous substrates, and kinase inhibition, using site-directed abl-specific antisera, suggested that c-abl P210 could function similarly to v-abl in vivo. In addition, c-abl P210 possessed an associated serine kinase activity in immunoprecipitates. The serine kinase activity was not inhibited by site-directed, abl-specific antisera that inhibit the tyrosine kinase activity, suggesting that the serine kinase activity is not an intrinsic property of c-abl P210. Thus, the activation of the c-abl gene in a human leukemia cell line may have functional consequences analogous to activation of the c-abl gene in Abelson murine leukemia virus.     

Abelson murine leukemia virus induces platelet-derived growth factor-independent fibroblast growth: correlation with kinase activity and dissociation from full morphologic transformation.

Abelson murine leukemia virus (A-MuLV) encodes a single protein product, a tyrosine-specific protein kinase, whose activity is necessary for cell transformation by this retrovirus. Using a defined medium culture system, we demonstrate that transformation of NIH 3T3 fibroblasts by A-MuLV abrogates their normal requirement for platelet-derived growth factor (PDGF) for cell growth. Analysis of constructed insertional mutant viruses revealed an absolute correlation between A-MuLV-encoded tyrosine kinase activity and PDGF-independent fibroblast growth. Sequences of the provirus not required for kinase activity appeared unnecessary for abrogating the fibroblast requirement for PDGF. Conversely, sequences required for kinase activity appeared necessary, suggesting that induction of PDGF-independent fibroblast growth, like cell transformation, is a function of this tyrosine kinase. Fibroblasts transformed by a partially transformation-defective mutant demonstrated incomplete morphological transformation but were still independent of PDGF for growth. Thus, the processes of full morphological transformation and growth factor independence can be partially dissociated.     

A strain of Fujinami sarcoma virus which is temperature-sensitive in protein phosphorylation and cellular transformation

Cells infected by one strain of Fujinami sarcoma virus (FSV) are transformed at 38 degrees C but are phenotypically normal at 41.5 degrees C. FSV encodes a 140,000 molecular weight protein (P140) with gag gene-related and FSV-specific peptide sequences. At 41.5 degrees C, P140 is weakly phosphorylated at serine residues, and is inactive in the immune complex protein kinase assay. At 38 degrees C, P140 is highly phosphorylated, contains phosphotyrosine in addition to phosphoserine, and in the immune complex kinase assay becomes phosphorylated at three tyrosine residues. Phosphorylation of cellular polypeptides at tyrosine residues in FSV-infected cells is also temperature-sensitive. These observations indicate that P140 is the transforming protein of FSV and that protein phosphorylation at tyrosine residues is involved in transformation by this virus.     

Characterization of protein kinase activity associated with the transforming gene product of Fujinami sarcoma virus

Fujinami sarcoma virus (FSV), a newly characterized avian sarcoma virus, produces a protein of 140,000 daltons (p140) in infected cells. p140 is the product of a fused gene consisting of a part of the gag gene of avian retrovirus and FSV-unique sequences which are not related to the src sequences of Rous sarcoma virus. In vivo, p140 was found to be phosphorylated at both serine and tyrosine residues. Immunoprecipitates of p140 with antiserum against gag gene-coded proteins had a cyclic nucleotide-independent protein kinase activity which phosphorylated p140 itself, rabbit IgG of the immune complex and alpha-casein, an externally added soluble protein substrate. The phosphorylation was specific to tyrosine of the substrate proteins. p140 was phosphorylated in vitro at the same two tyrosine residues that were phosphorylated in vivo. The phosphate transferred to tyrosine residues of p140 forms a stable bond: it does not turn over during the kinase reaction, and the 32P-phosphate of p140 labeled in vitro or in vivo is not transferred to alpha-casein. FSV-p140 differs from p60src, the transforming protein of Rous sarcoma virus, in its marked preference of Mn2+ to Mg2+ ions, and in its inability to use GTP instead of ATP as the donor of gamma-phosphate.     

Tyrosine kinase expression profiles of chicken erythro-progenitor cells and oncogene-transformed erythroblasts

Tyrosine kinases are implicated in the growth and differentiation of erythroid cells. Aberrant expression and structural alterations of certain tyrosine kinases, such as erbB and sea, are known to trigger erythroleukemia development. To facilitate our understanding of the signal transduction pathways involved in erythroid differentiation and leukemic transformation, we have applied a recently developed tyrosine kinase profile technique to identify the tyrosine kinases and some novel serine/threonine kinases expressed in normal chicken erythroid progenitor cells that respond to TGF (TGF-EB), and erythroblasts transformed by viruses encoding v-erbB (v-erbB-EB) and v-sea (v-sea-EB). Our results reveal that the non-receptor tyrosine kinases, Abl, Fyn, Lyn, Btk and Csk, are expressed in all three cell types. The expression level of Btk, a tyrosine kinase implicated in Bruton's syndrome, is exceptionally high in the erythroblastoid cell line 6C2, transformed by the v-erbB carrying avian erythroblastosis virus, AEV-ES4. We have also uncovered a new STE-20-related serine/threonine kinase, KFC, which is abundantly expressed in both the TGF-stimulated erythroid progenitor cells and v-sea-transformed erythroblasts. Based on sequence homology of the kinase domain, KFC appears to be the first member of a new subfamily of STE-20-like kinases.     

A membrane-associated, carbohydrate-modified form of the v-abl protein that cannot be phosphorylated in vivo or in vitro.

Abelson murine leukemia virus encodes a transforming protein which contains tyrosine kinase activity and is phosphorylated in vivo and in vitro. We found that P160 and P160-derived virus strains expressed an additional, altered v-abl protein which could not be phosphorylated. The altered v-abl protein (L-v-abl) differed from the phosphorylated form (K-v-abl) in that it was glycosylated and localized exclusively to the membrane fraction. Tunicamycin inhibition of N-linked carbohydrate addition did not restore phosphorylation. It did, however, reveal that L-v-abl had additional sequences relative to K-v-abl. The coding sequences required for this region and for the expression of L-v-abl were identified by replacing sequences in the P120 virus genome, which did not express L-v-abl, with sequences from the P160 virus genome. The necessary sequences were localized to the Moloney murine leukemia virus-derived gag gene. Comparison between the in vitro altered P120 and wild-type P120 virus strains indicated that expression of L-v-abl did not increase the efficiency of lymphoid transformation. Although the biological role of L-v-abl is not clear, our analyses have revealed that a specific amino terminal gag sequence can prevent v-abl from acting as a kinase substrate and can alter the cellular localization and modification of v-abl. These properties distinguish L-v-abl from previously reported v-abl proteins.     

Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation

Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV.     

Intracellular localization and processing of pp60v-src proteins expressed by two distinct temperature-sensitive mutants of Rous sarcoma virus.

The transforming protein of Rous sarcoma virus, pp60v-src, is known to be a tyrosine protein kinase, but the mechanism of cell transformation remains unclear. In further investigating pp60v-src structure and function, we have analyzed two temperature-sensitive (ts) Rous sarcoma virus src gene mutants, tsLA29 and tsLA32. The mutations in tsLA29 and tsLA32 map in the carboxy-terminal region and the amino-terminal half of pp60v-src, respectively, and encode mutant proteins with either temperature-labile (tsLA29) or -stable (tsLA32) kinase activities. Here we examined the intracellular processing and localization of these pp60v-src mutants and extended our characterization of transformation parameters expressed by cells infected by the Rous sarcoma virus variants. No obvious defects in functional integrity of the tsLA32 pp60v-src could yet be demonstrated, whereas the tsLA29 pp60v-src was perturbed not only in kinase activity, but also in aspects of protein processing and localization. Analysis of transformation parameters expressed by infected cells demonstrated the complete temperature lability of both mutants.