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Transduction of oncogenes by ALVs and generation of acute transforming viruses is common in natural viral infections. In order to understand the molecular basis for the rapid oncogenicity of Fu-J, an acutely transforming avian leukosis virus isolated from fibrosarcomas in crossbreed broilers infected with subgroup J avian leukosis virus (ALV-J) in China, complete genomic structure of Fu-J virus was determined by PCR amplification and compared with those of Fu-J1, Fu-J2, Fu-J3, Fu-J4, and Fu-J5 reported previously. The results showed that the genome of Fu-J was defective, with parts of gag gene replaced by the complete v-fps oncogene and encoded a 137 kDa Gag-fps fusion protein. Sequence analysis revealed that Fu-J and Fu-J1 to Fu-J5 were related quasi-species variants carrying different lengths of v-fps oncogenes generated from recombination between helper virus and c-fps gene. Comparison of virus carrying v-fps oncogene also gave us a glimpse of the molecular characterization and evolution process of the acutely transforming ALV.

The c-fps/fes protooncogene encodes a 92-kDa protein tyrosine kinase that is involved in myeloid cell development and immune responses of granulocytes and macrophages. To help define its biological role and mechanism of action, we have developed a gain of function allele of Fes that has potent biological activity in myeloid cells. Introduction of constitutively active Fes into myeloid progenitors induced the appearance of fully differentiated macrophages or granulocytes depending on the lineage commitment of the transduced cells. We found that Fes-induced macrophage differentiation correlated with activation of the ets family transcription factor PU.1, which is essential for macrophage development. On the other hand, granulocyte differentiation by Fes was mediated through activation of CCAAT/enhancer-binding protein alpha (C/EBP-alpha) and STAT3, two transcription factors that are critical for granulocytic differentiation. We postulate that Fes transduces inductive signals for terminal macrophage and granulocyte differentiation, and that this biological activity is mediated through the activation of lineage-specific transcription factors.

Relatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A-mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A-independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell-derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.

Objective: The fps/fes proto-oncogene is abundantly expressed in myeloid cells, and the Fps/Fes cytoplasmic protein-tyrosine kinase is implicated in signaling downstream from hematopoietic cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). Studies using leukemic cell lines have previously suggested that Fps/Fes contributes to granulomonocytic differentiation, and that it might play a more selective role in promoting survival and differentiation along the monocytic pathway. In this study we have used a genetic approach to explore the role of Fps/Fes in hematopoiesis., Methods: We used transgenic mice that tissue-specifically express a mutant human fps/fes transgene (fps(MF)) that was engineered to encode Fps/Fes kinase that is activated through N-terminal myristoylation (MFps). Hematopoietic function was assessed using lineage analysis, hematopoietic progenitor cell colony-forming assays, and biochemical approaches., Results: fps(MF) transgenic mice displayed a skewed hematopoietic output reflected by increased numbers of circulating granulocytic and monocytic cells and a corresponding decrease in lymphoid cells. Bone marrow colony assays of progenitor cells revealed a significant increase in the number of both granulomonocytic and multi-lineage progenitors. A molecular analysis of signaling in mature monocytic cells showed that MFps promoted GM-CSF-induced STAT3, STAT5, and ERK1/2 activation., Conclusions: These observations support a role for Fps/Fes in signaling pathways that contribute to lineage determination at the level of multi-lineage hematopoietic progenitors as well as the more committed granulomonocytic progenitors.

Objective: The highly related protein-tyrosine kinases Fps (also called Fes) and Fer are sole members of a subfamily of kinases. In this study, knock-in mice harboring kinase-inactivating mutations in both fps and fer alleles were used to assess functional redundancy between Fps and Fer kinases in regulating hematopoiesis., Methods: Mice harboring kinase-inactivating mutations in fps and fer alleles were generated previously. Compound homozygous mice were bred that lack both Fps and Fer kinase activities and progeny were analyzed for potential defects in viability and fertility. Potential differences in hematopoiesis were analyzed by lineage analysis of bone marrow cells, peripheral blood counts, and hematopoietic progenitor cell colony-forming assays., Results: Mice devoid of both Fps and Fer kinase activities were viable and displayed reduced fertility. Circulating levels of neutrophils, erythrocytes, and platelets were elevated in compound mutant mice compared to wild-type controls, suggesting that hematopoiesis is deregulated in the absence of Fps and Fer kinases. Compound mutant mice also showed reduced overall bone marrow cellularity, and lineage analysis revealed elevated CD11b(hi)Ly-6G(lo) myeloid cells, which may reflect increased granulocyte progenitors. Although no differences in the overall number of granulocyte/monocyte colony-forming progenitors were observed, qualitative differences in myeloid colonies from compound mutant mice suggested a role for Fps and Fer kinases in regulating cell-cell adhesion or a skewing in cellularity of colonies., Conclusions: Mice lacking both Fps and Fer kinase activities develop normally, show reduced fertility, and display defects in hematopoiesis, thus providing evidence for functional redundancy between Fps and Fer kinases in regulating hematopoiesis.

The fps/fes proto-oncogene encodes a cytoplasmic protein tyrosine kinase implicated in growth factor and cytokine receptor signaling and thought to be essential for the survival and terminal differentiation of myeloid progenitors. Fps/Fes-null mice were healthy and fertile, displayed slightly reduced numbers of bone marrow myeloid progenitors and circulating mature myeloid cells, and were more sensitive to lipopolysaccharide (LPS). These phenotypes were rescued using a fps/fes transgene. This confirmed that Fps/Fes is involved in, but not required for, myelopoiesis and that it plays a role in regulating the innate immune response. Bone marrow-derived Fps/Fes-null macrophages showed no defects in granulocyte-macrophage colony-stimulating factor-, interleukin 6 (IL-6)-, or IL-3-induced activation of signal transducer and activator of transcription 3 (Stat3) and Stat5A or LPS-induced degradation of I kappa B or activation of p38, Jnk, Erk, or Akt.

Fps/Fes and Fer are the only known members of a distinct subfamily of the non-receptor protein-tyrosine kinase family. Recent studies indicate that these kinases have roles in regulating cytoskeletal rearrangements and inside out signalling that accompany receptor ligand, cell matrix and cell cell interactions. Genetic analysis using transgenic mouse models also implicates these kinases in the regulation of inflammation and innate immunity.

The c-fps/fes proto-oncogene encodes a 92-kDa protein tyrosine kinase that is preferentially expressed in myeloid and endothelial cells. Fes is believed to play a role in vascular development and myelopoiesis and in the inflammatory responses of granulocytes and macrophages. To help define the biological role of this kinase and identify its downstream targets, we have developed a gain-of-function allele of Fes that has potent biological activity in myeloid cell progenitors. Introduction of constitutively active Fes into bipotential U937 cells induced the appearance of fully differentiated macrophages within 6 to 12 days. The Fes-expressing differentiated cells became adherent, had distinctive macrophage morphology, and exhibited increased expression of myelomonocytic differentiation markers, including CD11b, CD11c, CD18, CD14, and the macrophage colony-stimulating factor receptor. These cells acquired phagocytic properties and exhibited NADPH oxidase and nonspecific esterase activities, confirming that they were functionally active macrophages. Concomitantly, there was downregulation of the granulocytic marker granulocyte colony-stimulating factor receptor, indicating that the biological activity of Fes was coordinated in a lineage-specific manner. A constitutively active Src did not induce macrophage morphology or upregulation of myelomonocytic markers in U937 cells, suggesting that the biological activity we observed was not a general consequence of expression of an activated nonreceptor tyrosine kinase. Analysis of possible downstream targets of Fes revealed that this kinase activated the ets family transcription factor PU.1, which is essential for macrophage development. Our results strongly implicate Fes as a key regulator of terminal macrophage differentiation and identify PU.1 as a transcription factor that may mediate some of its biological activities in myeloid cells.

Autophosphorylation of Tyr-1073 in the activation loop of the oncoprotein v-Fps enhances the phosphoryl transfer reaction without influencing substrate, ATP, or metal ion binding affinities [Saylor, P., et al. (1998) Biochemistry 37, 17875-17881]. A structural model of v-Fps, generated from the insulin receptor, indicates that pTyr-1073 chelates two arginines. Mutation of these residues to alanine (R1042A and R1066A) results in weakly phosphorylated enzymes, indicating that one electropositive center is insufficient for attaining maximum loop phosphorylation and concomitant high catalytic activity. While the turnover rate for R1066A is similar to that for a mutant lacking a phosphorylatable residue in the activation loop, the rate for R1042A is 50-fold slower. While solvent perturbation studies suggest that the former is due to a slow phosphoryl transfer step, the latter effect results from a slow conformational change in the mutant, potentially linked to motions in the catalytic loop. Binding of a stoichiometric quantity of Mg(2+) is essential for ATP binding and catalysis, while binding of an additional Mg(2+) ion activates further the wild-type enzyme. The affinity of the R1066A enzyme for the second Mg(2+) ion is 23-fold higher than that of the phosphorylated or unphosphorylated form of wild-type v-Fps, with substrate binding unaffected. Conversely, the affinity of R1066A for a substrate mimic lacking a phosphorylation site is 12-fold higher than that for the phosphorylated or unphosphorylated form of wild-type v-Fps, with binding of the second Mg(2+) ion unaffected. A comparison of these enzyme-independent parameters indicates that Arg-1042 and Arg-1066 induce strain in the active site in the repressed form of the enzyme. While this strain is not relieved in the phosphorylated form, the improvements in catalysis in activated v-Fps compensate for reduced metal and substrate binding affinities.

The c-fes proto-oncogene encodes a Mr 93,000 protein-tyrosine kinase (Fes) that is strongly expressed in myeloid cells and has been implicated in myelomonocytic differentiation. Fes autophosphorylation and transforming activity are highly restrained after ectopic expression in fibroblasts, indicating tight negative regulation of Fes kinase activity in vivo. Here we investigated the regulatory role of the Fes Src homology 2 (SH2) domain by producing a series of chimeric constructs in which the Fes SH2 domain was replaced with those of the transforming oncogenes v-Fps and v-Src or by the NH2-terminal SH2 domain of the Ras GTPase-activating protein. Wild-type and chimeric Fes proteins readily underwent tyrosine autophosphorylation in vitro and produced identical cyanogen bromide phosphopeptide cleavage patterns, indicating that the SH2 substitutions did not influence overall kinase activity or autophosphorylation site selection. However, metabolic labeling of Rat-2 fibroblasts expressing each construct showed that only the Fes/Src SH2 chimera was active in vivo. Consistent with this result, the Fes/Src SH2 domain chimera exhibited potent transforming activity in fibroblasts and enhanced differentiation-inducing activity in K-562 myeloid leukemia cells. In addition, the Fes/Src SH2 chimera exhibited constitutive localization to focal adhesions in Rat-2 fibroblasts and induced the attachment and spreading of TF-1 myeloid cells. These data demonstrate a central role for the SH2 domain in the regulation of Fes kinase activity and biological function in vivo.

The three glycine residues in the glycine-rich loop of the oncoprotein, v-Fps, were mutated to determine the function of these highly conserved residues in catalysis. The kinase domains of six mutants (G928A,S, G930A,S, and G933A,S) and the wild-type enzyme were expressed and purified as fusion proteins of glutathione-S-transferase in Escherichia coli, and their catalytic properties were assessed using steady-state kinetic, inhibition, viscosity and autophosphorylation studies. Although both G928A and G930A had no detectable activity toward the substrate peptide (EAEIYEAIE), the other mutants had apparent, but varying activities. G930S lowered the rate of phosphoryl transfer by 130-fold while G928S and G933S had smaller (6-9-fold) reductions in this step. These effects on catalytic function parallel the reductions in turnover and autophosphorylation but, for G933S and G933A, net product release is still rate limiting at saturating substrate and ATP concentrations. On the basis of K(I) measurements, the effects on turnover for these mutants may be due to improved ADP affinity. While ADP affinity is reduced 2- and 3-fold for G928S and G930S, the affinity of this product is increased by 22- and 7-fold for G933S and G933A. In contrast, ATP affinity is enhanced by 5-fold for G928S and G933S and is reduced by less than 2-fold for G930S. These complex, differential effects on nucleotide binding indicate that the glycines influence the relative affinities of ADP and ATP. On the basis of the results of serine replacements, Gly-928 and Gly-930 enhance ADP affinity by 9- and 2-fold compared to ATP affinity whereas Gly-933 diminishes ADP affinity by approximately 4-fold compared to ATP affinity. These findings demonstrate that the functions of the loop lie not only in modulating the rate of the phosphoryl transfer step but also in balancing the relative affinities of ATP and ADP. These effects on nucleotide specificity may be a contributing element for the stabilization of the phosphoryl transition state and may also facilitate quick release of bound products.

Our preliminary studies suggested that the novel gag-truncated mos (tmos) open reading frame (ORF) of R7, a spontaneous deletion mutant of Moloney murine sarcoma virus 124 (MoMuSV124), may be responsible for R7's unique ability to induce brain lesions in all R7-injected mice. However, when we replaced the gag-tmos ORF with either the MoMuSV124 or the homologous myeloproliferative sarcoma virus env-mos gene, we found that both recombinant viruses also induced brain lesions in all injected mice. Although these studies suggested that the critical determinants for brain lesion induction may reside in the tmos sequence common to all three viruses, they did not demonstrate if the N-terminus of Mos was dispensable for this activity. By inserting the FLAG sequence at the 3' end of the R7 gag-tmos ORF, we demonstrated that R7 does synthesize a Gag-tMos fusion protein. Using R7 gag deletion mutants with and without the FLAG sequence, we further demonstrated that (i) deletion of the entire gag sequence abolished R7's transforming activity; (ii) the ability of the virus to transform cultured NIH/3T3 cells was significantly reduced only when most of gag was deleted; (iii) the ability of the virus to induce brain lesions was inversely proportional to the extent of its gag deletions; and (iv) the insertion of FLAG at the Mos C-terminus did not reduce the in vitro transforming activity of the FLAG-tagged viruses but did reduce their ability to induce brain lesions. Thus, we have demonstrated that altering the N- or C-terminus of the R7 Gag-tMos fusion protein can affect disease manifestation.

Fes is a nonreceptor tyrosine kinase expressed at the highest level in macrophages. We previously showed that the overexpression of c-fes in murine macrophages of the BAC-1.2F5 cell line renders these cells independent of macrophage colony-stimulating factor (MCSF) for survival and proliferation, although no direct relationship could be established between tyrosine-phosphorylated substrates of Fes- and MCSF receptor-dependent signaling and mitogenesis. In this study, we investigated whether the mitogen-activated protein kinase (MAPK) pathway is involved in the growth factor-independent growth of v-fes-overexpressing macrophages. We found a constitutively increased phosphorylation of extracellularly regulated kinase (ERK) in v-fes-overexpressing macrophages as compared with mock-infected cells. This finding was associated with activation of mitogen/extracellular signal-regulated kinase (MEK) and with constitutive localization of ERK in the nucleus. Treatment of v-fes-overexpressing cells with the MEK-specific inhibitor PD98059 markedly reduced cell growth, hyperphosphorylation, and nuclear localization of ERK, indicating that the MAPK pathway mediates the mitogenic effect of v-fes. (Blood. 2000;95:3959-3963)

Based on the X-ray structure of the insulin receptor kinase [Hubbard, S. R. (1997) EMBO J. 16, 5572-5581], Arg-1130 in the oncoprotein v-Fps, a nonreceptor tyrosine protein kinase, is predicted to interact with the P+1 glutamate in substrate peptides. To determine whether this residue is an important recognition element in v-Fps, Arg-1130 was substituted with leucine (R1130L) and glutamic acid (R1130E). The ability of these mutants to phosphorylate the peptide EAEIYXAIE, where X is glutamic acid, alanine, or lysine, was assessed. A comparison of the rates of peptide phosphorylation under limiting substrate concentrations (i.e., k(cat)/K(m) conditions) indicates that substrate specificity is altered by the electrostatic environment of the P+1 pocket. When the pocket displays a positive charge (Arg-1130; wild type), no charge (R1130L), or a negative charge (R1130E), v-Fps prefers to phosphorylate the glutamate peptide over the lysine peptide by a 200:1, 9:1, or 1:1 margin. While k(cat)/K(m) for the glutamate peptide is 50-fold higher for wild type compared to R1130E, k(cat)/K(m) for the lysine peptide is 3-fold higher for R1130E compared to wild type, a 150-fold change in relative substrate specificity. Analysis of the individual steps in the kinetic mechanism using viscosometric techniques indicates that the wild-type enzyme binds the glutamate peptide 3-fold better than the alanine peptide and, at least, 10-fold better than the lysine peptide. For R1130L, this margin range is reduced substantially, and for R1130E, no binding preference is observed. Nonetheless, the lysine peptide binds, at least, 4-fold better to R1130E than to wild type, and the glutamate peptide binds 3-fold poorer to R1130E than to wild type. The mutants lower the phosphoryl transfer rate by 4-30-fold for the three peptides, suggesting that Arg-1130 helps to position the tyrosine for optimum catalysis. The data indicate that a single mutation in v-Fps can alter significantly the relative substrate specificity by about 2 orders of magnitude with, at least, 50% of this effect occurring through relative changes in peptide binding affinity.

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.

The human c-fes protooncogene encodes a protein-tyrosine kinase (c-Fes) distinct from c-Src, c-Abl and other nonreceptor tyrosine kinases. Although originally identified as the cellular homolog of several transforming retroviral oncoproteins, Fes was later found to exhibit strong expression in myeloid hematopoietic cells and to play a direct role in their differentiation. Recent work has shown that Fes exhibits a more widespread expression pattern in both developing and adult tissues, suggesting a general physiological function for this kinase and its closely related homolog, Fer. This review highlights the unique aspects of Fes structure, regulation, and function that set it apart from other tyrosine kinase families.

Concentrations of the bioactive lipids, phosphatidate and diacylglycerol, increased with time in culture in ras- and tyrosine kinase (fps)-transformed fibroblasts but not in control fibroblasts. On Day 3, diacylglycerol and phosphatidate concentrations were about 3.3- and 5.5-fold higher respectively in the ras-transformed compared to control fibroblasts. These concentrations in fps-transformed fibroblasts were increased about twofold. The changes in phosphatidate and diacylglycerol resulted from enhanced phospholipid turnover rather than from synthesis de novo. The increased ratio of phosphatidate to diacylglycerol is explained by decreased activities of two distinct phosphatidate phosphohydrolases and increased diacylglycerol kinase in ras-transformed fibroblasts. Ceramide concentrations were about 2.5- and threefold higher in the fps- and ras-transformed cells respectively on Day 3 compared to the controls. Incubating control fibroblasts from Days 1 to 3 with phosphatidylcholine-specific phospholipase C increased diacylglycerol, phosphatidate and ceramide concentrations, and decreased Mg2+-independent-phosphatidate phosphohydrolase activity. 8-(4-chlorophenylthio)-cAMP had a cytostatic effect in ras-transformed cells, it decreased the concentrations of phosphatidate and diacylglycerol, but increased that of ceramide. The consequences of increased ceramide and phosphatidate concentrations in ras-transformed cells are discussed in relation to signal transduction, cell division and the transformed phenotype.

The rate-determining steps in the phosphorylation of four tyrosine-containing peptides by the kinase domain of the nonreceptor tyrosine protein kinase v-fps were measured using viscosometric methods. The peptides were phosphorylated by a fusion protein of glutathione-S-transferase and the kinase domain of v-fps (GST-kin) and the initial velocities were determined by a coupled enzyme assay. Peptides I (EEEIYEEIE), II (EAEIYEAIE), and III (DADIYDAID) were phosphorylated by GST-kin with similar kinetic constants. The viscosogens, glycerol and sucrose, were found to have intermediate effects on kcat and no effect on kcat/Kpeptide for the phosphorylation of these three peptides. The data are interpreted according to the Stokes-Einstein equation and a simple three-step mechanism involving substrate binding, phosphoryl group transfer, and net product release. Two competitive inhibitors (EAEIFEAIE and DADIFDAID) exhibited K1 values that are 6-10-fold higher than the Kpeptide values for their analogous peptide substrates. The data imply that peptides I-III are in rapid equilibrium with the enzyme and that kcat is partially limited by both phosphoryl group transfer (40-100 s-1) and product release (17-22 s-1). GST-kin phosphorylates peptide IV (R5AENLEYamide) with a low Km (100 microM) and a kcat that is 40-fold lower than that for peptide I. No effect of solvent viscosity was observed for the phosphorylation of this peptide on either kcat or kcat/Kpeptide. This suggests that highly viscous solutions do not perturb structure and that the rate-determining step for this poor substrate is phosphoryl group transfer. The data indicate that the kinase domain of v-fps phosphorylates its best substrate with a chemical rate constant that is at least 5-fold lower than that for the serine-specific cAMP-dependent protein kinase and its best substrate LRRASLG (Adams & Taylor, 1992). Interestingly, both enzymes exhibit a similar affinity for their substrates and both enzymes release their products at a similar rate. This implies that the differences in catalytic efficiency between serine- and tyrosine-specific protein kinases lie exclusively in the rate constants for phosphoryl group transfer and not in substrate absorption or product desorption.

In sarcomagenesis in rats infected neonatally with feline sarcoma virus (ST-FeSV), v-fes product (P85) was previously shown by us to be a predictive and preventive determinant. In order to explore the part played by P85 in tumor suppression, DNA was extracted from precancerous granulomas and from slow or rapid growing sarcomas induced by neonatal injection of the virus. The v-fes signal from extracted DNA was analyzed by PCR-SSCP. The prototype v-fes gene signal was detected in most lesions and found to be generally amplified in rapid growing sarcomas and in some granulomas. Several v-fes homologs showing varying mobilities in gel were seen in most sarcomas and some granulomas with or without the prototype v-fes signal. In slow growing sarcomas and granulomas induced in hosts that were immunized with ST-FeSV induced syngeneic sarcoma and proved to carry IgG antibody to P85, the prototype v-fes gene was found to be down-regulated and v-fes homologs were found to be reduced in number or eliminated. These results suggest that the development of v-fes mutations is associated with the growth potential of cells carrying the v-fes gene, and that host immunity to v-onc product influences the development of virogene rearrangements and results in slow and suppressed growth of tumors caused by neonatal infection with retrovirus.

To determine whether the v-fes oncogene can malignantly transform human fibroblasts that have acquired an infinite life span and are partially growth-factor-independent, we transfected cell strain MSU-1.1 with a plasmid containing the v-fes oncogene and a bacterial histidinol dehydrogenase gene. Of the 60 independent histidinol-resistant clones isolated and assayed for v-fes expression using a fes-specific monoclonal antibody, 6 were found to express the v-fes protein at a detectable level. When progeny cells from these 6 clones were further characterized, 3 of the 6 clonal populations exhibited a significant increase in the ability to form medium-sized colonies in agarose, but none were tumorigenic in athymic mice. However, when the 6 populations were propagated for many generations, the same 3 populations acquired the ability to form very large colonies in agarose ( > or = 100 microM in diameter) at a frequency of 2% to 17%, and formed malignant tumors in athymic mice. This suggests that an additional genetic change required for malignant transformation had been spontaneously acquired in 3 of the v-fes -transformed cell strains. To determine whether the change or changes were the equivalent of an activated sis or ras proto-oncogene, we transfected the v-fes oncogene into derivative strains of MSU-1.1 that express a transfected v-sis, c-H-ras or c-N-ras oncogene, but that do not form tumors, and assayed the v-fes-expressing transfectants for tumorigenicity. The results showed that when complemented either by a ras oncogene expressed at a somewhat enhanced level or by the v-sis oncogene, v-fes can supply the additional change required for malignant transformation.

The gene coding for the tyrosine protein kinase domain of v-fps was subcloned into a plasmid vector expressing glutathione-S-transferase (GST). This new vector expresses a fusion protein in Escherichia coli composed of the kinase domain linked with GST at the N-terminus (GST-kin). A portion of the total expressed protein was soluble upon cell lysis and was purified by affinity chromatography using glutathione cross-linked agarose. GST-kin (M(r) 57,000) is a phosphoprotein as judged by 32P autoradiography, consistent with the known autophosphorylation site within the kinase core [Weinmaster et al. (1984) Cell, 37, 559-568]. Cleavage of the fusion protein with thrombin and purification on phosphocellulose resin yielded the pure kinase domain (M(r) 33,000). The activity of the kinase domain is indistinguishable from that of GST-kin using the peptide substrate EEEIYEEIE, indicating that N-terminal fusion has no effect on the kinase domain. GST-kin phosphorylates a second peptide, EAEIYEAIE, with improved catalytic efficiency. Initial velocity data are consistent with a random bireactant mechanism with no substrate synergism observed in the ternary complex. Steady-state kinetic analyses reveal that this peptide is phosphorylated, with a kcat of 3.6 s-1, a Kpeptide of 500 microM and a KATP of 250 microM. The expression, purification and preliminary kinetic analysis of the kinase domain of v-fps provide the first step in the application of structure-function studies for this oncoprotein.

The pp60v-src tyrosine kinase disrupts gap junctional communication in transformed fibroblasts and induces the phosphorylation of the gap junction protein, connexin43, on tyrosine. We report here that the p130gag-fps tyrosine kinase also profoundly disrupted gap junctional communication and markedly increased the phosphorylation of connexin43 which appeared to result from an accumulation of phosphotyrosine and phosphoserine. The disruption of gap junctional communication by pp60v-src and p130gag-fps did not appear to result from the gross alteration of gap junction plaques. Furthermore, two-dimensional phosphotryptic peptide mapping showed that the v-Src and V-Fps kinases stimulated the phosphorylation of multiple connexin43 peptides which contained phosphotyrosine and/or phosphoserine. Phosphotyrosine was detected in two connexin43 phosphotryptic peptides from v-src-tranformed cells which suggested that more than one connexin43 tyrosine site may be recognized by pp60v-src in fibroblasts. The apparent higher levels of phosphoserine-containing connexin43 peptides in the oncogene-transformed cells pointed to the possibility that pp60v-src and p130gag-fps may also modulate connexin43 function through mechanism(s) involving the activation of signaling serine kinases. Taken together, these results suggested that connexin43 is a common target of the v-Src and v-Fps tyrosine kinase oncoproteins.

The activity of N-ethylmaleimide-insensitive phosphatidate phosphohydrolase (PAP-2) was characterized in control, ras-transformed, and tyrosine kinase-(fps) transformed rat fibroblasts. PAP-2 was assayed in two different ways: 1) within its natural membrane using liposomes of phosphatidate and 2) in the presence of sufficient Triton X-100 to solubilize PAP-2, and to form mixed micelles with the phosphatidate. Harvesting the fibroblasts in medium containing orthovanadate and Zn2+ gave up to 3-fold higher PAP-2 activities when measured in the absence, but not in the presence, of Triton X-100. PAP-2-specific activities from both assays increased in the control fibroblasts as the cells reached confluence. Both specific activities were lower in the oncogenically transformed fibroblasts than in controls at all cell densities tested. The specific activities of PAP-2 did not increase with time in culture in transformed cells which continued to divide. The relative increase in activity of phospholipase D after stimulation with serum or phorbol myristate acetate was lower in the transformed fibroblasts compared to control cells. This indicates a coordinated decrease in the phospholipase D/phosphatidate phosphohydrolase pathway at the level of both enzymes in ras and fps transformed fibroblasts. The ratio of the production of diacylglycerol relative to phosphatidate, after stimulation with serum, or phorbol ester, was lower in both transformed fibroblasts relative to the controls. This is compatible with the decreased specific activity of PAP-2 and indicates functional significance for the differences in PAP-2 activity in regulating the balance between the two mitogenic lipids, phosphatidate and diacylglycerol. Control of PAP-2 activity could be an important factor in regulating appropriate signals for cell division.

NIH3T3 cells carrying a dominant negative H-ras mutant 116Y acquired resistance to transformation by some PTK oncogenes, i.e., v-fes, v-abl, and v-fms, but were sensitive to viral ras and serine threonine kinase oncogenes, v-raf and v-mos. One clone, designated 1-20, infected with v-fes (1-20 fes) exhibited flat morphology and anchorage-dependent cell growth, as did noninfected 1-20 cells. The 1-20 fes cells expressed v-fes oncogene and produced transforming viruses, although these levels were much lower than those in NIH3T3 cells infected with v-fes (NIH3T3 fes). v-fes mRNAs in NIH3T3 fes cells rapidly increased after infection, while accumulation of the v-fes transcripts in 1-20 fes cells was significantly prolonged. Total tyrosine phosphorylation in both NIH3T3 fes and 1-20 fes cells was correlated with the amounts of pp110v-fes. A few proteins were phosphorylated only in NIH3T3 fes but not in 1-20 fes cells. These results suggest that the cellular ras is involved in a signaling pathway from pp110v-fes and this signal stimulates v-fes expression. Inhibition of the ras function may down-regulate this pathway and result in resistance to transformation by v-fes.

XD4 is a host range deletion mutant (delta 77-225) of the v-src transforming gene. This mutant transforms chicken embryo fibroblasts (CEF) but not Rat-2 cells. It encodes a product (XD4-src) that is phosphorylated at tyrosine and active as a tyrosine kinase in CEF, but is neither phosphorylated at tyrosine nor active as a kinase in Rat-2 cells. We report here that the XD4-src kinase activity in Rat-2 cells can be restored by co-expression with the tyrosine kinase encoded by v-fps, but not by co-expression with activated Ha-ras. Mutation of the major tyrosine autophosphorylation site (Y416) in XD4-src reduced but did not abolish the reactivation of XD4 tyrosine phosphorylation and kinase activity by v-fps. These results suggest that deletion of the SH2 and SH3 domains renders v-src kinase activity dependent on tyrosine phosphorylation at Y416 and other sites. The reactivation of XD4-src may result either from direct phosphorylation by the v-fps gene product or may be an indirect consequence of v-fps expression.

The Tec kinase was initially identified as a novel cytoplasmic protein tyrosine kinase that is preferentially expressed in the liver and is highly homologous to the Drosophila Dsrc28C src-related tyrosine kinase. In screening of interleukin 3 (IL-3)-dependent myeloid leukemia cells for protein tyrosine kinases, we observed that all cell lines examined expressed high levels of Tec transcripts. However, characterization of Tec cDNAs indicated that they differed significantly from the published sequence. Most strikingly, an insertion of 41 bp in the 5' region affects the initiation codon and results in replacing the published 13 amino acid amino-terminal sequences with 94 amino acids. Using polymerase chain reaction (PCR) analysis, only the form containing the insertion was detected in hematopoietic cells. In addition, we found an in-frame insertion of 66 bp that introduces an additional 22 amino acids into the SH3 domain. This insertion restores conserved SH3 sequences that are found in the src gene family and in the Dsrc28C gene. By PCR analysis, approximately equal levels of Tec transcripts containing the intact SH3 domain and containing the 22 amino acid deletion were found in hematopoietic cells. Lastly, by interspecies backcross analysis, we show that the Tec gene is tightly linked to the c-Kit gene on mouse chromosome 5.

It has been reported that loss of the tumorigenic potential of attenuated Marek's disease virus (MDV) is strongly associated with amplification of the 132-bp repeat sequences found within the BamHI-D and BamHI-H fragments contained within the long terminal repeat and the long internal repeat, respectively. The expansion of this region results in loss of transcripts that are 3.8, 3.0, and 1.8 kbp long that are produced by tumorigenic strains of MDV. This evidence suggests that production of one or more of these three RNAs is strongly associated with the tumorigenic potential of the virus. In this study, we have cloned and sequenced 1.69-, 1.5-, 1.9-, and 2.2-kbp cDNAs from the BamHI-H gene family RNAs associated with tumorigenicity. The 1.69- and 2.2-kbp cDNAs are derived from nonspliced transcripts, whereas the 1.5- and 1.9-kbp cDNAs are from single spliced mRNAs spanning the BamHI-H and BamHI-I2 fragments of MDV DNA. Sequence analysis has shown two potential open reading frames in each of the cDNAs. The putative 63-amino-acid protein encoded by the first open reading frame in the 1.69-kbp cDNA and a putative 75-amino-acid protein encoded by the first open reading frame in the 1.5-kbp cDNA showed limited homology with the mouse T-cell lymphoma oncogene and the fes/fps family of kinase-related transforming proteins.

There is increasing evidence that protein-tyrosine kinases play pivotal roles in the response to growth-factor signals. The cytoplasmic tyrosine kinase c-fps/fes, due to its restricted expression in hematopoietic tissue, is likely to participate in hematopoietic growth-factor signalling. We have introduced a retrovirus containing an activated fps gene (encoding P130gag-fps) into the growth factor-dependent myeloid cell line FDC-P1. Clonal cell lines were derived by selection for a marker gene coding for G418 resistance in the absence or presence of the hematopoietic growth factor IL-3. G418 resistant clones expressed P130gag-fps and its associated protein-tyrosine kinase activity and displayed either a factor-independent or IL-3 hypersensitive phenotype and were tumorigenic in syngeneic recipients. Thus, introduction of the activated v-fps gene was able to circumvent the requirement for exogenous growth factors by FDC-P1 cells. Bioassay of conditioned medium from the various clones did not detect hematopoietic growth factor activity and PCR analysis for IL-3 transcripts were negative, suggesting that growth-factor independence was achieved by a mechanism other than autocrine production of a growth factor. We suggest that P130gag-fps is acting to directly stimulate a hematopoietic growth-factor signalling pathway, perhaps one that normally involves the endogenous c-fps/fes protein-tyrosine kinase of FDC-P1 cells.

Avian myelocytomatosis virus MC29 induces a wide variety of neoplastic diseases in infected birds and transforms cells of the macrophage lineage as well as fibroblasts and epithelial cells. A biological and biochemical analysis, carried out on a series of in-frame insertion and deletion mutations within the gag-myc gene of MC29, revealed several mutations within the 5' portion of the v-myc gene that encode proteins either completely defective for transformation or compromised in their ability to transform chicken embryo fibroblasts but not macrophages. Mutations within the 3' end of the v-myc gene which disrupt sequences encoding the basic/helix-loop-helix region were defective for transformation of both fibroblasts and macrophages. Eight variants were cloned into the replication-competent avian expression vector RCAS. Analysis of cells infected with transformation-defective, replication-competent viruses confirmed the expression of functionally defective Myc proteins. Further, expression of the transformation defective variant dl91-137 in chicken fibroblasts inhibited subsequent transformation by wild-type MC29. The results reported herein support the hypothesis that Myc proteins function as regulators of transcription in a variety of cell types and clearly point out the necessity of putative regulatory domains within the amino-terminal half of the Myc protein.

Important cellular nutrients, including nucleosides and hexose sugars, are rapidly taken up by cells, largely through mediated carrier systems. The present study examined nucleoside and hexose transport activity in normal Rat-2 fibroblasts and clonal derivatives that expressed either the wild-type (C10) or a temperature-sensitive mutant (NA9) form of v-fps, a transforming protein-tyrosine kinase. Initial uptake rates (transport) of adenosine, thymidine, 3-O-methylglucose and 2-deoxyglucose were greater in v-fps-transformed cells than in normal cells. Elevated transport rates were seen in cells that expressed the temperature-sensitive mutant v-fps only after growth at a temperature that was permissive for protein-tyrosine kinase activity. Nucleoside transport rates declined with increasing cell density in both normal and v-fps transformed cells. Analysis of the sensitivity of adenosine transport to inhibition by nitrobenzylthioinosine (NBMPR) indicated that Rat-2 fibroblasts, like many other rat cell types, possess at least two nucleoside transport systems, which can be distinguished by differences in sensitivity to NBMPR. Although both transport activities were elevated in v-fps-transformed cells, a greater increase was seen in the NBMPR-sensitive component than in the NBMPR-insensitive component. Mass law analysis of the binding of [3H]NBMPR indicated that transformed cells had either the same number (NA9) or a smaller number (C10) of NBMPR-binding sites than normal cells, and photolabelling of membrane proteins with [3H]NBMPR identified polypeptides with similar electrophoretic mobilities (55-75 kDa) in both normal and transformed cells. Thus transformation by v-fps resulted in an increase in NBMPR-sensitive transport activity which was not related to either the number of NBMPR-binding sites or the apparent molecular mass of NBMPR-binding polypeptides.

The myristylated v-fos product, FBR murine sarcoma virus (Gag-Fos) protein, exhibits a lower level of transrepression of the serum response element (SRE) than does c-fos protein (Fos). Mutation of the N-terminal myristylation site in FBR protein restored SRE transrepression. Replacement of N-terminal viral Gag sequences with the Fos N terminus also restored this activity, providing additional evidence that myristylation inhibits transrepression by FBR protein. However, the myristylated Gag domain did not inhibit SRE transrepression when fused to Fos, indicating that myristylation of a fos protein is not by itself sufficient to prevent SRE transrepression and that C-terminal mutation is necessary to inhibit transrepression by N myristylation. Comparison of transfection results with Fos C-terminal deletion mutants and the Fos/FBR chimeric mutant revealed that the FBR C terminus retained the potential for transrepression despite deletion of the normal Fos C terminus, whereas similar Fos deletion mutants did not. These results indicate that both N- and C-terminal mutations are required to inhibit transrepression by FBR protein and that multiple structural mutations accompanied by posttranslational protein modification alter gene regulation by FBR protein.

The stimulation of quiescent murine fibroblasts by growth factors and by phorbol esters results in a rapid and transient transcriptional activation of a large group of so-called immediate early genes. Several such genes were found to be induced in chicken embryo fibroblasts following activation of a temperature sensitive (ts) Rous sarcoma virus v-src mutant following temperature shift (Simmons et al., 1989). In contrast, the classical immediate early genes c-myc, c-fos and c-jun were essentially uninducible upon activation of a ts v-src mutant in rat-1 fibroblasts (Welham et al., 1990). We have cloned 9 cDNAs of genes that are rapidly and transiently inducible in rat fibroblasts by ts v-src mutants, and by a ts Fujinami sarcoma virus v-fps mutant. Six of these cDNAs are derived from the known immediate early genes NGFI-A, KC, c-fos, tissue factor, PC4 and ornithine decarboxylase; the other three cDNAs have not been described before. These 9 genes showed individual profiles of inducibility by fetal calf serum, epidermal growth factor (EGF) and by phorbol esters. Their response to the retroviral oncogenic protein-tyrosine kinases correlated best with the one to EGF, suggesting a common pathway of signal transduction. c-fos did not respond strongly to this pathway but was well induced by fetal calf serum. NGFI-A, however, was induced to a similar extent by all activators tested. Furthermore, we demonstrated that the induction of several of these genes by the retroviral oncogenic protein-tyrosine kinases is rapid, direct and occurs at the transcriptional level.

A set of genes is rapidly inducible when quiescent fibroblasts are stimulated by growth factors or by the activation of temperature-sensitive retroviral protein-tyrosine kinases. Most of these so-called immediate-early genes were cloned by differential cDNA hybridization. DNA sequence analysis identified many of them as putative members of the growth factor or of the transcription factor gene family, suggesting a role in signal transmission during the G0-to-G1 transition. In this study, we identified one of the genes that are rapidly inducible by the retroviral protein-tyrosine kinases v-Src and v-Fps of Rous sarcoma virus and Fujinami sarcoma virus, respectively, as the rhoB gene, a member of the ras gene superfamily whose products are GTP-binding proteins, rhoB is transiently activated at the transcriptional level by v-Fps and by epidermal growth factor. Its labile RNA is inducible in the presence of cycloheximide but not of actinomycin D. rhoB is strongly induced by epidermal growth factor and by platelet-derived growth factor both in subconfluent, serum-starved and in density-arrested Rat-2 fibroblasts. Fetal calf serum is a poor inducer, particularly in density-arrested cells, and phorbol esters do not increase rhoB expression at all. These data suggest that rhoB is inducible by protein-tyrosine kinases through a pathway not involving the activation of protein kinase C. Neither the closely related rhoC and rhoA genes nor the distantly related c-H-ras gene is rapidly inducible by mitogens. Thus, rhoB is the first known member of the small GTP-binding proteins among the immediate-early genes.

The pyruvate-stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450 microM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C-terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N-terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.

Transgenic mice that express v-fps protein-tyrosine kinase have severe cardiac or neurologic abnormalities and a high incidence of lymphoid or mesenchymal tumors. The cardiac lesions of v-fps transgenic mice were examined at less than 1, 2, 3, 6, 14, 26, and 43 weeks of age (total N = 19) and compared with nontransgenic littermate controls (N = 34). Three of eight transgenic animals 1 to 4 days old showed moderate proliferation of connective tissue elements most evident along the septal endocardium of the right ventricle. Variable cardiac hypertrophy was seen grossly in 2- to 14-week-old transgenic animals, and marked biventricular dilatation was present in those 6 to 43 weeks of age. Sections of all transgenic mice 3 weeks or older revealed characteristic lesions that consisted of cellular fibrosis in subendocardial, subepicardial, and perivascular sites, with associated proliferation of pericytes and fat cells. Atrophy, degeneration, and loss of entrapped myocytes were noted in transgenic animals as early as 2 weeks of age, but frank coagulative necrosis or myocytolysis was absent at all ages studied. Nonetheless, pleomorphic nuclei were found in occasional myocytes late in disease. Inflammatory cells were rare, as confirmed immunohistochemically (Thy-1 [pan-T], L3T4 [CD4], Lyt-2 [CD8], interleukin-2 receptor [activated lymphocyte], Mac-1 [macrophage], and B220 [pan-B]). Monoclonal immunoreactivity to the v-fps transgene product was positive predominantly in nonmyocyte cardiac constituents. Collectively, the data suggest a primary proliferative abnormality of connective tissue elements in the heart, accompanied by secondary myocyte damage.

FBR murine sarcoma virus (gag-fos) protein, a virally transduced Fos protein, exhibits decreased gene transactivation in comparison with the cellular Fos protein. Biochemical analysis suggests that myristylation of the virally encoded N-terminal gag region results in decreased DNA binding and transcriptional activation without affecting heterodimerization with Jun protein. These findings demonstrate that protein myristylation can modulate gene regulation by a DNA-binding protein.

The transforming protein of the avian sarcoma virus UR2 is a 68-kDa transmembrane tyrosine protein kinase. We examined the relationship between membrane localization and transforming activity of P68 by changing Val-168-Val-169 in its hydrophobic domain into Asp-168-Glu-169. The resulting transmembrane (TM) mutant (P68TM) lost transforming activity toward chicken embryo fibroblasts (CEF). We found that the mutant protein was expressed and rapidly degraded into a smaller form which was still membrane associated and kinase active. The instability of the TM mutant protein is a phenomenon only manifested in CEF, because the same mutant protein was expressed with efficiency and stability similar to those of the wild-type protein in a transient expression system in COS cells. However, there are several differences between the wild-type and the TM mutant proteins in COS cells. The wild-type protein is more heavily phosphorylated and associated with membrane fractions in a cotranslational manner. It is enzymatically active when recovered from membrane fractions. The TM mutant protein is less phosphorylated, more labile toward protease degradation, and delayed in membrane association, with a lag period of 30 min or longer, and has little kinase activity when recovered from membrane fractions. Most of the kinase-active TM mutant protein was found in the cytosol fractions. Despite the delay, most of the TM protein in COS cells was found to be membrane associated, and its orientation on the cell surface was similar to that of the wild-type protein. It is probable that loss of the CEF-transforming activity of the TM mutant protein is due to its susceptibility to protease degradation resulting from improper membrane association of the newly synthesized product. The differences in the kinetics of membrane association and the distribution of kinase activity in COS cells might not be directly applicable in explaining the inability of the TM mutant to transform CEF but are intriguing as regards protein biosynthesis and translocation. The difference between CEF and COS cells implies that different factors or pathways are involved in the biosynthesis and processing of the TM mutant protein in these two cellular environments. Changes of P68TM in the kinetics of membrane association indicate that the transmembrane domain of ros, besides functioning as a membrane anchor, also plays a role in directing initial membrane association.

The transforming protein P68gag-ros of avian sarcoma virus UR2 is a transmembrane tyrosine protein kinase molecule with the gag portion protruding extracellularly. To investigate the role of the gag moiety in the biochemical properties and biological functions of the P68gag-ros fusion protein, retroviruses containing the ros coding sequence of UR2 were constructed and analyzed. The gag-free ros protein was expressed from one of the mutant retroviruses at a level 10 to 50% of that of the wild-type UR2. However, the gag-free ros-containing viruses were not able to either transform chicken embryo fibroblasts or induce tumors in chickens. The specific tyrosine protein kinase activity of gag-free ros protein is about 10- to 20-fold reduced as judged by in vitro autophosphorylation. The gag-free ros protein is still capable of associating with membrane fractions including the plasma membrane, indicating that sequences essential for recognition and binding membranes must be located within ros. Upon passages of the gag-free mutants, transforming and tumorigenic variants occasionally emerged. The variants were found to have regained the gag sequence fused to the 5' end of the ros, apparently via recombination with the helper virus or through intramolecular recombination between ros and upstream gag sequences in the same virus construct. All three variants analyzed code for gag-ros fusion protein larger than 68 kDa. The gag-ros recombination junction of one of the transforming variants was sequenced and found to consist of a p19-p10-p27-ros fusion sequence. We conclude that the gag sequence is essential for the transforming activity of P68gag-ros but is not important for its membrane association.

The abilities of a series of six mutants of the human insulin receptor, an insulin receptor/v-ros hybrid (IR-ros) and the P68gag-ros transforming protein to stimulate S6 protein kinase have been assessed. Insulin receptor mutants in which either 1 or 2 tyrosine residues have been replaced with phenylalanine (YF1, YF3) have lost some or all of the capacity to mediate the activation of S6 kinase in response to insulin. None of the four mutants that contain deletions (spBam, spBamYF3, iBgl, T-t) elicit an insulin-dependent stimulation of S6 kinase. A previous study of the IRros hybrid receptor demonstrated that it was unable to cause either insulin-stimulated thymidine incorporation or glucose uptake (Ellis, L., Morgan, D. O., Jong, S.-M., Wang, L.-H., Roth, R. A., and Rutter, W. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5101-5105). In contrast, the IRros chimera appears to mediate the activation of S6 protein kinase by insulin. In further evaluating the biological activities of the IRros hybrid, we have examined its effects on a microtubule-associated protein-2 (MAP2) kinase that is thought to be an early target in the cascade of reactions leading to increased S6 phosphorylation (Sturgill, T. W., Ray, L. B., Erickson, E., and Maller, J. L. (1988) Nature 334, 715-718). We find that the IRros receptor stimulates the MAP2 protein kinase from 3- to 6-fold in insulin-treated cells, conferring more than a 30-fold increase in the insulin sensitivity of MAP2 kinase activation.

The critical pathways through which protein-tyrosine kinases induce cellular proliferation and malignant transformation are not well defined. As microinjection of antibodies against p21ras can block the biological effects of both normal and oncogenic tyrosine kinases, it is likely that they require functional p21ras to transmit their mitogenic signals. No biochemical link has been established, however, between tyrosine kinases and p21ras. We have identified a non-catalytic domain of cytoplasmic tyrosine kinases, SH2, that regulates the activity and specificity of the kinase domain. The presence of two adjacent SH2 domains in the p21ras GTPase-activating protein (GAP) indicates that GAP might interact directly with tyrosine kinases. Here we show that GAP, and two co-precipitating proteins of relative molecular masses 62,000 and 190,000 (p62 and p190) are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. The phosphorylation of these polypeptides correlates with transformation in cells expressing inducible forms of the v-src or v-fps encoded tyrosine kinases. Furthermore, GAP, p62 and p190 are also rapidly phosphorylated on tyrosine in fibroblasts stimulated with epidermal growth factor. Our results suggest a mechanism by which tyrosine kinases might modify p21ras function, and implicate GAP and its associated proteins as targets of both oncoproteins and normal growth factor receptors with tyrosine kinase activity. These data support the idea that SH2 sequences direct the interactions of cytoplasmic proteins involved in signal transduction.

Primary neonatal Balb/c keratinocyte (NEK) cultures grown, using 3T3 feeder cell support in high calcium, serum supplemented medium, were transfected with EJ-Ha-ras or v-fos DNA sequences and pSV2 neo. Several neo resistant clones were isolated and several established cell lines expressing the transfected gene products derived. Two of these lines, Ras 8 and Fos 1, have been examined in detail with respect to their self renewal capacity and differentiation potential in vitro and in vivo. In vitro, both lines (when compared to normal NEK) have an extended probably immortal phenotype, enhanced colony forming efficiency (a measure of in vitro self renewal capacity) and a reduction in growth factor and serum dependence. When grafted onto syngeneic recipients neither cell line is tumourigenic. Instead a histologically abnormal epithelium with no stratum corneum and with features specific to the oncogene expressed is formed. The extent of the histological atypia correlates with the in vitro alterations in cytoskeletal peptides as revealed by 2D PAGE. However despite the gross histological abnormality there is no alteration in the in vivo self renewal capacity (measured as the number of grafted cells required for epidermal reformation) between normal NEK and the Ras 8 or Fos 1 lines; in each case a minimum of 10(5) cells/1.14 cm2 is required before a full thickness epithelium forms.

We previously reported that v-fos transfer to a src-transformed rat 3Y1 cell line enhanced lung metastasis. To clarify the mechanism of this enhancement, we compared various biological factors related to metastatic potential between a fos-transferred highly metastatic cell line (fos-SR-3Y1-202) and the control cell line transferred with genetic marker (pSV2-neo) plus pBR322, neo-SR-3Y1-200. Lung arrest, the effect of lung extract on cell growth, or sensitivity to natural killer cells could not explain the higher metastasis of fos-SR-3Y1-202, compared to findings with neo-SR-3Y1-200. The invasiveness, assessed by penetration through a Matrigel-coated filter was about 5 times higher in fos-SR-3Y1-202 than in neo-SR-3Y1-200; high invasiveness in vitro was also observed in a fos-transferred mixed-population cell line (fos-SR-3Y1-200) and fos-transferred highly metastatic clones. Histopathological evidence of an in vivo tumor also showed the high invasiveness of fos-SR-3Y1-202 cells. To elucidate the causes of the increased invasiveness of fos-SR-3Y1-202, attachment of the cells to Matrigel and its components, such as laminin and type IV collagen, type IV collagenase activity, and motility were examined. Attachment of the cells to the substrate coated on Petri dishes or the activity of type IV collagenase did not differ significantly. On the other hand, cell motility, determined by a new method to directly quantitate alteration of cell shape continuously, using video image analysis and computer techniques, was higher in fos-SR-3Y1-202 than in neo-SR-3Y1-200. Thus, the fos-transferred cell line, fos-SR-3Y1-202 has a high invasiveness, in association with augmentation of motility, hence the enhancement of metastatic potential.

A conserved noncatalytic domain SH2 (for src homology region 2) is located immediately N terminal to the kinase domains of all cytoplasmic protein-tyrosine kinases. We found that the wild-type v-fps SH2 domain stimulated the enzymatic activity of the adjacent kinase domain 10-fold and functioned as a powerful positive effector of catalytic and transforming activities within the v-fps oncoprotein (P130gag-fps). Partial proteolysis of P130gag-fps and supporting genetic data indicated that the v-fps SH2 domain exerts its effect on catalytic activity through an intramolecular interaction with the kinase domain. Amino acid alterations in the SH2 domain that impaired kinase function interfered with association of the SH2 domain with the kinase domain. Deletion of a conserved octapeptide motif converted the v-fps SH2 domain from an activator to an inhibitor of tyrosine kinase activity. This latent inhibitory activity of v-fps SH2 has functional implications for phospholipase C-gamma and p21ras GTPase-activating protein, both of which have two distinct SH2 domains suggestive of complex regulation. In addition to regulating the specific activity of the kinase domain, the SH2 domain of P130gag-fps was also found to be required for the tyrosine phosphorylation of specific cellular proteins, notably polypeptides of 124 and 62 kilodaltons. The SH2 domain therefore appears to play a dual role in regulation of kinase activity and recognition of cellular substrates.