Excess early signaling activity inhibits cellular chemotaxis toward PDGF-BB.

Chemotaxis, directed migration toward a gradient of a soluble substance, requires a cell to spatially distinguish the concentration of a chemoattractant at one end relative to its opposite end. Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant. In the current study, we attempted to interfere with PDGF-BB mediated chemotaxis by abnormal expression of potential early components of the signaling cascade. We find that expression of the PDGF homolog v-Sis prevents cellular migration toward PDGF-BB, indicating that autocrine production of a PDGF receptor ligand will prevent the chemotactic response to exogenously added ligand. In addition, while it is known that PDGF receptor mutants incapable of activating tyrosine kinase activity cannot transduce a signal for mitogenesis or chemotaxis, the effects of excess tyrosine kinase activity on PDGF mediated chemotaxis have not been tested. We demonstrate that cells expressing constitutively active tyrosine kinase genes such as v-fms, v-fes, or v-src fail to migrate toward PDGF-BB whereas expression of the serine/threonine kinase v-mos does not block the chemotactic response. The results demonstrate that chemotaxis may be prevented by excess production of either ligand, receptor activity, or downstream signaling molecule. In addition, our results show that the signals that mediate chemotaxis are separable from those that regulate unstimulated random motility in the same cells.

An enhancer element responsive to ras and fms signaling pathways is composed of two distinct nuclear factor binding sites.

In order to precisely define the sequences that constitute the ras-responsive enhancers element present in the murine retrotransposon NVL3, point mutations were introduced into the previously defined minimal transcriptional enhancer DNA. Analyses of the effects of these point mutations in transient transfection experiments, in gel retention assays, and by methylation interference footprinting indicated that the enhancer element was composed of two binding sites for distinct nuclear factors. Both binding sites were required for activation of the enhancer by either ras or v-fms oncogenes, and the distinct nuclear factors were found in extracts from cells that contained either oncogene. UV cross-linking analysis revealed that the AP1-related binding site, TGACTCT, was recognized by a nuclear factor of apparent molecular size of 50 kilodaltons, that is probably c-jun. The other binding site, CAGGATAT, is very similar to sites recognized by the ets-family of transcription factors, and was recognized by the 120-kilodalton ras-responsive factor-1. Activation of the NVL3 element was reconstituted in an in vitro transcription assay. The ets-related binding site was necessary for this in vitro reconstitution of activity. Thus, the NVL3 enhancer is related to the previously described oncogene-responsive enhancer element present in polyoma virus and is also related to elements identified in several cellular genes known to be ras-responsive, including the transforming growth factor-beta 1 gene.

Induction of the fms proto-oncogene product in HL-60 cells by vitamin D: a flow cytometric analysis.

Agents which induce monocytic characteristics in HL-60 human acute promyelocytic leukemia cells induce mRNA for the fms proto-oncogene, which encodes the receptor for M-CSF. Previous studies of fms expression in HL-60 cells have characterized chiefly induction by phorbol esters of fms mRNA. Our studies of fms expression in HI-60 cells have characterized induction by vitamin D3 of the fms protein. We have used flow cytometry to correlate fms antigen with a monocyte-specific differentiation antigen recognized by antibody MO2 (CD14), with DNA content, and with the nuclear antigen Ki-67, a marker of cell cycling. HL-60 cells were cultured with or without 1 microM vitamin D for 7 days. fms antigen was found on 42 +/- 5.8% of the cells cultured without vitamin D, but on 63 +/- 4.3% of the cells cultured with vitamin D. MO2 binding was detected on only 2 +/- 0.5% of the cells without vitamin D, but on 59 +/- 9% with vitamin D. Cells cultured with vitamin D that were fms-positive were also predominantly (83%) MO2-positive. Analysis of DNA content, measured by propidium iodide staining, showed that 57 +/- 1.5% of cells cultured without vitamin D, but 93 +/- 0.5% of cells cultured with vitamin D, were in the G0/G1 cell cycle phase. Analysis of nuclear antigen Ki-67 revealed that, of the vitamin D-treated cells that were fms-positive, a significant proportion (37%) were still cycling. We conclude that (1) fms is demonstrable on some uninduced HL-60 cells, (2) when HL-60 cells are induced to develop monocytic characteristics by vitamin D, fms induction is part of the program for monocytic differentiation that includes MO2 expression, yet (3) some induced cells expressing fms are still cycling.

Reassessment of the v-fms sequence: threonine phosphorylation of the COOH-terminal domain.

The v-fms oncogene product of the McDonough strain of feline sarcoma virus is a member of the receptor tyrosine kinase family. Its cellular counterpart, the c-fms product, is the receptor for colony-stimulating factor 1 (CSF-1) of macrophages. We have reanalyzed the v-fms gene by direct sequencing of a biologically active clone. An additional A nucleotide was detected in position 2810 of the published v-fms sequence. The frameshift changed the COOH-terminal sequence of the v-fms protein from -R-937-G-P-P-L-COOH to -Q-937-R-T-P-P-V-A-R-COOH. Antibodies against a synthetic peptide representing this new sequence precipitated the v-fms proteins from transformed NRK cells as well as from feline sarcoma virus (McDonough)-infected feline fibroblasts. We show by tryptic peptide mapping that threonine 939 present in the new sequence is phosphorylated by a yet unknown serine/threonine kinase in vivo. In chicken fibroblasts expressing the v-fms gene, this phosphorylation clearly depended on the addition of exogenous CSF-1. Furthermore, addition of CSF-1 appeared to activate the serine/threonine kinase, as judged by phosphorylation of the synthetic peptide QRTPPVAR.

Transforming mechanism of the feline sarcoma virus encoded v-fms oncogene product.

The v-fms oncogene product encoded by the McDonough strain of feline sarcoma virus (SM-FeSV) is a transmembrane glycoprotein which belongs to the tyrosine kinase receptor family. The cellular counterpart, the c-fms product, is the receptor for macrophage colony stimulating factor (M-CSF or CSF-1). The v-fms and the c-fms product differ structurally only in seven point mutations and in their C-terminal domains. We have corrected the published sequence of the v-fms product and found that the new C-terminal end contains a threonine phosphorylation site (Thr939). This site is phosphorylated in vivo leading to an enhancement of the v-fms-specific tyrosine kinase activity. The extracellular domain of the v-fms product contains 11 N-glycosylation sites. Glycosylation and transport of the v-fms molecules to the plasma membrane are prerequisites for the transforming potential of the virus. Phosphorylation of the v-fms molecules in tyrosine, serine and threonine residues takes place only at the plasma membrane. Coexpression showed that the overexpression of M-CSF and c-fms in fibroblasts leads to cell transformation by an autocrine loop mechanism. This interaction between M-CSF and the c-fms protein also takes place at the plasma membrane. To study the v-fms transforming mechanisms, we have expressed the v-fms oncogene in chicken fibroblasts which are free of the cross-reactive M-CSF. The expression of the v-fms oncogene alone did not cause transformation. However, upon addition of M-CSF, these cells became completely transformed.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular structures of CPF-dipeptides, inhibitors for the binding of CD4 with gp120.

The dipeptides N-carbomethoxycarbonyl-prolyl-phenylalanyl-benzyl esters (CPFs) play a significant role in the prevention of HIV-1 virus infection by interacting with the glycoprotein gp120, one of the envelope proteins of the HIV-1 virus. Of the four possible isomers of CPF, (L-pro-L-phe), (L-pro-D-phe), (D-pro-L-phe) and (D-pro-D-phe), herein denoted LL etc., the crystal structures of LL, stereoisomeric LD and the racemic mixture of LD/DL have been determined. All three peptides crystallize in the orthorhombic system and they all have similar cell dimensions: (i) LL, P2(1)2(1)2(1), a = 13.699(2), b = 25.893(5), c = 6.155(1) A, Z = 4, Dcale = 1.333 g cm-3, R = 0.070 for 1247 observed reflections; (ii) LD, P2(1)2(1)2(1), a = 11.663(2), b = 26.557(2), c = 7.281(1) A, Z = 4, Dcalc = 1.290 g cm-3, R = 0.054 for 1918 observed reflections; (iii) LD/DL, Pbc2(1), a = 11.953(2), b = 24.208(8), c = 7.782(2) A, Z = 4, Dcalc = 1.292 g cm-3, R = 0.080 for 894 observed reflections. Both the enantiomeric LD and the LD in racemic LD/DL have a similar conformation, an extended peptide chain with phi 1 = -76, -73 degrees; psi 1 = 160, 158 degrees, psi 2 = 123, 131 degrees and psi 2 = -172, -167 degrees, while peptide LL adopts a bent conformation at the Phe residue, phi 1 = -69 degrees, psi 1 = 158 degrees, phi 2 = -60 degrees and psi 2 = -34 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine phosphorylation of the juxtamembrane domain of the v-Fms oncogene product is required for its association with a 55-kDa protein.

Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high affinity binding sites for cellular proteins with Src homology 2 (SH2) domains that are involved in various signal cascades. Tryptic digestion of the autophosphorylated v-Fms and of its cellular counterpart, the feline c-Fms polypeptide, gave rise to at least six common major phosphopeptides, four of which have been characterized previously. Employing site-directed mutagenesis and phosphopeptide mapping of in vitro phosphorylated glutathione S-transferase v-Fms fusion proteins as well as full-length v-Fms molecules expressed in various cells, we show here that Tyr543 of the juxtamembrane domain and Tyr696 of the kinase insert domain constitute major autophosphorylation sites. Recombinant fusion proteins containing the tyrosine-phosphorylated kinase insert domain bind the growth factor receptor bound protein 2 and the p85 and p110 subunits of phosphatidylinositol 3'-kinase. In contrast, fusion proteins containing the juxtamembrane domain phosphorylated on Tyr543 fail to bind any of the known SH2 domain-containing cellular proteins but associate specifically with an as yet undefined 55-kDa cellular protein that by itself is phosphorylated on tyrosine.

cDNA microarray analysis of invasive and tumorigenic phenotypes in a breast cancer model.

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets.

Tyrosine 807 of the v-Fms oncogene product controls cell morphology and association with p120RasGAP.

Expression of the v-fms oncogene of feline sarcoma virus in fibroblasts causes surface exposure of an activated receptor tyrosine kinase, v-Fms, that is autophosphorylated at multiple sites within its cytoplasmic domain. Cellular proteins interacting with this part of v-Fms modulate the mitogenic activity and morphology of the cells. We show here that the tyrosine residue in position 807 (Y-807) of the v-Fms molecule constitutes a major autophosphorylation site. The replacement of this residue by phenylalanine (Y807F mutation) allowed us to functionally dissect v-Fms-specific mitogenic and morphogenic cascades. Cells expressing the mutant v-Fms molecule resembled wild-type (wt) v-Fms-transformed (wt-v-Fms) cells in terms of [3H]thymidine uptake rates and activation of the Ras/Raf-1 mitogenic cascade. Such cells showed, however, a flat morphology and contained intact actin cables and fibronectin network. Our studies indicate that the v-Fms molecule controls cell morphology by a cascade that involves a direct interaction with p120RasGAP and p190RhoGAP: (i) in contrast to wt v-Fms molecules, the Y807F v-Fms protein failed to associate with and phosphorylate p120RasGAP; (ii) tight complexes between p120RasGAP and p190RhoGAP as well as detectable RhoGAP activity were present exclusively in wt-v-Fms cells; and (iii) p190RhoGAP was dispersed throughout the cytoplasm of wt-v-Fms cells, whereas its distribution was restricted to perinuclear regions of cells expressing the mutant v-Fms gene.

c-Cbl associates directly with the C-terminal tail of the receptor for the macrophage colony-stimulating factor, c-Fms, and down-modulates this receptor but not the viral oncogene v-Fms.

The receptor for the macrophage colony-stimulating factor (CSF-1, also termed M-CSF), the tyrosine kinase c-Fms, was originally determined to be the oncogene product of the McDonough strain of feline sarcoma virus, v-Fms. The structural difference between c-Fms and v-Fms amounts to only five point mutations in the extracellular domain, two mutations in the cytoplasmic domain, and the replacement of 50 amino acids by 14 unrelated amino acids at the C-terminal tail. Here, we have identified c-Cbl as the direct binding partner for c-Fms. c-Cbl binds to phosphotyrosine residue 977 at the C-terminal end of feline c-Fms, which is absent in v-Fms. The replacement of the C-terminal end of v-Fms by the corresponding part of c-Fms (vc-Fms) restored the binding potential. As a result, vc-Fms reduced the transforming potency of v-Fms. The overexpression of Cbl did not influence the v-Fms-transformed phenotype, although c-Cbl forms a complex with v-Fms indirectly. In contrast, the expression of Cbl drastically reduced the vc-Fms-transformed phenotype and the activation of Erk and enhanced Fms ubiquitination via phosphotyrosine residue 977. Furthermore, the replacement of tyrosine 977 into phenylalanine in feline c-Fms and vc-Fms reduced the Cbl-dependent ubiquitination. These data suggest that an indirect association of c-Cbl via multimeric complex induced a different signaling pathway from the pathway induced by c-Cbl direct interaction.

Epidermal growth factor (EGF) modulation of feline sarcoma virus fms tyrosine kinase activity, internalization, degradation, and transforming potential in an EGF receptor/v-fms chimera.

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.