Functional specificity of cytoplasmic and transmembrane tyrosine kinases: identification of 130- and 75-kilodalton substrates of c-fps/fes tyrosine kinase in macrophages.

c-fps/fes encodes a 92-kDa protein-tyrosine kinase (NCP92) that is expressed at the highest levels in macrophages. To determine if c-fps/fes can mediate the action of the colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) and to identify potential targets of c-fps/fes in macrophages, we have overexpressed c-fps/fes in a CSF-1-dependent macrophage cell line. A 30- to 50-fold overexpression of c-fps/fes partially released these cells from their factor dependence by a nonautocrine mechanism, and this correlated with the tyrosine phosphorylation of two proteins of 130 and 75 kDa (P130 and P75). c-fps/fes did not cause tyrosine phosphorylation or activation of CSF-1 dependent targets, including CSF-1R, Shc, and phosphatidylinositol 3-kinase, and conversely, CSF-1 did not induce tyrosine phosphorylation of P130 and P75. P75 appears to be a novel phosphotyrosyl protein, whereas P130 cross-reacts with a known substrate of v-src. P130 and P75 may be direct substrates of c-fps/fes: P130 was tightly associated with NCP92, and the src homology 2 domain of NCP92 specifically bound phosphorylated P130 and P75 but not the CSF-1-induced phosphotyrosyl proteins, consistent with the possibility that P130 and P75 are physiological targets of c-fps/fes. We conclude that although c-fps/fes can functionally substitute for CSF-1R to a certain extent, these tyrosine kinases act largely independently of each other and that P130 and P75 are novel targets whose mechanisms of action may be unrelated to the signalling pathways utilized by receptor tyrosine kinases.     

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

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

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

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

The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.     

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.     

Characterization of a plasmid carrying the Escherichia coli K-12 entD, fepA, fes, and entF genes

Abstract The plasmid pMS101 carries Escherichia coli K-12 genes ( entD, fes, entF ) essential for enterochelin-mediated iron transport [Laird, A.J. and Young, I.G., Gene 11 (1980) 359–366]. We have further characterized pMS101 and shown that it also contains the gene ( fepA ) for the 81 000 Da outer membrane ferrienterochelin receptor. Subcloning experiments in conjunction with complementation and maxicell studies demonstrated the gene order to be entD fepA fes entF . The entF - and fes -encoded polypeptides were found to be 115 000 and 42 000-Da soluble proteins, respectively. Plasmid-borne enterochelin cluster genes were overexpressed in iron-deficient conditions and their products were undetectable under iron-replete conditions.     

Relationship between the transport of iron and the amount of specific colicin Ia membrane receptors in Escherichia coli.

Strains of Escherichia coli K-12 defective in their ability to utilize exogenously supplied iron due to genetic defects in the entF, tonB, fes, or fep gene exhibited elevated levels of the specific outer-membrane receptor for colicin Ia when compared with parental strains. Although entF, fes, and fep strains showed a higher degree of Ia sensitivity than did the parental strains, tonB strains were resistant to colicin action. The colicin insensitivity of tonB strains was not due to hyperproduction of enterochelin. Growth in medium containing 101.8 muM Fe2+ led to a lowering of receptor levels in all the above strains and resulted in decreased colicin Ia sensitivity in all strains except tonB, which was already at maximal resistance. Growth in citrate plus iron (1.8 muM) or in ferrichrome resulted in a substantial reduction in both receptor levels and Ia sensitivity in ent, fes, and fep strains but had no effect on receptor levels in tonB strains. Growth in citrate did not lead to an alteration in receptor levels in a mutant specifically defective in citrate-mediated iron transport. The presence of enterochelin during growth led to a reduction in the number of receptors in the parental and ent strains but not in tonB, fes, or fep strains. Thus, in all cases examined, there was an inverse relationship between the number of colicin receptors per cell and the ability of the strain to take up iron from the growth medium. This suggests that under conditions of iron limitation there is a derepression of colicin Ia receptor biosynthesis. These results may point to a role of the colicin I receptor in iron uptake.     

Purification and characterization of p93fes- and p60src-related tyrosine protein kinase activities in differentiated HL-60 leukemia cells

Two tyrosine protein kinase activities have been identified previously to be present in HL-60 leukemia cells during induction of granulocytic and monocytic differentiation with a variety of differentiating agents. We have copurified a membrane-associated tyrosine kinase (p93) and an activity associated with both the cytosol and membrane fractions (p60). Triton X-100 extracts from HL-60 cells treated with dimethyl sulfoxide were subjected to tyrosine-agarose chromatography, polypropyl aspartamide high performance liquid chromatography (HPLC), and HPLC using an antiphosphotyrosine IgG-derivatized column. Overall purification was 2700-fold for p93 and 1800-fold for p60. p60 and p93 are phosphorylated exclusively on tyrosine residues and can use poly(Glu,Tyr)4:1, histone H1 and vasoactive intestinal peptide as substrates. Poly(Glu,Tyr)1:1 and poly(Glu,Ala,Tyr)6:3:1 were less effective substrates for p60 and p93. The activity of p93 was dependent on Mg2+ or Mn2+, whereas p60 was dependent on Mg2+; however, the activity of p60 was stimulated in a synergistic manner by the presence of both Mg2+ and Mn2+, whereas the activity of p93 was not enhanced further by the combination of divalent ions. Both p60 and p93 were immunoprecipitated by an anti-v-src monoclonal antibody but only p93 was immunoprecipitated by an anti-v-fps/fes antibody. V8 protease digestion of p60 revealed one major proteolytic fragment containing phosphotyrosine, whereas V8 protease digestion of p93 produced two major peptides that were phosphorylated on tyrosine residues. These results suggest that, although p93 and p60 may possess some epitopic similarities, they have distinguishing phosphorylation sites. Moreover, p93, in contrast to p60, appears to be strictly associated with granulocytic/monocytic differentiation and related to the cellular fps/fes protooncogene.     

Targeted Disruption of the Murine fps/fes Proto-Oncogene Reveals that Fps/Fes Kinase Activity Is Dispensable for Hematopoiesis

The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase that is functionally implicated in the survival and terminal differentiation of myeloid progenitors and in signaling from several members of the cytokine receptor superfamily. To gain further insight into the physiological function of fps/fes, we targeted the mouse locus with a kinase-inactivating missense mutation. Mutant Fps/Fes protein was expressed at normal levels in these mice, but it lacked detectable kinase activity. Homozygous mutant animals were viable and fertile, and they showed no obvious defects. Flow cytometry analysis of bone marrow showed no statistically significant differences in the levels of myeloid, erythroid, or B-cell precursors. Subtle abnormalities observed in mutant mice included slightly elevated total leukocyte counts and splenomegaly. In bone marrow hematopoietic progenitor cell colony-forming assays, mutant mice gave slightly elevated numbers and variable sizes of CFU-granulocyte macrophage in response to interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tyrosine phosphorylation of Stat3 and Stat5A in bone marrow-derived macrophages was dramatically reduced in response to GM-CSF but not to IL-3 or IL-6. This suggests a distinct nonredundant role for Fps/Fes in signaling from the GM-CSF receptor that does not extend to the closely related IL-3 receptor. Lipopolysaccharide-induced Erk1/2 activation was also reduced in mutant macrophages. These subtle molecular phenotypes suggest a possible nonredundant role for Fps/Fes in myelopoiesis and immune responses.     

Nucleotide exchange factor for the yeast Hsp70 molecular chaperone Ssa1p

We report on the identification of Fes1p (yBR101cp) as a cytosolic homologue of Sls1p, an endoplasmic reticulum (ER) protein previously shown to act as a nucleotide exchange factor for yeast BiP (M. Kabani, J.-M. Beckerich, and C. Gaillardin, Mol. Cell. Biol. 20:6923-6934, 2000). We found that Fes1p associates preferentially to the ADP-bound form of the cytosolic Hsp70 molecular chaperone Ssa1p and promotes nucleotide release. Fes1p activity was shown to be compartment and species specific since Sls1p and Escherichia coli GrpE could not substitute for Fes1p. Surprisingly, whereas Sls1p stimulated the ATPase activity of BiP in cooperation with luminal J proteins, Fes1p was shown to inhibit the Ydj1p-mediated activation of Ssa1p ATPase activity in steady-state and single-turnover assays. Disruption of FES1 in several wild-type backgrounds conferred a strong thermosensitive phenotype but partially rescued ydj1-151 thermosensitivity. The Delta fes1 strain was proficient for posttranslational protein translocation, as well as for the ER-associated degradation of two substrates. However, the Delta fes1 mutant showed increased cycloheximide sensitivity and a general translational defect, suggesting that Fes1p acts during protein translation, a process in which Ssa1p and Ydj1p are known to be involved. In support of this hypothesis, Fes1p was found to be associated with ribosomes.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

Analysis of cellular expression of gangliosides by gene transfection. II: Rat 3Y1 cells transformed with several DNAs containing oncogenes (fes, fps, ras & src) invariably express sialosylparagloboside

The transfection of several DNAs, containing oncogenes ras, src, fes or fps, into rat 3Y1 cells invariably induced the neosynthesis of sialosylparagloboside (SPG; IV3 alpha NeuAc nLcOse4Cer), with a concomitant decrease in GM3. All these oncogenes are 'extranuclear' type oncogenes, of which the products are expressed in the cytoplasm or on the cell surface membrane. These results are in striking contrast to those on the transfection of 'intranuclear' type oncogenes (ex., adeno E1) into the same 3Y1 cells, where GD3 neosynthesis was specifically brought about by the transfection.