Tyrosine-protein kinase that acts downstream of cell surface receptors and plays a role in the regulation of the actin cytoskeleton, microtubule assembly, cell attachment and cell spreading. Plays a role in FCER1 (high affinity immunoglobulin epsilon receptor)-mediated signaling in mast cells. Acts down-stream of the activated FCER1 receptor and the mast/stem cell growth factor receptor KIT. Plays a role in the regulation of mast cell degranulation. Plays a role in the regulation of cell differentiation and promotes neurite outgrowth in response to NGF signaling. Plays a role in cell scattering and cell migration in response to HGF-induced activation of EZR. Phosphorylates BCR and down-regulates BCR kinase activity. Phosphorylates HCLS1/HS1, PECAM1, STAT3 and TRIM28.
c-Fes is a proto-oncogene encoded non-receptor protein-tyrosine kinase (PTK). However, genetic studies have indicated that it has anti-tumorigenic effects in certain cancers. The pathological and clinical significance of c-Fes in prostate cancer are unknown.
KIT is a tyrosine kinase receptor that is aberrantly activated in several neoplasms. In human pathologies, the most frequent mutation of KIT occurs at codon 816. The resulting KIT mutant protein is activated in the absence of ligand and is resistant to the clinically available inhibitors of KIT. In this report, we provide evidence for an essential function of the cytoplasmic tyrosine kinase FES downstream of KIT(D816V). FES is phosphorylated on tyrosine residues in cells that carry KIT(D816V) mutation, and this phosphorylation is KIT dependent. Reduction of FES expression using RNA interference results in decreased cell proliferation in human or murine cells harboring KIT(D816V) or the homologous mouse mutation KIT(D814Y). The reduced cell growth can be rescued using another cytokine (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and is not observed when the closely related fer gene is targeted. Finally, signaling downstream of KIT(D816V) is altered in cells lacking FES expression. This study shows a major function of FES downstream of activated KIT receptor and thereby points to FES as a novel target in KIT-related pathologies.
The human c-fes locus encodes a non-receptor protein-tyrosine kinase implicated in myeloid, vascular endothelial, and neuronal cell differentiation. A recent analysis of the tyrosine kinome in colorectal cancer identified c-fes as one of only seven genes with consistent kinase domain mutations. Although four mutations were identified (M704V, R706Q, V743M, S759F), the consequences of these mutations on Fes kinase activity were not explored. To address this issue, Fes mutants with these substitutions were co-expressed with STAT3 in human 293T cells. Surprisingly, the M704V, R706Q, and V743M mutations substantially reduced Fes autophosphorylation and STAT3 Tyr-705 phosphorylation compared with wild-type Fes, whereas S759F had little effect. These mutations had a similar impact on Fes kinase activity in a yeast expression system, suggesting that they inhibit Fes by affecting kinase domain structure. We have also demonstrated for the first time that endogenous Fes is strongly expressed at the base of colonic crypts where it co-localizes with epithelial cells positive for the progenitor cell marker Musashi-1. In contrast to normal colonic epithelium, Fes expression was reduced or absent in colon tumor sections from most individuals. Fes protein levels were also low or absent in a panel of human colorectal cancer cell lines, including HT-29 and HCT 116 cells. Introduction of Fes into these lines with a recombinant retrovirus suppressed their growth in soft agar. Together, our findings strongly implicate the c-Fes protein-tyrosine kinase as a tumor suppressor rather than a dominant oncogene in colorectal cancer.
This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.
The FES locus encodes a unique nonreceptor protein-tyrosine kinase (FES) traditionally viewed as a proto-oncogene but more recently implicated as a tumor suppressor in colorectal cancer (CRC). Recent studies have demonstrated that while FES is expressed in normal colonic epithelium, expression is lost in tumor tissue and colorectal cancer cell lines, a finding common among tumor suppressors. Here we provide compelling evidence that promoter methylation is an important mechanism responsible for downregulation of FES gene expression in colorectal cancer cells. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in the expression of functional FES transcripts in all CRC cell lines examined, including Caco-2, COLO 320, DLD-1, HCT 116, SNU-1040, SW-480, and HT-29. Bisulfite sequencing of genomic DNA isolated from 5-aza-2'-deoxycytidine-treated HT-29 cells identified methylated CpG dinucleotides immediately upstream from the FES transcription initiation sites. In contrast, this region of the FES promoter was hypomethylated in genomic DNA from normal colonic epithelium. In addition, methylation completely blocked the activity of the FES promoter in reporter gene assays. Promoter methylation is a previously unrecognized mechanism by which FES expression is suppressed in CRC cell lines, and is consistent with a tumor suppressor role for FES in this tumor site despite its tyrosine kinase activity.
The closely related non-receptor tyrosine kinases FEline Sarcoma (FES) and FEs Related (FER) are activated by cell surface receptors in hematopoietic cells. Despite the early description of oncogenic viral forms of fes, v-fes, and v-fps, the implication of FES and FER in human pathology is not known. We have recently shown that FES but not FER is necessary for oncogenic KIT receptor signaling. Here, we report that both FES and FER kinases are activated in primary acute myeloid leukemia (AML) blasts and in AML cell lines. FES and FER activation is dependent on FLT3 in cell lines harboring constitutively active FLT3 mutants. Moreover, both FES and FER proteins are critical for FLT3-internal tandem duplication (ITD) signaling and for cell proliferation in relevant AML cell lines. FER is required for cell cycle transitions, whereas FES seems necessary for cell survival. We concluded that FES and FER kinases mediate essential non-redundant functions downstream of FLT3-ITD.
The c-Fes protein-tyrosine kinase is associated with growth and differentiation of hematopoietic, neuronal, vascular endothelial and epithelial cell types. In this study, we investigated whether small interfering RNA (siRNA)-mediated knockdown of c-Fes expression affected proliferation of the human renal carcinoma cell lines, ACHN and VMRC-RCW. Immunofluorescence microscopy showed that c-Fes was expressed in both the cytosol and nuclei of these cells, and siRNA treatment preferentially downregulated c-Fes expression in the cytosol. Knock-down of c-Fes inhibited cellular proliferation in a dose-dependent manner with minimal increase in cell death. c-Fes siRNA treatment also downregulated the phosphorylation of Akt1 on S473 and IKKalpha on T23, and cyclin D1 expression, enhanced the expression of IkappaBalpha, and prevented the nuclear localization of NFkappaB. Treatment with an NFkappaB inhibitory peptide (SN50) also blocked the proliferation and nuclear localization of NFkappaB in these cells. The effect of SN50 treatment was not enhanced by c-Fes siRNA, suggesting that downregulation of c-Fes expression inhibited cell cycle progression through the Akt1/NFkappaB pathway. In contrast to siRNA-mediated knockdown, ectopic expression of either wild-type or kinase-inactive c-Fes in renal carcinoma cells failed to alter their proliferation in vitro and in vivo. Thus, suppression of proliferation resulting from siRNA-mediated knockdown may depend upon an expression of c-Fes protein rather than its kinase activity. Taken together, our results indicate that downregulation of c-Fes expression may be a potential therapeutic strategy for advanced human renal cell carcinoma and inhibition of its kinase activity as an antiangiogenic therapy does not seem to induce the growth of human renal carcinoma cells.
J. Biol. Chem. 271, 32930-32936 (1996)[PubMed:8955135]
The human BCR gene encodes a protein with serine/threonine kinase activity and regulatory domains for the small G-proteins RAC and CDC42. Previous work in our laboratory has established that BCR is a substrate for c-FES, a non-receptor tyrosine kinase linked to myeloid growth and differentiation. Tyrosine phosphorylation led to the association of BCR with the RAS guanine nucleotide exchange complex GRB2-SOS in vivo via the GRB2 SH2 domain, linking BCR to RAS signaling (Maru, Y., Peters, K. L., Afar, D. E. H., Shibuya, M., Witte, O. N., and Smithgall, T. E. (1995) Mol. Cell. Biol. 15, 835-842). In the present study, we demonstrate that BCR Tyr-246 and at least one of the closely spaced tyrosine residues, Tyr-279, Tyr-283, and Tyr-289 (3Y cluster), are phosphorylated by FES both in vitro and in 32Pi-labeled cells. Mutagenesis of BCR Tyr-177 to Phe completely abolished FES-induced BCR binding to the GRB2 SH2 domain, identifying Tyr-177 as an additional phosphorylation site for FES. Co-expression of BCR and FES in human 293T cells stimulated the tyrosine autophosphorylation of FES. By contrast, tyrosine phosphorylation of BCR by FES suppressed BCR serine/threonine kinase activity toward the 14-3-3 protein and BCR substrate, BAP-1. These data show that tyrosine phosphorylation by FES affects the interaction of BCR with multiple signaling partners and suggest a general role for BCR in non-receptor protein-tyrosine kinase regulation and signal transduction.
The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.
J. Biol. Chem. 264, 10276-10281 (1989)[PubMed:2656706]
Expression of the proto-oncogene p93c-fes and its associated tyrosine kinase activity is marked in mature granulocytes, monocytes, differentiated HL-60 leukemia cells, and leukemia cell lines KG-1, THP-1, HEL, and U-937, which can be induced to differentiate along the granulocyte/monocyte pathway. Conversely, p93-c-fes expression is absent in the K562 cell line, which is resistant to myeloid differentiation. Upon transfection and clonal selection of K562 cells using a mammalian expression vector containing the 13-kilobase pair c-fes gene, c-fes mRNA was transcribed and p93-c-fes tyrosine activity kinase was expressed. Clones expressing c-fes underwent myeloid differentiation as assessed by the appearance of phagocytic activity, Fc receptors, nitro blue tetrazolium reduction, Mac-1 immunofluorescence, and lysozyme production. These results indicate that the expression of the c-fes protooncogene and its associated tyrosine kinase activity plays a major role in the initiation of myeloid differentiation.
The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane-cytoskeleton linker ezrin plays a major role in HGF-induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF-mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell-cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell-cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell-cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion.
The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.
The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.
This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.
The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.
Interacting selectively and non-covalently with any inositol-containing glycerophospholipid, i.e. phosphatidylinositol (PtdIns) and its phosphorylated derivatives.
This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.
The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.
Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.
The biological process whose specific outcome is the progression of a multicellular organism over time from an initial condition (e.g. a zygote or a young adult) to a later condition (e.g. a multicellular animal or an aged adult).
Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.
The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.
The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.
The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.
The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.
J. Biol. Chem. 264, 10276-10281 (1989)[PubMed:2656706]
Expression of the proto-oncogene p93c-fes and its associated tyrosine kinase activity is marked in mature granulocytes, monocytes, differentiated HL-60 leukemia cells, and leukemia cell lines KG-1, THP-1, HEL, and U-937, which can be induced to differentiate along the granulocyte/monocyte pathway. Conversely, p93-c-fes expression is absent in the K562 cell line, which is resistant to myeloid differentiation. Upon transfection and clonal selection of K562 cells using a mammalian expression vector containing the 13-kilobase pair c-fes gene, c-fes mRNA was transcribed and p93-c-fes tyrosine activity kinase was expressed. Clones expressing c-fes underwent myeloid differentiation as assessed by the appearance of phagocytic activity, Fc receptors, nitro blue tetrazolium reduction, Mac-1 immunofluorescence, and lysozyme production. These results indicate that the expression of the c-fes protooncogene and its associated tyrosine kinase activity plays a major role in the initiation of myeloid differentiation.
Any process that increases the rate, frequency or extent of neuron projection development. Neuron projection development is the process whose specific outcome is the progression of a neuron projection over time, from its formation to the mature structure. A neuron projection is any process extending from a neural cell, such as axons or dendrites (collectively called neurites).
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.
The c-fes proto-oncogene product is expressed predominantly in hematopoietic cells of the myeloid lineage and has been implicated in the regulation of myeloid differentiation. The c-fes locus encodes a 93-kDa protein tyrosine kinase (p93c-fes) that possesses several structural features characteristic of the cytoplasmic class of protein tyrosine kinases, including a consensus sequence for autophosphorylation surrounding Tyr-713 and a src homology 2 (SH2) domain. To assess the effect of each of these potential regulatory sites on p93c-fes protein tyrosine kinase activity, we specifically deleted the c-fes SH2 domain using the polymerase chain reaction and replaced Tyr-713 with phenylalanine by oligonucleotide-directed mutagenesis (Y713F mutant). The resulting mutants were expressed in Escherichia coli and assayed for changes in protein tyrosine kinase activity using an immune complex kinase assay. Both mutations produced a marked decrease in the rate and extent of autophosphorylation and phosphorylation of the model substrate, enolase. To test whether the c-fes SH2 domain could interact with the autophosphorylated kinase domain, the SH2 domain was expressed as a fusion protein with glutathione S-transferase and immobilized on glutathione-agarose. The recombinant c-fes SH2 domain precipitated p93c-fes as readily as a monoclonal antibody. Binding of the SH2 domain to p93c-fes was completely dependent upon autophosphorylation, as a kinase-defective mutant of p93c-fes was not precipitated by the SH2 domain. High-affinity binding was also observed with recombinant SH2 domains from v-src and v-fps, raising the possibility of protein-protein interactions between various members of the cytoplasmic PTK family. These results indicate that the c-fes SH2 domain and consensus autophosphorylation site (Tyr-713) play major roles in the positive regulation of p93c-fes tyrosine kinase activity, possibly through intramolecular interaction.
The nucleotide sequences encoding the transforming polyproteins of the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV) have been determined. These sequences include a viral transforming gene (v-fes), derived from cellular proto-oncogene sequences (c-fes) of domestic cats by recombination with feline leukemia virus (FeLV). The v-fes sequences are predicted to encode a polypeptide domain strikingly similar to that specified by the transforming gene (v-fps) of the avian Fujinami sarcoma virus. In addition, the 3' 0.8 kilobase pairs of v-fes encode amino acid sequences homologous to the carboxy-terminal portion of pp60src, the transforming protein encoded by the avian Rous sarcoma virus src gene. Thus different feline and avian retroviral transforming genes, all of which encode functionally related proteins with associated tyrosine-specific kinase activities, must be derived from divergent members of the same proto-oncogene family.
The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.
Any process that modulates the frequency, rate or extent of cell differentiation, the process in which relatively unspecialized cells acquire specialized structural and functional features.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.
The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane-cytoskeleton linker ezrin plays a major role in HGF-induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF-mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell-cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell-cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell-cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion.
The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.
The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane-cytoskeleton linker ezrin plays a major role in HGF-induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF-mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell-cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell-cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell-cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion.
Evidence
2:
Inferred from Mutant PhenotypeUniProtKB
The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.
This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.
Any process that modulates the rate, frequency, or extent of vesicle-mediated transport, the directed movement of substances, either within a vesicle or in the vesicle membrane, into, out of or within a cell.
The subcellular localizations of the Fps/Fes and closely related Fer cytoplasmic tyrosine kinases were studied using green fluorescent protein (GFP) fusions and confocal fluorescence microscopy. In contrast to previous reports, neither kinase localized to the nucleus. Fer was diffusely cytoplasmic throughout the cell cycle. Fps/Fes also displayed a diffuse cytoplasmic localization, but in addition it showed distinct accumulations in cytoplasmic vesicles as well as in a perinuclear region consistent with the Golgi. This localization was very similar to that of TGN38, a known marker of the trans Golgi. The localization of Fps/Fes and TGN38 were both perturbed by brefeldin A, a fungal metabolite that disrupts the Golgi apparatus. Fps/Fes was also found to colocalize to various extents with several Rab proteins, which are members of the monomeric G-protein superfamily involved in vesicular transport between specific subcellular compartments. Using Rabs that are involved in endocytosis (Rab5B and Rab7) or exocytosis (Rab1A and Rab3A), we showed that Fps/Fes is localized in both pathways. These results suggest that Fps/Fes may play a general role in the regulation of vesicular trafficking.
The c-fes proto-oncogene product is expressed predominantly in hematopoietic cells of the myeloid lineage and has been implicated in the regulation of myeloid differentiation. The c-fes locus encodes a 93-kDa protein tyrosine kinase (p93c-fes) that possesses several structural features characteristic of the cytoplasmic class of protein tyrosine kinases, including a consensus sequence for autophosphorylation surrounding Tyr-713 and a src homology 2 (SH2) domain. To assess the effect of each of these potential regulatory sites on p93c-fes protein tyrosine kinase activity, we specifically deleted the c-fes SH2 domain using the polymerase chain reaction and replaced Tyr-713 with phenylalanine by oligonucleotide-directed mutagenesis (Y713F mutant). The resulting mutants were expressed in Escherichia coli and assayed for changes in protein tyrosine kinase activity using an immune complex kinase assay. Both mutations produced a marked decrease in the rate and extent of autophosphorylation and phosphorylation of the model substrate, enolase. To test whether the c-fes SH2 domain could interact with the autophosphorylated kinase domain, the SH2 domain was expressed as a fusion protein with glutathione S-transferase and immobilized on glutathione-agarose. The recombinant c-fes SH2 domain precipitated p93c-fes as readily as a monoclonal antibody. Binding of the SH2 domain to p93c-fes was completely dependent upon autophosphorylation, as a kinase-defective mutant of p93c-fes was not precipitated by the SH2 domain. High-affinity binding was also observed with recombinant SH2 domains from v-src and v-fps, raising the possibility of protein-protein interactions between various members of the cytoplasmic PTK family. These results indicate that the c-fes SH2 domain and consensus autophosphorylation site (Tyr-713) play major roles in the positive regulation of p93c-fes tyrosine kinase activity, possibly through intramolecular interaction.
J. Biol. Chem. 264, 10276-10281 (1989)[PubMed:2656706]
Expression of the proto-oncogene p93c-fes and its associated tyrosine kinase activity is marked in mature granulocytes, monocytes, differentiated HL-60 leukemia cells, and leukemia cell lines KG-1, THP-1, HEL, and U-937, which can be induced to differentiate along the granulocyte/monocyte pathway. Conversely, p93-c-fes expression is absent in the K562 cell line, which is resistant to myeloid differentiation. Upon transfection and clonal selection of K562 cells using a mammalian expression vector containing the 13-kilobase pair c-fes gene, c-fes mRNA was transcribed and p93-c-fes tyrosine activity kinase was expressed. Clones expressing c-fes underwent myeloid differentiation as assessed by the appearance of phagocytic activity, Fc receptors, nitro blue tetrazolium reduction, Mac-1 immunofluorescence, and lysozyme production. These results indicate that the expression of the c-fes protooncogene and its associated tyrosine kinase activity plays a major role in the initiation of myeloid differentiation.
Fps/Fes proteins were among the first members of the protein tyrosine kinase family to be characterized as dominant-acting oncoproteins. Addition of retroviral GAG sequences or other experimentally induced mutations activated the latent transforming potential of Fps/Fes. However, activating mutations in fps/fes had not been found in human tumors until recently, when mutational analysis of a panel of colorectal cancers identified four somatic mutations in sequences encoding the Fps/Fes kinase domain. Here, we report biochemical and theoretical structural analysis demonstrating that three of these mutations result in inactivation, not activation, of Fps/Fes, whereas the fourth mutation compromised in vivo activity. These results did not concur with a classic dominant-acting oncogenic role for fps/fes involving activating somatic mutations but instead raised the possibility that inactivating fps/fes mutations might promote tumor progression in vivo. Consistent with this, we observed that tumor onset in a mouse model of breast epithelial cancer occurred earlier in mice targeted with either null or kinase-inactivating fps/fes mutations. Furthermore, a fps/fes transgene restored normal tumor onset kinetics in targeted fps/fes null mice. These data suggest a novel and unexpected tumor suppressor role for Fps/Fes in epithelial cells.
The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.
The c-fes locus encodes a 93-kDa non-receptor protein tyrosine kinase (Fes) that regulates the growth and differentiation of hematopoietic and vascular endothelial cells. Unique to Fes is a long N-terminal sequence with two regions of strong homology to coiled-coil oligomerization domains. We introduced leucine-to-proline substitutions into the coiled coils that were predicted to disrupt the coiled-coil structure. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. We observed that a point mutation in the first coiled-coil domain (L145P) dramatically increased Fes tyrosine kinase and transforming activities in this cell type. In contrast, a similar point mutation in the second coiled-coil motif (L334P) was without effect. However, combining the L334P and L145P mutations reduced transforming and kinase activities by approximately 50% relative to the levels of activity produced with the L145P mutation alone. To study the effects of the coiled-coil mutations in a biologically relevant context, we expressed the mutant proteins in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid leukemia cell line TF-1. In this cellular context, the L145P mutation induced GM-CSF independence, cell attachment, and spreading. These effects correlated with a marked increase in L145P protein autophosphorylation relative to that of wild-type Fes. In contrast, the double coiled-coil mutant protein showed greatly reduced kinase and biological activities in TF-1 cells. These data are consistent with a role for the first coiled coil in the negative regulation of kinase activity and a requirement for the second coiled coil in either oligomerization or recruitment of signaling partners. Gel filtration experiments showed that the unique N-terminal region interconverts between monomeric and oligomeric forms. Single point mutations favored oligomerization, while the double point mutant protein eluted essentially as the monomer. These data provide new evidence for coiled-coil-mediated regulation of c-Fes tyrosine kinase activity and signaling, a mechanism unique among tyrosine kinases.
The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.
Kinase activity is tightly regulated. Activated in response to signaling from a cell surface receptor. Activation probably requires binding of a substrate via the SH2 domain, plus autophosphorylation at Tyr-713. Present in an inactive form in the absence of activating stimuli.
The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.
J. Biol. Chem. 271, 32930-32936 (1996)[PubMed:8955135]
The human BCR gene encodes a protein with serine/threonine kinase activity and regulatory domains for the small G-proteins RAC and CDC42. Previous work in our laboratory has established that BCR is a substrate for c-FES, a non-receptor tyrosine kinase linked to myeloid growth and differentiation. Tyrosine phosphorylation led to the association of BCR with the RAS guanine nucleotide exchange complex GRB2-SOS in vivo via the GRB2 SH2 domain, linking BCR to RAS signaling (Maru, Y., Peters, K. L., Afar, D. E. H., Shibuya, M., Witte, O. N., and Smithgall, T. E. (1995) Mol. Cell. Biol. 15, 835-842). In the present study, we demonstrate that BCR Tyr-246 and at least one of the closely spaced tyrosine residues, Tyr-279, Tyr-283, and Tyr-289 (3Y cluster), are phosphorylated by FES both in vitro and in 32Pi-labeled cells. Mutagenesis of BCR Tyr-177 to Phe completely abolished FES-induced BCR binding to the GRB2 SH2 domain, identifying Tyr-177 as an additional phosphorylation site for FES. Co-expression of BCR and FES in human 293T cells stimulated the tyrosine autophosphorylation of FES. By contrast, tyrosine phosphorylation of BCR by FES suppressed BCR serine/threonine kinase activity toward the 14-3-3 protein and BCR substrate, BAP-1. These data show that tyrosine phosphorylation by FES affects the interaction of BCR with multiple signaling partners and suggest a general role for BCR in non-receptor protein-tyrosine kinase regulation and signal transduction.
Cellular homolog of retroviral oncogenes. In constrast to the viral oncoproteins, the kinase activity of cellular FSP/FES is tightly regulated, and the kinase is inactive in normal cells in the absence of activating stimuli (PubMed15485904).
The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.
Enzyme which catalyzes the transfer of the terminal phosphate of ATP to a specific tyrosine residue on its target protein. Many of these kinases play significant roles in development and cell division. Tyrosine-protein kinases can be divided into two subfamilies: receptor tyrosine kinases, which have an intracellular tyrosine kinase domain, a transmembrane domain and an extracellular ligand-binding domain; and non-receptor (cytoplasmic) tyrosine kinases, which are soluble, cytoplasmic kinases.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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