Fps oncogene analog: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - FER ortholog (H. sapiens)
Synonyms - Fps oncogene analog
Cytological map position- 85D13-85D15
Function - kinase
Symbol - FER
FlyBase ID: FBgn0000723
Genetic map position - 3R
Cellular location - cytoplasmic
Fes/Fer non-receptor tyrosine kinases regulate cell adhesion and cytoskeletal reorganisation through the modification of adherens junctions. Unregulated mammalian Fes/Fer kinase activity has been shown to lead to tumours in vivo. Drosophila Fer localises to adherens junctions in the dorsal epidermis and regulates a major morphological event, dorsal closure. Mutations in Src42A cause defects in dorsal closure similar to those seen in dfer mutant embryos. Furthermore, Src42A mutations enhance the dfer mutant phenotype, suggesting that Src42A and DFer act in the same cellular process. DFer is required for the formation of the actin cable in leading edge cells and for normal rates of dorsal closure. A gain-of-function mutation in dfer (dfergof) expresses an N-terminally fused form of the protein, similar to oncogenic forms of vertebrate Fer. dfergof blocks dorsal closure and causes axon misrouting. In dfer loss-of-function mutants ß-catenin (Armadillo) is hypophosphorylated, whereas in dfergof ß-catenin is hyperphosphorylated. Phosphorylated ß-catenin is removed from adherens junctions and degraded, thus implicating DFer in the regulation of adherens junctions (Murray, 2006),
Fes/Fer non-receptor tyrosine kinases regulate numerous cellular processes, including cell adhesion, cytoskeletal reorganisation and intracellular signalling (Greer, 2002). Mammalian Fer has been implicated in the regulation of adherens junctions (AJs) in tissue culture (Kim, 1995; Piedra, 2003; Rosato, 1998; Xu, 2004). Adherens junctions (AJ) are responsible not only for adhesion between neighbouring cells, but also for providing a link between the plasma membrane and the actin cytoskeleton. AJs are rich in phosphotyrosine residues, and several tyrosine kinases and phosphatases are known to regulate their assembly, stability and function (Murray, 2006),
Cell adhesion and cytoskeletal reorganisation are the driving force behind many morphogenetic movements of embryonic development. For example, during dorsal closure in the Drosophila embryo, the epidermal sheets on each side of the embryo extend dorsally to meet and fuse at the dorsal midline, thereby enclosing the amnioserosa and yolk sac. Cell adhesion in the dorsal-most row of cells, the leading edge cells, is regulated in part through the modification of adherens junctions (Murray, 2006),
Intercellular adhesion is mediated by the transmembrane protein E-Cadherin, a homophilic adhesion molecule that binds to E-Cadherin on neighbouring cells. ß-catenin binds to the cytoplasmic tail of E-Cadherin. ß-catenin can also interact with the F-actin binding protein α-catenin. α-catenin acts as a molecular switch regulating F-actin assembly, binding alternately to ß-catenin and to F-actin. p120-catenin (p120ctn) binds to the juxtamembrane domain (JMD) of E-Cadherin, and is thought to act as a regulator of adherens junction assembly and disassembly (Murray, 2006),
Both ß-catenin and p120-catenin are regulated by phosphorylation. Fer binds constitutively to p120-catenin and can phosphorylate it in vitro (Kim, 1995). Phosphorylation of p120-catenin increases its affinity for Cadherin. When overexpressed or activated, Fer can also phosphorylate ß-catenin and thereby disrupt adhesion (Piedra, 2003; Rosato, 1998): phosphorylation on Tyr654 disrupts its association with E-Cadherin; phosphorylation on Tyr142 blocks interaction with α-catenin. In C. elegans, the Fer orthologue associates with ß-catenin in vivo and depends upon ß-catenin for localisation to cell-cell junctions (Putzke, 2005). Fer can also stabilise the cadherin complex by phosphorylating and activating the phosphatase PTP1B, which in turn keeps ß-catenin (Tyr654) dephosphorylated (Xu, 2004). Thus Fer has the capacity to both positively and negatively regulate cadherin complex stability (Murray, 2006),
Several of the putative substrates of Fer are also substrates of the Src family of cytoplasmic tyrosine kinases (SFKs). Both p120ctn and Cortactin have been identified as being substrates of Src. Like Fer, the SFK Fyn binds constitutively to p120-catenin, and can phosphorylate ß-catenin at Tyr142. Src is able to phosphorylate ß-catenin at Tyr654. Given the overlap in substrate specificity, Fer and SFKs may play overlapping or redundant roles in the regulation of cell adhesion and motility. Functional redundancy between different SFKs has been demonstrated in mammals and in insects. In mice, double mutations in SFKs lead to overt phenotypes, where single mutations do not. In Drosophila, members of the Src kinase family cooperate to regulate JUN kinase (JNK) activity: double mutations in Src42A;tec29, and Src42A;Src64 give a dorsal open phenotype, whereas single mutations do not (Murray, 2006),
Only a single member of the Fes/Fer family is found in Drosophila, DFer (Fps85D). The canonical form of DFer, p92dfer, was identified by similarity to other family members (Katzen, 1991), and shares equal homology with Fes and Fer. Subsequently, a second cDNA encoding a short isoform, p45dfer, was discovered (Paulson, 1997). Paulson showed that DFer can transform vertebrate cells, suggesting that the molecular pathways through which Fer signals are likely to be conserved (Murray, 2006),
This study shows that DFer acts in conjunction with Src42A in the process of dorsal closure. dfer mRNA is specifically upregulated in the leading edge cells of the dorsal epidermis. DFer localises to adherens junctions and is required for the formation of the F-actin cable in leading edge cells, and for normal rates of dorsal closure. When mutations in dfer are combined with a mutation in Src42A, dorsal closure fails completely. A gain-of-function dfer mutant (dfergof) blocks dorsal closure and causes axon misrouting. The dfergof mutant expresses an N-terminally fused form of DFer, similar to oncogenic forms of Fer. ß-catenin phosphorylation levels are reduced in dfer loss-of-function mutants and increased in dfergof mutants. This supports a role for DFer in the regulation of AJs and cell-cell adhesion, and may begin to explain its role in oncogenesis (Murray, 2006),
This study shows that DFer localises to adherens junctions during Drosophila embryogenesis, where it regulates leading edge morphology and dorsal closure. In dfer null mutants, P-Tyr staining is reduced at the leading edge, in particular at the actin-nucleating centers. Two potential substrates of DFer, p120ctn and ß-catenin, are localised to adherens junctions. It was found that in dferΔ1 mutants ß-catenin phosphorylation is reduced. Conversely, ß-catenin is more highly phosphorylated in dfergof mutants, demonstrating that the role of Fer in the phosphorylation of ß-catenin is conserved in Drosophila. Interestingly, the overall level of ß-catenin at cell-cell junctions is lower in dfergof mutants, suggesting that phosphorylated ß-catenin is lost from AJs and subsequently degraded (Murray, 2006),
dfer mutants also exhibit a disorganised and reduced F-actin cable at the leading edge. Formation of the F-actin cable appears to depend on adherens junctions, as F-actin nucleation begins at the level of the AJs and the F-actin cable is disrupted in DE-Cadherin mutants. It has been suggested that elevated levels of cytoplasmic α-catenin near stable AJs could favour the formation of F-actin bundles. DFer may contribute to the formation of the F-actin cable by phosphorylating ß-catenin, reducing its affinity for α-catenin, and thereby increasing the local levels of cytoplasmic α-catenin. If DFer promotes stable F-actin bundles then the regulated loss of DFer from the leading edge at stage 14 may enable the more motile Arp2/3 regulated F-actin filopodia to form and complete dorsal closure by 'zipping up' (Murray, 2006),
DFer and Src42A cooperate during dorsal closure. DFer localises to AJs and regulates ß-catenin phosphorylation. In Drosophila, Src42A binds and phosphorylates ß-catenin, although this may not be direct. Consequently, the more severe phenotypes seen in the dfer;Src42A loss-of-function mutants are most likely due to a combined loss of phosphorylation on at least two different tyrosine residues of ß-catenin (Murray, 2006),
dfer mRNA is upregulated in leading edge cells. This, together with reports that vertebrate v-Fps and Fes mediate JNK pathway activation (Li, 1998), suggested that dfer might activate the JNK pathway during dorsal closure. Although DFer itself cannot induce dpp expression, it does play a supporting role in the maintenance of dpp levels, as revealed in the Src42A mutant background. A similar failure in the maintenance of dpp, as opposed to its induction, is seen in mutants of the Wnt pathway. Given the comparable phenotypes, and the fact that phosphorylated ß-catenin is reduced in dfer mutants, it is possible that DFer contributes to the maintenance of dpp via the Wnt pathway (Murray, 2006),
This study isolated a novel, gain-of-function mutation, dfergof, in which a fragment of the White protein is fused to the N terminus of Dfer. This protein, Wex1-DFerRB, is analogous to oncogenic forms of Fps in which part of the viral GAG protein is fused to the N terminus of the endogenous proto-oncogene, generating an activated kinase. Although dfergof mutants express DFer at higher levels, this alone seems unlikely to account for the observed defects, as overexpression of DFerRB gives no obvious embryonic phenotype (Murray, 2006),
In dfergof mutants, the leading edge cells fail to elongate and the F-actin-rich filopodia are greatly reduced. The overall levels of the AJ junction components DE-Cadherin and ß-catenin are reduced, and ß-catenin is hyperphosphorylated. This suggests that AJs are disrupted in dfergof mutants. By contrast, it is interesting that the morphology of amnioserosal cells is shifted to a more motile appearance: F-actin is reduced at the cortex and there is an increase in the number of filopodia, perhaps because of a disruption of cell-cell junctions. In vertebrates, Fer has the capacity to both positively and negatively regulate cadherin-complex stability. This dual function may reflect a difference in binding partners present at AJs in different tissues (Murray, 2006),
Although loss of dfer does not appear to affect axon guidance, dfergof mutants have a clear CNS phenotype in which axons aberrantly cross the midline. A similar phenotype is seen with overexpression of the Abelson tyrosine kinase, which antagonises the receptor Robo. dfergof mutants also disrupt axon guidance in the PNS, with some general misrouting of motor nerves and some overly large synapses. In vertebrates, Fer associates with N-Cadherin in elongating neurites, where it can coordinately regulate N-Cadherin and integrin adhesion (Arregui, 2000). Fer has been shown to be concentrated in growth cones of stage 2 hippocampal neurons and is required for neuronal polarisation and neurite development (Lee, 2005). Similar to the leading edge, DFer may be required at growth cones to regulate filopodial extensions. In chick retinal cells, the phosphatase PTP1B when phosphorylated by Fer, localises to the catenin-binding domain of N-Cadherin (Xu, 2004). Interestingly, the Drosophila homologue of PTP1B, DPTP61F, is expressed in the CNS and binds to the axon guidance molecule Dock (Murray, 2006),
Strikingly, all of the phenotypes associated with dfergof mutants are rescued by expression of the Puckered tyrosine phosphatase. Given that JNK pathway activity appears normal in dfergof mutants, Puckered may target DFer itself, or its substrates, at least one of which is hyperphosphorylated in dfergof mutants. A role for DFer during Drosophila embryonic development in the regulation of AJ stability, in the formation of the contractile leading edge during dorsal closure, and in axon guidance. It cooperates with Src42A to regulate ß-catenin phosphorylation at AJs. A gain-of-function mutant with structural similarity to oncogenic forms of vertebrate Fer was isolated. Unregulated Fer activity leads to oncogenesis, possibly through unregulated epidermal to mesenchymal transition. This study has shown that activated DFer, or loss of DFer together with Src42A, disrupts AJs. This may provide a model for studying oncogenesis in the whole organism (Murray, 2006).
The vertebrate gene FER encodes two protein-tyrosine kinases with molecular weights of 51,000 and 94,000 and distinctive aminotermini. The larger kinase is expressed ubiquitously among vertebrate tissues, whereas expression of the smaller kinase appears to be limited to spermatogenic cells in the testes. Drosophila contains an apparent ortholog of FER (DFer) that also produces two mRNAs by separate initiation of transcription, and two proteins with molecular weights of 45,000 and 92,000. Both proteins are in part loosely associated with cytoplasmic membranes. Both can transform avian and rodent cells with roughly equal potency, when expressed from retroviral vectors. Fusing the myristoylation signal from the SRC protein-tyrosine kinase to the aminoterminus of the DFer protein increases the strength of attachment to membranes but augments transformation only marginally. The results provide the first demonstration of neoplastic transformation by a protein-tyrosine kinase of Drosophila and by FER from any species. The products of Drosophila and vertebrate FER may be part of similar signaling pathways in the two species (Paulson, 1997).
In wild-type embryos, dorsal closure is initiated by activation of the JNK pathway in leading edge cells, resulting in the transcription of two JNK pathway targets, decapentaplegic, a TGF-ß homologue, and puckered, a dual specificity phosphatase. Dpp signals to the neighbouring epidermal cells, causing them to elongate dorsoventrally. Puc inhibits Jun kinase, initiating a negative-feedback loop. Given that both Src and DFer function in dorsal closure, and that Src is an upstream regulator of the JNK pathway, whether DFer also regulates the JNK pathway was tested (Murray, 2006).
Initially assayed was whether the activity of the JNK pathway in leading edge cells is altered in dfer loss-of-function mutants. In dferΔ1 and dferΔex1 mutants, dpp expression levels appear normal. In Src42A mutants, dpp expression is slightly reduced, becoming patchy from stage 13 onwards. In Src42A;dferΔ1 double mutants, dpp expression in the leading edge is reduced further and is almost abolished by stage 13. This suggests that DFer facilitates Src42A-mediated JNK signalling. However, neither DFerRB nor wex1-DFerRB is able to induce ectopic expression of dpp, suggesting that DFer is not itself sufficient to activate the pathway. Furthermore, JNK activation is normal in leading edge cells of dfergof mutants (Murray, 2006).
dfer transcription is upregulated in LE cells, as are dpp and puc. Although dfer transcription occurs at a later stage than that of dpp and puc, whether dfer might nonetheless be a transcriptional target of the JNK pathway was tested. In loss-of-function mutants for the Jun kinase kinase hemipterous, dpp is lost in leading edge cells. dfer, however, is still expressed. When a constitutively active form of Hep, UAS-hepCA, is expressed in the engrailed pattern, dpp is upregulated in the posterior half of each segment, but dfer is not. Therefore, dfer is not a JNK target (Murray, 2006).
Surprisingly, although dfer is not regulated by the JNK pathway, it was found that the dfergof phenotype is rescued by expression of the JNK inhibitor Puckered (Puc). UASpuc,dfergof embryos close at normal rates, resulting in a regular arrangement of epidermal cells. Axon misrouting at the ventral midline is also rescued. Since DFer is unlikely to act through the JNK pathway, Puc may suppress the dfergof phenotype by dephosphorylating either DFergof itself or its downstream targets (Murray, 2006).
DFer localises to adherins junctions (AJs) where it may regulate the phosphorylation state, and hence stability, of AJ proteins. To test whether DFer regulates the phosphorylation of ß-catenin, the extent of ß-catenin tyrosine phosphorylation was determined in dfer mutants. ß-catenin was immunoprecipitated from yw,dferΔ1 and dfergof embryos, and probed with anti-Armadillo (ß-catenin) and anti-phospho-tyrosine. In dferΔ1 embryos, ß-catenin tyrosine phosphorylation is reduced nearly fivefold with respect to control embryos. Conversely, tyrosine phosphorylation is significantly increased in dfergof embryos. Much less ß-catenin is recovered from dfergof embryos, suggesting that hyperphosphorylated ß-catenin is removed from AJs and degraded. This is confirmed by whole-mount staining of dfergof embryos, where the levels of ß-catenin are decreased in the epidermis of stage 13 embryos. DE-Cadherin levels may also be slightly reduced in stage 13 dfergof embryos, but the result is more variable. Western blots on total extracts from late stage dfergof embryos also show a reduction in the levels of ß-catenin (Murray, 2006).
A Drosophila gene (dfps85D) has been identified whose product resembles the proteins encoded by vertebrate fps/fes and the closely related gene fer. dfps85D is located at chromosomal position 85D10-13 and is unlikely to correspond to any previously defined genetic locus in Drosophila. Expression of the gene is entirely zygotic in origin and occurs throughout the life cycle. But hybridization in situ revealed that the pattern of expression is specialized and evolves in a provocative manner. The most notable feature of expression is the diversity of developmental periods, tissues, and cells in which it occurs. In some tissues, expression is transient; in others, it is continuous. Expression occurs in both mitotic and terminally differentiated tissue and, at various times in development, is prominent in imaginal disks, gut, muscle, testes, ovaries, retina, and other neural tissues. It appears that the use of dfps85D is more diversified than that of other Drosophila protein-tyrosine kinases reported to date and contrasts sharply with the restricted expression of fps itself in vertebrates (Katzen, 1991).
In situ hybridization revealed that dfps85D expression is first detectable at several positions in the late cellular blastoderm, including the yolk nuclei (vitellophages), where expression is especially strong and maintained into early gastrulation. Expression was also observed in dorsomedial, dorsolateral, and posterior positions. These regions encompass the anlage for the amnioserosa, dorsal epidermis, and proctodeum. During germ band extension, expression continues in the amnioserosa and the dorsal epidermis and becomes pronounced in the proctodeum. In the fully extended embryo, expression also appears in the ventral ectoderm, clypeolabrum, invaginating stomadeum, and mesoderm. Subsequent to germ band shortening, expression becomes more general but is still not universal. Sites of expression included the clypeolabrum, foregut, visceral mesoderm, somatic mesoderm, ventral epidermis, procephalic lobe, amnioserosa, and the dorsal ridge. Expression is notably absent from most of the developing nervous system, including the supraesophageal ganglia, the subesophageal ganglia, and the majority of lateral cell bodies of the ventral nerve cord. Expression is detected, however, in cells located at the midline of the ventral nervous system (Katzen, 1991).
In the final stages of embryogenesis, expression appears transiently in somatic muscle, pharyngeal muscles, tracheal epithelium, and spiracles and persistently in the frontal sac, esophagus, and proventriculus. In third-instar larvae, expression is detected in all imaginal disks. Transcripts were distributed unevenly within the disks and were especially prominent in the adepithelium. In the eye portion of the eye-antennal discs, expression is weak anterior to the morphogenetic furrow but becomes strong in both the apical and basal levels immediately posterior to the furrow; in more posterior positions, expression is predominantly in the basal portion of the tissue. Expression is also apparent in neural tissue, specifically the cellular cortices of the midbrain and ventral ganglia. Expression levels in the optic lobe are much lower and more discrete. Other tissues expressing dfps85D are the testes, immature blood cells of the lymph glands, and the polyploid epithelial cells of the midgut. There is no detectable expression in the ovaries or in the polyploid tissue of salivary glands, fat body, and larval muscles (Katzen, 1991).
The pattern of expression established in larvae persists into the early pupal stage but is supplemented by the appearance of expression in the tracheal epithelium and abdominal histoblasts and in precursors for visceral muscle. Later in pupal development, expression in most epithelial tissues diminishes appreciably but is prominent in developing skeletal muscle. Expression in muscle varies as development progresses. For example, 48 h into the pupal stage, expression is high in the direct flight muscles and low in the indirect flight muscles. Later, expression in both types of muscle is at the same low level (Katzen, 1991).
dfps85D is expressed in the developing testes throughout pupal development. Midway through the pupal period, expression declined to undetectable levels in the cortex of the midbrain but is strong in regions of the optic lobe. Subsequently, two distinct layers of the optic lamina expressed dfps85D strongly. The more distal hybridization (away from the brain, toward the eye) is located at the base of the lamina cellular cortex and is likely to represent expression from either the L4 or L5 cells. The more proximal expression is in cells located near the base of the lamina neuropile. Their location suggests that they are neuroglial cells. Also expressing dfps85D are a small group of cells of undetermined identity, located at the junction of the medulla, lobula, and lobula plate neuropiles (Katzen, 1991).
Expression in adult flies was especially strong in the retina, more so at the base than at the apex of the tissue, a layering that suggests expression in photoreceptor cells. The widespread expression detected in the thoracic muscles of pupae has now disappeared but is observed instead in the lateral tergosternal muscles of the abdomen. Expression continues in some regions of the testes and appears for the first time in the ovary, localized to the follicular epithelium, particularly at stages 10 to 11 of oogenesis. As in the larval gut, expression is detectable in the epithelium throughout the gut and in specific regions of the proventriculus. Expression is not detected in any portion of the adult brain (Katzen, 1991).
dfer expression is highly dynamic in a wide range of tissues, including the epidermis, central nervous system and developing trachea (Katzen, 1991). In the epidermis, dfer is strongly expressed in the leading edge cells, beginning at late stage 13 and continuing throughout dorsal closure to the end of stage 15. In the CNS, dfer is expressed in the midline glia at stage 13, and later in a segmentally repeated subset of cells and in the dMP2 neurons. To investigate the distribution and localisation of DFer protein, an antibody was raised against an N-terminal fragment of DFer that excludes the SH2 and kinase domains. This fragment overlaps the predicted protein for the intermediate isoform DFerRC by 30 residues. The antibody recognises ectopic expression of DFer-RB using the GAL4/UAS system, and staining is lost in the dferΔex1 mutant, which confirms the specificity of the antibody (Murray, 2006).
DFer protein is ubiquitously expressed at relatively uniform levels throughout embryogenesis. Prior to dorsal closure, during stages 9-10, DFer is predominantly localised to the cytoplasm, although some weak staining is seen around the perimeter of epidermal cells. During stages 11-12, DFer becomes localised to cell-cell junctions in the epidermis. DFer becomes polarised in the leading edge cells as dorsal closure proceeds, as is seen for several other cell-cell junction proteins (e.g. Canoe). Although initially present around the entire circumference of the cell, DFer is lost from the border with the amnioserosa during stages 13-14. DFer protein is also apically enriched in other epithelial sheets such as the gut and trachea, and is expressed in a subset of cells within the CNS (Murray, 2006).
In vertebrates, Fer associates with the adherens junction components p120-catenin and ß-catenin (Kim, 1995; Rosato, 1998). To test whether DFer also localises to adherens junctions, embryos were co-stained for DFer and DE-Cadherin (Drosophila E-Cadherin). In the dorsal epidermis, DFer extensively colocalises with DE-Cadherin, although the distribution is not identical. At the leading edge, DE-Cadherin can be seen around the apical circumference at stage 14, whereas at this stage DFer is lost from the leading edge itself. In the amnioserosa, DFer is only occasionally detected at cell-cell junctions, where again it colocalises with DE-Cadherin. At the leading edge, DFer is apical to the more basal septate junction proteins, Fasciclin 3 and Discs large (Murray, 2006).
Expression of DFer in leading edge cells suggests that it might play a role in dorsal closure. In the GAL4 insertion line MZ465, a P-element has inserted upstream of the first, non-coding, exon of dfer. Mutations in dfer were generated by imprecise excision of the P-element and by male recombination, and screened for loss of DFer protein expression. Nine recombinant lines were generated. In one line, dferΔex1, the deletion removes only the promoter and first exon of the dfer gene. In the other eight lines, a region of over 50 kb is deleted, encompassing the entire dfer locus and the four genes proximal to dfer (CG8129, a threonine dehydratase; CG18473, a phosphotriesterase; CG33936, a zinc finger protein; and CG33937). One of these lines, Df(3R)dferΔ1 (hereafter dferΔ1), was selected for further analysis. Both dferΔ1 and dferΔex1 still express functional GAL4 in patterns similar to that of MZ465. dferΔ1 homozygotes are embryonic lethal in 53% of cases with the remainder dying during larval and pupal development. dferΔex1 homozygotes are both viable and fertile. All results in this study using dferΔex1 are from embryos derived from dferΔex1 homozygous parents (Murray, 2006).
dferΔ1 is an RNA and protein null for all DFer isoforms. In dferΔex1 mutants, dfer mRNA is lost from the CNS and leading edge, but tracheal expression is still evident, as is a low level of ubiquitous staining in the epidermis. DFer protein is not detected in the CNS, but some cell-cell junction staining is faintly visible in the epidermis. The first, non-coding, exon and promoter are deleted in dferΔex1 mutants; however, mRNA transcripts starting at the second exon, which encodes the translational start, are present. Western blots show that full-length DFer protein is produced in dferΔex1 mutants. Thus, dferΔex1 is a hypomorphic mutation in which dfer mRNA expression is lost in a subset of tissues and DFer protein levels in the dorsal epidermis are reduced (Murray, 2006).
During dorsal closure in wild-type embryos, the leading edge and neighbouring cells elongate along the dorsoventral axis. The profile of the leading edge changes from an irregular scalloped to a straightened edge as the actomyosin contractile cable forms. Phosphotyrosine (P-Tyr) levels increase along the leading edge, particularly at the contact points between neighbouring cells or actin nucleating centres (ANC). As closure continues, leading edge cells extend filopodial and lamellipodial processes that zip up the epidermal sheets that meet at the dorsal midline (Murray, 2006).
In dferΔ1 mutants, the actomyosin cable is reduced and the leading edge maintains an irregular profile during closure. P-Tyr levels at the leading edge are also decreased, consistent with the loss of a cytoplasmic tyrosine kinase. These morphological differences are accompanied by a slower rate of closure. In wild-type embryos, dorsal closure is complete by the end of stage 15. dferΔ1 mutant embryos are still open dorsally three hours later, at the end of stage 16. Closure eventually completes, although 2% of cuticles from dferΔ1 mutants exhibit an anterior hole. By contrast, dferΔex1 mutants appear to close normally and exhibit normal leading edge morphology, F-actin and P-Tyr staining (Murray, 2006).
Vertebrate Fes/Fer kinases and members of the Src family kinases share some substrates, such as p120ctn, ß-catenin (Piedra, 2003) and the Arp2/3 activator Cortactin (Kim, 1998; Wu, 1993), and play similar roles in regulating cell-cell adhesion. Src family kinases are functionally redundant in several developmental processes, including dorsal closure: single mutations in Src42A, tec29A and Src64C do not exhibit dorsal holes, whereas double mutants, such as tec29A,Src42A, do. Therefore, whether Src42A and DFer act together in dorsal closure was tested (Murray, 2006).
Src42A single mutants do not exhibit dorsal holes, but show defects in mouthpart formation and epithelial organisation following closure. Src42A embryos also exhibit defects in leading edge cells that are similar to, but less severe than, dferΔ1 mutants: the actomyosin cable is disrupted, P-Tyr staining is weaker than in wild type, and dorsal closure is slightly defective. Eight percent of embryos show a very small dorsal hole at late stage 16 when analysed by confocal microscopy, and the remainder show an irregular arrangement of epidermal cells that is reflected later in the arrangement of dorsal hairs. Embryonic lethality is 63%, with 60% of the unhatched embryos showing malformed mouthparts and a small anterior hole (Murray, 2006).
When the Src42A and dferΔex1 mutants are combined, leading edge cells have a more irregular profile and P-Tyr staining is weaker. When analysed by confocal microscopy, most embryos are still undergoing dorsal closure by late stage 16. Embryonic lethality is 100%, and embryos have breaks and irregularities in the dorsal hair pattern, and a small anterior hole near the mouthparts. In the remaining embryos, dorsal closure fails completely, leaving a large anterior hole. When the dfer deficiency, dferΔ1, and Src42A are combined, these defects are further enhanced. The leading edge becomes highly irregular with a complete loss of the F-actin cable and a substantial reduction in P-Tyr staining. When analysed by confocal microscopy, embryos exhibit a large dorsal hole at late stage 16. Cuticle preparations from these embryos show a large anterior hole (30%) or a small anterior hole (59%), often with small scabs along the dorsal midline. The remaining embryos do not secrete a cuticle (11%). Therefore, when either DFer or Src42A expression is reduced, leading edge cell morphology is compromised and closure is delayed, but when both are removed dorsal closure fails completely (Murray, 2006).
Fes/Fer family non-receptor tyrosine kinases were first identified as the retroviral oncogenes, v-fps and v-fes, from avian and feline sarcomas, respectively (Shibuya, 1980; Snyder, 1969). In v-fes and v-fps, a fragment of the viral GAG protein is fused to the N terminus of the endogenous protein. The N terminus of Fes/Fer is implicated in the regulation of autophosphorylation (Orlovsky, 2000), and N-terminal fusions result in a constitutively active kinase. Activated kinases have also been created by the introduction of an N-terminal myristoylation sequence (Greer, 1994), and by point mutations in the first coiled-coil domain (Cheng, 2001). These are thought to disrupt intramolecular autoregulatory interactions (Murray, 2006).
A third dfer mutant, dfergof, was isolated that behaves as a gain-of-function mutant. Homozygous dfergof mutants are embryonic lethal and exhibit a number of embryonic defects, including a large dorsal hole and an aberrant midline crossing of axons in the CNS. DFer protein is expressed at higher levels than in wild-type embryos, and when the levels of DFerRB are further increased in dfergof mutants, the midline-crossing defect is enhanced. By contrast, expression of DFerRB in a wild-type background has no effect on dorsal closure or CNS development. dfergof mutants express an N-terminally modified form of DFerRB, similar to the activated forms of Fes/Fer kinases, such as v-fps. This appears as an extra band, slightly larger than the canonical DFer isoform (Murray, 2006).
In dfergof mutants, dorsal closure starts to arrest at stage 13, with only the most anterior and posterior segments meeting at the dorsal midline at stage 16. The leading edge and dorsal epidermal cells fail to elongate. The actomyosin cable still forms and creates a straightened leading edge, albeit one reduced in thickness. The F-actin-rich filopodia that extend from the leading edge are also much less extensive (Murray, 2006).
The amnioserosa is also affected in dfergof mutant embryos. In wild-type embryos, F-actin staining becomes increasingly strong at the perimeter of amnioserosal cells as they progressively contract. In dfergof mutants, F-actin staining is much less concentrated at cell-cell junctions, and the cell cortices are irregular. Accelerated contraction of isolated amnioserosal cells still occurs (Murray, 2006).
To characterise further dfergof mutants, GFP-actin was expressed ubiquitously in wild-type and dfergof backgrounds. The leading edge actomyosin cable and filopodia are reduced in dfergof mutants, and less GFP-Actin is concentrated at the cell-cell junctions of amnioserosal cells. In addition, the amnioserosal cells exhibit more lamellipodia (Murray, 2006).
In dfergof mutants the P-element, pGawB, has undergone a rearrangement, duplicating and inverting the GAL4 gene, deleting pBluescript, and all but the promoter and first exon of the mini-white gene. As predicted from this map, dfergof still expresses GAL4. In fact, GAL4 is expressed at higher levels and in more tissues, such as the epidermis and the amnioserosa, than in the original starting line, GAL4MZ465. Three new fusion transcripts were detected in which the first exon of the mini-white gene is spliced to the beginning of the second exon of dfer, (wex1-DFerRB), to the beginning of the third exon (wex1_stop1), or to an alternate splice acceptor in intron2 (wex1_stop2). The second and third of these transcripts encode short proteins comprising the first 24 residues of White, followed shortly thereafter by stop codons. The first transcript encodes a predicted fusion protein in which the first 24 residues of White (MGQEDQELLIRGGSKHPSAEHLNN) are followed by 12 novel amino acids (RAATQIGSNESI) and the entire DFerRB protein. This chimaeric protein is strikingly similar to the oncogenic forms of Fes, such as the Fujinami sarcoma virus protein GAG-Fps (Shibuya, 1980), in which part of the retroviral GAG sequence is fused to the N terminus of the entire Fps gene (Murray, 2006).
p94fer and p51ferT are two tyrosine kinases that are encoded by differentially spliced transcripts of the FER locus in the mouse. The two tyrosine kinases share identical SH2 and kinase domains but differ in their NH2-terminal amino acid sequence. Unlike p94fer, the presence of which has been demonstrated in most mammalian cell lines analyzed, the expression of p51ferT is restricted to meiotic cells. The two related tyrosine kinases also differ in their subcellular localization profiles. Although p51ferT accumulates constitutively in the cell nucleus, p94fer is cytoplasmic in quiescent cells and enters the nucleus concomitantly with the onset of S phase. The nuclear translocation of the FER proteins is driven by a nuclear localization signal (NLS), which is located within the kinase domain of these enzymes. The functioning of that NLS depends on the integrity of the kinase domain but is not affected by inactivation of the kinase activity. The NH2 terminus of p94fer dictates the cell cycle-dependent functioning of the NLS of FER kinase. This process is governed by coiled-coil forming sequences that are present in the NH2 terminus of the kinase. The regulatory effect of the p94fer NH2-terminal sequences is not affected by kinase activity but is perturbed by mutations in the kinase domain ATP binding site. Ectopic expression of the constitutively nuclear p51ferT in CHO cells interfers with S-phase progression in these cells. This is not seen in p94fer-overexpressing cells. The FER tyrosine kinases seem, thus, to be regulated by novel mechanisms that direct their different subcellular distribution profiles and may, consequently, control their cellular functioning (Ben-Dor, 1999).
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. Leucine-to-proline substitutions that were predicted to disrupt the coiled-coil structure were introduced into the coiled coils. The resulting mutant proteins, together with wild-type Fes, were fused to green fluorescent protein and expressed in Rat-2 fibroblasts. 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, the mutant proteins were expressed 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 (Cheng, 2001).
p94(fer) and p51(ferT) are two tyrosine kinases which share identical SH2 and kinase domains but differ in their N-terminal regions. While p94(fer) is expressed in most mammalian cells, the accumulation of p51(ferT) is restricted to meiotic spermatocytes. The different N-terminal tails of p94(fer) and p51(ferT) direct different autophosphorylation states of these two kinases in vivo. N-terminal coiled-coil domains cooperated to drive the oligomerization and autophosphorylation in trans of p94(fer). Moreover, the ectopically expressed N-terminal tail of p94(fer) can act as a dominant negative mutant and associates with the endogenous p94(fer) protein in CHO cells. This increases significantly the percentage of cells residing in the G0/G1 phase, thus suggesting a role for p94(fer) in the regulation of G1 progression. Unlike p94(fer), overexpressed p51(ferT) is not autophosphorylated in COS1 cells. However, removal of the unique N-terminal 43 aa of p51(ferT) or the replacement of this region by a parallel segment from p94(fer) endows the modified p51(ferT) with the ability to autophosphorylate. The unique N-terminal sequences of p51(ferT) thus interfere with its ability to autophosphorylate in vivo. These experiments indicate that the N-terminal sequences of the FER tyrosine kinases direct their different cellular autophosphorylation states, thereby dictating their different cellular functions (Orlovsky, 2000).
The FER gene encodes a cytoplasmic tyrosine kinase with a single SH2 domain and an extensive amino terminus. In order to understand the cellular function of the FER kinase, the effect of growth factor stimulation was analyzed on the phosphorylation and activity of FER. Stimulation of A431 cells and 3T3 fibroblasts with epidermal growth factor or platelet-derived growth factor results in the phosphorylation of FER and two associated polypeptides. The associated polypeptides were shown to be the epidermal growth factor receptor or the platelet-derived growth factor receptor and a previously identified target, pp120. Since pp120 has been shown to interact with components of the cadherin-catenin complex, these results implicate FER in the regulation of cell-cell interactions. The physical association of FER with pp120 is constitutive and is mediated by a 400-amino-acid sequence in the amino terminus of FER. Analyses of that sequence revealed that it has the ability to form coiled coils and that it oligomerizes in vitro. The identification of a coiled coil sequence in the FER kinase and the demonstration that the sequence mediates association with a potential substrate suggest a novel mechanism for signal transduction by cytoplasmic tyrosine kinases (Kim, 1995).
The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels has suggested a strong negative selection against too much Fer protein and points to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually leads to cell death. Induction of Fer expression coincides with increased complex formation between Fer and the cadherin/src-associated substrate p120(cas) and elevated tyrosine phosphorylation of p120(cas). beta-Catenin also exhibits clearly increased phosphotyrosine levels, and Fer and beta-catenin are found to be in complex. Significantly, although the levels of alpha-catenin, beta-catenin, and E-cadherin are unaffected by Fer overexpression, decreased amounts of alpha-catenin and beta-catenin are coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 is also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion (Rosato, 1998).
Cadherins and integrins must function in a coordinated manner to effectively mediate the cellular interactions essential for development. It was hypothesized that exchange of proteins associated with their cytoplasmic domains may play a role in coordinating function. To test this idea, Trojan peptides were used to introduce into cells and tissues peptide sequences designed to compete for the interaction of specific effectors with the cytoplasmic domain of N-cadherin, and their effect on cadherin- and integrin-mediated adhesion and neurite outgrowth were assayed. A peptide mimicking the juxtamembrane (JMP) region of the cytoplasmic domain of N-cadherin results in inhibition of N-cadherin and β1-integrin function. The effect of JMP on β1-integrin function depends on the expression of N-cadherin and is independent of transcription or translation. Treatment of cells with JMP results in the release of the nonreceptor tyrosine kinase Fer from the cadherin complex and its accumulation in the integrin complex. A peptide that mimics the first coiled-coil domain of Fer prevents Fer accumulation in the integrin complex and reverses the inhibitory effect of JMP. These findings suggest a new mechanism through which N-cadherin and β1-integrins are coordinately regulated: loss of an effector from the cytoplasmic domain of N-cadherin and gain of that effector by the β1-integrin complex (Arregui, 2000).
β-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and alpha-catenin. Interaction of β-catenin with alpha-catenin is regulated by the phosphorylation of β-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupts the association between both catenins. Whether these kinases are involved in the regulation of this interaction by K-ras was examined. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little alpha-catenin-β-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of β-catenin-alpha-catenin interaction (Piedra, 2003).
The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by alpha- and β-catenin. The phosphorylation state of β-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. The phosphorylation of β-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of β-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. The interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated β-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a β-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains β-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and β-catenin, as well as localization of cadherin and β-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts (Xu, 2004).
Cortactin regulates the strength of nascent N-cadherin-mediated intercellular adhesions through a tyrosine phosphorylation-dependent mechanism. Currently, the functional significance of cortactin phosphorylation and the kinases responsible for the regulation of adhesion strength are not defined. The nonreceptor tyrosine kinase Fer phosphorylates cadherin-associated cortactin and this process is involved in mediating intercellular adhesion strength. In wild-type fibroblasts N-cadherin ligation induces transient phosphorylation of Fer, indicating that junction formation activates Fer kinase. Tyrosine phosphorylation of cortactin after N-cadherin ligation is strongly reduced in fibroblasts expressing only catalytically inactive Fer (D743R), compared with wild-type cells. In wild-type cells, N-cadherin-coated bead pull-off assays induce fourfold greater endogenous N-cadherin association than in D743R cells. Fluorescence recovery after photobleaching showed that GFP-N-cadherin mobility at nascent contacts is 50% faster in wild-type than D743R cells. In shear wash-off assays, nascent intercellular adhesion strength is twofold higher in wild-type than D743R cells. Cortactin recruitment to adhesions is independent of Fer kinase activity, but is impacted by N-cadherin ligation-provoked Rac activation. It is concluded that N-cadherin ligation induces Rac-dependent cortactin recruitment and Fer-dependent cortactin phosphorylation, which in turn promotes enhanced mobilization and interaction of surface expressed N-cadherin in contacting cells (El Sayegh, 2005).
Cell migration is regulated by focal adhesion (FA) turnover. Fibroblast growth factor-2 (FGF-2) induces FA disassembly in the murine brain capillary endothelial cell line IBE, leading to FGF-2-directed chemotaxis. Activation of Src and Fes by FGF-2 was involved in chemotaxis of IBE cells. This study examined the interplay between Src and Fes. FGF-2 treatment decreases the number of FA in IBE cells, but not in cells expressing dominant-negative Fes (denoted KE5-15 cells). FGF-2 induces the activation of Src and subsequent binding to and phosphorylation of Cas in IBE cells, but not in KE5-15 cells. Focal adhesion kinase (FAK) activation and tyrosine phosphorylation by Src were also delayed in KE5-15 cells compared to parental cells. FGF-2 induces activation of Src within FA in IBE cells, but not in KE5-15 cells. Downregulation of Fes or FAK using small interfering RNA diminishes Src activation by FGF-2 within FA. These findings suggest that activation of Fes by FGF-2 enhances FAK-dependent activation of Src within FA, promoting FGF-2-induced disassembly of focal adhesions (Kanda, 2006).
Nonreceptor tyrosine kinase FER exhibits a tight physical association with the catenin pp120; this has led to the suggestion that FER may be involved in cell-cell signaling. To further understand the function of FER, interaction of FER with pp120 and other proteins was analyzed. The majority of FER is localized to the cytoplasmic fraction where it forms a complex with the actin-binding protein cortactin. The Src homology 2 sequence of FER is required for directly binding cortactin, and phosphorylation of the FER-cortactin complex is up-regulated in cells treated with peptide growth factors. Using a dominant-negative mutant of FER, evidence is provided that FER kinase activity is required for the growth factor-dependent phosphorylation of cortactin. These data suggest that cortactin is likely to be a direct substrate of FER. These observations provide additional support for a role of FER in mediating signaling from the cell surface, via growth factor receptors, to the cytoskeleton. The nature of the FER-cortactin interaction, and their putative enzyme-substrate relationship, support the proposal that one of the functions of the Src homology 2 sequences of nonreceptor tyrosine kinases is to provide a binding site for their preferred substrates (Kim, 1998).
The F-actin-binding protein cortactin is an important regulator of cytoskeletal dynamics, and a prominent target of various tyrosine kinases. Tyrosine phosphorylation of cortactin has been suggested to reduce its F-actin cross-linking capability. This study investigated whether a reciprocal relationship exists, i.e. whether the polymerization state of actin impacts on the cortactin tyrosine phosphorylation. Actin depolymerization by LB (latrunculin B) induces robust phosphorylation of C-terminal tyrosine residues of cortactin. In contrast, F-actin stabilization by jasplakinolide, which redistributes cortactin to F-actin-containing patches, preventes cortactin phosphorylation triggered by hypertonic stress or LB. Using cell lines deficient in candidate tyrosine kinases, it was found that the F-actin depolymerization-induced cortactin phosphorylation is mediated by the Fyn/Fer kinase pathway, independent of Src and c-Abl. LB causes modest Fer activation and strongly facilitates the association between Fer and cortactin. Interestingly, the F-actin-binding region within the cortactin N-terminus is essential for the efficient phosphorylation of C-terminal tyrosine residues. Investigating the structural requirements for the Fer-cortactin association, it was found that (1) phosphorylation-incompetent cortactin still binds to Fer; (2) the isolated N-terminus associates with Fer; and (3) the C-terminus alone is insufficient for binding. Thus the cortactin N-terminus participates in the Fer-cortactin interaction, which cannot be fully due to the binding of the Fer Src homology 2 domain to C-terminal tyrosine residues of cortactin. Taken together, F-actin stabilization prevents cortactin tyrosine phosphorylation, whereas depolymerization promotes it. Depolymerization-induced phosphorylation is mediated by Fer, and requires the actin-binding domain of cortactin. These results define a novel F-actin-dependent pathway that may serve as a feedback mechanism during cytoskeleton remodelling (Fan, 2004).
Fes/Fps (Fes) tyrosine kinase is involved in Semaphorin3A-mediated signaling. This study reports a role for Fes tyrosine kinase in microtubule dynamics. A fibrous formation of Fes was observed in a kinase-dependent manner, which associated with microtubules and functionally correlated with microtubule bundling. Microtubule regeneration assays revealed that Fes aggregates colocalized with gamma-tubulin at microtubule nucleation sites in a Fes/CIP4 homology (FCH) domain-dependent manner and that expression of FCH domain-deleted Fes mutants blocks normal centrosome formation. In support of these observations, mouse embryonic fibroblasts derived from Fes-deficient mice display an aberrant structure of nucleation and centrosome with unbundling and disoriented filaments of microtubules. These findings suggest that Fes plays a critical role in microtubule dynamics including microtubule nucleation and bundling through its FCH domain (Takahashi, 2003).
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, it was 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 shows a diffuse cytoplasmic localization in this system, which correlates 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 is not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain is essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, it was observed that purified c-Fes also catalyzes 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 (Laurent, 2004b).
The small GTP-binding proteins Ras, Rac, and Cdc42 link protein-tyrosine kinases with mitogen-activated protein kinase (MAPK) signaling cascades. Ras controls the activation of extracellular signal-regulated kinases (ERKs), while Rac and Cdc42 regulate the c-Jun N-terminal kinases (JNKs). This study investigated whether small G protein/MAPK cascades contribute to signal transduction by transforming variants of c-Fes, a nonreceptor tyrosine kinase implicated in cytokine signaling and myeloid differentiation. First, the effects of dominant-negative small G proteins were investigated on Rat-2 fibroblast transformation by a retroviral homolog of c-Fes (v-Fps) and by c-Fes activated via N-terminal addition of the v-Src myristylation signal (Myr-Fes). Dominant-negative Ras, Rac, and Cdc42 inhibit v-Fps- and Myr-Fes-induced growth of Rat-2 cells in soft agar, indicating that activation of these small GTP-binding proteins is required for fibroblast transformation by Fps/Fes tyrosine kinases. To determine whether MAPK pathways are activated downstream of these small G proteins, ERK and JNK activity were measured in the v-Fps- and Myr-Fes-transformed Rat-2 cells. Both ERK and JNK activities were elevated in the transformed cells, suggesting that these pathways are involved in cellular transformation. Dominant-negative mutants of Ras (but not Rac or Cdc42) specifically inhibit ERK activation by v-Fps and Myr-Fes, demonstrating that ERK activation occurs exclusively downstream of Ras. All three dominant-negative small G proteins inhibit JNK activation by v-Fps and Myr-Fes, indicating that JNK activation by these tyrosine kinases requires both Ras and Rho family GTPases. These data demonstrate that multiple small G protein/MAPK cascades are involved in downstream signal transduction by Fps/Fes tyrosine kinases (Li, 1998).
Morphogenesis requires coordination of cell surface activity and cytoskeletal architecture. During the initial stage of morphogenesis in C. elegans, the concerted movement of surface epithelial cells results in enclosure of the embryo by the epidermis. Fer-related kinase-1 (FRK-1), an ortholog of the mammalian non-receptor tyrosine kinase Fer, is necessary for embryonic enclosure and morphogenesis in C. elegans. Expression of FRK-1 in epidermal cells is sufficient to rescue a chromosomal deficiency that removes the frk-1 locus, demonstrating its autonomous requirement in the epidermis. The essential function of FRK-1 is independent of its kinase domain, suggesting a non-enzymatic role in morphogenesis. Localization of FRK-1 to the plasma membrane requires ß-catenin, but not cadherin or alpha-catenin, and muscle-expressed ß-integrin is non-autonomously required for this localization; in the absence of these components FRK-1 becomes nuclear. Mouse FerT rescues the morphogenetic defects of frk-1 mutants and expression of FRK-1 in mammalian cells results in loss of adhesion, implying a conserved function for FRK-1/FerT in cell adhesion and morphogenesis. Thus, FRK-1 performs a kinase-independent function in differentiation and morphogenesis of the C. elegans epidermis during embryogenesis (Putzke, 2005).
The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase known to be highly expressed in hematopoietic cells. To investigate fps/fes biological function, an activating mutation was introduced into the human fps/fes gene; the mutation directs amino-terminal myristylation of the Fps/Fes protein. This mutant, myristylated protein induces transformation of Rat-2 fibroblasts. The mutant fps/fes allele was incorporated into the mouse germ line and was found to be appropriately expressed in transgenic mice, in a tissue-specific pattern indistinguishable from that of the endogenous mouse gene. These mice displayed widespread hypervascularity, progressing to multifocal hemangiomas. High levels of both the transgenic human and endogenous murine fps/fes transcripts were detected in vascular tumors by using RNase protection, and fps/fes transcripts were localized to endothelial cells of both the vascular tumors and normal blood vessels by in situ RNA hybridization. Primary human umbilical vein endothelial cultures were also shown to express fps/fes transcripts and the Fps/Fes tyrosine kinase. These results indicate that fps/fes expression is intrinsic to cells of the vascular endothelial lineage and suggest a direct role of the Fps/Fes protein-tyrosine kinase in the regulation of angiogenesis (Greer, 1994).
Mast cells express the high affinity IgE receptor FcepsilonRI, which upon aggregation by multivalent antigens elicits signals that cause rapid changes within the mast cell and in the surrounding tissue. FcepsilonRI aggregation causes a rapid increase in phosphorylation of both Fer and Fps/Fes kinases in bone marrow-derived mast cells. FcepsilonRI aggregation leads to increased Fer/Fps kinase activities and Fer phosphorylation downstream of FcepsilonRI is independent of Syk, Fyn, and Gab2 but requires Lyn. Activated Fer/Fps readily phosphorylate the C terminus of platelet-endothelial cell adhesion molecule 1 (Pecam-1) on immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a non-ITIM residue (Tyr(700)) in vitro and in transfected cells. Mast cells devoid of Fer/Fps kinase activities display a reduction in FcepsilonRI aggregation-induced tyrosine phosphorylation of Pecam-1, with no defects in recruitment of Shp1/Shp2 phosphatases observed. Lyn-deficient mast cells display a dramatic reduction in Pecam-1 phosphorylation at Tyr(685) and a complete loss of Shp2 recruitment, suggesting a role as an initiator kinase for Pecam-1. Consistent with previous studies of Pecam-1-deficient mast cells, an exaggerated degranulation response is observed in mast cells lacking Fer/Fps kinases at low antigen dosages. Thus, Lyn and Fer/Fps kinases cooperate to phosphorylate Pecam-1 and activate Shp1/Shp2 phosphatases that function in part to limit mast cell activation (Udell, 2006).
The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. This study 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 is completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induces constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and leads to dramatic actin reorganization and spontaneous neurite extension. The breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, was investigated 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 suppresses neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induces 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 (Laurent, 2004a).
The neuronal cytoskeleton is essential for establishment of neuronal polarity, but mechanisms controlling generation of polarity in the cytoskeleton are poorly understood. The nonreceptor tyrosine kinase, Fer, has been shown to bind to microtubules and to interact with several actin-regulatory proteins. Furthermore, Fer binds p120 catenin and has been shown to regulate cadherin function by modulating cadherin-β-catenin interaction. Fer is involved in neuronal polarization and neurite development. Fer is concentrated in growth cones together with cadherin, β-catenin, and cortactin in stage 2 hippocampal neurons. Inhibition of Fer-p120 catenin interaction with a cell-permeable inhibitory peptide (FerP) increases neurite branching. In addition, the peptide significantly delays conversion of one of several dendrites into an axon in early stage hippocampal neurons. FerP-treated growth cones also exhibit modified localization of the microtubule and actin cytoskeleton. Together, this indicates that the Fer-p120 interaction is required for normal neuronal polarization and neurite development (Lee, 2005).
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 reduce Fes autophosphorylation and STAT3 Tyr-705 phosphorylation compared with wild-type Fes, whereas S759F has little effect. These mutations have a similar impact on Fes kinase activity in a yeast expression system, suggesting that they inhibit Fes by affecting kinase domain structure. 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 is reduced or absent in colon tumor sections from most individuals. Fes protein levels are 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 suppresses their growth in soft agar. Together, these findings strongly implicate the c-Fes protein-tyrosine kinase as a tumor suppressor rather than a dominant oncogene in colorectal cancer (Delfino, 2006).
Fer is a nuclear and cytoplasmic intracellular tyrosine kinase. This study shows that Fer is required for cell-cycle progression in malignant cells. Decreasing the level of Fer using the RNA interference (RNAi) approach impedes the proliferation of prostate and breast carcinoma cells and leads to their arrest at the G0/G1 phase. At the molecular level, knockdown of Fer results in the activation of the retinoblastoma protein (pRB), and this is reflected by profound hypo-phosphorylation of pRB on both cyclin-dependent kinase CDK4 and CDK2 phosphorylation sites. Dephosphorylation of pRB is not seen upon the direct targeting of either CDK4 or CDK2 expression, and is only partially achieved by the simultaneous depletion of these two kinases. Amino-acid sequence analysis revealed two protein phosphatase 1 (PP1) binding motifs in the kinase domain of Fer and the association of Fer with the pRB phosphatase PP1alpha was verified using co-immunoprecipitation analysis. Downregulation of Fer potentiates the activation of PP1alpha and overexpression of Fer decreases the enzymatic activity of that phosphatase. These findings portray Fer as a regulator of cell-cycle progression in malignant cells and as a potential target for cancer intervention (Pasder, 2006).
Search PubMed for articles about Drosophila Fps oncogene analog
Arregui, C., Pathre, P., Lilien, J. and Balsamo, J. (2000). The non receptor tyrosine kinase fer mediates cross-talk between N-cadherin and β 1-integrins. J. Cell Biol. 149: 1263-1274. 10851023
Ben-Dor, I., Bern, O., Tennenbaum, T. and Nir, U. (1999). Cell cycle-dependent nuclear accumulation of the p94fer tyrosine kinase is regulated by its NH2 terminus and is affected by kinase domain integrity and ATP binding. Cell Growth Differ. 10(2): 113-29. 10074905
Cheng, H. Y., Schiavone, A. P. and Smithgall, T. E. (2001). A point mutation in the N-terminal coiled-coil domain releases c-Fes tyrosine kinase activity and survival signaling in myeloid leukemia cells. Mol. Cell. Biol. 21: 6170-6180. 11509660
Delfino, F. J., Stevenson, H. and Smithgall, T. E. (2006). A growth-suppressive function for the c-fes protein-tyrosine kinase in colorectal cancer. J. Biol. Chem. 281(13): 8829-35. 16455651
El Sayegh, T. Y., et al. (2005). Phosphorylation of N-cadherin-associated cortactin by Fer kinase regulates N-cadherin mobility and intercellular adhesion strength. Mol. Biol. Cell 16(12): 5514-27. 16176974
Fan, L., et al. (2004). Actin depolymerization-induced tyrosine phosphorylation of cortactin: the role of Fer kinase. Biochem J. 380(Pt 2): 581-91. 15030313
Greer, P., Haigh, J., Mbamalu, G., Khoo, W., Bernstein, A. and Pawson, T. (1994). The Fps/Fes protein-tyrosine kinase promotes angiogenesis in transgenic mice. Mol. Cell. Biol. 14: 6755-6763. 7523858
Greer, P. (2002). Closing in on the biological functions of Fps/Fes and Fer. Nat. Rev. Mol. Cell Biol. 3: 278-289. 11994747
Kanda, S., Miyata, Y., Kanetake, H. and Smithgall, T. E. (2006). Fibroblast growth factor-2 induces the activation of Src through Fes, which regulates focal adhesion disassembly. Exp. Cell Res. 312(16): 3015-22. 16884713
Katzen, A. L., Montarras, D., Jackson, J., Paulson, R. F., Kornberg, T. and Bishop, J. M. (1991). A gene related to the proto-oncogene fps/fes is expressed at diverse times during the life cycle of Drosophila melanogaster. Mol. Cell. Biol. 11: 226-239. 1898762
Kim, L. and Wong, T. W. (1995). The cytoplasmic tyrosine kinase FER is associated with the catenin-like substrate pp120 and is activated by growth factors. Mol. Cell. Biol. 15: 4553-4561. 7623846
Kim, L. and Wong, T. W. (1998). Growth factor-dependent phosphorylation of the actin-binding protein cortactin is mediated by the cytoplasmic tyrosine kinase FER. J. Biol. Chem. 273: 23542-23548. 9722593
Laurent, C. E. and Smithgall, T. E. (2004a). The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner. Exp. Cell Res. 299(1): 188-98. 15302586
Laurent, C. E., Delfino, F. J., Cheng, H. Y. and Smithgall, T. E. (2004b). The human c-Fes tyrosine kinase binds tubulin and microtubules through separate domains and promotes microtubule assembly. Mol. Cell. Biol. 24(21):9351-8. 15485904
Lee, S. H. (2005). Interaction of nonreceptor tyrosine-kinase Fer and p120 catenin is involved in neuronal polarization. Mol. Cells 20: 256-262. 16267401
Li, J. and Smithgall, T. E. (1998). Fibroblast transformation by Fps/Fes tyrosine kinases requires Ras, Rac, and Cdc42 and induces extracellular signal-regulated and c-Jun N-terminal kinase activation. J. Biol. Chem. 273: 13828-13834. 9593727
Murray, M. J., Davidson, C. M., Hayward, N. M. and Brand, A. H. (2006). The Fes/Fer non-receptor tyrosine kinase cooperates with Src42A to regulate dorsal closure in Drosophila. Development 133(16): 3063-73. 16831834
Orlovsky, K., Ben-Dor, I., Priel-Halachmi, S., Malovany, H. and Nir, U. (2000). N-terminal sequences direct the autophosphorylation states of the FER tyrosine kinases in vivo. Biochemistry 39: 11084-11091. 10998246
Pasder, O., et al. (2006). Downregulation of Fer induces PP1 activation and cell-cycle arrest in malignant cells. Oncogene 25(30): 4194-206. 16732323
Paulson, R., Jackson, J., Immergluck, K. and Bishop, J. M. (1997). The DFer gene of Drosophila melanogaster encodes two membrane-associated proteins that can both transform vertebrate cells. Oncogene 14: 641-652. 9038371
Piedra, J., Miravet, S., Castano, J., Palmer, H. G., Heisterkamp, N., Garcia de Herreros, A. and Dunach, M. (2003). p120 Catenin-associated Fer and Fyn tyrosine kinases regulate β-catenin Tyr-142 phosphorylation and βcatenin-alpha-catenin interaction. Mol. Cell. Biol. 23: 2287-2297. 12640114
Putzke, A. P., Hikita, S. T., Clegg, D. O. and Rothman, J. H. (2005). Essential kinase-independent role of a Fer-like non-receptor tyrosine kinase in Caenorhabditis elegans morphogenesis. Development 132: 3185-3195. 15958510
Rosato, R., Veltmaat, J. M., Groffen, J. and Heisterkamp, N. (1998). Involvement of the tyrosine kinase fer in cell adhesion. Mol. Cell. Biol. 18: 5762-5770. 9742093
Shibuya, M., Hanafusa, T., Hanafusa, H. and Stephenson, J. R. (1980). Homology exists among the transforming sequences of avian and feline sarcoma viruses. Proc. Natl. Acad. Sci. 77: 6536-6540. 6256742
Snyder, S. P. and Theilen, G. H. (1969). Transmissible feline fibrosarcoma. Nature 221: 1074-1075. 5774407
Takahashi, S., et al. (2003). Role for Fes/Fps tyrosine kinase in microtubule nucleation through is Fes/CIP4 homology domain. J. Biol. Chem. 278(49): 49129-33. 14551201
Udell, C. M., Samayawardhena, L. A., Kawakami, Y., Kawakami, T. and Craig, A. W. (2006). Fer and Fps/Fes participate in a Lyn-dependent pathway from FcepsilonRI to platelet-endothelial cell adhesion molecule 1 to limit mast cell activation. J. Biol. Chem. 281(30): 20949-57. 16731527
Wu, H. and Parsons, J. T. (1993). Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex. J. Cell Biol. 120(6): 1417-26. 7680654
Xu, G., Craig, A. W., Greer, P., Miller, M., Anastasiadis, P. Z., Lilien, J. and Balsamo, J. (2004). Continuous association of cadherin with β-catenin requires the non-receptor tyrosine-kinase Fer. J. Cell Sci. 117: 3207-3219. 15226396
date revised: 10 December 2006
Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.