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 dfer^Δ1 and dfer^Δex1 mutants, dpp expression levels appear normal. In Src42A mutants, dpp expression is slightly reduced, becoming patchy from stage 13 onwards. In Src42A;dfer^Δ1 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 dfer^gof 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-hep^CA, 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 dfer^gof phenotype is rescued by expression of the JNK inhibitor Puckered (Puc). UASpuc,dfer^gof 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 dfer^gof phenotype by dephosphorylating either DFer^gof 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,dfer^Δ1 and dfer^gof embryos, and probed with anti-Armadillo (ß-catenin) and anti-phospho-tyrosine. In dfer^Δ1 embryos, ß-catenin tyrosine phosphorylation is reduced nearly fivefold with respect to control embryos. Conversely, tyrosine phosphorylation is significantly increased in dfer^gof embryos. Much less ß-catenin is recovered from dfer^gof embryos, suggesting that hyperphosphorylated ß-catenin is removed from AJs and degraded. This is confirmed by whole-mount staining of dfer^gof 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 dfer^gof embryos, but the result is more variable. Western blots on total extracts from late stage dfer^gof embryos also show a reduction in the levels of ß-catenin (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, dferΔ^ex1, 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)dfer^Δ1 (hereafter dfer^Δ1), was selected for further analysis. Both dfer^Δ1 and dfer^Δex1 still express functional GAL4 in patterns similar to that of MZ465. dfer^Δ1 homozygotes are embryonic lethal in 53% of cases with the remainder dying during larval and pupal development. dfer^Δex1 homozygotes are both viable and fertile. All results in this study using dfer^Δex1 are from embryos derived from dfer^Δex1 homozygous parents (Murray, 2006).

dfer^Δ1 is an RNA and protein null for all DFer isoforms. In dfer^Δex1 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 dfer^Δex1 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 dfer^Δex1 mutants. Thus, dfer^Δex1 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 dfer^Δ1 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. dfer^Δ1 mutant embryos are still open dorsally three hours later, at the end of stage 16. Closure eventually completes, although 2% of cuticles from dfer^Δ1 mutants exhibit an anterior hole. By contrast, dfer^Δex1 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, dfer^Δ1 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 dfer^Δex1 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, dfer^Δ1, 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, dfer^gof, was isolated that behaves as a gain-of-function mutant. Homozygous dfer^gof 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 dfer^gof 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. dfer^gof 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 dfer^gof 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 dfer^gof mutant embryos. In wild-type embryos, F-actin staining becomes increasingly strong at the perimeter of amnioserosal cells as they progressively contract. In dfer^gof 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 dfer^gof mutants, GFP-actin was expressed ubiquitously in wild-type and dfer^gof backgrounds. The leading edge actomyosin cable and filopodia are reduced in dfer^gof 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 dfer^gof 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, dfer^gof 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, GAL4^MZ465. 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).