InteractiveFly: GeneBrief

kekkon-1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - kekkon-1

Synonyms -

Cytological map position - 33F1--2

Function - transmembrane receptor

Keywords - cns, oogenesis, egf and ras pathway

Symbol - kek1

FlyBase ID: FBgn0015399

Genetic map position - 2-

Classification - immunoglobulin-C2-type domain protein, leucine-rich repeat protein

Cellular location - cell surface



Precomputed BLAST | Entrez Gene
Recent literature
Vonesch, S.C., Lamparter, D., Mackay, T.F., Bergmann, S. and Hafen, E. (2016). Genome-wide analysis reveals novel regulators of growth in Drosophila melanogaster. PLoS Genet 12: e1005616. PubMed ID: 26751788
Summary:
Organismal size depends on the interplay between genetic and environmental factors. Genome-wide association (GWA) analyses in humans have implied many genes in the control of height but suffer from the inability to control the environment. Genetic analyses in Drosophila have identified conserved signaling pathways controlling size; however, how these pathways control phenotypic diversity is unclear. This study performed GWA of size traits using the Drosophila Genetic Reference Panel of inbred, sequenced lines. It was found that the top associated variants differ between traits and sexes; do not map to canonical growth pathway genes, but can be linked to these by epistasis analysis; and are enriched for genes and putative enhancers. A cluster of associations close to the kek1 locus, a well-characterized growth regulator, was identified but otherwise most variants are located in or close to genes that do not belong to the conserved pathways but may interact with these in a biological network. 33 novel growth regulatory genes that participate in diverse cellular processes were validated, most notably cellular metabolism and cell polarity. Performing GWA on well-studied developmental traits under controlled conditions expands the understanding of developmental processes underlying phenotypic diversity. 


BIOLOGICAL OVERVIEW

Kekkon-1 was identified in a search for genes that may play a role in axonal outgrowth in the central nervous system (CNS). A collection of enhancer trap insertion lines was examined for beta-galactosidase expression in subsets of CNS cells. Among these the line 15A6 was selected because it expresses lacZ initially in a segmentally repeated set of cells at the CNS midline at early stages. At later stages, lacZ eventually becomes expressed in a large set of CNS cells and some peripheral nervous system cells. Kekkon2, an unlinked, independent gene, was isolated in a search for genes whose sequence resembles that of Kek1. Like Kek1, Kek2 is expressed in the CNS (Musacchio, 1996).

The same enhancer trap line, 15A6, was identified in a search for enhancer trap lines that exhibit expression patterns in follicle cells in a pattern consistent with the marked gene being a regulatory target of the epidermal growth factor receptor (Egfr) pathway, since the pattern of expression undergoes a transition during stages 8-10 when it relocalizes from a posterior to a dorsal-anterior gradient. Kekkon-1 was identified as an inhibitor of the Egfr; Kek1 acts in a negative feedback loop to modulate the activity of the Egfr tyrosine kinase. During oogenesis, kek1 is expressed in response to the Gurken/Egfr signaling pathway, and loss of kek1 activity is associated with an increase in Egfr signaling. Consistent with loss-of-function studies, ectopic overexpression of kek1 mimics a loss of Egfr activity. The extracellular and transmembrane domains of Kek1 can inhibit and physically associate with the Egfr, suggesting potential models for this inhibitory mechanism (Ghiglione, 1999).

In the complete absence of kek1 gene activity, flies are viable, fertile, and do not exhibit any overt morphological defects (Musacchio, 1996). Kek1 has the features of cell adhesion molecules (CAMs), and loss-of-function mutations of many CAMs have subtle mutant phenotypes. However, when ectopically overexpressed, some CAM molecules can generate striking mutant phenotypes that are revealing of their functions. Thus, to gain insights into the possible function of kek1 during oogenesis, the effect of overexpressing kek1 in the follicle cells was examined using the GAL4/UAS system When kek1 is expressed under the control of the GAL4 line T155, which drives expression all over the follicle cell epithelium beginning at stage 9, 100% of the resulting eggs are longer than the WT eggs and show a reduction or a complete loss of dorsal appendages. This loss of dorsal appendage material is due to a ventralization of the eggshell, a phenotype associated with egfr, or grk loss-of-function mutations. A similar ventralized eggshell phenotype is obtained when a dominant-negative form of the Egfr, EgfrDN, is expressed under the control of T155 (Ghiglione, 1999).

In addition to its role in establishing the dorsal characteristics of the eggshell, the Grk/Egfr pathway controls embryonic dorsoventral patterning by restricting a ventralizing signal. As a result, loss of Egfr function leads to ventralized embryos. Embryonic cuticles derived from T155; UAS-kek1 females shows a ventralized cuticle phenotype. To analyze the extent of this ventralization, embryos were stained for Twist (Twi) mRNA and protein that label a ventral domain ten cells wide. Overexpression of kek1 in follicle cells results in an expansion of the Twi expression domain. Similarly, embryos derived from T155; UAS-egfrDN females are also strongly ventralized. Altogether, these results indicate that overexpression of kek1 in follicle cells ventralizes both the eggshell and the embryo by most likely interfering with the activity of the Grk/Egfr signaling pathway. Interestingly, kek1 is expressed in the eye and wing imaginal discs in a pattern that is highly suggestive of induction by the Egfr (Musacchio, 1996). Although no obvious phenotypes have been described in these tissues in kek1 mutants, overexpression of kek1 in these tissues also generate phenotypes reminiscent of loss of Egfr activity. This suggests that the negative regulation of the Egfr by Kek1 is not only restricted to oogenesis (Ghiglione, 1999).

To determine whether Kek1 blocks the signaling activity of the Egfr, a test was performed to see if ectopic expression of kek1 blocks the transcriptional activation of target genes regulated by the Egfr/Ras/Raf/MEK/MAPK pathway in follicle cells. Since kek1 is a target of this pathway, kek-lacZ was used as a reporter for this experiment. Overexpression of kek1 using the Gal4 driver T155 is associated with a strong but not complete reduction of kek-lacZ expression. Using a stronger Gal4 line, CY2, a complete disappearance of kek-lacZ expression is observed. These data are consistent with the model that Kek1 downregulates the activity of the Ras/Raf/MEK/MAPK pathway. Consistent with this, epistasis experiments indicate that the inhibitory effect of Kek1 is overridden by the constitutive activation of the Egfr or Draf. In addition, the interaction between the transmembrane protein Rhomboid (Rho) and Kek1 was tested. Rho has been proposed to play a role in the activation of the Egfr, and overexpression of Rho leads to dorsalized eggshells. Overexpression of rho does not override the ventralization phenotype of UAS-kek1, indicating that Kek1 can block the effect of Rho on Egfr activation. Altogether, these results place Kek1 upstream of the Egfr and downstream of Rho (Ghiglione, 1999).

Because of the severe inhibition of Egfr activity by Kek1, revealed by the overexpression experiment, the effect of loss of kek1 function during oogenesis was reexamined. A subtle egg morphology phenotype may have been missed by simply using fertility as an assay (Musacchio, 1996). Indeed, the spacing between the dorsal appendages of eggs derived from kek1 mutant females is increased when compared to WT. Further, these eggs are also mildly shorter and rounder, a phenotype consistent with a hyperactivation of the Grk/Egfr pathway. These features do not interfere with hatching rates and patterning that is consistent with the normal Twi expression found in kek1 mutant embryos. Interestingly, when kek1 mutant flies are raised at 29°C, 5% (n = 257) of the embryos derived from kek1 mutant females showed a mild reduction in Twi expression. It is concluded that loss of kek1 activity during oogenesis leads to mildly dorsalized eggs (Ghiglione, 1999).

During oogenesis, Grk derived from the oocyte activates in a paracrine fashion the Egfr in dorsal follicle cells. Recent work (Wasserman, 1998) has shown that this paracrine signaling leads to the activation of a second phase of signaling, whereby the Egfr activity is amplified among follicle cells themselves. During this second phase, the Egfr activates a number of target genes that include both positive (rhomboid and vein) and negative (argos) regulators of the pathway. Activation of Rho in follicle cells presumably leads to the activation of the Spitz ligand for Egfr, while activation of Aos within the peak of Egfr activity at the dorsal anterior leads to repression of Egfr, effectively splitting in two the initial peak of Egfr activity. This splitting of Egfr activity eventually defines the domains where the dorsal appendages will form.

The identification of Kek1, together with the studies of Aos, indicates that there are at least two different negative regulators of Egfr activity in follicle cells. However, the regulation and function of Kek1 is distinct from those of Aos. aos is expressed only in response to high levels of Egfr activity, while kek1 is expressed in a graded fashion. Further, loss-of-function phenotypes of kek1 and aos in follicle cells are different. In the absence of kek1 activity, the spacing between the dorsal appendages is increased, while in the absence of aos, the appendages are fused dorsally. Aos has been proposed to split the initial peak of Egfr activity into two (Wasserman, 1998). It is proposed that the function of Kek1 is to restrict the lateral spreading of Egfr activation by Spi. Thus, in the absence of kek1 activity, Rho/Spi activation could spread more laterally, explaining the enhancement of the spacing between the two dorsal appendages (Ghiglione, 1999).

Thus, Kek1 acts as a potent negative regulator of Egfr activity when overexpressed. Further, it has been shown that the extracellular and transmembrane domains of Kek1 are sufficient for this inhibition. The extracellular domain of Kek1 contains one Ig-like domain and five LRRs, both of which can mediate protein-protein interactions. A number of mechanisms can underlie the mechanism by which this extracellular domain acts as an inhibitor. For example, the Kek1 extracellular domain could mask the accessibility of the extracellular domain of the Egfr to all ligands. Conversely, it could form a heterodimer with Egfr monomers and block their dimerization. Dimerization is a prerequisite to the activation of downstream signaling events by the RTK. Alternatively, Kek1 could be involved in bringing a transmembrane tyrosine phosphatase to the vicinity of the Egfr and thus lead to its deactivation. Consistent with these models, it has been observed that Kek1 can inhibit mammalian Egfr molecules from becoming tyrosine phosphorylated in response to growth factor treatment in infected insect cells (L. T. Amundadottir, et al., unpublished observations cited in Ghiglione, 1999). Finally, it is envisioned that Kek1 could target the Egfr to a degradation pathway through endocytosis or bind to additional proteins involved in their subcellular localization (Ghiglione, 1999).

In Drosophila, two other putative Kek molecules that share extensive homologies with kek1 have been identified: kek2 (Musacchio, 1996) and kek3 (unpublished data of Ghiglione, 1999). The function of these additional Kek-like proteins is not known. However, it is interesting to note that despite extensive saturation of the region containing kek3, no mutant alleles have been recovered. Whether or not this and the subtle effect of loss of Kek1 activity is due to redundancy within the Kekkon family remains to be determined. Thus, it will be important to characterize the expression patterns as well as the loss-of-function and overexpresssion phenotypes of kek2 and kek3 in order to evaluate any potential abilities to modulate the Egfr activity. Further, chimeric proteins between these molecules may help to further define the Kek1 domain(s) required for the inhibitory mechanism. Finally, there are putative transmembrane proteins in vertebrates (Suzuki, 1996) and invertebrates that show similar arrangements of LRRs and Ig motifs. This raises the possibility that Kek1 is a member of a family of structurally related Egfr inhibitors. Because any alteration in the activity of the various members of the human Egfr/ErbB family has strong links to oncogenesis, it will be important to determine if vertebrate LRR/Ig molecules share functional, in addition to sequence, similarities to Kek1. It is anticipated that the continued characterization of the Kek and related molecules will provide novel approaches to the design of inhibitors of the Egfr/ErbB family for therapeutic use in oncogenesis (Ghiglione, 1999).


REGULATION

Transcriptional Regulation

To establish a regulatory link between the transcriptional regulation of kek1 in the follicle cell epithelium and the Grk/Egfr signaling pathway, kek-lacZ expression was examined in mutants for grk, egfr, and the kinase Draf that is both necessary and sufficient for Egfr signaling in follicle cells. In each case, the dorsal-anterior gradient of kek-lacZ expression is abolished or severely diminished, revealing that kek-lacZ expression is transcriptionally regulated by the Grk/Egfr/Draf pathway. Expression of an activated form of Draf results in the ubiquitous expression of kek-lacZ within the follicular epithelium (Ghiglione, 1999).

To further analyze the role of the Grk/Egfr pathway in the transcriptional control of kek-lacZ, the subcellular localization of GRK mRNAs was mislocalized by disrupting the oocyte cytoskeletal network. Treatment of the oocyte with colchicine, an inhibitor of microtubule polymerization, leads to mislocalization of the oocyte nucleus and its associated GRK mRNAs, which correlates with ectopic kek-lacZ expression as well as KEK1 mRNA expression. Altogether, these results demonstrate that Grk is not only necessary but sufficient to regulate the spatial expression of kek1 (Ghiglione, 1999).

Signaling by the Drosophila EGF receptor (Egfr) is modulated by four known EGF-like proteins: the agonists Vein (Vn), Spitz (Spi), and Gurken (Grk) and the antagonist Argos (Aos). Egfr is broadly expressed and thus tissue-specific regulation of ligand expression and activity is an important mechanism for controlling signaling. The tissue-specific regulation of Vn signaling was investigated by examining vn transcriptional control and Vn target gene activation in the embryo and the wing. The results show a complex temporal and spatial regulation of vn transcription involving multiple signaling pathways and tissue-specific activation of Vn target genes. In the embryo, vn is a target of Spi/Egfr signaling mediated by the ETS transcription factor PointedP1 (PntP1). This establishes a positive feedback loop in addition to the negative feedback loop involving Aos. The simultaneous production of Vn provides a mechanism for dampening Aos inhibition and thus fine-tunes signaling. In the larval wing pouch, vn is not a target of Spi/Egfr signaling but is expressed along the anterior-posterior boundary in response to Hedgehog (Hh) signaling. Repression by Wingless (Wg) signaling further refines the vn expression pattern by causing a discontinuity at the dorsal-ventral boundary. The potential for vn to activate Egfr target genes correlates with its roles in development: vn has a minor role in embryogenesis and does not induce Egfr target genes such as aos and pntP1 in the embryo. Conversely, vn has a major role in wing development and Vn/Egfr signaling is a potent inducer of Egfr target genes in the wing disk. Spi also has the potential to induce Egfr target genes in the wing disk. However, the ligands appear to evoke specific responses that result in different patterns of target gene expression. Other factors modulate the potential of Vn so that induction of Vn/Egfr target genes in the wing pouch is cell specific (Wessells, 1999).

Differences between Vn and Spi are apparent in the patterns of target gene induction resultant from the ectopic expression of Vn and soluble Spitz (sSpi) in the wing pouch. Effects have been noted for three Egfr target genes: aos, pntP1, and kekkon-1 (kek1). In each case, a different response to the ligands is seen. Both ligands induce ectopic aos expression but Vn does so in a broader domain than sSpi, however, neither induces aos in the L3/L4 intervein region. In the embryo, the Egfr target gene pntP1 mediates aos induction by sSpi. Likewise in the wing, ectopic sSpi induces pntP1 expression in cells that also expressed aos. However, following ectopic Vn no detectable change in pntP1 expression is seen using in situ hybridization and only very weak induction of pntP1-lacZ is seen in a domain that does not correspond fully with ectopic aos expression. This suggests either that another transcription factor mediates the induction of aos in response to Vn or that PntP1 is capable of inducing aos, even when changes in its own expression level are too low to be detected by current methods. Both Vn and sSpi induce ectopic expression of kek1, but predominantly in posterior cells rather than throughout the domain of their ectopic expression. Thus the action of both ligands appears limited by the presence or absence of some other factor in anterior cells. There is also a difference in the level of induction: Vn, which functions to induce kek1 expression in normal development, is a potent inducer of high levels of ectopic kek1 expression, whereas sSpi induces low levels of ectopic kek1 expression and appears to reduce expression of endogenous kek1 (Wessells, 1999).

During Drosophila oogenesis, asymmetrically localized Gurken activates the EGF receptor (Egfr) and determines dorsal follicle cell fates. Using a mosaic follicle cell system a mutation has been identified in the Cbl gene that causes hyperactivation of the Egfr pathway. Cbl is required in ventral follicle cells to ensure that ventral patterning occurs correctly in the embryo. Cbl proteins are known to downregulate activated receptors. The abnormal Egfr activation is ligand dependent. These results show that the precise regulation of Egfr activity necessary to establish different follicle cell fates requires two levels of control. The localized ligand Gurken activates Egfr to different levels in different follicle cells. In addition, Egfr activity has to be repressed through the activity of Cbl to ensure the absence of signaling in the ventral most follicle cells (Pai, 2000).

To examine the Cbl expression pattern in the ovary, in situ hybridization experiments were performed. Cbl mRNA is detected both in nurse cells and in follicle cells that are associated with the oocyte. The high levels of expression in nurse cells may indicate a maternal contribution of Cbl to the embryo: this is consistent with its presence at the blastoderm stage. To test the requirement for Cbl in embryonic development, germline clones for Cbl were generated using heat shock Flipase and the ovoD1 system. A distinct head defect is observed in embryos lacking both maternally and zygotically contributed Cbl. Zygotic Cbl is able to rescue the maternal lack of Cbl. This results in normal embryos that hatch. A small percentage of embryos had dorsalization phenotypes, which is likely due to the simultaneous generation of follicle cell clones induced by the heat shock Flipase. Egfr signaling is also required for ventral ectoderm development during embryogenesis. However, there were no other cuticle phenotypes detected in Cbl germline clone embryos, which suggests that Egfr signaling in cuticle development is not severely affected by loss of Cbl. In particular, no obvious segmentation defects, that would have suggested hyperactivity of the torso pathway, were detected. Taken together, these results demonstrate that in some signaling pathways involving receptor tyrosine kinases, loss of Cbl has no visible phenotypes, but in processes that are very sensitive to levels of receptor activity, such as in the follicle cells, a mutation of Cbl has dramatic effects (Pai, 2000).

To confirm that the dorsalized embryonic phenotype is caused by follicle cell clones homozygous for Cbl, and to determine in which follicle cells Cbl is required, the Cbl mutant cells were marked with a defective chorion 1 (dec) mutation. dec mutant follicle cells produce an abnormal egg shell resulting in an almost transparent appearance of the follicle cell imprints on the egg shell, in contrast to the opaque appearance of wild-type follicle cell imprints. Dorsalized embryos were observed only within mosaic egg shells. Furthermore, by correlating the position of the mutant clones on the egg shell with the region of dorsalization in the embryo, the spatial requirement for F165 (a Cbl mutation) activity in the establishment of ventral cell fates in the embryo could be determined. Surprisingly, only clones at ventral positions within the follicle cell epithelium result in a dorsalized embryo phenotype. Clones that were confined to the dorsal half of the egg shell do not produce a visible embryonic mutant phenotype. From these observations, it is concluded that Cbl is required in ventral follicle cells to ensure that ventral patterning occurs correctly in the embryo (Pai, 2000).

Although Cbl mutant dorsal follicle cell clones do not alter the pattern of the embryo, an effect of these clones was observed on the pattern of the egg shell when the clones occupied the dorsoanterior regions adjacent to the dorsal appendages. When dorsolateral cells are mutant for Cbl, dorsal appendages are shifted to more lateral positions. This shift of dorsal appendage could be due to an expansion of midline cell fates caused by higher Egfr activity at the dorsal-lateral position. This possibility was tested by staining for the expression of argos, a dorsal midline, cell-specific gene. Argos expression was expanded in several mosaic egg chambers with dorsal Cbl mutant clones. When cells situated more laterally, adjacent to the dorsal appendages, are mutant for Cbl, extra or wider dorsal appendages are produced. These egg shell phenotypes suggest that Egfr signaling is elevated in these dorsal Cbl mutant follicle cell populations (Pai, 2000).

To directly analyze the effects of the Cbl mutation on the Egfr pathway, the expression of a primary downstream target gene, kekkon 1 (kek), was examined in mutant clones. kek expression is induced in follicle cells in response to Egfr activation triggered by the germline specific ligand, Gurken. In grk mutants, kek expression is lost; conversely, expression of a constitutively active form of Egfr in all follicle cells results in kek expression in all follicle cells. In order to detect kek expression, a kek enhancer trap line, BB142, was used in which LacZ is expressed under the control of the kek promoter, and the ovaries were stained with anti-ß-galactosidase antibody. To visualize the Cbl mutant cells, Cbl was placed in trans to a c-myc-tagged chromosome. Homozygous mutant clones were detected by the absence of c-myc expression, and the wild-type cells were detected by staining positively with anti-c-myc antibody. In wild-type stage 7 and 8 egg chambers, kek is highly expressed in the posterior follicle cells that overlie the oocyte, and in a small number of the most anterior follicle cells. When Cbl mutant clones are induced in the anterior half of the egg chamber, these mutant cells ectopically express kek. In wild-type egg chambers at stages 9 to 10, after the oocyte nucleus has migrated to the dorsal anterior corner of the oocyte, kek expression becomes restricted to the dorsal cells that are exposed to Gurken signal. Ectopic kek expression is detected in mutant clones localized at the ventral side of stage 10 egg chambers. At both stages, this ectopic expression of kek is cell autonomous: all the mutant cells express kek, even at the clone boundary. The induction of kek expression in mutant clones demonstrates that the Cbl phenotype reflects activation of the Egfr pathway and indicates that this activation occurs in a cell autonomous manner. These results are consistent with the molecular nature of Cbl, given that Cbl downregulates receptor activity in signal receiving cells (Pai, 2000).

The ectopic expression of kek indicates that in the F165 mutant clones, the Egfr pathway is active in ventral follicle cells. This raises the question whether the effect of F165 on Egfr activity requires Gurken. To answer this question, F165 mutant clones were generated in a gurken mutant background and examined for kek expression. Surprisingly, no kek expression was detected in ventral Cbl mutant clones in gurken mutant egg chambers. The fact that ectopic kek expression was lost from the mutant clones in the absence of Gurken strongly suggests that ectopic activation of the Egfr pathway in Cbl mutant cells requires the presence of ligand to activate the receptor (Pai, 2000).

The wide-ranging defects in dendrites and axons indicate that sequoia functions to regulate axonal and dendritic morphogenesis in most neurons. Alternatively, it is conceivable that sequoia regulates the expression of genes generally required for neuronal differentiation. To gain mechanistic insight into sequoia function, the transcript profiles in wild-type and sequoia mutant embryos were compared based on microarray analyses of over 3,000 genes or ESTs, corresponding to about 25% of the Drosophila genome. The vast majority of these genes show comparable expression levels, including genes for cytoskeletal elements, genes that specify neuronal cell fates, and genes generally required for neurite outgrowth such as cdc42. Interestingly, a small fraction of the genes/ESTs analyzed showed clearly distinct expression ratios in sequoia mutants. Of these, 93 (3.1%) different transcripts were reduced by at least one-third of the wild-type level, and 34 (1.1%) different transcripts were increased by at least 75% of the wild-type level. A number of genes that appear to be regulated by sequoia, directly or indirectly, correspond to genes implicated in the control of axon morphogenesis rather than neuronal fate. These include known genes such as connectin, frazzled, roundabout 2, and longitudinals lacking, in addition to novel molecules with homology to axon guidance molecules including slit/kekkon-1 and neuropilin-2. It is noteworthy that two of the genes showing increased transcript ratios, roundabout 2 and CG1435, a novel calcium binding protein, were both also identified in a gain-of-function screen affecting motor axon guidance and synaptogenesis. In addition to genes that have clearly been implicated in axon development based on previous studies or sequence similarity, microarray data reveal that other genes potentially regulated by sequoia include peptidases, lipases, and transporters, as well as novel zinc finger proteins. It should be noted that transcripts that are broadly expressed and increased or decreased in sequoia mutants may actually be altered to a greater extent within neurons, because sequoia likely functions cell autonomously and is only expressed in the nervous system (Brenman, 2001).

Protein Interactions

To test whether the extracellular domain of kekkon-1, which contains five LRR and one Ig motif, is required for the inhibition of the Egfr activity by Kek1, transgenic lines were generated that contain either UAS-kek1extra or UAS-kek1intra. No phenotype is observed by overexpressing kek1intra in the follicle cells using the T155 or CY2 GAL4 lines. In contrast, overexpression of kek1extra using the same drivers led to eggs with reduced dorsal appendage materials. These observations indicate that the extracellular domain of Kek1 is sufficient to inhibit the activity of the Egfr. Although the phenotypes obtained following kek1extra overexpression are very similar to those obtained with full-length kek1, these overexpression phenotypes are weaker. One possibility is that Kek1extra is expressed at a lower level than the WT protein. Weaker effects were observed with 12 independent transgenic lines, as well as when multiple copies of the transgenes were added. This suggests that Kek1extra is less stable than the full-length Kek1 molecule or that the cytoplasmic domain of Kek1 participates to some extent in the inhibition. Finally, a test was performed to see whether the inhibition of the Egfr activity by kek1extra requires the presence of the endogenous Kek1 protein. When kek1extra is overexpressed in kek1 mutant females, the eggs are ventralized, which excludes a model whereby kek1extra blocks the Egfr by forming a heterodimer with the endogenous Kek1 protein (Ghiglione, 1999).

Having demonstrated that Kek1 acts to inhibit Egfr signaling, it was important gain insight into the mechanism of inhibition of the Egfr by Kek1. A test was performed to see whether Egfr and Kek1 physically associate. Following coexpression of a Myc-tagged version of Kek1 and the Egfr or the Drosophila Torso RTK in Sf9 cells, Kek1 was immunoprecipitated from the cell lysates using an anti-Myc antibody. Coprecipitation of the Egfr is observed by probing the resulting blot with the anti-Egfr antibody, suggesting that Kek1 associates physically with the Egfr and that this interaction is responsible for the inhibitory effect. When both Kek1 and Torso are coexpressed in Sf9 cells, no Torso is coprecipitated by Kek1, suggesting a selectivity of Kek1 for binding to the Egfr. Because the extracellular and transmembrane portions of Kek1 are essential for the inhibitory effect, an examination was carried out to see whether these portions of Kek1 would be able to bind to the Egfr. The extracellular domain, but not the intracellular domain, of Kek1 can bind to the Egfr. Thus, it is proposed that the inhibitory effect of the Egfr by Kek1 is mediated through direct association of the extracellular and transmembrane domains of Kek1 with the Egfr (Ghiglione, 1999).

Bipartite inhibition of Drosophila epidermal growth factor receptor by the extracellular and transmembrane domains of Kekkon1

In Drosophila, signaling by the epidermal growth factor receptor (EGFR) is required for a diverse array of developmental decisions. Essential to these decisions is the precise regulation of the receptor's activity by both stimulatory and inhibitory molecules. To better understand the regulation of EGFR activity inhibition of EGFR by the transmembrane protein Kekkon1 (Kek1) was investigated. Kek1 encodes a molecule containing leucine-rich repeats (LRR) and an immunoglobulin (Ig) domain and is the founding member of the Drosophila Kekkon family. This study demonstrates with a series of Kek1-Kek2 chimeras that while the LRRs suffice for EGFR binding, inhibition in vivo requires the Kek1 juxta/transmembrane region. It is demonstrated directly, and using a series of Kek1-EGFR chimeras, that Kek1 is not a phosphorylation substrate for the receptor in vivo. In addition, EGFR inhibition is shown to be unique to Kek1 among Kek family members, and this function is not ligand or tissue specific. Finally, a unique class of EGFR alleles has been identified that specifically disrupts Kek1 binding and inhibition, but preserves receptor activation. Interestingly, these alleles map to domain V of the Drosophila EGFR, a region absent from the vertebrate receptors. Together, these results support a model in which the LRRs of Kek1 in conjunction with its juxta/transmembrane region direct association and inhibition of the Drosophila EGFR through interactions with receptor domain V (Alvarado, 2004b).

Throughout development, EGFR activity specifies distinct cellular responses. Essential to this ability is the existence of an integrated network of regulatory molecules that direct receptor activity. Kek1, a member of a family of LRR- and Ig-containing molecules, represents a component of this network through its role as a feedback inhibitor of receptor activity. Deletion and mutagenesis studies have now demonstrated that the LRRs of Kek1, specifically LRR2 and G160, are essential for its association with, and consequently inhibition of, the receptor. The Kek1 cytoplasmic domain and associated Kek1 tail (KT) box have also been implicated (Alvarado, 2004b).

While it is clear that the Kek1 LRRs are essential for EGFR binding and inhibition, secreted forms of Kek1 are nonfunctional, indicating that membrane anchoring is likely to be an essential element to the inhibitory mechanism. Directly testing this, Kek1-Kek2 swaps demonstrate that while the Kek1 LRRs are sufficient for binding in vitro, they provide only minimal inhibition in vivo. Indeed, full inhibition is restored only when the entire extracellular and transmembane regions of Kek1 are placed in the context of a Kek2 backbone. This result supports an active role for the Kek1 jt/tm domain in inhibition, as a chimera containing the Kek1 LRRs in a Kek2 backbone is membrane tethered, but a weak inhibitor. This indicates that LRR-mediated binding alone is insufficient for receptor inhibition. Rather, the results suggest that Kek1-mediated inhibition of EGFR signaling is a bipartite process, in which the LRRs dictate EGFR binding and the jt/tm region facilitates inhibition. Phylogenetic analysis has indicated that this region is well conserved in Kek1 orthologs, supporting an important functional role for this region. Given this requirement for the Kek1 jt/tm region in inhibition, it was interesting to note that the SOK alleles identify three amino acids present in domain V of the receptor. Alteration of these three residues renders the receptor refractory to inhibition by Kek1 and activation by KEDeltaCG, respectively. Moreover, two of the changes, R738Q and E718K, represent viable alleles of the receptor, capable of ligand binding and receptor activation. Together with these binding data, these results assign a role for domain V in mediating regulation by Kek1. It is interesting to note that EGFR domain V represents a third cysteine-rich domain in Drosophila, which is absent in the vertebrate ErbBs. This raises intriguing structural and evolutionary questions, as Kek1 has been reported to associate with all human ErbBs. It will be important in the future to define those elements in the receptor that suffice for its inhibition by Kek1 and determine if additional distinctions in the interactions between Kek1 and the different receptor family members exist (Alvarado, 2004b).

Both direct (absence of phosphorylation) and indirect evidence (chimeras) is provided that Kek1 is not a phosphorylation substrate for the receptor. This was somewhat surprising, as structural work with the vertebrate receptor has indicated that the EGFR kinase domain is in a catalytically open configuration. Such a configuration is unique in that receptor tyrosine kinases normally require activation loop phosphorylation to relieve autoinhibitory interactions that prevent substrate binding and phosphorylation. In light of a distinct mechanism for activation of EGFR, one proposal put forth is the rotation twist model, in which ligand binding induces dimerized receptors to pivot in or near the transmembrane domain, thereby reorienting the kinase domains to their substrates. One potential explanation for the inability of the receptor to phosphorylate Kek1 is that the structure of the Kek1 jt/tm region might act to hinder such a rotation (Alvarado, 2004b).

Considering that kek1 knockouts exhibit subtle and dose-dependent phenotypes, one important question remaining is, what is the role of Kek1 in a cellular and developmental context? An initial explanation for the subtle LOF phenotype of kek1 with respect to EGFR inhibition was the possibility of functional redundancy between members of the Kek family. However, data for Kek2, Kek4, Kek5, and Kek6 indicate that EGFR inhibition is not a common feature of the Kek family. Alternatively, Kek1's inhibitory activity might reflect a recently acquired trait and not an ancestral or conserved role. However, analysis of kek1 orthologs in Drosophila virilis, D. pseudoobscura, and Anopheles gambiae argues against such a notion (Alvarado, 2004b)

Although it is not a common feature within the Drosophila Kek family, it is unclear whether inhibition of EGFR by Kek1 represents a more widely conserved regulatory mechanism for receptors. For instance, the LRR-containing transmembrane protein Decorin binds to the human EGFR and has been implicated in the regulation of receptor activity. It has been reported, however, that the motifs in the Decorin LRRs required for binding EGFR differ from those of Kek1, suggesting these two LRR molecules are unlikely to represent comparable regulatory modes (Alvarado, 2004b).

Finally, a role for Kek1 in the nervous system has also been reported. Expression of most kek family members is observed in the nervous system and recently three molecules that are structurally similar to Kek1, AMIGO1-3, have been implicated in neuronal development in vertebrates. It will be interesting to determine if Kek1 functions in neuronal development in an EGFR-independent manner and if such a role underlies its ancestral function (Alvarado, 2004b).


DEVELOPMENTAL BIOLOGY

Embryonic

Both kek1 and kek2 are expressed in neurons as they are differentiating in the embryonic central nervous system (CNS). kek1 is also expressed in other patterned epithelia, such as the follicle cells of the developing egg chamber, where it is found in a dorsal-ventral gradient around the oocyte. The homology of the kek genes to other known adhesion and signaling molecules, together with their expression patterns, suggests that both genes are involved in interactions at the cell surface. The coexpression of kek2 in the CNS leads to a suggestion that Kek1 is part of a family of cell surface proteins with redundant function (Musacchio, 1996).

During embryogenesis, the 15A6 lacZ insertion (marking kek1 expression) is first strongly detected at the beginning of germ band retraction (stage10) in a few cells in each segment at the dorsal position in the midline of the CNS. By early stage 12, the dorsal midline staining evolves to a ventral position, and some midline-staining cells begin to double-stain with mAB22C10, indicating a neuronal identity. This, together with their apparent migration from dorsal to ventral, suggests that these cells include the ventral unpaired medial cells (VUMs), which are among the first CNS neurons to differentiate. lacZ expression evolves by stage 12 to include many neurons of the CNS, and is maintained throughout larval development. In the peripheral nervous system, a small subset of ventral and lateral cell clusters express lacZ by stage 14. In abdominal segments, lacZ is also expressed in the cap cells, which are nonneuronal support cells that contact the chordotonal neurons and are identified by their distinct morphology and postion. In the head region, both neuronal and nonneuronal cells of the antennomaxillary complex begin expressing lacZ by stage 14 (Musacchio, 1996).

Kekkon2 expression was examined using RNA in situ hybridization. Like KEK1 mRNA, KEK2 mRNA is first expressed at stage 11 in a small, segmentally repeated group of cells at the dorsal midline of the CNS; it is then expressed in a ventral group of midline cells as it expands to many cells in the CNS. The pattern of this expression appears slightly different than that of KEK1, with several prominent dorsolateral cells staining at earlier stages. However, by stage 15, KEK2 is expressed in many cells of the CNS in a pattern similar to Kek1, although Kek2 appears to be expressed in a much larger subset of neurons than Kek1. In the PNS, a small group of lateral cell clusters and a group of ventral cell clusters also express KEK2. KEK2 transcript expression differs from the KEK1 pattern in its tissue specificity. Notably, at early stages (11 to early 12) it is transiently expressed in a lateral patch in the head, and at stage 14, it is expressed in segmentally repeated ventrolateral patches outside the CNS, which correspond to the postion of extending ventrolateral muscles (Musacchio, 1996)

Larval

The 15A6 lacZ insertion is expressed during larval development in all imaginal discs while those tissues undergo patterning. In the eye-antennal disc, lacZ is expressed throughout the differentiating retinal epithelium, from the posterior border up until several cells behind the morphogenetic furrow. lacZ is also expressed in a large patch that includes the presumptive ocelli and ocellar bristles (Musacchio, 1996).

Adult

The 15A6 line shows dynamic lacZ expression during the patterning of the follicle cell epithelium in the ovary. During oogenesis, lacZ is weakly expressed in the nurse cells and more strongly in the columnar follicle cells surrounding the developing oocyte. By stage 7 of oogenesis, lacZ expression is apparent in follicle cells at the posterior pole surrounding the oocyte. By stage 9, this posterior expression disappears and lacZ is found heavily expressed in follicle cells above the anteriorly placed oocyte nucleus. Interestingly, this pattern of lacZ expression reflects the onset of dorsoventral polarity in the egg chamber, and at stage 9 lacZ is distributed in a gradient. The follicle cells at this position later differentiate into the dorsal filaments and lacZ is expressed during their formation in the 15A6 line. Other follicle cells that express lacZ include the border cells, which migrate extensively throughout oogenesis (Masacchio, 1996).


EFFECTS OF MUTATION

kekkon-1 null mutants were generated. Axon tracts in the kek1 mutant CNS appear normal and CNS neurons expressing kek1 are present and in their normal positions. These results indicate that kek1 is not essential for viability and that removal of kek1 alone does not grossly affect the morphological development of the cells that express it (Musacchio, 1996).

Because of the severe inhibition of Egfr activity by Kek1, as revealed by the overexpression experiment, the effect of loss of kek1 function during oogenesis was reexamined. The spacing between the dorsal appendages of eggs derived from kek1 mutant females is increased when compared to WT. Further, these eggs are also mildly shorter and rounder, a phenotype consistent with a hyperactivation of the Grk/Egfr pathway. To substantiate the functional relationship between Kek1 and the Grk/Egfr signaling pathway during oogenesis, the effects of a loss of Kek1 activity were examined in two different genetic backgrounds. First, a test was performed to see if an increase in the level of Grk molecules could generate a stronger phenotype in the absence of kek1. Females carrying four copies of a transgene expressing grk (P[grk]), in addition to the two endogenous copies, are known to lay a significant fraction of partially or severely dorsalized eggs. In contrast, females that carry only one copy of P[grk] lay WT eggs. Eggs laid by females carrying one copy of P[grk] in the absence of kek1 are significantly dorsalized: the spacing between the dorsal appendages is increased and the eggs are rounder and smaller. Further, Twi expression that is normally expressed in embryos laid by females carrying one copy of P[grk] is repressed ventrally in 15% of the embryos derived from kek1 mutant females that carry a single copy of P[grk]. When raised at 29°C, 22% of the embryos derived from eggs laid by females carrying one copy of P[grk] in the absence of kek1 show a dorsalized phenotype readily detectable by aberrant Twi expression. There is a variability in the reduction of the Twi domain, ranging from embryos in which Twi-expressing cells are missing in the middle regions of the embryo to the most severe cases in which only the poles express the Twi protein (Ghiglione, 1999).

It was reasoned that if loss of Kek1 activity leads to a hyperactivation of Egfr activity, then loss of Kek1 activity should rescue a decrease in Egfr activity. This hypothesis was tested by generating flies that simultaneously lacked Kek1 activity and had reduced activity of the Egfr. Consistent with the hypothesis that Kek1 modulates Egfr signaling, loss of Kek1 activity results in partial suppression of the Egfr phenotype. Eggs laid by females mutant for Egfr alone have a ventralized phenotype. They exhibit either no or only a single dorsal appendage, and the eggs are significantly longer than WT. In contrast, the majority of eggs laid by the kek1- egfr- double mutant females have two dorsal appendages and their length is shorter, consistent with a reversion to a more WT phenotype (Ghiglione, 1999).

The lack of widespread axonal defects in the CNS of neurotactin mutants suggests that the function of Nrt in CNS morphogenesis might be largely replaced by functionally related molecules. If so, embryos lacking Nrt as well as one of these other molecules may display synergistic mutant phenotypes. To test this possibility, embryos lacking function of both nrt and one of several genes encoding neural CAMs were examined. Embryos of some double mutant combinations of neurotactin and other genes encoding adhesion/signaling molecules, including neuroglian, derailed, and kekkon-1, display phenotypic synergy. This result provides evidence for functional cooperativity in vivo between the adhesion and signaling pathways controlled by neurotactin and the other three genes (Speicher, 1998).

Knockouts of Kekkon1 define sequence elements essential for EgfR inhibition

Throughout development, cells utilize feedback inhibition of receptor tyrosine kinase (RTK) signaling as an important means to direct cellular fates. In Drosophila, epidermal growth factor receptor (EGFR) activity is tightly regulated by a complex array of autoregulatory loops, involving an assortment of inhibitory proteins. One inhibitor, the transmembrane protein Kekkon1 (Kek1) functions during oogenesis in a negative feedback loop to directly attenuate EGFR activity. Kek1 contains both leucine-rich repeats (LRRs) and an immunoglobulin (Ig) domain, two of the most prevalent motifs found within metazoan genomes. Kek1 is shown to inhibit EGFR activity during eye development and this role has been used to identify kek1 loss-of-function mutations that implicate the LRRs in directing receptor inhibition. Using a GMR-GAL4, UAS kek1-GFP misexpression phenotype, missense mutations were isolated in the kek1 transgene, affecting its ability to inhibit EGFR signaling. Genetic, molecular, and biochemical characterization of these alleles indicate that they represent two functionally distinct classes. Class I alleles directly diminish Kek1's affinity for EGFR, while class II alleles disrupt Kek1's subcellular localization, thereby indirectly affecting its ability to associate with and inhibit the receptor. All class I alleles map to the first and second LRRs of Kek1, suggesting a primary role for these two repeats in specifying association with and inhibition of EGFR. This analysis implicates glycine 160 of the second LRR in regulating EGFR binding (Alvarado, 2004a).

Typical LRRs consist of stretches of 21-25 amino acids and are defined by repeats of the conserved sequence LxxLxLxxN/CxL, where conservative substitutions of leucine for similar hydrophobic residues are common. However, the rest of the repeat can be highly divergent. Structurally, these motifs are composed of a ß-sheet, defined by the conserved sequence, which is connected to an alpha-helix. The entire set of LRRs is thought to form a horseshoe structure, with the hydrophobic ß-sheets lining the inside of the structure and the alpha-helices exposed to the outer surface (Alvarado, 2004a and references therein).

The class I allele kek196 disrupts a conserved leucine in the first LRR and is a partial suppressor. L136 is conserved in all Kek family members, suggesting that it does not directly dictate EGFR-binding specificity. In addition, the amino acids surrounding L136 at the n - 2, n - 1, and n + 1 positions are highly conserved in all Kek proteins. This reinforces the notion that L136 plays a structural rather than a direct role in association with the receptor. Consistent with a partial loss of activity in vivo, L136F also displays a reduction in EGFR-binding affinity as shown by co-immunoprecipitation. This partial loss-of-function phenotype could be due to the fairly conservative nature of the substitution, where leucine is changed to phenylalanine, a bulkier hydrophobic residue. Alternatively, the first LRR may meet only a small structural requirement for Kek1 function (Alvarado, 2004a).

The three remaining class I alleles change G160 in the second LRR and may represent the most functionally relevant mutations uncovered in the screen. Several lines of evidence support this suggestion: (1) glycine 160 is mutated in three separate suppressors: to serine in kek182 and kek153B and to aspartic acid in kek17C; (2) these alleles display strong suppression of the Kek1-GFP misexpression phenotype, in both the eye and the ovary, while exhibiting correct subcellular localization(3) G160 is conserved in Kek1 orthologs from Drosophila virilis and Anopheles gambiae, but is divergent in the other Kek family members; (4) these changes reduce the affinity of Kek1 for EGFR, but not for itself in co-immunoprecipitation experiments. These lines of evidence demonstrate that G160 is likely to play an instructive rather than a permissive role in mediating EGFR binding and inhibition. Together, the data from class I alleles suggest that the first and second LRRs function together to direct EGFR binding, consistent with recent findings that the LRRs are essential for inhibition of EGFR. The first repeat consequently may be required for the correct positioning of the second repeat in which G160 specifies EGFR binding (Alvarado, 2004a).

Notably, all class II alleles alter Kek1 subcellular localization and involve changes in proline residues that are conserved, with one exception, throughout the Kek family. Whereas EGFR, Kek1-GFP, and class I mutants localize primarily to the apical membrane of polarized follicle cells, class II alleles localize more uniformly throughout the cell and appear cytoplasmic in their distribution. Furthermore, within class II, strong suppressors display higher degrees of mislocalization than do intermediate suppressors. The class II allele kek1137 (P187S) affects the third LRR and is a suppressor with intermediate activity. This proline is conserved in all Kek1 orthologs and Kek family members, with the exception of Kek6. Two alleles of moderate strength, kek165 and kek1118, both mapped to a single residue (P309) located in the C-flank. N-terminal and C-terminal cysteine-rich flanks are capping motifs commonly associated with LRRs and are defined by the conserved positioning of cysteine residues. All Kek family members contain a proline at the same relative position as P309. kek165 (P309L) behaves as a slightly stronger suppressor than kek1118 (P309S), consistent with the higher degree of subcellular mislocalization in kek165. This minor difference in protein localization is likely caused by the nature of the substituting amino acid. The two remaining class II alleles are mutations in the Ig domain. Proteins with Ig domains constitute a superfamily of molecules with varied function in which the Ig domain confers protein-binding properties. kek182A (P329S) is the result of a change in the first amino acid of the Ig domain, which is conserved in all Kek family members. The allele with this change was the strongest suppressor identified in the screen and localizes uniformly throughout the cell. The mutation encoded by kek1176V (P356S) is also within the Ig domain, but it represents a moderate suppressor. Consistent with only a partial LOF in Kek1, this mutant protein, although localized abnormally, displayed a slight bias for the apical surface of follicle cells. Finally, co-immunoprecipitation experiments between class II alleles and EGFR reveals that most class II alleles have the intrinsic ability to bind the receptor with wild-type affinity. This strongly suggests that the suppression observed in vivo is due to reduced apical membrane localization of Kek1, consequently limiting its ability to interact with and inhibit the receptor. Thus, class II alleles define a set of distinct proline residues that promote Kek1 function through effects on subcellular localization (Alvarado, 2004a).

The cytoplasmic region of Kek1 is dispensable for EGFR binding and inhibition. No mutations affecting Kek1 function were recovered in this region. Interestingly, however, the C-terminal tail (48 amino acids) represents the most highly conserved portion of Kek1 between Drosophila and Anopheles. This portion of Kek1, like Kek2 and Kek5, contains a putative type 1 binding site (S/T-X-I/V/G) for proteins containing PDZ domains. Supporting this, deletion of the cytoplasmic domain of Kek1 can disrupt its trafficking. However, the fact that no mutations in the cytoplasmic domain were recovered in the screen suggests that loss of the cytoplasmic domain does not compromise Kek1 function in the eye. This is consistent with observations and suggests that this region may contribute to Kek1's inhibitory function in a more refined or tissue-specific fashion. Alternatively, the basis for this conservation might lie in an EGFR-independent role (Alvarado, 2004a).

In conclusion, inhibition of EGFR signaling by Kek1 occurs in multiple developmental processes and is mediated by the extracellular portion of Kek1. Mutations affecting Kek1's inhibitory activity are spread throughout the extracellular region, but reflect different LOF mechanisms. Specificity for EGFR binding is likely to reside to a large degree in the second LRR at G160. This residue was mutated in three different suppressors, affects the affinity of Kek1 for EGFR, and is unique to Kek1 among Kek family members. On the basis of this finding it is proposed that the second LRR underlies the binding specificity of Kek1 for EGFR and therefore its inhibitory function. Given this and the plethora of secreted and transmembrane molecules containing LRRs within the Drosophila genome, it will be important to determine if this sequence represents an EGFR interaction motif present in additional LRR-containing molecules and to decipher their contributions to EGFR signaling. Likewise, it will be interesting to determine if the analogous region in other Kek family members directs their function and if they act in a related manner on distinct receptors (Alvarado, 2004a).

Control of bract formation in Drosophila: poxn, kek1, and the EGF-R pathway

In Drosophila, the sensory organs are formed by cells that derive from a precursor cell through a fixed lineage. One exception to this rule is the bract cell that accompanies some of the adult bristles. The bract cell is derived from the surrounding epidermis and is induced by the bristle cells. On the adult tibia, bracts are associated with all mechanosensory bristles, but not with chemosensory bristles. The differences between chemosensory and mechanosensory lineages are controlled by the selector gene pox-neuro (poxn). This study shows that poxn is also involved in suppressing bract formation near the chemosensory bristles. The gene kek1, described as an inhibitor of the EGF-R signaling pathway, has been identified in a screen for poxn downstream genes. kek1 can suppress bract formation and can interfere with other steps of sensory development, including SMC determination and shaft differentiation (Layalle, 2004).

Misexpression of poxn at a late stage of mechanosensory bristle development has no effect on the morphology of the organ, but results in a suppression of bract formation. Misexpression of poxn and alteration of the EGF-R pathway affect bract formation during the same time window. It is concluded that poxn is responsible for the absence of a bract near the organs where it is expressed (Layalle, 2004).

kek1, a gene defined as an inhibitor of the EGF-R signaling pathway, is represented in a subtractive library enriched in genes that are specifically expressed in the chemosensory lineage. kek1 is not expressed in cells of the mechanosensory lineage at the time when bract induction takes place, and is expressed at a high level by the outer cells of the chemosensory organs. Its presence in the subtractive library, and differential pattern of expression between mechanosensory and chemosensory lineages, make kek1 a putative target of poxn (Layalle, 2004).

This point was confirmed by demonstrating that the expression of kek1 is modified following ectopic expression of poxn. Specifically, the ubiquitous expression of poxn results in the activation of kek1 expression in the outer cells of the mechanosensory lineage, where kek1 is normally silent. The activation of kek1 in mechanosensory cells is not complete in the experimental conditions that were used. It should be noted, however, that the repression of bract formation is also partial, suggesting that the overexpression of poxn is not complete. Altogether, this dataset reveals that kek1 is a target of poxn (although not necessarily a direct one) (Layalle, 2004).

The difference of expression of kek1 in the chemosensory and mechanosensory lineages, and the role of kek1 in modulating the EGF-R pathway, suggest a role for this gene in the control of bract formation. kek1 mutants do not show any abnormality in bract formation or in sensory organ development, however. More generally, the complete viability and wildtype phenotype of flies deleted for kek1 is a surprise, given the importance of the EGF-R pathway in many aspects of development (Layalle, 2004).

One obvious explanation for this absence of phenotype in kek1 mutant flies would be the existence of a functional redundancy between kek1 and another inhibitor of the EGF-R pathway. This possibility is supported by the identification of five kekkon-like genes in the Drosophila genome. Therefore this study relied on a gain-of-function analysis to decide whether kek1 might play a role in the control of bract induction (Layalle, 2004).

The EGF-R pathway has been implicated in the formation of the precursor cells for at least some of the macrochaetae on the notum. Since kek1 acts as an inhibitor of the EGF-R signaling pathway in ovary development, it might also inhibit this pathway in the notum and thereby interfere with the determination of macrochaetae. The overexpression of kek1 eliminates those macrochaetae that are most dependent on EGF-R signaling (Layalle, 2004).

Macrochaetae suppression was observed when the expression of kek1 was forced in the proneural cluster (using the sca-Gal4 driver), but not when its expression was forced after the SMC had been determined (using the neu-Gal4 driver). This shows that kek1 interferes with the formation of the precursor cells but not with subsequent steps of the lineage. It is concluded that, with respect to SMC determination, kek1 acts as an inhibitor of the EGF-R pathway, much as it does in the ovary. kek1 is expressed in the notum region of third instar wing discs. It may be, therefore, that kek1 plays a role in defining the position where SMCs are formed, or in defining the time window during which they are determined. The expression of kek1 in wing discs is very dynamic, however, and it has not been possible to determine whether this expression overlaps that of the proneural genes during normal development (Layalle, 2004).

The overexpression of kek1 induces a loss of mechanosensory shafts in the legs. At the latest step of the lineage the socket cell was found to express kek1 at a high level, whereas the shaft cell does not. The loss of shafts could therefore be due to a transdetermination of shaft towards socket fate. No socket duplication was observed however, and anti-Cut labeling demonstrated the absence of one of the two support cells at a frequency similar to that of shaft disappearance. It is concluded that the shaft cell has been lost rather than transformed (Layalle, 2004).

The ectopic expression of kek1 can prevent bract induction near mechanosensory bristles. This observation is entirely consistent with the idea that the control of kek1 expression contributes to the control of bract formation. In the sca-Gal4 line, bracts may be absent even when a shaft is formed, suggesting a direct effect of kek1 on bract formation. Since in this line kek1 expression is driven not only in the mechanosensory lineage but also in epidermal cells, it may be that this epidermal expression contributes to bract suppression. Whatever the case, the effect demonstrates that kek1 is capable of interfering with bract formation (Layalle, 2004).

The effect of Kek1 on EGF-R signaling has been shown to involve a direct interaction between the extracellular domains of the two proteins. At least part of the effect may be mediated by heterodimerization, implying that the two genes are expressed in the same cell. The observation that kek1 is expressed in the chemosensory support cells and affects bract formation by ectodermal cells suggests that the Kek1 protein may also interfere with the functioning of EGF-R proteins carried by an adjacent cell (Layalle, 2004).

Bract induction involves the activation of the EGF-R pathway in an epidermal cell, presumably through the expression of the EGF-R ligand, Spitz, by the outer cells of the sensory lineage. In the case of chemosensory lineages, or after ectopic expression of poxn at a late stage of the mechanosensory lineage, the presence of Poxn protein activates the expression of kek1 (and presumably of other members of the kek family). The Kek1 protein binds to the EGF-R and prevents the formation of a bract. The expression of a dominant-negative form of the receptor mimics this effect. When the dominant-negative is expressed both in bristle cells and in epidermal cells the inhibition of bract formation could be due to the inactivation of the EFG-R in the cells that receive the Spitz signal, i.e., in the epidermal cells. An absence of bracts is also observed when the dominant-negative form of the EGF-R is overexpressed only in bristles cells. In this case, it is proposed that the supernumerary receptors sequester the ligand and thereby prevent bract induction. Ligand sequestration would also account for the absence of bract cells when the normal EGF-R is overexpressed in the bristle outer cells (Layalle, 2004).

Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-β and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought (Kucherenko, 2008).

Interestingly, in the genetic screens several genes were found that showed phenotypes in oocyte development. One of these genes is kek1, a transmembrane protein of the Drosophila Kekkon family that has been reported to be a negative regulator of the EGFR receptor. It has been shown that EGFR signaling regulates the expression pattern of Dystroglycan to establish anterior-posterior polarity of oocyte. Further study is required to determine if kek1, as an EGFR regulator controls Dg expression in the germ line (Kucherenko, 2008).


EVOLUTIONARY HOMOLOGS

A cDNA encoding a protein designated as LIG-1 has been cloned and characterized. A fragment of this cDNA was found previously in a screen for genes up-regulated during neural differentiation in mouse P19 embryonal carcinoma cells. Comparative sequence analysis reveals LIG-1 to be a novel integral membrane glycoprotein (1091 amino acids) containing an extracellular region (794 amino acids) with a potential signal peptide, 15 leucine-rich repeats, 3 immunoglobulin-like domains (and 7 potential N-glycosylation sites), a transmembrane region of 23 amino acids, and a cytoplasmic region of 274 amino acids. This protein, therefore, is a new member of both the leucine-rich repeat and the immunoglobulin superfamilies. Furthermore, Northern blot and in situ hybridization analyses have shown LIG-1 gene expression to be predominantly in the brain, restricted to a small subset of glial cells such as Bergmann glial cells of the cerebellum and glial cells in the nerve fiber layer of the olfactory bulb. On the basis of its structural features and expression pattern, it is proposed that LIG-1 functions as a cell type-specific adhesion molecule or receptor at the glial cell surface, and plays a role in the nervous system, for example in neuroglial differentiation, development, and/or maintenance of neural functions where it is expressed (Suzuki, 1996).

cDNAs have been isolated for a novel protein with a calculated molecular mass of 46 kDa, containing a leucine-rich repeat (LRR) with conserved flanking sequences and a C2-type immunoglobulin (Ig)-like domain. This novel protein is considered to be a new member of the Ig superfamily and was named ISLR (immunoglobulin superfamily containing LRR). These domains are known to be important for protein-protein interaction or cell adhesion, and therefore it is possible that the novel protein ISLR may also interact with other proteins or cells. Northern blot analysis shows the presence of a 2.4-kb transcript in various human tissues including retina, heart, skeletal muscle, prostate, ovary, small intestine, thyroid, adrenal cortex, testis, stomach, and spinal cord as well as fetal lung and fetal kidney. The ISLR gene was mapped on human chromosome 15q23-q24 by fluorescence in situ hybridization (Nagasawa, 1997).

The isolation and characterization of a Xenopus sequence, XNLRR-1, is reported that is closely related to a gene for mouse neuronal leucine-rich repeat protein (NLRR-1). The cDNA clone is 4179 bp long and encodes a putative transmembrane glycoprotein of 718 amino acids, containing 12 leucine-rich repeats followed by one C2-type immunoglobulin-like domain and one fibronectin type-III repeat. XNLRR-1 is transcribed mainly in the developing eye area and the ventricular zone from diencephalon to hindbrain and slightly in spinal cord in Xenopus tadpoles. The similarity of the XNLRR-1 gene to other known cell adhesion molecules, together with the expression pattern, suggests that XNLRR-1 is involved in interactions at the neuronal cell surface (Hayata, 1998).

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila development. Whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4, was tested. In co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. Co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, it was observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. These observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function (Laederich, 2004).

Regulation of epidermal growth factor receptor (EGFR) signaling requires the concerted action of both positive and negative factors. While the existence of numerous molecules that stimulate EGFR activity has been well documented, direct biological inhibitors appear to be more limited in number and phylogenetic distribution. Kekkon1 (Kek1) represents one such inhibitor. Kek1 was initially identified in Drosophila melanogaster and appears to be absent from vertebrates and the invertebrate Caenorhabditis. To further investigate Kek1's function and evolution, kek1 orthologs have been identified within dipterans. In D. melanogaster, kek1 is a transcriptional target of EGFR signaling during oogenesis, where it acts to attenuate receptor activity through an inhibitory feedback loop. The extracellular and transmembrane portion of Kek1 is sufficient for its inhibitory activity in D. melanogaster. Consistent with conservation of its role in EGFR signaling, interspecies comparisons indicate a high degree of identity throughout these regions. During formation of the dorsal-ventral axis Kek1 is expressed in dorsal follicle cells in a pattern that reflects the profile of receptor activation. D. virilis Kek1 (DvKek1) is also expressed dynamically in the dorsal follicle cells, supporting a conserved role in EGFR signaling. Confirming this, biochemical and transgenic assays indicate that DvKek1 is functionally interchangeable with DmKek1. Strikingly, the cytoplasmic domain contains a region with the highest degree of conservation; this region has been implicated in EGFR inhibition and has been dubbed the Kek tail (KT) box (Derheimer, 2004).

Consistent with the known requirement of the extracellular and transmembrane domains of Kek1 for EGFR binding and inhibition, interspecies comparison has indicated a high degree of conservation throughout this region, with the exception of the N-terminal insert. The conservation of the transmembrane domain relative to the signal peptide (62% vs. 14% identity) was particularly striking and supports the notion of an essential function for this region. It will be interesting to determine if this conservation is the result of an EGFR-dependent or -independent function. The transmembrane and juxtamembrane portion of Kek1 displays limited identity with some transmembrane receptor-like kinases from Arabidopsis, also of the LRR superfamily. Future functional studies will be required to directly assess the relevance of such conservation and the contribution of this region to the in vivo function of Kek1. Finally, the highest degree of conservation detected in Kek1 was in the KT box. This striking conservation (92% identity across 48 aa) over 250 MYA argues strongly for an essential role in Kek1 function. This conservation might be due in part to its role in enhancing Kek1's ability to inhibit EGFR signaling. Whether conservation is solely representative of a contribution to Kek1's role in EGFR signaling or of an alternative function, perhaps contributing to Kek1's role in neuronal pathfinding, awaits further analysis (Derheimer, 2004).


REFERENCES

Search PubMed for articles about Drosophila kekkon-1

Alvarado, D., Rice, A. H. and Duffy, J. B. (2004a). Knockouts of Kekkon1 define sequence elements essential for Drosophila Epidermal growth factor receptor inhibition. Genetics 166: 201-211. 15020418

Alvarado, D., Rice, A. H. and Duffy, J. B. (2004b). Bipartite inhibition of Drosophila epidermal growth factor receptor by the extracellular and transmembrane domains of Kekkon1. Genetics. 2004 May;167(1):187-202. 15166146

Brenman, J. E., Gao, F.-B., Jan, L. Y. and Jan, Y. N. (2001). Sequoia, a Tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila. Dev. Cell 1: 667-677. 11709187

Derheimer, F. A., et al. (2004). Conservation of an inhibitor of the Epidermal growth factor receptor, Kekkon1, in dipterans. Genetics 166: 213-224. 15020419

Ghiglione, C., et al. (1999). The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis. Cell 96(6): 847-56. 99200395

Hayata, T., Uochi, T. and Asashima, M. (1998). Molecular cloning of XNLRR-1, a Xenopus homolog of mouse neuronal leucine-rich repeat protein expressed in the developing Xenopus nervous system. Gene 221(1): 159-66

Kucherenko, M. M., et al. (2008). Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex. PLoS ONE 3(6): e2418. PubMed Citation: 18545683

Laederich, M. B., et al. (2004). The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases. J. Biol. Chem. 279(45): 47050-6. 15345710

Layalle, S., et al. (2004). Control of bract formation in Drosophila: poxn, kek1, and the EGF-R pathway. Genesis 39: 246-255. Medline abstract: 15286997

Musacchio, M. and Perrimon, N. (1996). The Drosophila kekkon genes: novel members of both the leucine-rich repeat and immunoglobulin superfamilies expressed in the CNS. Dev. Biol. 178(1): 63-76. PubMed Citation: 8812109

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Biological Overview

date revised: 20 September 2008

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