Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study shows that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless, a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing (Jaiswal, 2006).
Cells of the dorsoventral (DV) boundary in the wing imaginal disc synthesize Wg. The DV boundary marks the distal end of the growing appendage, while the future hinge region, displaying Wg expression in two concentric rings, marks the proximal wing. The lacZ reporter of the quadrant enhancer of vestigial (vg), Q-vg-lacZ marks the entire distal wing [i.e. the presumptive wing blade (pouch) (Jaiswal, 2006).
In optical sections of the imaginal disc epithelium, AJs are visualized in the XY or XZ planes based on immunolocalization of DE-Cad and ß-catenin/Arm, besides binding with fluorochrome conjugated Phalloidin to F-actin. Both ß-catenin/Arm and DE-Cad display characteristic upregulation across the DV boundary along the PD axis of the wing imaginal disc. Optical sections along the XY plane reveal higher levels of DE-Cad localization and narrower apical circumferences in the AJs of cells flanking the DV boundary when compared with those of the more proximally located cells. Optical sections along the XZ plane further confirmed upregulation of DE-Cad. Thus, along the PD axis of the wing disc, cell shapes and DE-Cad levels are graded (Jaiswal, 2006).
Whether the PD gradient of cell shape and DE-Cad levels are linked to Wg signaling was tested. Somatic clones displaying constitutive Wg signaling (induced by overexpression of Dsh or of a degradation resistant variant of ß-catenin/Arm, ArmS10) induce cell-autonomous upregulation in the levels of DE-Cad and apical cell constrictions. Somatic clones expressing secreted Wg, however, are expected to induce non-cell-autonomous effects. Indeed, these clones induced non-cell autonomous and graded upregulation in the levels of DE-Cad in the AJs and changes in apical cell shapes. In the presumptive hinge region, Wg overexpression produces a more striking pattern of non-cell autonomous changes in cell shapes: cells neighboring the Wg-expressing cells appear to organize as whorls around the former and display epithelial misfolding (Jaiswal, 2006).
Furthermore, expression of GPI-anchored DFz2 receptor GPI-DFz2, which compromises Wg signaling, obliterates the characteristic PD gradient in the levels of DE-Cad and F-actin in the AJs. Finally, loss of Wg expression in the DV boundary of wing imaginal disc of Nts mutants grown at a restricted temperature also abolishes the PD gradient of DE-Cad and apical cell shapes. To further test if apical cell constrictions are linked to elevated levels of DE-Cad in AJs, DE-Cad was expressed in somatic clones. These clones were apically constricted, consistent with the role of DE-Cad/E-Cad in remodeling cell shape and tissue architecture. These results thus link Wg signaling to the PD gradient in the levels of DE-Cad and apical cell shapes in the wing imaginal discs (Jaiswal, 2006).
Somatic clones with altered cell-cell adhesion sort out from their neighbors and display smooth clone borders. Indeed, somatic clones displaying gain of Wg signaling owing to Dsh or ArmS10 misexpression sort out from their neighbors and display smooth clone borders, akin to those misexpressing DE-Cad. Wg signaling may alter cell-cell adhesion by enhancing recruitment of ß-catenin/Arm to the AJs and/or by its transcriptional input. In many cell types, for example, expression of cadherins rather than the levels of catenins appears to be the rate-limiting step of Catenin-Cadherin complex formation at AJs and cell-cell adhesion. Wild type ß-catenin/Arm (ArmS2), when overexpressed, does not transduce Wg signaling. Somatic clones overexpressing ArmS2 display 'wiggly' clone borders, unlike those expressing Dsh or ArmS10. Thus, expression of ß-catenin/Arm alone, without a concomitant enhancement of Wg signaling, fails to alter cell-cell adhesion. Cell-cell adhesion in wing imaginal disc epithelium is therefore likely to be regulated by transcriptional input from Wg signaling (Jaiswal, 2006).
To test if canonical Wg signaling regulates DE-Cad expression, the response of its lacZ reporter, DE-Cad-lacZ, was examined. Cells receiving high threshold of Wg signaling in the wing imaginal discs, as in those flanking the DV boundary, displayed higher levels of DE-Cad-lacZ reporter activity when compared with those further away from the source of Wg expression. Furthermore, somatic clones expressing ArmS10 or Dsh display cell-autonomous activation of the DE-Cad-lacZ. Finally, clones expressing the secreted Wg induce non-cell-autonomous activation of DE-Cad-lacZ: i.e., in cells within and surrounding the clones. Together, these results suggest that regulation of DE-Cad by the long-range activity of the Wg morphogen sets up the PD gradient of cell-cell adhesion and cell shape in the distal wing (Jaiswal, 2006).
Somatic clones lacking Ft (ft-/ft-), marked by loss of GFP, display overgrowth and altered cell-cell adhesion with characteristic circular and smooth clone borders, unlike the 'wiggly' borders of their wild type (ft+/ft+) twins that are marked by brighter GFP. Furthermore, cells lacking Ft displayed upregulation of DE-Cad in their AJs and DE-Cad-lacZ. By contrast, when Ft was overexpressed, levels of both DE-Cad or DE-Cad-lacZ were downregulated. Besides, following overexpression of Ft in the posterior wing compartment, cells flanking the DV boundary displayed wider apical circumferences when compared with those of the anterior wing compartment. These results suggest that Ft regulates DE-Cad expression, cell-cell adhesion and apical cell shapes in the distal wing (Jaiswal, 2006).
The results suggest that by regulating DE-Cad expression, Wg signaling integrates cell-cell adhesion with tissue growth and pattern. Regulation of DE-Cad expression could be a prevalent mechanism for coordination of the emerging pattern in an organ primordium with the spatial control of its cell-cell adhesion. For example, DE-Cad levels are also upregulated in cells flanking the stripe of cells along the AP boundary that express the morphogen Decapentaplegic (Dpp); misregulation of Dpp signaling also affects DE-Cad expression. The Ft tumor suppressor, by contrast, negatively regulates DE-Cad expression in the distal wing. This may also explain the inverse correlation between the levels of DE-Cad in AJs and the activity of Ft. Thus, besides its heterophilic binding with Ds, Ft controls cell-cell adhesions at AJs by regulating DE-Cad expression (Jaiswal, 2006).
Apart from cell-cell adhesion, DE/E-Cad regulation may impact a variety of other cellular processes and developmental mechanisms. E-Cad has been shown to mark the sites of actin assembly on cell surface. Cadherin complexes regulate cytoskeletal networks and cell polarity, while disruption of AJ associated components affects asymmetric cell division. Fat1, a mammalian homolog of Drosophila Ft, modulates actin dynamics. Interestingly, Ft also regulates orientated cell division (OCD) in imaginal epithelium, which is mirrored by orientation of the spindles of the dividing cells; OCD may also regulate organ shape along the PD axis. Misregulation of DE-Cad may thus affect the cytoskeleton and produce OCD phenotype in ft mutant discs (Jaiswal, 2006).
In both loss- and gain-of-function assays, this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained to be the direct targets of Wg, all available evidence so far suggests that these targets positively respond to Wg signaling. These results also show that Ft and Wg signaling intersect and control distal wing growth and pattern, presumably through their opposing regulation of a common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling, Dpp signaling also regulates Q-vg-lacZ; however, its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by its intersection with Wg signaling (Jaiswal, 2006).
The results show that Ft negatively regulates Wg signaling. Loss or gain of Ft induces a telltale sign of perturbations in Wg signaling, namely, changes in the cellular pool of ß-catenin/Arm, consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP) Wnt signaling pathways (Jaiswal, 2006).
One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).
Drosophila wing growth is under dynamic spatial and temporal regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact cell proliferation in their characteristic ways and activate distinct sets of PD markers. Although at a very high threshold, Wg signaling inhibits cell proliferation, at a modest threshold it has been shown to stimulate growth. It is noted that loss of Ft fails to activate Wg targets that demand a high threshold of Wg signaling, e.g., Ac, which is required for wing margin specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ, which responds to a high threshold of Wg signaling, is also not upregulated by loss of Ft. Dll responds to a higher threshold of Wg signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).
Over-proliferation in ft mutant imaginal discs is induced by perturbation of as yet unidentified disc-intrinsic mechanisms that determine the discs' characteristic final sizes. The imaginal discs of ft mutants continue to grow and the extent of their over-proliferation appears to be constrained only by the developmental time available during the extended periods of their larval life. By contrast, growth in wild-type imaginal discs is determinate, which ceases after they attain their predetermined sizes even under conditions of unlimited developmental time; for example, on transplantation into wild-type adult host abdomen that can sustain development. ft mutant imaginal discs thus acquire unlimited proliferative potential, akin to immortalization, a crucial step during tumorigenesis. It is significant that the Ft tumor suppressor downregulates Wg/Wnt signaling, a pathway implicated in cancers. Several orthologs of Ft have been identified in vertebrates with diverse functions. It will thus be interesting to explore if these orthologs of Ft in higher vertebrates also interact with Wnt signaling and thereby behave as tumor suppressors (Jaiswal, 2006).
Fat is an atypical cadherin that controls both cell growth and planar polarity. Atrophin (Grunge) is a nuclear co-repressor that is also essential for planar polarity; however, it is not known what genes Atrophin controls in planar polarity, or how Atrophin activity is regulated during the establishment of planar polarity. Atrophin is shown to bind to the cytoplasmic domain of Fat and Atrophin mutants show strong genetic interactions with fat. Both Atrophin and fat clones in the eye have non-autonomous disruptions in planar polarity that are restricted to the polar border of clones and there is rescue of planar polarity defects on the equatorial border of these clones. Both fat and Atrophin are required to control four-jointed expression. In addition mosaic analysis demonstrates an enhanced requirement for Atrophin in the R3 photoreceptor. These data suggest a model in which fat and Atrophin act twice in the determination of planar polarity in the eye: first in setting up positional information through the production of a planar polarity diffusible signal, and later in R3 fate determination (Fanto, 2003).
Mosaic analysis of ft mutant clones demonstrates a strong bias for the cell that retains ft function to become the R3 cell. This has been interpreted to indicate that Fat directly biases the cell to become an R3 cell. Atro clones also show a bias for the R3 cell to retain Atro function, supporting the model that Atro, like Fat, works in R3 fate determination (Fanto, 2003).
However, an extensive mosaic analysis found that all anterior cells (R1, R2 and R3) tend to be ft+, and all posterior cells (R4, R5 and R6) tend to be ft-. It has been suggested that this bias is due to spatial considerations, cells that are polar in the precluster, undergo a 90° rotation, leaving them in a posterior position in the adult. At the polar border of ft clones, ommatidia rotate in the opposite direction to wild type, therefore the bias is reversed, leading to an increase in ft- anterior cells. Therefore, it as been concluded that additional data are required to show that ft function is specifically needed in R3. To determine if ft and Atro are specifically required in the R3 photoreceptor, mosaic analysis of Atro, ft and wild-type clones has been undertaken. In wild-type clones, marked only by white, ommatidia at the polar border of the clone show a weak preference for posterior photoreceptors to be wild-type. This bias is strictly due to the spatial constraints of recruitment in clones. In ft clones, it has been found that at the equatorial border (where polarity is unaltered) there is no discernable difference between anterior photoreceptors subclass types; 85% of all photoreceptors that retain ft are anterior class. The increase from ~61% to 85% is probably due to the adhesive properties of ft, which result in smooth edged clones (Fanto, 2003).
By contrast, at the polar border, which is where planar polarity is altered, there is a marked tendency for ft function to be retained specifically in the R3 photoreceptor; 100% of R3 cells retained ft, whereas only 83% of all anterior photoreceptors retained ft. Mosaic analysis of Atro clones shows that the bias introduced by planar polarity (PP) alterations at the polar border of clones (similar to ft) introduces a general bias for anterior photoreceptors. However, again, the bias is stronger in the R3 cell than for other anterior photoreceptors. This increased bias in the R3 photoreceptor over the other anterior class member suggests that ft and Atro are important in R3 fate (Fanto, 2003).
The conclusion that Atro function is important for the R3 cell fate is also strongly supported by the observation that loss of Atro often results in symmetric ommatidia with two R4 cells. This is reflected by the increase in R4/R4 ommatidia seen in Atro clones in the eye disc marked by expression of the R4 marker, md-lacZ. In addition the overexpression of Atro in R3 and R4 generates symmetric ommatidia with two R3 cells. Together, these data support the proposal that Atro is needed for the R3 fate (Fanto, 2003).
The non-autonomous nature of the PP defects associated with ft and Atro mutant clones could have presented some problems to mosaic analysis. It might be expected that non-autonomous alterations in polarity would equally affect all photoreceptors, yet the data clearly show enhanced requirements for ft and Atro function in the R3 photoreceptor over other photoreceptors. In addition, the proposal that Atro is needed for the R3 cell fate is supported by analysis of the R4 marker in eye discs. Interestingly, the tendency to lose the R3 cell fate in Atro clones is seen throughout the clone, and does not appear to participate in the phenomena of equatorial rescue or polar nonautonomy (Fanto, 2003).
Because Frizzled (Fz) is also needed for R3 fate decisions, it has been suggested that Fat positively affects Fz signaling. The observation that Atro acts with Fat and also biases towards the R3 fate suggests that the regulation of Fz by Fat may not be direct. It is proposed instead that Atro is necessary for the ft-dependent bias to an R3 cell fate and for the production of a diffusible PP molecule that controls Fz activity (Fanto, 2003).
The proposal that Fat increases Fz activity, and thereby biases a cell towards the R3 fate, does not explain the non-autonomous disruptions of wild-type tissue on the polar side of ft and Atro clones, or the rescue of ft and Atro mutant tissue from wild-type tissue on the equatorial side of the clone. There are several models that could explain the non-autonomous disruptions of planar polarity. One model suggests that planar polarity is established through a 'domino effect'. This model is suggested by the striking accumulation of planar polarity components, such as Fz and Dsh on the distal edge of every cell in the wing. This observation, coupled with genetic data that suggest that high Fz activity on one side of the cell forces low Fz activity on the other side, leads to a model in which accumulation or loss of polarity in a cell leads to templating of that state onto the next cell, non-autonomously propogating PP defects. However in the eye, Fz and Dsh show differential distribution on only a subset of ommatidial precursor cells, and, importantly, intervening cells show no altered accumulation. These data argue against a simple templating model for PP in the eye (Fanto, 2003).
An alternative model suggests that the juxtapositioning of ft+ and ft- tissue contributes to midline determination and emphasizes the role of Fat in inhibiting DV signaling away from the equator. This inhibition would be relieved at the equator by an unidentified molecule that would inhibit Fat function. If this model were correct, a small ft clone should mimic the situation at the equator, where Fat function is predicted to be locally inhibited. One would therefore be expected to find an ectopic equator in the middle of the clone. Instead, however, the opposite phenotype is found, since the ommatidia on the two sides of the clone point toward the middle of the clone, rather than away from it (Fanto, 2003).
The model that best explains both the equatorial rescue and polar nonautonomy of ft and Atro clones is that Fat and Atro together control expression of a planar polarity morphogen, here called 'factor X'. It is imagined that factor X is in a gradient with high levels at the equator and low levels at the poles, thus all ommatidia will appear to 'point' down this gradient. If Fat and Atro are essential to the production of factor X, then the ft/Atro mutant tissue will be void of factor X, producing a sink in the gradient. The gradient will still be pointing in the same direction initially, explaining the wild-type polarity of ommatidia at the equatorial side of the clone and 'equatorial rescue' seen in ft and Atro clones. For ommatidia at the polar edge of the clone, the gradient will be reversed, and ommatidia will point in the opposite direction. The gradient will also be disrupted outside of the clone, leading to inversions of the polarity of wild-type tissue on the polar side of the clone and 'polar nonautonomy' seen in these mutant clones. In large clones, there will be a region in the center of the clone where there is no detectable factor X, and as a result polarity will be randomized. All of these predictions are met in ft and Atro clones. Loss of Ds, which inhibits Fat function, should increase factor X. As predicted by this model, ds clones show disruptions in wild-type tissue on the equatorial side of the clone, and rescue of mutant tissue on the polar side of the clone. Without ft or ds function there would be no gradient and, consistent with this prediction, complete loss of planar polarity is seen in eyes that are homozygous for strong alleles of ft or ds (Fanto, 2003).
A gradient of Wg protein (which is high at the poles and low at the equator) initially establishes a gradient of Ds protein over the eye field. This gradient of Ds protein in turn produces a gradient of Fat activity, which, it is believed, creates a gradient of Atro activity. It is proposed that each cell will produce factor X at a level that is proportionate to the level of Atro activity, which varies according to the position of that cell in the ds and ft activity gradients. This model assumes that Factor X is a short-range diffusible molecule, which provides polarity information to ommatidial preclusters to direct their rotation. Since Fat has been shown to be upstream of Fz, it is speculated that the Atro-dependant Factor X is a ligand for Fz (Fanto, 2003).
Both ft and Atro also act in other, apparently unrelated, pathways. One of the prominent features of ft mutant larvae is the loss of growth control, which leads to dramatically overgrown discs and mutant clones that are markedly larger than their sister twin spots. However, Atro- clones do not display overgrowth in the eye, suggesting that ft restricts growth via an Atro-independent pathway. In addition, in the adult eye Atro clones (unlike ft clones) show severe defects in photoreceptor number and type, suggesting Atro has additional roles in photoreceptor specification and/or survival that are not shared by Fat. One particularly surprising result was the finding that Atro- clones are markedly smooth before the furrow, and that this smoothness is lost after the furrow passes. This suggests that Atro may function in a cell adhesion process that is lost upon cell differentiation (Fanto, 2003).
Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominantly inherited neuronal degenerative disease characterized by the variable combination of ataxia, choreoathetosis, myoclonus, epilepsy and dementia. This disease is caused by the expansion of a polyglutamine tract within the Atrophin 1 protein. Atro is the sole fly homolog of human atrophins. Atro has been shown to act as a transcriptional co-repressor in vivo in Drosophila. Atro interacts genetically with even skipped, a transcriptional repressor, and is required for the in vivo repressive activity of even skipped. The transcriptional repressor activity of Atro has been localized to the highly conserved C-terminal region of Atro. This C-terminal region can bind to Even skipped in vitro and interacts with the minimal repression domain of Even skipped (Fanto, 2003).
This study has shown that the intracellular domain of Fat binds the C-terminal domain of Atro. The cytoplasmic expression of Atro and its interaction with Fat raises the possibility that instead of acting as a simple co-repressor, Atro functions in a more complex manner. Other transcriptional co-repressors are known to be converted to transcriptional activators upon cell signaling, and future work will determine if the interaction of Fat with Ds alters the transcriptional activity of Atro (Fanto, 2003).
Owing to the fact that Atro binds the cytoplasmic domain of Fat, a model is favored in which Atro acts downstream of Fat, possibly relaying a Fat-dependant signal to the nucleus. However, the similarity of the ft and atro loss-of-function phenotypes makes classical epistasis experiments difficult, therefore a model in which Atro acts upstream of ft cannot be excluded. Examination of the amount or subcellular distributions of Fat and Atro, suggest that Atro does not control Fat expression or localization, nor does ft control the levels or subcellular localization of Atro (Fanto, 2003).
The atypical cadherin Fat acts as a receptor for a signaling pathway that regulates growth, gene expression, and planar cell polarity. Genetic studies in Drosophila identified the four-jointed gene as a regulator of Fat signaling. This study shows that four-jointed encodes a protein kinase that phosphorylates serine or threonine residues within extracellular cadherin domains of Fat and its transmembrane ligand, Dachsous. Four-jointed functions in the Golgi and is the first molecularly defined kinase that phosphorylates protein domains destined to be extracellular. An acidic sequence motif (Asp-Asn-Glu) within Four-jointed is essential for its kinase activity in vitro and for its biological activity in vivo. These results indicate that Four-jointed regulates Fat signaling by phosphorylating cadherin domains of Fat and Dachsous as they transit through the Golgi (Ishikawa, 2008).
The Fat and Hippo signaling pathways intersect at multiple points and influence growth and gene expression through regulation of the transcriptional coactivator Yorkie. Fat signaling also influences planar cell polarity (PCP). Fat acts as a transmembrane receptor, and is a large (5147 amino acids) atypical cadherin protein, with 34 extracellular cadherin domains. Dachsous (Ds) is also a large (3503 amino acids) transmembrane protein with multiple cadherin domains and is a candidate Fat ligand because it appears to bind Fat in a cultured cell assay, acts non-cell autonomously to influence Fat pathway gene expression, and acts genetically upstream of fat in the regulation of PCP. A second protein, Four-jointed (Fj), also acts non-cell autonomously to influence Fat pathway gene expression and acts genetically upstream of fat in the regulation of PCP. However, Fj is a type II transmembrane protein that functions in the Golgi. Thus, Fj might influence Fat signaling by posttranslationally modifying a component of the Fat pathway (Ishikawa, 2008).
To investigate the possibility of modification of Fat or Ds, FLAG epitope-tagged fragments of their extracellular domains together were coexpressed with Fj in cultured Drosophila S2 cells. When the first 10 cadherin domains of Ds (Ds1-10) were coexpressed with Fj, a shift in mobility was observed. A common posttranslational modification of secreted and transmembrane proteins as they pass through the Golgi is glycosylation. Most glycosyltransferases contain a conserved sequence motif, Asp-X-Asp (DXD; X, any amino acid), which is essential for their activity. Because a related sequence motif [Asp-Asn-Glu (DNE) at amino acids 490 to 492] is present in Fj and its vertebrate homologs, a mutant form of Fj was created in which DNE was changed to GGG (FjGGG; G, glycine). The expression levels and Golgi localization of FjGGG appear normal, but FjGGG expression did not shift Ds1-10 mobility (Ishikawa, 2008).
To identify modified cadherin domains, smaller fragments of Ds1-10 were expressed. The smallest fragments whose mobility was shifted in cells expressing Fj were two-cadherin-domain polypeptides: Ds2-3, Ds5-6, and Ds8-9. Ds2-3 and Ds5-6 appeared to be stoichiometrically modified in cells expressing Fj, whereas Ds8-9 was only partially modified. Fat4-5 was also partially shifted by Fj coexpression. The mobility shifts of these two-cadherin-domain polypeptides were not observed with FjGGG. To identify potential sites of modification, their sequences were aligned. This identified four sites at which a Ser or Thr residue was conserved, whose hydroxyl groups could potentially be sites of posttranslational modification. To evaluate their influence, each was mutated in turn to Ala within the Ds2-3 polypeptide. Three of the four mutants had no effect; however, one, Ds2-3S236A (mutation of Ser236 to Ala), completely eliminated the Fj-dependent mobility shift. Introduction of an analogous mutation into Ds8-9 also eliminated its mobility shift. Thus, a Ser reside at a specific location within the second of the two cadherin domains was essential for the Fj-dependent mobility shift. This amino acid was a Ser in each of these dicadherin domains, but Thr was also compatible with the Fj-dependent modification. In a structurally solved cadherin domain, this Ser is the seventh amino acid and predicted to be located on the surface near the middle of the cadherin domain (fig. S2) (Ishikawa, 2008).
To identify posttranslational modifications associated with this mobility shift, Ds2-3 was purified from S2 cells expressing or not expressing Fj, the proteins were digested with trypsin, andthe resulting peptides were analyzed by mass spectrometry. One peptide from Fj-expressing cells was stoichiometrically shifted by 80 daltons relative to the same peptide from cells not expressing Fj, and it also eluted earlier on high-performance liquid chromatography (HPLC). Mass and tandem mass spectrometry (MS/MS) fragmentation patterns identified this peptide as amino acids 215 to 237 of Ds and refined the site of modification to within amino acids 232 to 237. The mass of the equivalent peptide from Ds2-3S236A was not altered by Fj expression. Most of the peptides corresponding to Ds2-3 cadherin domains were identified, and none of the others were detectably modified in cells expressing Fj. Thus, the Fj-dependent modification of Ds2-3 comprises an addition of 80 daltons, which is attached to Ser236. An 80-dalton mass does not correspond to that of any known glycans, but does correspond to the mass associated with addition of a phosphate group. Incubation of Fj-modified Ds fragments with either calf intestinal alkaline phosphatase (CIP) or Antarctic phosphatase (AnP) reversed the Fj-dependent mobility shifts of Ds2-3, Ds8-9, and Fat 4-5. Thus, Ds and Fat cadherin domains are subject to Fj-dependent phosphorylation at a specific Ser residue (Ishikawa, 2008).
To investigate whether Fj itself has kinase activity, a secreted, epitope-tagged Fj (sFj:V5) was purified from the medium of cultured S2 cells. Purified sFj:V5 was then incubated with affinity-purified Ds2-3 and [γATP (adenosine 5'-triphosphate)] in buffer. Transfer of 32P onto Ds2-3 was observed in the presence of sFj, but not in its absence, and not when sFjGGG was used as the enzyme. Moreover, Ds2-3S236A was not detectably phosphorylated by sFj. The activity of Fj expressed in a heterologous system was also characterized by expressing a glutathione S-transferase:Fj (GST:Fj) fusion protein in Escherichi coli and partially purifying it on glutathione beads. GST:Fj, but not GST:FjGGG, catalyzed the transfer of 32P onto Ds2-3. Thus, Fj is a protein kinase (Ishikawa, 2008).
The generic kinase substrates myelin basic protein and casein were not detectably phosphorylated by sFj. Thus, Fj appears to have a limited substrate specificity. Only a few proteins have been identified as being phosphorylated in the secretory pathway, and none of the responsible kinase(s) have been molecularly identified. A Golgi kinase activity, referred to as Golgi casein kinase, preferentially phosphorylates Ser or Thr residues within a S/T-X-E/D/S(Phos) consensus sequence. Because Fj does not phosphorylate casein, and the Ser residues within cadherin domains targeted by Fj do not conform to Golgi casein kinase sites, Fj is not Golgi casein kinase. Fj autophosphorylation was detected, but this reaction was weak compared to phosphorylation of Ds2-3. The autophosphorylation reaction is apparently unimolecular, because GST:Fj and sFj:V5 did not phosphorylate each other and the fraction of Fj phosphorylated was independent of concentration (fig. S5) (Ishikawa, 2008).
Some cadherin domain polypeptides that include a Ser as the seventh amino acid were not detectably shifted, but the mobility shift on Ds2-3 might reflect a conformational effect. To examine the ability of Fj to phosphorylate other cadherin domains, in vitro kinase reactions were performed with [γ-32P]ATP. This identified phosphorylation sites on polypeptides that were not gel shifted, including Fat2-3, Fat10-11, and Fat12-13. The in vitro kinase reactions also identified differences in the efficiency with which different cadherin domains were phosphorylated by Fj, with Ft3, Ds3, and Ds6 being the best substrates (Ishikawa, 2008).
If the presence of a Ser or Thr at the seventh amino acid of a cadherin domain is taken as the minimal requirement for Fj-mediated phosphorylation, there are nine potential sites in Ds and 11 in Fat. However, Fat10, Ds2, Ds11, Ds13, and Ds18 were not detectably phosphorylated, despite the presence of Ser or Thr at this position. Presumably, there are other structural features important for recognition by Fj. This was also emphasized by the detection of phosphorylation of our Ds2-3 polypeptide, but not the Ds3-4 polypeptide, even though both contain Ser236. The dicadherin constructs were based on published annotations, but in comparing Ds cadherin domains to structurally solved cadherin domains, it was realized that these misposition the intercadherin domain boundary, and consequently these constructs lacked three amino acids of the first cadherin domain. Addition of these amino acids, together with the intercadherin domain linker sequence, enabled phosphorylation of a Ds3 single-cadherin domain construct (Ishikawa, 2008).
A weak similarity between Fj and the bacterial kinase HipA, and between Fj and the mammalian lipid kinase phosphatidylinositol 4-kinase II (PI4KII), has been suggested previously on the basis of bioinformatic analyses in which HipA or PI4KII were used as the starting point for PSI-BLAST searches. Asp residues play critical roles in catalysis and in the coordination of Mg2+ in these and other kinases, and the loss of Fj kinase activity associated with mutation of the conserved DNE motif is thus consistent with the inference that Fj is related to other kinases. A single Fj ortholog, Fjx1, is present in a range of vertebrate species, including humans (Ishikawa, 2008).
To investigate the biological requirement for Fj kinase activity, the catalytically inactive fjGGG mutant was assayed in vivo. A V5 epitope-tagged form of this gene was expressed in transgenic Drosophila. At the same time, V5-tagged wild-type fj was contructed. To ensure that both forms were expressed in similar amounts, site-specific integration was used to insert transgenes at the same chromosomal location. Immunostaining confirmed that FjGGG:V5 and Fj:V5 both exhibited normal Golgi localization and were expressed in similar amounts. Uniform overexpression of fj reduces the growth of legs and wings and interferes with normal PCP. Fj:V5 exhibited phenotypes consistent with previous studies, but FjGGG:V5 was completely inactive. Thus, mutation of the DNE motif in Fj abolishes its biological activity (Ishikawa, 2008).
The identification of Fj's cadherin domain kinase activity provides a biochemical explanation for the influence of Fj on Fat signaling and supports a model in which Fj directly phosphorylates Fat and Ds as they transit through the Golgi to influence their activity, presumably by modulating interactions between their cadherin domains. Because there was a substantial difference in the efficiency with which individual cadherin domains could be modified by Fj, both in cell-based and in vitro assays, it is also possible that differences in the extent of Fat and Ds phosphorylation normally occur in vivo and might differentially modify their binding or activity (Ishikawa, 2008).
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