InteractiveFly: GeneBrief

GDI interacting protein 3: Biological Overview | References

The Wingless (Wg/Wnt) signaling pathway is essential for metazoan development, where it is central to tissue growth and cellular differentiation. Deregulated Wg pathway activation underlies severe developmental abnormalities, as well as carcinogenesis. Armadillo/β-Catenin plays a key role in the Wg transduction cascade; its cytoplasmic and nuclear levels directly determine the output activity of Wg signaling and are thus tightly controlled. In all current models, once Arm is targeted for degradation by the Arm/β-Catenin destruction complex, its fate is viewed as set. This study identified a novel Wg/Wnt pathway component, Armless (Als; CG5469) that is required for Wg target gene expression in a cell-autonomous manner. Genetic and biochemical analyses showed that Als functions downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. In the absence of Als, Arm levels are severely reduced. Biochemical and in vivo studies showed that Als interacts directly with Ter94, an AAA ATPase known to associate with E3 ligases and to drive protein turnover. It is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, and it is proposed that Als promotes Wg signaling by rescuing Arm from proteolytic degradation, spotlighting an unexpected step where the Wg pathway signal is modulated (Reim, 2014).

The wingless (wg) gene was found nearly forty years ago with the characterization of a Drosophila mutant without wings. The gene encodes a secreted glycoprotein, the founding member of the Wnt family of signaling proteins. In the decades following its discovery, Wg/Wnt signaling has been shown to be essential during embryogenesis. Indeed, it is important throughout an organism's life, controlling also the homeostasis of different organs, for example, regeneration of epithelial cells in the intestine - the aberrant behavior of these cells in cancer is caused by constitutive Wg/Wnt signaling, which is consequently a key focus of medical and translational research (Reim, 2014).

The relay of the Wg signal is controlled at different levels. However, the pivotal step is the regulation of the levels of Armadillo (Arm)/β-Catenin, the key transducer of the Wg/Wnt pathway. A multiprotein complex consisting of the scaffold proteins Axin and APC and the kinases Shaggy/GSK3β and Casein kinase I (CKI) recruits and phosphorylates Arm/β-Catenin. This marks Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 ubiquitin ligase and subsequent degradation by the ubiquitin-proteasome system (UPS). When Wg/Wnt binds its receptors at the cell membrane, degradation of Arm/β-Catenin is prevented, presumably by protein interactions that lead to the dissociation of the E3 ubiquitin ligase from the Arm/β-Catenin destruction complex. As a consequence, Arm/β-Catenin translocates into the nucleus, where it adopts its role as a transcriptional effector of Wg/Wnt signaling. Although this step is crucial, and is a potential point of regulation, little is known about the players involved in the processing of Arm/β-Catenin and its ultimate degradation (Reim, 2014).

In a genome-wide RNA interference (RNAi) screen Armless (Als) was isolated as a regulator of proximodistal growth of Drosophila limbs, and has been shown in subsequent analyses to exert its function in the Wg pathway. Detailed genetic studies demonstrate that Als acts downstream of the destruction complex, at the level of the SCF/Slimb/βTRCP E3 Ub ligase. Cells depleted for Als exhibit strongly reduced Arm protein levels. Importantly, the activity of a constitutively active form of Arm, which cannot be phosphorylated and hence escapes ubiquitination and proteasomal degradation, is insensitive to depletion of Als. Using immunopurification and mass spectrometry analysis this study found that Ter94 interacts with Als. Ter94 is an AAA ATPase associated with protein turnover and proteasomal degradation. In sum, these data suggest that Als acts downstream of the Arm/β-Catenin destruction complex to positively regulate Arm protein levels, possibly by rescuing Arm from ubiquitination via Slimb. The human ortholog of Als, UBXN6, can substitute for Als in Drosophila, and Wnt target gene expression was impaired upon knock-down of UBXN6 in HEK-293 cells. It is thus infered that Als and UBXN6 represent regulators of a conserved mechanism that ensures appropriate levels of Armadillo/β-Catenin by antagonizing its entry into the UPS (Reim, 2014).

A prevalent mechanism for controlling information flow in signaling pathways is the alteration of the protein levels of key components. In the Wg/Wnt pathway, the Arm/β-Catenin destruction complex targets Arm/β-Catenin for ubiquitination by the SCF/Slimb/βTRCP E3 Ub ligase, resulting in proteasomal degradation and low cytoplasmic levels of Arm/β-Catenin in the Wnt pathway off state. If the pathway is turned on, Slimb-mediated ubiquitination is prevented, thus rescuing Arm from its proteasomal fate and causing a concomitant increase in Arm protein levels. This study describes Als as a new component of this control system; Als was found to be required to prevent the degradation of Arm/β-Catenin (Reim, 2014).

This study has identified als in a genome-wide in vivo RNAi screen in Drosophila. Because no EMS- or P-element-induced null allele was isolated, and because another gene overlaps with als, particularly thorough evidence validating als gene function was obtained. (1) The als phenotypes could be reproduced by nine different UAS-RNAi transgenes encoding independent RNA target sites. Together with an extended off target analysis, unintentional RNAi was ruled out as a cause for the als phenotypes. (2) RNAi-mediated inhibition of als expression was ascertained by monitoring als mRNA expression via real-time PCR and antisense mRNA in situ hybridization. (3) Expression of Als with different RNAi-insensitive rescue transgenes, as well as with its human ortholog UBXN6, rescued als phenotypes (Reim, 2014).

These analyses show that als encodes an essential positive Wg signaling component. This conclusion is based on the following evidence. als depletion caused wings with notched wing margins and loss of sensory bristles, which is characteristic of impaired Wg signaling. The distal wing region is most sensitive to als levels, as is the case for other positive components of Wg signaling. In agreement with this, increased als expression was found in the central wing pouch, at least in earlier L3 larval stages. Stimulation of the Wg pathway in wing imaginal discs or Kc-167 cells caused higher als expression, suggesting that als can be positively controlled by Wg signaling. However, Als levels must be precisely controlled since already mild overexpression of UAS-als elicits a dominant-negative effect on Wg signaling. The function of als for Wg signaling is not restricted to the wing: also in other tissues, such as the thorax, eyes, legs, and the embryo, als phenotypes are identical to those seen when Wg signaling is disturbed. Also in human HEK-293 cells UBXN6/UBXD1, the ortholog of Als, was found to be required for Wnt signaling, and human UBXN6 largely rescues the als phenotypes in Drosophila, which suggests their functional conservation. Depletion of als also enhanced Wg-sensitized phenotypes, further supporting the notion that its product is a Wg pathway component. Moreover, the expression of positively regulated Wg target genes is reduced or abolished upon loss of als function, while Wg-repressed target gene expression is ectopically activated. Importantly, while interfering with als function suppressed Wg signaling, it did not affect other pathways, such as Notch and Hh, Jak/Stat, or EGFR signaling. However, it cannot be ruled out that als is not required in another pathway in a different biological context. In humans, UBXN6 is reported to play a role in diverse scenarios: for example, it was shown to play a role in Caveolin turnover in human osteosarcoma U2OS cells. This might indicate a broader role of UBXN6 in mammalians (Reim, 2014).

The data show that Als regulates Armadillo protein levels. Based on epistasis experiments, Als acts downstream of Shaggy/GSK3β and upstream of the SCF/Slimb/βTRCP E3 Ub ligase, which is known to ubiquitinate Arm, a prerequisite for proteasomal degradation. Consistent with this, the degradation-resistant form of Arm could completely bypass the requirement for als, in contrast to the wild-type form of Arm. This suggests that proteasomal degradation acts downstream of als; however, this cannot be taken as an unambiguous proof. Importantly, depletion of ubiquitin and overexpression of CSN6, a negative regulator of SCF/Slimb/βTRCP E3 Ub ligase, could ameliorate the als phenotype (as well as phenotypes based on the overexpression of Axin or Shaggy, which overactivate the destruction complex, thus resulting in enhanced Arm degradation). In contrast, altering these factors did not ameliorate the Lgs phenotype, which is caused by interfering more downstream in the Wg pathway. These findings suggest that als works upstream of proteasomal degradation. A further informative experiment was monitoring Wg pathway components with respect to protein levels: Arr, Fz, Axin, APC, Sgg, and Arm. The only change in the absence of Als function was Arm: its levels were strongly reduced upon als depletion. The effects on Arm levels could be due either to a direct effect on Arm or to an indirect effect on a negative component. Importantly, the rate-limiting factor Axin as well as other key negative components of the Arm/β-Catenin destruction complex were unaltered uponals depletion (Reim, 2014).

Some further mechanistic insight was obtained with the finding that Ter94 interacts in vitro and in vivo with Als. Interestingly, Als-Ter94 was found to localize at the cell cortex, as was similarly observed for the Arm/β-Catenin destruction complex. The studies are consistent with earlier work that showed that the human ortholog of Ter94, p97, interacts with UBXN6 (Madsen, 2008). Ter94/p97/Cdc48 is a conserved and highly abundant AAA ATPase that was found to associate with SCF/Slimb/βTRCP E3 Ub ligases or proteasomal shuttle factors to mediate UPS-mediated protein degradation. Specifications of the diverse activities of Ter94/p97 and the fate of its substrates are mainly exerted by UBX domain protein co-factors, which eventually either promote or hinder p97's function in protein turnover; an example of the latter involves the dissociation of the SCF/Slimb/βTRCP E3 Ub ligase complex, eventually leading to its inactivation. Interestingly, it was recently reported that inactivation of the E3 ligase complex upon Wnt signaling is achieved by its dissociation from the destruction complex. Based on the current experiments and what is known about Ter94/p97, a possible mechanism is suggested that Als antagonizes Ter94's positive effect on E3 ligase function, thereby rescuing Arm levels. No increased protein levels were observed of Slimb, Axin, Shaggy, or APC in this analyses; thus, the results favor a model in which Als antagonizes Ter94 to hinder the transfer of Arm to the proteasome by interfering with the SCF/Slimb/βTRCP E3 Ub ligase function or its assembly. Importantly, no interaction was found between Arm and Als. This is consistent with the finding that UBX domain family members lacking an UBA domain, such as UBXN6/Als, do not directly interact with substrate proteins (Beskow, 2009), but are necessary for the activity or fate of the Ter94/p97 (Reim, 2014).

Interestingly, Zhang (2013) found that ter94 depletion affected the partial proteolysis of Ci. However, that study observed neither any typical consequence of disturbed Hh signaling per se (i.e., no alteration of Hh target gene expression in genes such as ptc) nor any phenotypical consequence upon overexpression of a dominant negative form of Ter94 (i.e., aberrant wing patterning and growth typical for Hh signaling). This is consistent with the current data that neither Ci target expression nor Hh signaling was affected upon als or ter94 depletion (Reim, 2014).

p97/Ter94 is known as a highly pleiotropic AAA ATPase associated with many cellular functions. Further, p97/Ter94 acts in multifaceted and large protein–protein complexes, and it is its regulatory co-factors, including UBX domain proteins, that render p97/Ter94 specific for a certain task in a particular cellular context. For example, p47/Shp1 is a co-factor of p97/Ter94 that blocks other co-factors from Ter94 binding (Kondo, 1997; Bruderer, 2004). Interestingly, in Kc-167 cell mass spectroscopy experiments, this study found p47 in Ter94/Als protein complexes, but only in the absence of Wg stimulation. On the other hand, als transcript and Als protein levels were elevated upon Wg signaling. These findings suggest a dynamic regulation of the Ter94 complex upon signaling inputs. The identification and functional analysis of all key components of the Als-Ter94 complex will be needed to obtain a refined insight into Als-Ter94's molecular mechanism (Reim, 2014).

Critically, this work spotlights an underappreciated facet in the control of the output of the entire canonical Wg/Wnt pathway - how Arm/β-Catenin is handed over to the proteasome - and the potential for regulating this step; this works also indicates that this step, in contrast to the conventional wisdom, is tunable. The identification and characterization of the UBX protein Als as a positive regulator of Wg/Wnt signaling contributes to this layer of pathway control (Reim, 2014).

Search PubMed for articles about Drosophila Armless

Beskow, A., Grimberg, K. B., Bott, L. C., Salomons, F. A., Dantuma, N. P. and Young, P. (2009). A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation. J Mol Biol 394: 732-746. PubMed ID: 19782090

Bruderer, R. M., Brasseur, C. and Meyer, H. H. (2004). The AAA ATPase p97/VCP interacts with its alternative co-factors, Ufd1-Npl4 and p47, through a common bipartite binding mechanism. J Biol Chem 279: 49609-49616. PubMed ID: 15371428

Kondo, H., Rabouille, C., Newman, R., Levine, T. P., Pappin, D., Freemont, P. and Warren, G. (1997). p47 is a cofactor for p97-mediated membrane fusion. Nature 388: 75-78. PubMed ID: 9214505

Madsen, L., Andersen, K. M., Prag, S., Moos, T., Semple, C. A., Seeger, M. and Hartmann-Petersen, R. (2008). Ubxd1 is a novel co-factor of the human p97 ATPase. Int J Biochem Cell Biol 40: 2927-2942. PubMed ID: 18656546

Reim, G., Hruzova, M., Goetze, S. and Basler, K. (2014). Protection of Armadillo/beta-Catenin by Armless, a novel positive regulator of Wingless signaling. PLoS Biol 12: e1001988. PubMed ID: 25369031

Zhang, Z., Lv, X., Yin, W. C., Zhang, X., Feng, J., Wu, W., Hui, C. C., Zhang, L. and Zhao, Y. (2013). Ter94 ATPase complex targets k11-linked ubiquitinated ci to proteasomes for partial degradation. Dev Cell 25: 636-644. PubMed ID: 23747190

date revised: 20 December 2014

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