Regulation of Toll Signaling and Inflammation by b-Arrestin and the SUMO Protease Ulp1

The Toll signaling pathway has a highly conserved function in innate immunity and is regulated by multiple factors that fine tune its activity. One such factor is b-arrestin Kurtz (Krz), which we previously implicated in the inhibition of developmental Toll signaling in the Drosophila melanogaster embryo. Another level of controlling Toll activity and immune system homeostasis is by protein sumoylation. In this study, we have uncovered a link between these two modes of regulation and show that Krz affects sumoylation via a conserved protein interaction with a SUMO protease, Ulp1. Loss of function of krz or Ulp1 in Drosophila larvae results in a similar inflammatory phenotype, which is manifested as increased lamellocyte production; melanotic mass formation; nuclear accumulation of Toll pathway transcriptional effectors, Dorsal and Dif; and expression of immunity genes, such as Drosomycin. Moreover, mutations in krz and Ulp1 show dosage-sensitive synergistic genetic interactions, suggesting that these two proteins are involved in the same pathway. Using Dorsal sumoylation as a readout, we found that altering Krz levels can affect the efficiency of SUMO deconjugation mediated by Ulp1. Our results demonstrate that b-arrestin controls Toll signaling and systemic inflammation at the level of sumoylation. THE establishment of the immune system during development and subsequent maintenance of its homeostasis in the adult is under the control of several conserved signaling pathways (Evans et al. 2003; Lemaitre and Hoffmann 2007). A proper balance of activating and inhibitory mechanisms ensures a robust defense response to infection and at the same time prevents inappropriate activation of the immune pathways that can lead to inflammation, tissue damage, and cancer. In both insects and mammals, Toll and related receptors are central for the function of the immune system (Lemaitre and Hoffmann 2007; Ganesan et al. 2011). The core components of the Toll pathway in Drosophila include the ligand Spätzle, the receptor Toll, the intracellular adaptors MyD88 and Tube, and the kinase Pelle. Toll signaling regulates the nuclear accumulation of the NF-kB homologs Dorsal (Dl) and Dif. Inappropriate activation of the Toll pathway often leads to a systemic inflammation phenotype, which is defined as activation of blood cells and elevated expression of innate immunity genes in the absence of infection (Paddibhatla et al. 2010; Ganesan et al. 2011). An emerging control mechanism involved in the regulation of the core components of Toll/NF-kB signaling is sumoylation. Modification of proteins by SUMO (small ubiquitin-related modifier) has been shown to play a role in controlling multiple cellular functions, such as nucleocytoplasmic transport, transcriptional regulation, protein stability, signal transduction, and cell cycle progression (Geiss-Friedlander and Melchior 2007). In mammalian systems, the effects of sumoylation on NF-kB signaling are diverse and complex, including regulation of IkBa degradation, modulation of NEMO/IKKg activity, and both activation and repression of transcription by NF-kB (Mabb and Miyamoto 2007). In the Drosophila genome, components of sumoylationmachinery are mostly represented by single genes, making it an attractive system to dissect the role of sumoylation in Toll/NF-kB signaling (Talamillo et al. 2008a; Smith et al. 2012). Copyright © 2013 by the Genetics Society of America doi: 10.1534/genetics.113.157859 Manuscript received December 18, 2012; accepted for publication September 24, 2013; published Early Online September 27, 2013. Supporting information is available online at http://www.genetic.org/lookup/suppl/ doi:10.1534/genetics.113.157859/-/DC1. These authors contributed equally to this work. Corresponding author: Biology Department, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA 02125. E-mail: [email protected] Genetics, Vol. 195, 1307–1317 December 2013 1307 Genetic analyses as well as experiments in cultured cells showed the important but conflicting roles of sumoylation in regulating Toll signaling in Drosophila. Loss-of-function mutations in lesswright (lwr), which encodes the Drosophila E2 SUMO-conjugating enzyme Ubc9, were found to cause overproliferation of hematopoietic precursors, production of abnormally high levels of the differentiated blood cells called lamellocytes, and formation of lamellocyte-containing melanotic masses (Chiu et al. 2005; Huang et al. 2005; Paddibhatla et al. 2010; Kalamarz et al. 2012). Loss of lwr was also associated with increased expression of Drosomycin (Drs) as well as nuclear accumulation of the Toll transcriptional effector Dl in the hemocytes, suggesting that Ubc9 exerts an inhibitory effect on Toll signaling in larvae and protects the organism from abnormal inflammation (Chiu et al. 2005; Huang et al. 2005). However, in other reports, Ubc9/Lwr was shown to bind Dl directly and promote its sumoylation, which increased Dl nuclear retention and potentiated Dl-dependent transcriptional activation in cultured cells (Bhaskar et al. 2000, 2002). Moreover, components of the sumoylation machinery such as SUMO/Smt3 and Ubc9/ Lwr were required for expression of antimicrobial peptide genes in Drosophila larvae and cultured cells after exposure to microbial elicitors (Bhaskar et al. 2002), suggesting a positive role for sumoylation in Toll activation. Recently, sumoylation was shown to be required for antimicrobial gene expression in the second major regulator of immunity in Drosophila, the immune deficiency (IMD) pathway (Fukuyama et al. 2013). Therefore, further investigation at the genetic and molecular levels is required to elucidate the complex role of sumoylation in Toll signaling, immunity, and inflammation. Another level of control over the Toll/NF-kB pathway is exerted by b-arrestins. b-Arrestins were initially characterized as mediators of G protein coupled receptor (GPCR) desensitization and endocytosis (Pierce and Lefkowitz 2001). More recently, our knowledge of their signaling functions has dramatically broadened to involve a wide variety of signaling pathways and modes of regulation (Kovacs et al. 2009). The Drosophila genome encodes a single ortholog of b-arrestins, Kurtz (Krz) (Roman et al. 2000), which has been implicated in the regulation of GPCR signaling, as well as Notch, Hedgehog, receptor tyrosine kinase, and Toll pathways (Mukherjee et al. 2005; Ge et al. 2006; Tipping et al. 2010; Molnar et al. 2011; Li et al. 2012). Mammalian b-arrestin proteins can down-regulate NF-kB signaling by binding and stabilizing IkBa (Gao et al. 2004; Witherow et al. 2004) and by preventing autoubiquitination of TRAF6 (Wang et al. 2006). We have found that Krz limits the extent of Toll activation during embryogenesis (Tipping et al. 2010); however, the molecular details of this regulation are unknown. Here, we show for the first time that b-arrestin can control Toll signaling and systemic inflammation at the level of sumoylation. We demonstrate that loss of krz results in an up-regulation of Toll downstream effectors and that Krz exerts its functions by binding to Ulp1, a SUMO protease. Knockdown of Ulp1 leads to an increase in global sumoylation in vivo and causes Toll hyperactivity phenotypes that are similar to the effects of loss of krz. Using Dl as a target for Ulp1-mediated desumoylation, we show that Krz is required for the optimal SUMO protease activity of Ulp1, and that both proteins are necessary for limiting the Toll pathway activity and preventing an inappropriate inflammatory response. Materials and Methods Plasmid construction Drosophila Ulp1, Dorsal, SUMO/Smt3, and human SENP1 (Origene) open reading frames were amplified by PCR using tag and/or restriction site-containing primers and cloned into pMT/V5-His series vectors (Invitrogen) to generate carboxy-terminally tagged Ulp1-V5, Ulp1-SBP, and Dorsal-V5, and amino-terminally tagged Flag-SUMOGG (active processed form of SUMO) and Flag-SENP1. For in vitro translation, HA-Krz and Ulp1-V5 full-length open reading frames and fragments were cloned into the pSP73 vector (Promega). Dl-K382R-V5 was generated using site-directed mutagenesis. HA-Krz, HA-ARRB1, and HA-ARRB2 constructs were described previously (Tipping et al. 2010). Ulp1-V5 was cloned into the pUAST-attB vector (Bischof et al. 2007) for transgenic expression in flies.