Negative feedback regulation of Wnt signaling by Gβγ-mediated reduction of Dishevelled

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 41, No. 10, 695-706, October 2009 Negative feedback regulation of Wnt signaling by Gβγ-mediated reduction of Dishevelled 1 2 1 Hwajin Jung , Hyun Joon Kim , Suk Kyung Lee , 1 3 2 Rokki Kim , Will Kopachik , Jin-Kwan Han 1,4 and Eek-hoon Jho 1 Department of Life Science The University of Seoul Seoul 130-743, Korea 2 Division of Molecular and Life Sciences Pohang University of Science and Technology Pohang 790-784, Korea 3 Department of Zoology Michigan State University E. Lansing, MI 48824, USA 4 Corresponding author: Tel, 82-2-2210-2681; Fax, 82-2-2210-2888; E-mail, DOI 10.3858/emm.2009.41.10.076 Accepted 26 May 2009 Abbreviations: APC, adenomatous polyposis coli; GPCR, G protein coupled receptors; GSK3β, glycogen synthase kinase 3β; Gβ2, guanine nucleotide binding protein β2; NSCLC, non small cell lung cancer; PP2A, protein phosphatase type 2A; Iso, isoproterenol; β2AR, β2 adrenergic receptor Abstract Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gβ2) bound to Axin and Gβ2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gβ2γ2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gβ2γ2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by β-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gβ2γ2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gβ2γ2 is required because a mutant of Gβ2, Gβ2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gβ1 and Gβ2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled → Gβγ → +2 PLC → Ca /PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gβ2γ2 inhibits Wnt signaling by degradation of Dishevelled. Keywords: dishevelled proteins; feedback, biochemical; Frizzled receptors; heterotrimeric GTP-binding proteins; type C phospholipases; Wnt proteins Introduction Wnt signaling plays a pivotal role in diverse biological processes in embryonic development and in adult organisms (Clevers, 2006; Logan and Nusse, 2004; White et al., 2007). Mammals have 19 different Wnt ligands, 10 Frizzled receptors which are similar to serpentine G protein coupled receptors (GPCR) (Schulte and Bryja, 2007), plus other co-receptors such as LRP5/6 (He, 2003; Logan and Nusse, 2004). Binding of Wnt to its receptors activates downstream signaling events, such as the "canonical" pathway, mainly mediated by controlling the level of β-catenin via the ubiquitination/proteasome degradation pathway, or the "non-canonical" pathway which is controlled either +2 via Rac/Rho or Ca signaling (Kohn and Moon, 2005; Veeman et al., 2003). In some specific combinations of Wnt/Frizzled signaling the intracellular protein Dishevelled determines whether signaling proceeds by β-catenin-dependent or independent pathways. It is obvious that tight regulation of the signaling is critical since mis-regulation of Wnt signaling during embryonic development or postnatal life leads to different types of developmental defects, or diseases such as cancer, Alzheimer's disease or osteoporosis and others (http://www. stanford.edu/~rnusse/wntwindow.html). Axin is a scaffold protein that interacts with a number of proteins to regulate Wnt/β-catenin signaling (Lee et al., 2003; Tolwinski and Wieschaus, 2004; Kikuchi et al., 2006). In the absence of Wnt, 696 Exp. Mol. Med. Vol. 41(10), 695-706, 2009 Axin is part of a complex with adenomatous polyposis coli (APC) and glycogen synthase kinase 3β (GSK3β) whose action is to phosphorylate cytoplasmic β-catenin and thus target it for ubiquitinand proteasome-mediated degradation. When, however, Wnt binds to Frizzled and Dishevelled is activated then β-catenin degradation is inhibited, and after accumulating in the cytoplasm translocates to the nucleus where it binds to and activates TCF/LEF transcription factor (Logan and Nusse, 2004; Kikuchi et al., 2006). Although several mechanisms have been proposed how Dishevelled is activated in the presence of Wnt (Li et al., 1999; Chen et al., 2001), it is not clear how the activated Dishevelled signaling is later turned off. One way to turn off activated Dishevelled signaling is by activating the expression of its antagonist naked cuticle which causes down-regulation of Dishevelled (Zeng et al., 2000; Creyghton et al., 2005). The involvement of trimeric G protein signaling in the regulation of Wnt signaling was suggested by the finding that Frizzled interacts with the Gαsubunit of trimeric G proteins in mammalian cells and that G protein signaling directly transduces Frizzled signaling in Drosophila (Katanaev et al., 2005; Liu et al., 2005; Wang et al., 2006). Gα-protein signaling synergistically activates the Wnt/β-catenin pathway in colon cancer cells (Castellone et al., 2005; Yang et al., 2005), and bone formation is promoted by non-canonical Wnt-mediated G-protein signaling (Tu et al., 2007). We have found a role of Gβγ in Wnt signaling through the discovery that in yeast two-hybrid screening Axin binds to Guanine nucleotide binding protein, beta 2 (Gnb2; Gβ2). Here, we provide evidence for a novel negative feedback loop for Wnt signaling in which activated Gβγ signaling can turn off Wnt signaling via degradation of Dishevelled. Results Gβ interacts with Axin To confirm that the interaction between Axin and Gβ2 occurs in mammalian cells, Myc-tagged Axin Figure 1. Gβ interacts with Axin. (A) Co-immunoprecipitation of myc-Axin and Flag-Gβ2. Myc-tagged Axin and FLAG-tagged Gβ2 were transfected into HEK293T cells. The lysates were first subjected to immunoprecipitation (IP) using anti-Myc antibody, followed by western blotting (WB) using the antibodies indicated on the left side. β-catenin and GSK3β were used for positive controls. (B) Myc-tagged Axin with FLAG-tagged isoforms of Gβ or EYFP were transfected into HEK293T cells. Immunoprecipitation followed by western blotting was performed to test the specificity of interaction between Axin and isoforms of Gβ. (C) Deletion constructs of Axin with HA-tagged Gβ2 and Gγ2 were transfected into HEK293T cells and the expres (...truncated)