The Ubiquitin specific protease USP34 regulates Axin stability and Wnt

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Mar 7, 2011 ... Our results indicate that USP34 functions downstream of the -. 2 8 ... AXIN homeostasis during Wnt signaling, interfering with USP34 function by ...

MCB Accepts, published online ahead of print on 7 March 2011 Mol. Cell. Biol. doi:10.1128/MCB.01094-10 Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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The Ubiquitin specific protease USP34 regulates Axin stability and Wnt/ -catenin signaling.

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Avais M. Daulat1, Stephane Angers*1,3

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Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of

Toronto, Canada 2

Division of Research and Development, Progenra, Inc., Malvern, Pennsylvania 19355, USA

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Department of Biochemistry, Faculty of Medicine, University of Toronto, Canada

*Corresponding author: Stephane Angers, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, M5S 3M2, Toronto, Ontario, Canada. Phone: 416-978-4939, Fax: 416-978-8511, E-mail: [email protected]

Running title: USP34 regulates Axin stability

Character count (Abstract, introduction, results, discussion, figure legends): 36325

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Tony TH Lui1, Celine Lacroix1, Syed M. Ahmed1, Seth J. Goldenberg2, Craig A. Leach2,

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Abstract Wnt proteins control multiple cell behaviors during development and tissue homeostasis. However, pathological activation of Wnt signaling is the underlying cause of various human diseases. The ubiquitin-proteasome system plays important regulatory functions within the Wnt pathway by regulating the activity of several of its core components. Hence, multiple E3 ubiquitin ligases have been implicated in its regulation. Less is known however about the role of Ubiquitin specific proteases in Wnt signaling. The analysis of purified AXIN-containing protein complexes by LC-MS/MS revealed the presence of the Ubiquitin protease USP34. Our results indicate that USP34 functions downstream of the CATENIN destruction complex to control the stability of AXIN and opposes its TANKYRASE-dependent ubiquitination. Reflecting on the requirement for tight control of AXIN homeostasis during Wnt signaling, interfering with USP34 function by RNA interference leads to the degradation of AXIN and to inhibition of

-catenin-mediated

transcription. Given the numerous human diseases exhibiting spurious Wnt pathway activation, the development of USP34 inhibitors may offer a novel therapeutic opportunity.

Keywords: Axin/ubiquitin/ubiquitin specific protease/Wnt signaling

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Introduction

During embryonic development and tissue homeostasis in adults, the Wnt family of secreted glycoproteins modulates several cell behaviors including differentiation, proliferation, cell movement and polarity (32, 37).

Malfunctioning Wnt-activated signaling pathways are

associated with multiple human diseases including cancer (10, 38). The etiology of colon carcinoma is a particularly striking example that reflects the critical importance of the integrity of this signaling cascade during intestinal epithelium homeostasis (45). Approximately 80% of all colon cancers are molecularly rooted in mutations of Wnt pathway components. These primarily consist of inactivating mutations in the gene coding for the tumor suppressor Adenomatous Polyposis Coli (APC) (44, 47, 51) but also of activating mutations in the transcription factor d-catenin (39) and loss of function mutations in the scaffolding protein Axin (22). APC and Axin are the core components of a cellular machinery dubbed the “destruction complex” that promotes the phosphorylation of the cytoplasmic pool of d-catenin (24). Axin, through binding to the destruction complex kinases Casein Kinase 1 alpha (CK1c) and Glycogen Synthase Kinase 3 (GSK3), orchestrates d-catenin phosphorylation (31). Phospho-d-catenin is in turns

recognized

by

the

SCFd-TrCP (Skp1-Cullin1-FBOX)

E3

ubiquitin

ligase

that

polyubiquitinates d-catenin and promotes its proteolysis by the proteasome (26, 59). The destruction complex thereby maintains low levels of cytosolic d-catenin in the absence of Wnt stimulation. The recognition of Wnt ligands by the cell surface receptor complex FrizzledLRP5/6 leads to the activation of Dishevelled (Dsh) (62), which promotes the GSK3- and CK1i3

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dependent phosphorylation of the LRP5/6 cytosolic domain (12, 63). The phosphorylated LRP5/6 cytosolic domain acts as a high affinity binding site for Axin (36, 53) that is suspected to inactivate the destruction complex and to lead to d-catenin accumulation. Stabilized d-catenin can then enter the nucleus and co-operate with LEF/TCF transcription factors to regulate Wntdependent transcriptional programs in a context dependent fashion (50). The ubiquitin proteasome system (UPS) is emerging as master regulator of Wnt signaling, controlling the pathway at multiple levels.

In addition to the well-characterized

function of the SCFd-TrCP E3 ligase for d-catenin ubiquitination in the absence of Wnt-driven signals (17, 26, 59), other proteins of the pathway are either targeted for degradation or regulated by the UPS. The ubiquitination of APC (9, 56) and Dishevelled (3, 54), for instance, leads to their proteasome-mediated degradation or to degradation-independent functional regulation. This dual regulation by the UPS depends on whether K48- or K63-linked ubiquitin chains are involved. Although the E3 ubiquitin ligase for APC has not been identified, this process is thought to involve Axin, at least for the situation where APC is degraded (56). Another example is the post-translational control of Dsh stability by the Cullin3-KLHL12 E3 ligase (3). Consistent with roles in both d-catenin-dependent and -independent Wnt pathways for Dsh, the activity of this E3 ligase was shown to impact both pathways in Xenopus and zebrafish embryos. Axin has also been postulated to be regulated through the modulation of its stability, which might be a necessary step for the activation of the d-catenin pathway (27, 58). The precise mechanisms regulating the degradation of Axin are however not known at present but its parsylation by Tankyrase and its sumoylation have recently been shown to control its ubiquitindependent degradation (20, 23).

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Due to the multiple roles of the UPS in Wnt signaling, it is likely that members of the ubiquitin specific proteases (USP) (also termed de-ubiquitinating enzymes or DUBs) regulate some of these events and could therefore have important functional roles in Wnt signaling. An estimated 79 USPs are present in humans that function to remove ubiquitin conjugates from target proteins (43). Supporting the possibility that USPs may regulate Wnt signaling, recent report have identified the ubiquitin protease Trabid (56) and USP4 (64) as novel regulators of this pathway. Trabid regulates APC function through the editing of its K63-conjugated chains whereas USP4 regulates TCF4 (64). A recurrent theme in Wnt signal transduction is the re-utilization of Wnt pathway components in different subcellular compartments, often to perform alternate functions. For example, Dsh has been localized to punctate structures within the cytoplasm (7, 49) or to the plasma membrane upon Wnt activation of the Frizzled-LRP receptor complex (5, 62). However, other studies have shown that Dsh is also translocated to the nucleus where it performs a required but ill-defined role during Wnt signaling (15, 21). d-catenin-independent Wnt signaling also likely involves the re-localization of Dsh to additional subcellular structures in order to modulate cytoskeleton-associated processes (4). Likewise, GSK3 acts primarily as a negative regulator of Wnt signaling by promoting the phosphorylation of d-catenin. However, as mentioned above, GSK3 also plays a positive role, at the plasma membrane, via the phosphorylation of the LRP5/6 Wnt co-receptor (12, 63) and has also been found to have nuclear roles (8). Similarly, in addition to its task in the destruction complex, a nuclear role has been proposed for APC in Wnt signaling. Indeed, APC contains bipartite nuclear localization and nuclear export signals that promote its nuclear cytoplasmic shuttling (18, 40, 46). Nuclear APC antagonizes d-cateninmediated transcription either by the modulation of d-catenin nuclear export (18), the 5

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sequestration of d-catenin away from an active transcription complex (41) or via its association with transcriptional repressors (16). In contrast, a recent genetic screen in Drosophila uncovered a positive functional role for APC homologs in Wg signaling (52). It is therefore a common theme in Wnt signaling that its effectors are re-utilized in a context-dependent manner. Axin, normally associated with the destruction complex, does not escape this trend as it is recruited to the activated and phosphorylated LRP5/6 co-receptor (36, 53) at the plasma membrane. Moreover, Axin is also known to shuttle between the nucleus and the cytoplasm (11, 57) and is greatly enriched in the nucleus of diverse cancer cell lines and tissues (1, 29, 48, 60). However the precise function of nuclear Axin in Wnt signaling is not well understood. Here, using a proteomic approach we show that Axin associates with Ubiquitin-Specific Protease 34 (USP34). Our results indicate that USP34 controls the levels of Axin and positively modulate Wnt signaling by acting downstream of d-catenin stabilization through controlling the nuclear accumulation of Axin.

Materials and methods

Plasmids Human AXIN1 and AXIN2 cDNAs were cloned by PCR from a human brain cDNA library into the pGLUE tandem-affinity purification plasmid (3) that contains streptavidin (SBP) and calmodulin binding peptides (CBP) to generate pGLUE-hAXIN1 and pGLUE-hAXIN2. AXIN1 was also cloned downstream of a cDNA coding for the Venus fluorescent protein in the pIRES-puro vector to generate the pIRES-puro-Venus-hAXIN1 plasmid. Human point mutant -CATENIN (pt.mutant-j ECVGPKP-CBP-HA-SBP) (34) and human DISHEVELLED-2 6

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(pGLUE-hDSH2) (3) were described previously. USP34core (residues 1892-2241) was expressed and purified as a HIS-tagged protein from E. coli. USP2core was expressed and purified as previously described (42). All PCR amplified regions were sequence validated. Detailed description of plasmid maps and sequences will be provided upon request and are posted on the lab web site (http://phm.utoronto.ca/angers/).

Reagents, tissue culture and transfection Human HEK293T, RKO colon carcinoma (ATCC: CRL-2577), SW480 colorectal adenocarcinoma (CCL-228), HCT116 colorectal carcinoma (CCL-247), and Mouse L cells (CRL-2647/CRL-2648) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO) in a 37°C humidified incubator with 5% CO2. HEK293T stable cell lines were generated by transfection with calcium phosphate followed by puromycin selection (2og/ml). Transient cDNA transfections were performed following the manufacturer’s recommendations using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). For siRNA experiments, cells were transfected with 20nM of siRNA with recommended amounts of Lipofectamine RNAiMax (Invitrogen). Previously validated siRNAs against

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CATENIN, AXIN1, AXIN2 (34), and Control-non targeting (Dharmacon, Lafayette, CO) were used, while a set of 4 siRNAs targeting USP34 was obtained from Dharmacon (cat# LQ-00608200-0002) and tested using western blotting. Within this set, USP34 siRNA “A” was the most effective and its target sequence was: 5’-GCAGGGAAGUUCUGACGAA-3’.

The target

sequences of the other USP34 siRNAs were: “B”: 5’-CAACAGAUCAGUAGUAAUU-3’; “C”: 5’-GCAGCUAUCCAGUAUAUUA-3’; “D”: 5’-CCAUGUGACUGGAGAUUUA-3’. 7

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For the epistasis experiments involving expression of pt.mutant- -CATENIN or DSH2 with a given siRNA, siRNA were first reverse transfected at the time of seeding cells, followed by replacement of media 24h after seeding and cDNA transfection using Lipofectamine 2000. Cells were then assayed 36h after cDNA transfection using the TopFlash reporter assay. pGIPZ based shRNA for USP34 were obtained from OpenBiosystems and screened for their efficiency by western blotting.

The target sequence of the most efficient USP34 shRNA was 5’-

CCTATGATGGTTGTTCAAATT-3’.

Wnt3A conditioned media Mouse L cells expressing Wnt3A (CRL-2647) were cultured until reaching 90% confluence, upon which media was collected and refreshed every two days for a total of 6 days. Media from different days was assayed using TopFlash assays to determine fractions with maximal activity and subsequently used for Wnt stimulation experiments. Conditioned media from parental Mouse L cells not producing Wnt3A (CRL-2648) was also collected to use as control.

Western Blotting/Antibodies Protein lysates were resolved with SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blots were stained with antibodies indicated in the Figure legends, then incubated with horseradish peroxidase-conjugated secondary antibody and detected by ejgoknwokpguegpeg0" Cpvkdqfkgu80 cells). Error bars represent S.E.M; Single star represents statistical significance compared to Ctl siRNA condition. (B) USP34 depletion also inhibits the nuclear accumulation of AXIN in HEK293T cells observed when the CRM1 dependent nuclear export is blocked with Leptomycin B.

Table 1. Representative LC-MS/MS analysis of (A) AXIN1 and (B) AXIN2 affinity-purified protein complexes. “Total Peptides” column denotes total number of peptides successfully matched to protein identity, while “Unique Peptides” signify number of distinct peptide fragments identified. Four independent purifications of AXIN1 and AXIN2 complexes were analyzed by LC-MS/MS, and one representative pull-down experiment is shown in the table. The frequency a protein was identified in the four pull-downs is noted in the ‘#n pulldowns’ column.

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