Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.

Abstract. We have studied the expression of fibulin in cultured fibroblasts and determined its primary structure by eDNA cloning. Our results show that fibulin is a secreted glycoprotein that becomes incorporated into a fibriUar extracellular matrix when expressed by cultured cells or added exogenously to cell monolayers. In addition, we find that fibulin is present in plasma at a level of 33 + 3/~g/ml. Sequencing of multiple fibulin cDNAs indicates that a process of alternative splicing results in the expression of three fibulin transcripts. The transcripts encode overlapping polypeptides differing only in carboxy-terminal segments. Common to the three predicted forms of fibulin is a unique 537-amino acid-long cysteine-rich polypeptide and a 29-residue signal peptide. The amino-terminal portion of fibulin contains a repeated element with potential disulfide loop structure resembling that of the complement component anaphylatoxins C3a, C4a, and C5a as well as proteins of the albumin gene family. The bulk of the remaining portion of the molecule is a series of nine EGF-like repeats.F mULIN is a recently described calcium-binding protein which has been shown to interact with a synthetic peptide representing the cytoplasmic domain of the integrin/~1 subunit as well as native ot5/3~ fibronectin receptor (Argraves et al., 1989). Indirect immunofluorescent staining of cultured fibroblasts revealed that fibulin colocalized with the integrin/3~ subunit, in vivo, at sites of cellular interaction with underlying fibronectin substratum. It was therefore suspected that fibulin might be an intracellular protein involved with mediating cytoplasmic connections of the /31 integrins. Herein we report the results of characterization of fibulin expression and structure. The results indicate that fibulin is glycosylated and secreted by cultured fibroblasts and becomes incorporated into an extracellular matrix in a fashion similar to fibronectin. Salient structural features deduced from eDNA clones reveal that fibulin is a multidomain protein with two types of repeat motifs, one of which is homologous to the anaphylatoxins C3a, C4a, and C5a, as well as elements of proteins of the albumin gene family, and the other which is homologous to EGF. Materials and Methods AntibodiesThe following antisera were used for the immunoprecipitation and immunofluorescent staining experiments described herein: mouse monoclonal anti-human fibronectin (not cross-reactive with the bovine fibroneetin used in slide coating) was purchased from Telios Pharmaceuticals, La Jolla, CA; Kenneth Dickerson's present address is La Jolla Cancer Research Foundation, La JoUa, CA 92037. mouse monoelonal anti-human integrin #1 subunit was provided by Dr. E. Ruoslahti, La Jolla Cancer Research Foundation, La Jolla, CA; and rabbit anti-human fibulin serum was prepared in this laboratory and has been described previously (Argraves et al., 1989). As a precaution, the antifibulin serum used in the immunofluorescent staining experiments was absorbed on columns of human flbron...

[Keywords: Wnt pathway; ␤-catenin; APC tumour suppressor; deubiquitylation; K63-linked ubiquitin; NZF zinc finger; transcription] Supplemental material is available at http://www.genesdev.org.

The mRNAs of urokinase plasminogen activator (uPA) and its receptor, uPAR, contain instability-determining AU-rich elements (AREs) in their 3 untranslated regions. The cellular proteins binding to these RNA sequences (ARE uPA/uPAR ) are not known. We show here that the mRNA-stabilizing factor HuR functionally interacts with these sequences. HuR stabilized an ARE uPA -containing RNA substrate in vitro and stabilized in HeLa Tet-off cells both endogenous uPA and uPAR mRNAs and a ␤-globin reporter mRNA containing the ARE uPA . RNAi-mediated depletion of HuR in BT-549 and MDA-MB-231 cells significantly reduced the steadystate levels of endogenous uPA and uPAR mRNAs. Furthermore, we show that a constitutively active form of mitogen-activated protein kinase-activated protein kinase 2 (MK2), MK2-EE, has an ARE-mRNA-stabilizing effect that correlates with its ability to enhance the cytoplasmic accumulation of endogenous HuR, but not in cells cotransfected with a dominant negative version of MK2, MK2-K76R. These effects were mimicked by hydrogen peroxide treatment (oxidative stress), which resulted in the phosphorylation of endogenous MK2. In addition, hydrogen peroxide treatment enhanced the cytoplasmic binding of HuR to the ARE uPA , which was abrogated in cells transfected with MK2-K76R. These results indicate a role for HuR and MK2 in regulating the expression of uPA and uPAR genes at the posttranscriptional level.