The FAM Deubiquitylating Enzyme Localizes to Multiple Points of Protein Trafficking in Epithelia, where It Associates with E-cadherin and β-catenin

Abstract

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, ␤-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with ␤-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with ␤-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with ␤-catenin and E-cadherin from a higher molecular weight complex (ϳ500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, ␤-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (ϳ2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased ␤-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and ␤-catenin during trafficking to the plasma membrane. INTRODUCTIONCorrect sorting of the E-cadherin cell-cell adhesion protein to the basolateral plasma membranes of epithelial cells is essential for polarization and maintenance of cell integrity and function. Sorting primarily occurs at the trans-Golgi network (TGN) and the route taken to the plasma membrane can either be direct, as observed in MDCKII cells, or indirect, as in hepatocytes (Maurice et al., 1994) and intestinal epithelia (Le Bivic et al., 1990;Matter et al., 1990). The indirect route entails initial transport to either the basolateral or apical surface, before internalization and sorting via endosomes and reinsertion into the lateral membrane. A dileucine repeat in the cytoplasmic domain of E-cadherin is necessary and sufficient for targeting E-cadherin to the basolateral membrane (Miranda et al., 2001). However, increased efficiency of E-cadherin delivery to the cell surface can be influenced by trans-acting factors, including the presence of the E-cadherin-binding protein ␤-catenin (Chen et al., 1999).Post-translational ubiquitylation can also influence protein trafficking (Katzmann et al., 2002;Schnell and Hicke, 2003). In the yeast Saccharomyces cerevisiae, mono-ubiquitylation of cytoplasmic domains is necessary and sufficient for the internalization of plasma membrane proteins (Hicke and Riezman, 1996;Shih et al., 2000) and has been suggested to be the primary endocytic signal for most, if not all, yeast proteins (Hicke, 2001). In cultured mammalian cells liganddependent ubiquitylation of cell surface receptors triggers their internaliza...

Identification of Ubiquitin-specific Protease 9X (USP9X) as a Deubiquitinase Acting on Ubiquitin-Peroxin 5 (PEX5) Thioester Conjugate

“…USP9X has been implicated in many different biological processes, including protein trafficking (45) and tight junction biogenesis in epithelial cells (70), regulation of the transforming growth factor-␤ pathway (51), and chromosome alignment and segregation (71). In addition, it has been shown that USP9X modulates the levels of proteins such as the following: ␣-synuclein (72), a protein involved in the pathogenesis of Parkinson disease; MCL1, an anti-apoptotic protein that is overexpressed in some types of cancer (73); some protein kinases involved in stress responses and cellular energy homeostasis (65,74); and some ubiquitin-ligases, such as MARCH7 and Itch (75,76).…”

“…45). Indeed, USP9X presents a Stokes radius of 7.3 nm, eluting from the SEC column as if it were a 550-kDa globular/spherical protein (see supplemental Fig.…”

The Ubiquitin Ligase RNF220 Enhances Canonical Wnt Signaling through USP7-Mediated Deubiquitination of β-Catenin

“…Although the deubiquitinase FAM (USP9X) is able to target and stabilize ␤-catenin, it has been suggested to be mainly involved in the trafficking of ␤-catenin and E-cadherin in epithelia (19). USP14 was also found to be overexpressed in lung adenocarcinoma and able to stabilize ␤-catenin; however, it was shown to work by directly targeting the upstream regulator Dishevelled (Dvl) (20,21).…”

Ubiquitin becomes ubiquitous in cancer

“…128 Later, it was established that USP9x positively regulates the TGFβ signaling pathway by targeting Smad4 for deubiquitination. 129 Monoubiquitination of Smad4 impedes its association with phosphorylated Smad2, which is a decisive step in the activation of the TGFβ signaling pathway.…”