USP9x-mediated deubiquitination of EFA6 regulates de novo tight junction assembly

Abstract

In epithelial cells, the tight junction (TJ) functions as a permeability barrier and is involved in cellular differentiation and proliferation. Although many TJ proteins have been characterized, little is known about the sequence of events and temporal regulation of TJ assembly in response to adhesion cues. We report here that the deubiquitinating enzyme USP9x has a critical function in TJ biogenesis by controlling the levels of the exchange factor for Arf6 (EFA6), a protein shown to facilitate TJ formation, during a narrow temporal window preceding the establishment of cell polarity. At steady state, EFA6 is constitutively ubiquitinated and turned over by the proteasome. However, at newly forming contacts, USP9x-mediated deubiquitination protects EFA6 from proteasomal degradation, leading to a transient increase in EFA6 levels. Consistent with this model, USP9x and EFA6 transiently co-localize at primordial epithelial junctions. Furthermore, knockdown of either EFA6 or USP9x impairs TJ biogenesis and EFA6 overexpression rescues TJ biogenesis in USP9x-knockdown cells. As the loss of cell polarity is a critical event in the metastatic spread of cancer, these findings may help to understand the pathology of human carcinomas.

Arf6 negatively controls the rapid recycling of the β2AR

“…We have previously shown that EFA6 is involved in actin cytoskeleton reorganization and in epithelial cell polarity (Derrien et al, 2002;Franco et al, 1999;Luton et al, 2004;Théard et al, 2010). In these two processes, the EFA6 C-terminal domain is strictly required presumably through its ability to directly interact and remodel F-actin organization (Macia et al, 2008), while Arf6GTP alone cannot reproduce the full function of EFA6 (Klein et al, 2008;Luton et al, 2004).…”

“…As the localization of EFA6 does not seem to be affected by agonist or serum exposure, the exchange activity could result from a structural conformational change of the protein. Specific serum-activated receptors would trigger post-traductional modifications of EFA6 such as phosphorylation or ubiquitylation (Théard et al, 2010). A PKC-induced phosphorylation of the Arf1-GEF Arno/cytohesin family has been previously described (Santy et al, 1999) and seems to negatively affect the exchange activity.…”

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).…”

Deubiquitinase FAM/USP9X Interacts with the E3 Ubiquitin Ligase SMURF1 Protein and Protects It from Ligase Activity-dependent Self-degradation

“…USP9X Deubiquitinates SMURF1 and Protects It from Proteasomal Degradation-Previous studies have characterized USP9X as a potent deubiquitinase that can deconjugate ubiquitin chains from a plethora of protein substrates (31)(32)(33)(34)(35). In this study, we identified USP9X as an interacting protein for SMURF1, which undergoes active autoubiquitination and selfdegradation (12,39).…”

“…The major consequence of DUB-initiated deubiquitination includes ubiquitin biogenesis/recycling, substrate stabilization, and cell signaling modulation through ubiquitin-chain editing (29,30). Among the ϳ100 DUBs encoded by the human genome, the ubiquitin-specific peptidase 9, X-linked (USP9X/FAM), is implicated in multiple physiological pathways through targeting a variety of substrates, including ␤-catenin, AF-6, and EFA6 (31)(32)(33). More recently, pro-survival factor MCL-1 and Parkinson disease pathogenic protein ␣-Synuclein were also reported as substrates for USP9X, expanding the role of USP9X into tumor cell apoptosis and neurodegenerative diseases (34,35).…”