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

Ubiquitination is an essential post-translational modification that mediates diverse cellular functions. SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) belongs to the Nedd4 family of HECT ubiquitin ligases that directly catalyzes ubiquitin conjugation onto diverse substrates. As a result, SMURF1 regulates a great variety of cellular physiologies including bone morphogenetic protein (BMP) signaling, cell migration, and planar cell polarity. Structurally, SMURF1 consists of a C2 domain, two WW domain repeats, and a catalytic HECT domain essential for its E3 ubiquitin ligase activity. This modular architecture allows for interactions with other proteins, which are either substrates or adaptors of SMURF1. Despite the increasing number of SMURF1 substrates identified, current knowledge regarding regulatory proteins and their modes of action on controlling SMURF1 activity is still limited. In this study, we employed quantitative mass spectrometry to analyze SMURF1-associated cellular complexes, and identified the deubiquitinase FAM/USP9X as a novel interacting protein for SMURF1. Through domain mapping study, we found the second WW domain of SMURF1 and the carboxyl terminus of USP9X critical for this interaction. SMURF1 is autoubiquitinated through its intrinsic HECT E3 ligase activity, and is degraded by the proteasome. USP9X association antagonizes this activity, resulting in deubiquitination and stabilization of SMURF1. In MDA-MB-231 breast cancer cells, SMURF1 expression is elevated and is required for cellular motility. USP9X stabilizes endogenous SMURF1 in MDA-MB-231 cells. Depletion of USP9X led to down-regulation of SMURF1 and significantly impaired cellular migration. Taken together, our data reveal USP9X as an important regulatory protein of SMURF1 and suggest that the association between deubiquitinase and E3 ligase may serve as a common strategy to control the cellular protein dynamics through modulating E3 ligase stability.     

USP2 is an SKP2 deubiquitylase that stabilizes both SKP2 and its substrates

The stability of a protein is regulated by a balance between its ubiquitylation and deubiquitylation. S-phase kinase-associated protein 2 (SKP2) is an oncogenic F-box protein that recognizes tumor suppressor substrates for targeted ubiquitylation by the E3 ligase SKP1-Cullin1-F-box and degradation by proteasome. SKP2 is itself ubiquitylated by the E3 ligases APC/C^CDH1 and SCF^FBXW2, and deubiquitylated by deubiquitylases (DUBs) USP10 and USP13. Given the biological significance of SKP2, it is likely that the other E3s or DUBs may also regulate its stability. Here, we report the identification and characterization of USP2 as a new DUB. We first screened a panel of DUBs and found that both USP2 and USP21 bound to endogenous SKP2, but only USP2 deubiquitylated and stabilized SKP2 protein. USP2 inactivation via siRNA knockdown or small-molecule inhibitor treatment remarkably shortened SKP2 protein half-life by enhancing its ubiquitylation and subsequent degradation. Unexpectedly, USP2-stabilized SKP2 did not destabilize its substrates p21 and p27. Mechanistically, USP2 bound to SKP2 via the leucine-rich repeat substrate-binding domain on SKP2 to disrupt the SKP2-substrate binding, leading to stabilization of both SKP2 and these substrates. Biologically, growth suppression induced by USP2 knockdown or USP2 inhibitor is partially mediated via modulation of SKP2 and its substrates. Our study revealed a new mechanism of the cross-talk among the E3–DUB substrates and its potential implication in targeting the USP2–SKP2 axis for cancer therapy.     

The Deubiquitylating Enzyme USP44 Counteracts the DNA Double-strand Break Response Mediated by the RNF8 and RNF168 Ubiquitin Ligases

Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect of individually overexpressing the majority of human DUBs on RNF8/RNF168-mediated 53BP1 retention at DSB sites, we found that USP44 and USP29 powerfully inhibited this response at the level of RNF168 accrual. Both USP44 and USP29 promoted efficient deubiquitylation of histone H2A, but unlike USP44, USP29 displayed nonspecific reactivity toward ubiquitylated substrates. Moreover, USP44 but not other H2A DUBs was recruited to RNF168-generated ubiquitylation products at DSB sites. Individual depletion of these DUBs only mildly enhanced accumulation of ubiquitin conjugates and 53BP1 at DSBs, suggesting considerable functional redundancy among cellular DUBs that restrict ubiquitin-dependent protein assembly at DSBs. Our findings implicate USP44 in negative regulation of the RNF8/RNF168 pathway and illustrate the usefulness of DUB overexpression screens for identification of antagonizers of ubiquitin-dependent cellular responses.     

USP4 inhibits SMAD4 monoubiquitination and promotes activin and BMP signaling

SMAD4 is a common intracellular effector for TGF‐β family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin‐specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand‐induced regulatory (R)‐SMAD–SMAD4 complex formation. Whereas the interaction of the negative regulator c‐SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c‐SKI‐SMAD2 complex and triggers c‐SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF‐β family signaling. An increase in monoubiquitinated SMAD4 in USP4‐depleted mouse embryonic stem cells (mESCs) decreased both the BMP‐ and activin‐induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin‐ and BMP‐mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity.     

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF^Cdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF^Cdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF^Cdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF^Cdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.     

USP13 enzyme regulates Siah2 ligase stability and activity via noncatalytic ubiquitin-binding domains

The RING finger E3 ubiquitin ligase Siah2 is implicated in control of diverse cellular biological events, including MAPK signaling and hypoxia. Here we demonstrate that Siah2 is subject to regulation by the deubiquitinating enzyme USP13. Overexpression of USP13 increases Siah2 stability by attenuating its autodegradation. Consequently, the ability of Siah2 to target its substrates prolyl hydroxylase 3 and Spry2 (Sprouty2) for ubiquitin-mediated proteasomal degradation is attenuated. Conversely, inhibition of USP13 expression with corresponding shRNA decreases the stability of both Siah2 and its substrate Spry2. Thus, USP13 limits Siah2 autodegradation and its ubiquitin ligase activity against its target substrates. Strikingly, the effect of USP13 on Siah2 is not mediated by its isopeptidase activity: mutations in its ubiquitin-binding sequences positioned within the ubiquitin-specific processing protease and ubiquitin-binding domains, but not within putative catalytic sites, abolish USP13 binding to and effect on Siah2 autodegradation and targeted ubiquitination. Notably, USP13 expression is attenuated in melanoma cells maintained under hypoxia, thereby relieving Siah2 inhibition and increasing its activity under low oxygen levels. Significantly, on melanoma tissue microarray, high nuclear expression of USP13 coincided with high nuclear expression of Siah2. Overall, this study identifies a new layer of Siah2 regulation mediated by USP13 binding to ubiquitinated Siah2 protein with a concomitant inhibitory effect on its activity under normoxia.     

Ubiquitylation of an internalized killer cell Ig-like receptor by Triad3A disrupts sustained NF-κB signaling

Killer cell Ig-like receptor (KIR) with two Ig-like domains and a long cytoplasmic domain 4 (2DL4; CD158d) is a unique KIR expressed on human NK cells, which stimulates cytokine production, but mechanisms regulating its expression and function are poorly understood. By yeast two-hybrid screening, we identified the E3 ubiquitin ligase, Triad3A, as an interaction partner for the 2DL4 cytoplasmic domain. The protein interaction was confirmed in vivo, and Triad3A expression induced polyubiquitylation and degradation of 2DL4. Overexpression of Triad3A selectively abrogated the cytokine-producing function of 2DL4, whereas Triad3A short hairpin RNA reversed ubiquitylation and restored cytokine production. Expression of Triad3A in an NK cell line did not affect receptor surface expression, internalization, or early signaling, but significantly reduced receptor turnover and suppressed sustained NF-κB activation. 2DL4 endocytosis was found to be vital to stimulate cytokine production, and Triad3A expression diminished localization of internalized receptor in early endosomes. Our results reveal a critical role for endocytosed 2DL4 receptor to generate sustained NF-κB signaling and drive cytokine production. We conclude that Triad3A is a key negative regulator of sustained 2DL4-mediated NF-κB signaling from internalized 2DL4, which functions by promoting ubiquitylation and degradation of endocytosed receptor from early endosomes.     

Ring Finger Protein RNF169 Antagonizes the Ubiquitin-dependent Signaling Cascade at Sites of DNA Damage

Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance.     

HERC2-USP20 axis regulates DNA damage checkpoint through Claspin

The DNA damage response triggers cell-cycle checkpoints, DNA repair and apoptosis using multiple post-translational modifications as molecular switches. However, how ubiquitination regulates ATR signaling in response to replication stress and single-strand break is still unclear. Here, we identified the deubiquitination enzyme (DUB) USP20 as a pivotal regulator of ATR-related DDR pathway. Through screening a panel of DUBs, we identified USP20 as critical for replication stress response. USP20 is phosphorylated by ATR, resulting in disassociation of the E3 ubiquitin ligase HERC2 from USP20 and USP20 stabilization. USP20 in turn deubiquitinates and stabilizes Claspin and enhances the activation of ATR-Chk1 signaling. These findings reveal USP20 to be a novel regulator of ATR-dependent DNA damage signaling.     

USP15 stabilizes TGF-β receptor I and promotes oncogenesis through the activation of TGF-β signaling in glioblastoma

In advanced cancer, including glioblastoma, the transforming growth factor β (TGF-β) pathway acts as an oncogenic factor and is considered to be a therapeutic target. Using a functional RNAi screen, we identified the deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) as a key component of the TGF-β signaling pathway. USP15 binds to the SMAD7-SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) complex and deubiquitinates and stabilizes type I TGF-β receptor (TβR-I), leading to an enhanced TGF-β signal. High expression of USP15 correlates with high TGF-β activity, and the USP15 gene is found amplified in glioblastoma, breast and ovarian cancer. USP15 amplification confers poor prognosis in individuals with glioblastoma. Downregulation or inhibition of USP15 in a patient-derived orthotopic mouse model of glioblastoma decreases TGF-β activity. Moreover, depletion of USP15 decreases the oncogenic capacity of patient-derived glioma-initiating cells due to the repression of TGF-β signaling. Our results show that USP15 regulates the TGF-β pathway and is a key factor in glioblastoma pathogenesis.     

Identification and proteomic analysis of distinct UBE3A/E6AP protein complexes

The E6AP ubiquitin ligase catalyzes the high-risk human papillomaviruses' E6-mediated ubiquitylation of p53, contributing to the neoplastic progression of cells infected by these viruses. Defects in the activity and the dosage of E6AP are linked to Angelman syndrome and to autism spectrum disorders, respectively, highlighting the need for precise control of the enzyme. With the exception of HERC2, which modulates the ubiquitin ligase activity of E6AP, little is known about the regulation or function of E6AP normally. Using a proteomic approach, we have identified and validated several new E6AP-interacting proteins, including HIF1AN, NEURL4, and mitogen-activated protein kinase 6 (MAPK6). E6AP exists as part of several different protein complexes, including the proteasome and an independent high-molecular-weight complex containing HERC2, NEURL4, and MAPK6. In examining the functional consequence of its interaction with the proteasome, we found that UBE3C (another proteasome-associated ubiquitin ligase), but not E6AP, contributes to proteasomal processivity in mammalian cells. We also found that E6 associates with the HERC2-containing high-molecular-weight complex through its binding to E6AP. These proteomic studies reveal a level of complexity for E6AP that has not been previously appreciated and identify a number of new cellular proteins through which E6AP may be regulated or functioning.     

Ubiquitylation of MEKK1 inhibits its phosphorylation of MKK1 and MKK4 and activation of the ERK1/2 and JNK pathways

MEKK1 is a MAPK kinase kinase that is activated in response to stimuli that alter the cytoskeleton and cell shape. MEKK1 phosphorylates and activates MKK1 and MKK4, leading to ERK1/2 and JNK activation. MEKK1 has a plant homeobox domain (PHD) that has been shown to have E3 ligase activity. (Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., and Hunter, T. (2002) Mol. Cell 9, 945-956). MEKK1 kinase activity is required for ubiquitylation of MEKK1. MEKK1 ubiquitylation is inhibited by mutation of cysteine 441 to alanine (C441A) within the PHD. The functional consequence of MEKK1 ubiquitylation is the inhibition of MEKK1 catalyzed phosphorylation of MKK1 and MKK4 resulting in inhibition of ERK1/2 and JNK activation. The C441A mutation within the PHD of MEKK1 prevents ubiquitylation and preserves the ability of MEKK1 to catalyze MKK1 and MKK4 phosphorylation. MEKK1 ubiquitylation represents a mechanism for inhibiting the ability of a protein kinase to phosphorylate substrates and regulate downstream signaling pathways.     

The Protein Stability of Axin,a Negative Regulator of Wnt Signaling,Is Regulated by Smad Ubiquitination Regulatory Factor 2 (Smurf2)

Axin is a negative regulator of Wnt/β-catenin signaling via regulating the level of β-catenin, which is a key effector molecule. Therefore, controlling the level of Axin is a critical step for the regulation of Wnt/β-catenin signaling. It has been shown that ubiquitination-mediated proteasomal degradation may play a critical role in the regulation of Axin; however, the E3 ubiquitin ligase(s), which attaches ubiquitin to a target protein in combination with an E2 ubiquitin-conjugating enzyme, for Axin has not yet been identified. Here, we show that Smurf2 is an E3 ubiquitin ligase for Axin. Transient expression of Smurf2 down-regulated the level of Axin and increased the ubiquitination of Axin. Conversely, shRNA specific to Smurf2 blocked Axin ubiquitination. Essential domains of Axin responsible for Smurf2 interaction as well as Smurf2-mediated down-regulation and ubiquitination were identified. In vitro ubiquitination assays followed by analysis using mass spectroscopy revealed that Smurf2 specifically ubiquitinylated Lys⁵⁰⁵ of Axin and that the Axin(K505R) mutant resisted degradation. Knockdown of endogenous Smurf2 increased the level of endogenous Axin and resulted in reduced β-catenin/Tcf reporter activity. Overall, our data strongly suggest that Smurf2 is a genuine E3 ligase for Axin.