The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed-Lineage Kinase 3

Mixed-lineage kinase 3 (MLK3) activates mitogen-activated protein kinase (MAPK) signaling pathways and has important functions in migration, invasion, proliferation, tumorigenesis, and apoptosis. We investigated the role of the E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) in the regulation of MLK3 protein levels. We show that CHIP interacts with MLK3 and, together with the E2 ubiquitin-conjugating enzyme UbcH5 (UbcH5a, -b, -c, or -d), ubiquitinates MLK3 in vitro. CHIP or Hsp70 overexpression promoted endogenous MLK3 ubiquitination and induced a decline in MLK3 protein levels in cells with Hsp90 inhibition. Furthermore, CHIP overexpression caused a proteasome-dependent reduction in exogenous MLK3 protein. Geldanamycin (GA), heat shock, and osmotic shock treatments also reduced the level of MLK3 protein via a CHIP-dependent mechanism. In addition, CHIP depletion in ovarian cancer SKOV3 cells increased cell invasion, and the enhancement of invasiveness was abrogated by small interfering RNA (siRNA)-mediated knockdown of MLK3. Thus, CHIP modulates MLK3 protein levels in response to GA and stress stimuli, and CHIP-dependent regulation of MLK3 is required for suppression of SKOV3 ovarian cancer cell invasion.     

Crystal Structure of the Complex between Pseudomonas Effector AvrPtoB and the Tomato Pto Kinase Reveals Both a Shared and a Unique Interface Compared with AvrPto-Pto

Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9Å and of the AvrPtoB_121-205–Pto complex at a resolution of 3.3 Å. AvrPtoB_121-205 exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.     

Lipopolysaccharide Primes the NALP3 Inflammasome by Inhibiting Its Ubiquitination and Degradation Mediated by the SCFFBXL2 E3 Ligase

The inflammasome is a multiprotein complex that augments the proinflammatory response by increasing the generation and cellular release of key cytokines. Specifically, the NALP3 inflammasome requires two-step signaling, priming and activation, to be functional to release the proinflammatory cytokines IL-1β and IL-18. The priming process, through unknown mechanisms, increases the protein levels of NALP3 and pro-IL-1β in cells. Here we show that LPS increases the NALP3 protein lifespan without significantly altering steady-state mRNA in human cells. LPS exposure reduces the ubiquitin-mediated proteasomal processing of NALP3 by inducing levels of an E3 ligase component, FBXO3, which targets FBXL2. The latter is an endogenous mediator of NALP3 degradation. FBXL2 recognizes Trp-73 within NALP3 for interaction and targets Lys-689 within NALP3 for ubiquitin ligation and degradation. A unique small molecule inhibitor of FBXO3 restores FBXL2 levels, resulting in decreased NALP3 protein levels in cells and, thereby, reducing the release of IL-1β and IL-18 in human inflammatory cells after NALP3 activation. Our findings uncover NALP3 as a molecular target for FBXL2 and suggest that therapeutic targeting of the inflammasome may serve as a platform for preclinical intervention.     

The Arabidopsis EDR1 Protein Kinase Negatively Regulates the ATL1 E3 Ubiquitin Ligase to Suppress Cell Death

Loss-of-function mutations in the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced programmed cell death under a variety of abiotic and biotic stress conditions. All edr1 mutant phenotypes can be suppressed by missense mutations in the KEEP ON GOING gene, which encodes a trans-Golgi network/early endosome (TGN/EE)-localized E3 ubiquitin ligase. Here, we report that EDR1 interacts with a second E3 ubiquitin ligase, ARABIDOPSIS TOXICOS EN LEVADURA1 (ATL1), and negatively regulates its activity. Overexpression of ATL1 in transgenic Arabidopsis induced severe growth inhibition and patches of cell death, while transient overexpression in Nicotiana benthamiana leaves induced cell death and tissue collapse. The E3 ligase activity of ATL1 was required for both of these processes. Importantly, we found that ATL1 interacts with EDR1 on TGN/EE vesicles and that EDR1 suppresses ATL1-mediated cell death in N. benthamiana and Arabidopsis. Lastly, knockdown of ATL1 expression suppressed cell death phenotypes associated with the edr1 mutant and made Arabidopsis hypersusceptible to powdery mildew infection. Taken together, our data indicate that ATL1 is a positive regulator of programmed cell death and EDR1 negatively regulates ATL1 activity at the TGN/EE and thus controls stress responses initiated by ATL1-mediated ubiquitination events.     

Ubiquitination and Degradation of CFTR by the E3 Ubiquitin Ligase MARCH2 through Its Association with Adaptor Proteins CAL and STX6

Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.     

Ubiquitination and Degradation of the Hominoid-Specific Oncoprotein TBC1D3 Is Mediated by CUL7 E3 Ligase

Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8.     

Structural Basis of the Activation and Degradation Mechanisms of the E3 Ubiquitin Ligase Nedd4L

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The E3 Ubiquitin Ligase IDOL Induces the Degradation of the Low Density Lipoprotein Receptor Family Members VLDLR and ApoER2

We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism.     

Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation

Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.     

The Type II Hsp40 Sis1 Cooperates with Hsp70 and the E3 Ligase Ubr1 to Promote Degradation of Terminally Misfolded Cytosolic Protein

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.     

The Hect Domain E3 Ligase Tom1 and the F-box Protein Dia2 Control Cdc6 Degradation in G1 Phase

The accurate replication of genetic information is critical to maintaining chromosomal integrity. Cdc6 functions in the assembly of pre-replicative complexes and is specifically required to load the Mcm2-7 replicative helicase complex at replication origins. Cdc6 is targeted for protein degradation by multiple mechanisms in Saccharomyces cerevisiae, although only a single pathway and E3 ubiquitin ligase for Cdc6 has been identified, the SCF^Cdc4 (Skp1/Cdc53/F-box protein) complex. Notably, Cdc6 is unstable during the G₁ phase of the cell cycle, but the ubiquitination pathway has not been previously identified. Using a genetic approach, we identified two additional E3 ubiquitin ligase components required for Cdc6 degradation, the F-box protein Dia2 and the Hect domain E3 Tom1. Both Dia2 and Tom1 control Cdc6 turnover during G₁ phase of the cell cycle and act separately from SCF^Cdc4. Ubiquitination of Cdc6 is significantly reduced in dia2Δ and tom1Δ cells. Tom1 and Dia2 each independently immunoprecipitate Cdc6, binding to a C-terminal region of the protein. Tom1 and Dia2 cannot compensate for each other in Cdc6 degradation. Cdc6 and Mcm4 chromatin association is aberrant in tom1Δ and dia2Δ cells in G₁ phase. Together, these results present evidence for a novel degradation pathway that controls Cdc6 turnover in G₁ that may regulate pre-replicative complex assembly.     

Direct and Indirect Control of Mitogen-activated Protein Kinase Pathway-associated Components,BRAP/IMP E3 Ubiquitin Ligase and CRAF/RAF1 Kinase,by the Deubiquitylating Enzyme USP15

The opposing regulators of ubiquitylation status, E3 ligases and deubiquitylases, are often found to be associated in complexes. Here we report on a novel interaction between the E3 ligase BRAP (also referred to as IMP), a negative regulator of the MAPK scaffold protein KSR, and two closely related deubiquitylases, USP15 and USP4. We map the interaction to the N-terminal DUSP-UBL domain of USP15 and the coiled coil region of BRAP. USP15 as well as USP4 oppose the autoubiquitylation of BRAP, whereas BRAP promotes the ubiquitylation of USP15. Importantly, USP15 but not USP4 depletion destabilizes BRAP by promoting its proteasomal degradation, and BRAP-protein levels can be rescued by reintroducing catalytically active but not inactive mutant USP15. Unexpectedly, USP15 depletion results in a decrease in amplitude of MAPK signaling in response to EGF and PDGF. We provide evidence for a model in which the dominant effect of prolonged USP15 depletion upon signal amplitude is due to a decrease in CRAF levels while allowing for the possibility that USP15 may also function to dampen MAPK signaling through direct stabilization of a negative regulator, the E3 ligase BRAP.     

Structural Basis of the Binding of Merlin FERM Domain to the E3 Ubiquitin Ligase Substrate Adaptor DCAF1

The tumor suppressor gene Nf2 product, Merlin, plays vital roles in controlling proper development of organ sizes by specifically binding to a large number of target proteins localized both in cytoplasm and nuclei. The FERM domain of Merlin is chiefly responsible for its binding to target proteins, although the molecular basis governing these interactions are poorly understood due to lack of structural information. Here, we report the crystal structure of the Merlin FERM domain in complex with its binding domain derived from the E3 ubiquitin ligase substrate adaptor DCAF1 (also known as VPRBP). Unlike target binding modes found in ERM proteins, the Merlin-FERM binding domain of DCAF1 folds as a β-hairpin and binds to the α1/β5-groove of the F3 lobe of Merlin-FERM via extensive hydrophobic interactions. In addition to providing the first structural glimpse of a Merlin-FERM·target complex, the structure of the Merlin·DCAF1 complex is likely to be valuable for understanding the interactions of Merlin with its binding partners other than DCAF1.