CD2AP/SHIP1 complex positively regulates plasmacytoid dendritic cell receptor signaling by inhibiting the E3 ubiquitin ligase Cbl

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA-induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.     

Ubiquitination of Inositol-requiring Enzyme 1 (IRE1) by the E3 Ligase CHIP Mediates the IRE1/TRAF2/JNK Pathway

Deciphering the inositol-requiring enzyme 1 (IRE1) signaling pathway is fundamentally important for understanding the unfolded protein response (UPR). The ubiquitination of proteins residing on the endoplasmic reticulum (ER) membrane has been reported to be involved in the UPR, although the mechanism has yet to be fully elucidated. Using immunoprecipitation and mass spectrometry, IRE1 was identified as a substrate of the E3 ligase CHIP (carboxyl terminus of HSC70-interacting protein) in HEK293 cells under geldanamycin-induced ER stress. Two residues of IRE1, Lys⁵⁴⁵ and Lys⁸²⁸, were targeted for Lys⁶³-linked ubiquitination. Moreover, in CHIP knockdown cells, IRE1 phosphorylation and the IRE1-TRAF2 interaction were nearly abolished under ER stress, which may be due to lacking ubiquitination of IRE1 on Lys⁵⁴⁵ and Lys⁸²⁸, respectively. The cellular responses were evaluated, and the data indicated that CHIP-regulated IRE1/TRAF2/JNK signaling antagonized the senescence process. Therefore, our findings suggest that CHIP-mediated ubiquitination of IRE1 contributes to the dynamic regulation of the UPR.     

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.     

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3.     

Differential regulation of the B cell receptor-mediated signaling by the E3 ubiquitin ligase Cbl

The E3 ubiquitin ligase Cbl has been implicated in intracellular signaling pathways induced by the engagement of the B cell antigen receptor (BCR) as a negative regulator. Here we showed that Cbl deficiency results in a reduction of B cell proliferation. Cbl-/- B cells show impaired tyrosine phosphorylation, reduced Erk activation, and attenuated calcium mobilization in response to BCR engagement. The phosphorylation of Syk and Btk is also down-modulated. Interestingly, Cbl-/- B cells display enhanced BCR-induced phosphorylation of CD19 and its association with phosphatidylinositol 3-kinase. Importantly, Lyn kinase activity is up-regulated in Cbl-/- B cells, which correlates inversely with the Cbl-mediated ubiquitination of Lyn. Because Lyn has both negative and positive roles in B cells, our results suggested that Cbl differentially modulates the BCR-mediated signaling pathways through targeting Lyn ubiquitination, which affects B cell development and activation.     

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2^S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg⁹⁷/Lys⁹⁸, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.     

The E3 Ubiquitin Ligase Triad3A Negatively Regulates the RIG-I/MAVS Signaling Pathway by Targeting TRAF3 for Degradation

The primary role of the innate immune response is to limit the spread of infectious pathogens, with activation of Toll-like receptor (TLR) and RIG-like receptor (RLR) pathways resulting in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin ligase that interacts with several components of TLR signaling and modulates TLR activity. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys⁴⁸-linked, ubiquitin-mediated degradation of the tumor necrosis factor receptor-associated factor 3 (TRAF3) adapter. Triad3A was induced following dsRNA exposure or virus infection and decreased TRAF3 levels in a dose-dependent manner; moreover, Triad3A expression blocked IRF-3 activation by Ser-396 phosphorylation and inhibited the expression of type 1 interferon and antiviral genes. Lys⁴⁸-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover, whereas reduction of Triad3A expression by stable shRNA expression correlated with an increase in TRAF3 protein expression and enhancement of the antiviral response following VSV or Sendai virus infection. Triad3A and TRAF3 physically interacted together, and TRAF3 residues Y440 and Q442—previously shown to be important for association with the MAVS adapter—were also critical for Triad3A. Point mutation of the TRAF-Interacting-Motif (TIM) of Triad3A abrogated its ability to interact with TRAF3 and modulate RIG-I signaling. TRAF3 appears to undergo sequential ubiquitin “immuno-editing” following virus infection that is crucial for regulation of RIG-I-dependent signaling to the antiviral response. Thus, Triad3A represents a versatile E3 ubiquitin ligase that negatively regulates RIG-like receptor signaling by targeting TRAF3 for degradation following RNA virus infection.     

Lysine 63-linked Polyubiquitination of the Dopamine Transporter Requires WW3 and WW4 Domains of Nedd4-2 and UBE2D Ubiquitin-conjugating Enzymes

RNA interference screen previously revealed that a HECT-domain E3 ubiquitin ligase, neuronal precursor cell expressed, developmentally down-regulated 4-2 (Nedd4-2), is necessary for ubiquitination and endocytosis of the dopamine transporter (DAT) induced by the activation of protein kinase C (PKC). To further confirm the role of Nedd4-2 in DAT ubiquitination and endocytosis, we demonstrated that the depletion of Nedd4-2 by two different small interfering RNA (siRNA) duplexes suppressed PKC-dependent ubiquitination and endocytosis of DAT in human and porcine cells, whereas knock-down of a highly homologous E3 ligase, Nedd4-1, had no effect on DAT. The abolished DAT ubiquitination in Nedd4-2-depleted cells was rescued by expression of recombinant Nedd4-2. Moreover, overexpression of Nedd4-2 resulted in increased PKC-dependent ubiquitination of DAT. Mutational inactivation of the HECT domain of Nedd4-2 inhibited DAT ubiquitination and endocytosis. Structure-function analysis of Nedd4-2-mediated DAT ubiquitination revealed that the intact WW4 domain and to a lesser extent WW3 domain are necessary for PKC-dependent DAT ubiquitination. Moreover, a fragment of the Nedd4-2 molecule containing WW3, WW4, and HECT domains was sufficient for fully potentiating PKC-dependent ubiquitination of DAT. Analysis of DAT ubiquitination using polyubiquitin chain-specific antibodies showed that DAT is mainly conjugated with Lys⁶³-linked ubiquitin chains. siRNA analysis demonstrated that this polyubiquitination is mediated by Nedd4-2 cooperation with UBE2D and UBE2L3 E2 ubiquitin-conjugating enzymes. The model is proposed whereby each ubiquitinated DAT molecule is modified by a single four-ubiquitin Lys⁶³-linked chain that can be conjugated to various lysine residues of DAT.     

Multiple domains of Ruk/CIN85/SETA/CD2BP3 are involved in interaction with p85alpha regulatory subunit of PI 3-kinase

Ruk/CIN85/SETA/CD2BP3 and CD2AP/CMS/METS-1 comprise a new family of proteins involved in such fundamental processes as clustering of receptors and rearrangement of the cytoskeleton in regions of specialised cell-cell contacts, ligand-activated internalisation and targeting to lysosome degradation pathway of receptor tyrosine kinases, and apoptotic cell death. As typical adapter proteins they execute these functions by interacting with other signalling molecules via multiple protein-protein interaction interfaces: SH3 domains, Pro-rich region and coiled-coil domain. It has been previously demonstrated that Ruk is able to interact with the p85alpha regulatory subunit of PI 3-kinase and that the SH3 domain of p85alpha is required for this interaction. However, later observations hinted at a more complex mechanism than simple one-way SH3-Pro-rich interaction. Because interaction with p85alpha was suggested to be important for pro-apoptotic activity of the long isoform of Ruk, Ruk(l)/CIN85, we carried out detailed studies of the mechanism of this interaction and demonstrated that multiple domains are involved; SH3 domains of Ruk are required and sufficient for efficient interaction with full-length p85alpha but the SH3 domain of p85alpha is vital for their "activation" by ousting them from intramolecular interaction with the Pro-rich region of Ruk. Our data also suggest that homodimerisation via C-terminal coiled-coil domain affects both intra- and intermolecular interactions of Ruk proteins.     

The high resolution NMR structure of the third SH3 domain of CD2AP

CD2 associated protein (CD2AP) is an adaptor protein that plays an important role in cell to cell union needed for the kidney function. CD2AP interacts, as an adaptor protein, with different natural targets, such as CD2, nefrin, c-Cbl and podocin. These proteins are believed to interact to one of the three SH3 domains that are positioned in the N-terminal region of CD2AP. To understand the network of interactions between the natural targets and the three SH3 domains (SH3-A, B and C), we have started to determine the structures of the individual SH3 domains. Here we present the high-resolution structure of the SH3-C domain derived from NMR data. Full backbone and side-chain assignments were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from high-resolution 600 and 800 MHz NOESY spectra, together with phi and psi torsion angle restraints based on the analysis of ¹HN, ¹⁵N, ¹Hα, ¹³Cα, ¹³CO and ¹³Cβ chemical shifts. Structures were calculated using CYANA and refined in water using RECOORD. The three-dimensional structure of CD2AP SH3-C contains all the features that are typically found in other SH3 domains, including the general binding site for the recognition of polyproline sequences.     

The single subunit transmembrane E3 ligase gene related to anergy in lymphocytes (GRAIL) captures and then ubiquitinates transmembrane proteins across the cell membrane

The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. GRAIL also contains an equally large luminal region consisting primarily of an uncharacterized protease-associated (PA) domain. Using two-hybrid technology to screen for proteins that bound the PA domain we identified CD151, a member of the tetraspanin family of membrane proteins. GRAIL bound to the luminal/extracellular portion of both CD151 and the related tetraspanin CD81 using its PA domain, which promoted ubiquitination of cytosolic lysine residues. GRAIL exhibited specificity for lysines only within the tetraspanin amino terminus even in the presence of other cytosolic lysine residues in the substrate. GRAIL-mediated ubiquitination promoted proteasomal degradation and cell surface down-regulation of tetraspanins via Lys-48 linkages. As a result, the juxtaposition of PA and RING finger domains across a lipid bilayer facilitates the capture of transmembrane substrates for subsequent ubiquitination. These findings identify for the first time a single subunit E3 ligase containing a substrate-binding domain spatially restricted by a membrane from its E2 recruitment domain as well as an E3 ligase for members of the tetraspanin family.     

A new mechanism of RhoA ubiquitination and degradation: Roles of SCFFBXL19 E3 ligase and Erk2

RhoA is a small GTPase multifunctional protein that regulates cell proliferation and cytoskeletal reorganization. Regulation of its protein stability plays an important role in its biological functions. We have shown that a Skp1-Cul1-F-box (SCF) FBXL19 E3 ubiquitin ligase targets Rac1, a related member of the Rho family for ubiquitination and degradation. Here, SCF^FBXL19 mediates RhoA ubiquitination and proteasomal degradation in lung epithelial cells. Ectopically expressed FBXL19 decreased RhoA wild type, active, and inactive forms. Cellular depletion of FBXL19 increased RhoA protein levels and extended its half-life. FBXL19 bound the small GTPase in the cytoplasm leading to RhoA ubiquitination at Lys¹³⁵. A RhoA^K135R mutant protein was resistant to SCF^FBXL19-mediated ubiquitination and degradation and exhibited a longer lifespan. Protein kinase Erk2-mediated phosphorylation of RhoA was both sufficient and required for SCF^FBXL19-mediated RhoA ubiquitination and degradation. Thus, SCF^FBXL19 targets RhoA for its disposal, a process regulated by Erk2. Ectopically expressed FBXL19 reduced phosphorylation of p27 and cell proliferation, a process mediated by RhoA. Further, FBXL19 cellular expression diminished lysophosphatidic acid (LPA)-induced phosphorylation of myosin light chain (MLC) and stress fiber formation. Hence, SCF^FBXL19 functions as a RhoA antagonist during cell proliferation and cytoskeleton rearrangement. These results provide the first evidence of an F-box protein targeting RhoA thereby modulating its cellular lifespan that impacts cell proliferation and cytoskeleton rearrangement.     

The E3 Ubiquitin Ligase WWP1 Selectively Targets HER4 and Its Proteolytically Derived Signaling Isoforms for Degradation

In general, epidermal growth factor receptor family members stimulate cell proliferation. In contrast, at least one HER4 isoform, JM-a/Cyt1, inhibits cell growth after undergoing a two-step proteolytic cleavage that first produces a membrane-anchored 80-kDa fragment (m80^HER4) and subsequently liberates a soluble 80-kDa fragment, s80^HER4. Here we report that s80^HER4 Cyt1 action increased the expression of WWP1 (for WW domain-containing protein 1), an E3 ubiquitin ligase, but not other members of the Nedd4 E3 ligase family. The HER4 Cyt1 isoform contains three proline-rich tyrosine (PY) WW binding motifs, while Cyt2 has only two. WWP1 binds to all three Cyt1 PY motifs; the interaction with PY2 found exclusively in Cyt1 was strongest. WWP1 ubiquitinated and caused the degradation of HER4 but not of EGFR, HER2, or HER3. The HER4-WWP1 interaction also accelerated WWP1 degradation. Membrane HER4 (full length and m80^HER4, the product of the first proteolytic cleavage) were the preferred targets of WWP1, correlating with the membrane localization of WWP1. Conversely s80^HER4, a poorer WWP1 substrate, was found in the cell nucleus, while WWP1 was not. Deletion of the C2 membrane association domain of WWP1 allowed more efficient s80^HER4 degradation, suggesting that WWP1 is normally part of a membrane complex that regulates HER4 membrane species levels, with a predilection for the growth-inhibitory Cyt1 isoform. Finally, WWP1 expression diminished HER4 biologic activity in MCF-7 cells. We previously showed that nuclear s80^HER4 is ubiquitinated and degraded by the anaphase-promoting complex, suggesting that HER4 ubiquitination within specific cellular compartments helps regulate the unique HER4 signaling capabilities.     

The Human COP9 Signalosome Protects Ubiquitin-conjugating Enzyme 3 (UBC3/Cdc34) from β-Transducin Repeat-containing Protein (βTrCP)-mediated Degradation

The COP9 signalosome (CSN) is an essential multisubunit complex that regulates the activity of cullin-RING ubiquitin ligases by removing the ubiquitin-like peptide NEDD8 from cullins. Here, we demonstrate that the CSN can affect other components of the ubiquitination cascade. Down-regulation of human CSN4 or CSN5 induced proteasome-mediated degradation of the ubiquitin-conjugating enzyme UBC3/Cdc34. UBC3 was targeted for ubiquitination by the cullin-RING ubiquitin ligase SCF^βTrCP. This interaction required the acidic C-terminal extension of UBC3, which is absent in ubiquitin-conjugating enzymes of the UBCH5 family. Conversely, the UBC3 acidic domain was sufficient to impart sensitivity to SCF^βTrCP-mediated ubiquitination to UBCH5 enzymes. Our work indicates that the CSN is necessary to ensure the stability of selected ubiquitin-conjugating enzymes and uncovers a novel pathway of regulation of ubiquitination processes.     

Critical contribution of the MDM2 acidic domain to p53 ubiquitination

MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Recent studies have shown, however, that the ring-finger domain (RFD) of MDM2, where the ubiquitin E3 ligase activity resides, is necessary but not sufficient for p53 ubiquitination, suggesting that an additional activity of MDM2 might be required. To test this possibility, we generated a series of MDM2/MDMX chimeric proteins to assess the contribution of each domain of MDM2 to the ubiquitination process. MDMX is a close structural homolog of MDM2 that nevertheless lacks the E3 ligase activity in vivo. We demonstrate here that MDMX gains self-ubiquitination activity and becomes extremely unstable upon introduction of the MDM2 RFD, indicating that the RFD is essential for self-ubiquitination. This MDMX chimeric protein, however, is unable to ubiquitinate p53 in vivo despite its E3 ligase activity and binding to p53, separating the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Significantly, fusion of the central acidic domain (AD) of MDM2 to the MDMX chimeric protein renders the protein fully capable of ubiquitinating p53, and p53 ubiquitination is associated with p53 degradation and nuclear export. Moreover, the AD mini protein expressed in trans can functionally rescue the AD-lacking MDM2 mutant, further supporting a critical role for the AD in MDM2-mediated p53 ubiquitination.     

SCFFbxw15 Mediates Histone Acetyltransferase Binding to Origin Recognition Complex (HBO1) Ubiquitin-Proteasomal Degradation to Regulate Cell Proliferation

Histone acetyltransferase binding to origin recognition complex (HBO1) plays a crucial role in DNA replication licensing and cell proliferation, yet its molecular regulation in cells is relatively unknown. Here an uncharacterized protein, Fbxw15, directly interacts with HBO1, a labile protein (t_½ = ∼3 h), to mediate its ubiquitination (Lys³³⁸) and degradation in the cytoplasm. Fbxw15-mediated HBO1 depletion required mitogen-activated protein kinase 1 (Mek1), which was sufficient to trigger HBO1 phosphorylation and degradation in cells. Mek1 ability to produce HBO1 degradation was blocked by Fbxw15 silencing. Lipopolysaccharide induced HBO1 degradation, an effect abrogated by Fbxw15 or Mek1 cellular depletion. Modulation of Fbxw15 levels was able to differentially regulate histone H3K14 acetylation and cellular proliferation by altering HBO1 levels. These studies authenticate Fbxw15 as a ubiquitin E3 ligase subunit that mediates endotoxin-induced HBO1 depletion in cells, thereby controlling cell replicative capacity.