Ubiquitin Ligase Itch Research Articles

1H, 13C, and 15N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin ligase ITCH

ITCH (aka Atrophin-1-interacting protein 4) is a prominent member of the NEDD4 HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family that regulates numerous cellular functions including inflammatory responses through T-cell activation, cell differentiation, and apoptosis. Known intracellular targets of ITCH-dependent ubiquitylation include receptor proteins, signaling molecules, and transcription factors. The HECT C-terminal lobe of ITCH contains the conserved catalytic cysteine required for the covalent attachment of ubiquitin onto a substrate and polyubiquitin chain assembly. We report here the complete experimentally determined 1H, 13C, and 15N backbone and sidechain resonance assignments for the HECT C-terminal lobe of ITCH (residues 784–903) using heteronuclear, multidimensional NMR spectroscopy. These resonance assignments will be used in future NMR-based studies to examine the role of dynamics and conformational flexibility in HECT-dependent ubiquitylation as well as deciphering the structural and biochemical basis for polyubiquitin chain synthesis and specificity by ITCH.

PO-128 Identification of the E3 ubiquitin ligase ITCH as a novel regulator of connexin 43 degradation and gap junction intercellular communication

IntroductionGlioblastoma(GBM) is an aggressive brain tumour arising from glial cells. Our lab has identified the oncogenic role of FAT1 gene in GBM, regulating inflammatory and hypoxic microenvironment of the tumour as well as migratory/invasive properties of the tumour cells. In Drosophila, fat, the ortholog of FAT1, is known to regulate the Salvador-Warts-Hippo (SWH) pathway, but its role in human is not clear. Here, we have analysed the effect of FAT1 on SWH pathway in glioma.Material and methodsGlioma cell lines (U87MG, U373, A172, GOS3 and SW1088) were transfected with FAT1 specific siRNA/control siRNA and analysed the expression of SWH pathway molecules by qPCR/Western blot. Protein-protein interactions were analysed by Co-immunoprecipitation (Co-IP) after overexpression of YAP1 (wild-type and mutated) and TEAD1 with and without FAT1 knockdown. Sub-cellular localization of proteins was analysed by Confocal microscopy.Results and discussionsThe mRNA expression of FAT1 and SWH pathway molecules (MST1, LATS1, LATS2, YAP1 and TEAD1) was highest in U87MG cells followed by A172, U373MG and GOS3. After FAT1 knockdown, the mRNA expression of MST1 and BIRC2 were significantly decreased with no change in the levels of LATS1, LATS2, YAP1, TEAD1 and BIRC5. At protein level, increased YAP1 and phospho-YAP1 was observed after FAT1 knockdown with increased total as well as phospho-YAP1 in the cytoplasmic extract as compared to the nuclear extract. There was significant reduction in the interaction between YAP1 and TEAD1 in siFAT1 treated cells as compared to siControl treated cells.ConclusionKnockdown of FAT1 (i) increases the YAP1 protein level, could be by increasing the protein stability as no change was observed at the mRNA level (ii) it relieves the inhibitory effect on YAP1 phosphorylation, thereby increasing the phospho-YAP1 level (iii) it affects the sub-cellular localization of YAP1 by retaining YAP1 in the cytosol and thereby (iv) decrease in the YAP1:TEAD1 interaction with decreased expression of their target gene Birc2.This finding of the effect of FAT1 on YAP1 in GBM is novel with features pointing towards the oncogenic role of FAT1 by regulating YAP1 sub-cellular localization and co-transcriptional activity independent of SWH pathway.

HIV-1 gag recruits PACSIN2 to promote virus spreading

The p2b domain of Rous sarcoma virus (RSV) Gag and the p6 domain of HIV-1 Gag contain late assembly (L) domains that engage the ESCRT membrane fission machinery and are essential for virus release. We now show that the PPXY-type RSV L domain specifically recruits the BAR domain protein PACSIN2 into virus-like particles (VLP), in addition to the NEDD4-like ubiquitin ligase ITCH and ESCRT pathway components such as TSG101. PACSIN2, which has been implicated in the remodeling of cellular membranes and the actin cytoskeleton, is also recruited by HIV-1 p6 independent of its ability to engage the ESCRT factors TSG101 or ALIX. Moreover, PACSIN2 is robustly recruited by NEDD4-2s, a NEDD4-like ubiquitin ligase capable of rescuing HIV-1 budding defects. The NEDD4-2s-induced incorporation of PACSIN2 into VLP correlated with the formation of Gag-ubiquitin conjugates, indicating that PACSIN2 binds ubiquitin. Although PACSIN2 was not required for a single cycle of HIV-1 replication after infection with cell-free virus, HIV-1 spreading was nevertheless severely impaired in T cell lines and primary human peripheral blood mononuclear cells depleted of PACSIN2. HIV-1 spreading could be restored by reintroduction of wild-type PACSIN2, but not of a SH3 domain mutant unable to interact with the actin polymerization regulators WASP and N-WASP. Overall, our observations indicate that PACSIN2 promotes the cell-to-cell spreading of HIV-1 by connecting Gag to the actin cytoskeleton.

Activation and auto‐destruction of the ubiquitin ligase Itch

Ubiquitin ligases of the Nedd4 family are maintained inactive by intramolecular interactions (1). We reported that, like Nedd4, the closely related ubiquitin ligase Itch strongly binds to the endosomal protein LITAF through its WW domains, that recognize two PPXY motifs in the N‐terminal half of LITAF. Unlike Nedd4, Itch binding to LITAF brings the ligase to the lysosomal compartments. We thus postulated that LITAF overexpression might bring Itch to the lysosome, where it would access different substrates and target them for degradation (2). Concomitantly, LITAF binding to Itch may increase Itch ligase activity, as other PPXY motif proteins such as NDFIP and LRAD3 (3,4). Here, we set up to determine if Itch ubiquitin ligase activity is indeed up regulated by LITAF binding, and if Itch most common substrate degradation is increased in response to LITAF binding to Itch. We find that Itch, WWP1, WWP2 and Nedd4 all bind to LITAF, and that the four ligases autoubiquitylation is greatly increased upon LITAF binding, albeit to different levels. Both LITAF overexpression and addition of recombinant LITAF to crude cell lisates lead to rapid degradation of Itch, and, to a lesser extent, Nedd4. Itch substrates cJun and Endophilin degradation was also increased. In vitro ubiquitylation assays show that Itch autoubiquitylation is enhanced in the presence of LITAF, but substrate ubiquitylation was not significantly increased. In conclusion, LITAF binding strongly activate Itch autoubiquitylation, as well as that of other ligases of the Nedd4 family. The consequence of this activation is Itch proteasomal degradation, with little effect on its substrates ubiquitylation. LITAF is thus likely to be a negative regulator of Itch in cells where it is strongly expressed.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of ITCH Suppresses Proliferation and Induces Apoptosis of Lung Cancer Cells

Background/Aims: The E3 ubiquitin ligase ITCH plays an important role in invasive and metastatic cancers. However, the role of ITCH in the progression of lung cancer has not been fully described. Methods: Real-time PCR was used to detect the expression of ITCH mRNA in the tumor tissues and paracarcinoma tissues from 32 patients with lung cancer. SiRNA was used to inhibit the expression of ITCH in two lung cancer cell lines, H1975 and Calu3 and the cell proliferation and apoptosis were measured by MTT and flow cytometric assay. In addition, to further investigate whether ITCH affected the apoptosis of cancer cells and its underlying mechanisms, the expression of important markers of apoptosis and invasion in lung cancer cells were detected by Western blot. Results: The study showed significant increments in the expression of ITCH in lung cancer tissues (p< 0.001). ITCH siRNA effectively inhibited the proliferation and invasion of the lung cancer cells and promoted cell apoptosis. Molecular analysis further showed significant reductions in the expression of Bcl2, MMP2, MMP9 and β-catenin and an increase in the expression of Bax and E-cadherin in the lung cancer cells with ITCH deficiency. Conclusions: Inhibition of ITCH might suppress lung cancer proliferation and invasion via regulation of MMPs, EMT and Bcl2/Bax signaling pathway.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells

Roquin proteins preclude spontaneous Tcell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional Tcells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3'UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient Tcells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional Tcells and specialization of Treg cells.

Tricellulin is a target of the ubiquitin ligase Itch.

Tricellulin, a member of the tight junction-associated MAGUK protein family, preferentially localizes to tricellular junctions in confluent polarized epithelial cell layers and is downregulated during the epithelial-mesenchymal transition. Posttranslational modifications are assumed to play critical roles in the process of downregulation of tricellulin at the protein level. Here, we report that the E3 ubiquitin ligase Itch forms a complex with tricellulin and thereby enhances its ubiquitination. Pull-down assays confirmed a direct interaction between tricellulin and Itch, which is mediated by the Itch WW domain and the N-terminus of tricellulin. Experiments in the presence of the proteasome inhibitor MG-132 did not show major changes in the levels of ubiquitinated tricellulin in epithelial cells, suggesting that ubiquitination is not primarily involved in proteasomal degradation of tricellulin, but it appears to be important for endocytosis or recycling. In contrast, in HEK-293 cells, MG-132 caused polyubiquitination. Moreover, we observed that well-differentiated RT-112 and de-differentiated Cal-29 bladder cancer cells show an inverse expression of tricellulin and Itch. We postulate that ubiquitination is an important posttranslational modification involved in the determination of the intracellular fate of tricellulin deserving of more detailed further investigations into the underlying molecular mechanisms and their regulation.

Dishevelled-3 phosphorylation is governed by HIPK2/PP1Cα/ITCH axis and the non-phosphorylated form promotes cancer stemness via LGR5 in hepatocellular carcinoma

Dishevelled-3 (Dvl3) is regarded as a binding hub with many different interacting partners. However, its regulation and mechanism on cancer stemness remain to be explored. In this study, we showed that Dvl3 was significantly overexpressed in human hepatocellular carcinomas (HCCs) and promoted cancer stemness both in vitro and in vivo. We found that the non-phosphorylated (NP)-Dvl3 was more stable than the phosphorylated form, more active in activating β-catenin transcriptional activity, and more potent in enhancing self-renewal ability in HCC cells. Mechanistically, we confirmed that the homeodomain-interacting protein kinase-2 (HIPK2) and E3 ubiquitin ligase ITCH were able to physically bind to Dvl3 protein. Knockdown of HIPK2 and the protein phosphatase regulatory unit C-alpha (PP1Cα) resulted in sustained Dvl3 phosphorylation and hence decrease in the NP form of Dvl3. On the other hand, knockdown of E3 ubiquitin ligase ITCH reduced the phosphorylation-induced degradation and stabilized the phosphorylated Dvl3 protein. Furthermore, the NP-Dvl3 enhanced the LGR5 promoter activity to upregulate LGR5 expression, which was associated with increased cancer stemness in HCC. Our findings established that HIPK2/PP1Cα/ITCH axis sustains the de-phosphorylation of Dvl3. This post-translational modification of Dvl3 in turn maintains LGR5 expression and enhances the cancer stemness properties in HCC.

ASPP2 suppresses invasion and TGF-β1-induced epithelial–mesenchymal transition by inhibiting Smad7 degradation mediated by E3 ubiquitin ligase ITCH in gastric cancer

ASPP2 regulates cell polarity and cell–cell adhesion by binding to, and co-localizing with PAR3 at tight junctions. Here we show a novel role of ASPP2 in suppressing gastric cancer (GC) invasiveness. Immunoprecipitation and immunofluorescence analyses showed that ASPP2 promoted the recruitment of PAR3 to cell–cell junctions in GC cells. Diminished expression of ASPP2 and loss of junctional PAR3 localization were significantly associated with diffuse-type histology, deeper invasion depth, positive peritoneal dissemination and worse prognosis in primary GC. ASPP2 suppressed migration and invasion of GC cells in vitro and peritoneal dissemination of GC cells in vivo in a mouse model. ASPP2 suppressed epithelial–mesenchymal transition (EMT) induced by TGF-β1-Smad2/3 signaling in GC cells through suppression of the degradation of Smad7, a negative regulator of TGF-β1-Smad2/3 signaling, by interacting with the E3 ubiquitin ligase ITCH. In conclusion, ASPP2 suppresses invasion, peritoneal dissemination and TGF-β1-induced EMT by inhibiting Smad7 degradation mediated by ITCH.

Molecular basis of interactions between SH3 domain-containing proteins and the proline-rich region of the ubiquitin ligase Itch

The ligase Itch plays major roles in signaling pathways by inducing ubiquitylation-dependent degradation of several substrates. Substrate recognition and binding are critical for the regulation of this reaction. Like closely related ligases, Itch can interact with proteins containing a PPXY motif via its WW domains. In addition to these WW domains, Itch possesses a proline-rich region (PRR) that has been shown to interact with several Src homology 3 (SH3) domain-containing proteins. We have previously established that despite the apparent surface uniformity and conserved fold of SH3 domains, they display different binding mechanisms and affinities for their interaction with the PRR of Itch. Here, we attempt to determine the molecular bases underlying the wide range of binding properties of the Itch PRR. Using pulldown assays combined with mass spectrometry analysis, we show that the Itch PRR preferentially forms complexes with endophilins, amphyphisins, and pacsins but can also target a variety of other SH3 domain-containing proteins. In addition, we map the binding sites of these proteins using a combination of PRR sub-sequences and mutants. We find that different SH3 domains target distinct proline-rich sequences overlapping significantly. We also structurally analyze these protein complexes using crystallography and molecular modeling. These structures depict the position of Itch PRR engaged in a 1:2 protein complex with β-PIX and a 1:1 complex with the other SH3 domain-containing proteins. Taken together, these results reveal the binding preferences of the Itch PRR toward its most common SH3 domain-containing partners and demonstrate that the PRR region is sufficient for binding.

Ndfip1 restricts Th17 cell potency by limiting lineage stability and proinflammatory cytokine production

While Th17 cells can protect against colonization by pathogenic organisms, they also have the potential to become pathogenic and promote autoimmune and inflammatory diseases. Mechanisms that control their pathogenic potential remain poorly understood. Here we show that Ndfip1, a co-activator of the E3 ubiquitin ligase Itch, restricts the frequency and pathogenicity of Th17 cells. Mice lacking Ndfip1 have increased numbers of Th17 cells, and this increase is cell intrinsic. We found that Ndfip1 restricts production of the proinflammatory cytokines in Th17 cells. Increased cytokine production correlated with reduced degradation and accumulation of RORγT. When transferred in vivo, Th17 cells lacking Ndfip1 were more likely to maintain their ability to make IL-17, were more potent proinflammatory cytokine producers, and were powerful inducers of colitis. Together our data support an essential role for Ndfip1 in degrading RORγT and suppressing Th17 lineage stability, proinflammatory cytokine production, and pathogenicity.

The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages.

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tail-anchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction.

ITCH E3 Ubiquitin Ligase Interacts with Ebola Virus VP40 To Regulate Budding

Ebola virus (EBOV) and Marburg virus (MARV) belong to the Filoviridae family and can cause outbreaks of severe hemorrhagic fever, with high mortality rates in humans. The EBOV VP40 (eVP40) and MARV VP40 (mVP40) matrix proteins play a central role in virion assembly and egress, such that independent expression of VP40 leads to the production and egress of virus-like particles (VLPs) that accurately mimic the budding of infectious virus. Late (L) budding domains of eVP40 recruit host proteins (e.g., Tsg101, Nedd4, and Alix) that are important for efficient virus egress and spread. For example, the PPxY-type L domain of eVP40 and mVP40 recruits the host Nedd4 E3 ubiquitin ligase via its WW domains to facilitate budding. Here we sought to identify additional WW domain host interactors and demonstrate that the PPxY L domain motif of eVP40 interacts specifically with the WW domain of the host E3 ubiquitin ligase ITCH. ITCH, like Nedd4, is a member of the HECT class of E3 ubiquitin ligases, and the resultant physical and functional interaction with eVP40 facilitates VLP and virus budding. Identification of this novel eVP40 interactor highlights the functional interplay between cellular E3 ligases, ubiquitination, and regulation of VP40-mediated egress. The unprecedented magnitude and scope of the recent 2014-2015 EBOV outbreak in West Africa and its emergence here in the United States and other countries underscore the critical need for a better understanding of the biology and pathogenesis of this emerging pathogen. We have identified a novel and functional EBOV VP40 interactor, ITCH, that regulates VP40-mediated egress. This virus-host interaction may represent a new target for our previously identified small-molecule inhibitors of virus egress.

The Ubiquitin Ligase Itch and Ubiquitination Regulate BFRF1-Mediated Nuclear Envelope Modification for Epstein-Barr Virus Maturation.

The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.

The Itch to degrade ROR-γt.

The E3 ubiquitin ligase Itch limits interleukin 17A (IL-17A)-mediated intestinal inflammation by targeting the transcription factor ROR-γt for proteasomal degradation.

Itch inhibits IL-17-mediated colon inflammation and tumorigenesis by ROR-γt ubiquitination.

Dysregulated expression of interleukin 17 (IL-17) in the colonic mucosa is associated with colonic inflammation and cancer. However, the cell-intrinsic molecular mechanisms by which IL-17 expression is regulated remain unclear. We found that deficiency in the ubiquitin ligase Itch led to spontaneous colitis and increased susceptibility to colon cancer. Itch deficiency in the TH17 subset of helper T cells, innate lymphoid cells and γδ T cells resulted in the production of elevated amounts of IL-17 in the colonic mucosa. Mechanistically, Itch bound to the transcription factor ROR-γt and targeted ROR-γt for ubiquitination. Inhibition or genetic inactivation of ROR-γt attenuated IL-17 expression and reduced spontaneous colonic inflammation in Itch(-/-) mice. Thus, we have identified a previously unknown role for Itch in regulating IL-17-mediated colonic inflammation and carcinogenesis.

TGF-β Targets the Hippo Pathway Scaffold RASSF1A to Facilitate YAP/SMAD2 Nuclear Translocation

Epigenetic inactivation of the Hippo pathway scaffold RASSF1A is associated with poor prognosis in a wide range of sporadic human cancers. Loss of expression reduces tumor suppressor activity and promotes genomic instability, but how this pleiotropic biomarker is regulated at the protein level is unknown. Here we show that TGF-β is the physiological signal that stimulates RASSF1A degradation by the ubiquitin-proteasome pathway. In response to TGF-β, RASSF1A is recruited to TGF-β receptor I and targeted for degradation by the co-recruited E3 ubiquitin ligase ITCH. RASSF1A degradation is necessary to permit Hippo pathway effector YAP1 association with SMADs and subsequent nuclear translocation of receptor-activated SMAD2. We find that RASSF1A expression regulates TGF-β-induced YAP1/SMAD2 interaction and leads to SMAD2 cytoplasmic retention and inefficient transcription of TGF-β targets genes. Moreover, RASSF1A limits TGF-β induced invasion, offering a new frameworkonhow RASSF1A affects YAP1 transcriptional output and elicits its tumor-suppressive function.

Correction to Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region.

Correction to Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region.

FADD regulates NF-κB activation and promotes ubiquitination of cFLIPL to induce apoptosis.

Tumor Necrosis Factor-α canonically induces the activation of NF-κB and associated gene product cellular FLICE-like inhibitory protein (cFLIPL) to promote cell survival. Previously, we demonstrated that ectopic expression of the Fas associated death domain (FADD) diminishes the expression of cFLIPL and transduces caspases-8 mediated apoptosis, independent of FasL stimulation in HEK 293T cells. However, the underlying molecular mechanism of FADD mediated ablation of cFLIP and NF-κB signaling to determining the fate of cell death or survival remains elusive. Here, we explored a novel molecular mechanism of FADD mediated apoptotic cell death that was directed by ubiquitination of cFLIPL and inhibition of NF-κB activation, independent of TNF-α stimulation. We found that induced expression of FADD firmly interacts with procaspase-8 and precludes cFLIPL to from the death inducing signaling complex (DISC). In addition, FADD negatively regulates cellular inhibitor of apoptosis protein 2 (cIAP2) and Bcl-2. Furthermore, FADD restrains cIAP2 expression and interacts with RIP1 and procaspase-8 to accomplish apoptotic cell death signaling. Interestingly, FADD was also found to promote JNK1 mediated activation of E3 ubiquitin ligase ITCH to degrade cFLIPL that may lead to commencement of apoptosis. Thus, FADD is an important regulator for determining the fate of cell death or survival.

Structural basis for a novel interaction between TXNIP and Vav2.

Thioredoxin-interacting protein (TXNIP) is a multifunctional protein involved in diverse cellular processes such as cell proliferation and apoptosis. TXNIP stability is controlled by the ubiquitin-proteasome pathway, and the E3 ubiquitin ligase Itch directly interacts with TXNIP via PPxY motifs of TXNIP. In a previously published study, we have shown that phosphorylation of the PPxY tyrosyl residue switches TXNIP selectivity between different binding partners. Here, we describe that tyrosine-phosphorylated PPxY motifs also bind to SH2 domains of Vav2 and Src with dissociation constants around 10 μm and that phosphorylation is indispensable for these interactions as well. The crystal structure of the complex between a phosphorylated PPxY motif, and the SH2 domain of Vav2 reveals a conserved recognition mechanism.