Deubiquitylase USP9X maintains centriolar satellite integrity by stabilizing pericentriolar material 1 protein.

Centriolar satellites are small cytoplasmic granules that play important roles in regulating the formation of centrosomes and primary cilia. Ubiquitylation of satellite proteins, including the core satellite scaffold protein pericentriolar material 1 (PCM1), regulates centriolar satellite integrity. Currently, deubiquitylases that control centriolar satellite integrity have not been identified. In this study, we find that the deubiquitylase USP9X binds PCM1, and antagonizes PCM1 ubiquitylation to protect it from proteasomal degradation. Knockdown of USP9X in human cell lines reduces PCM1 protein levels, disrupts centriolar satellite particles and causes localization of satellite proteins, such as CEP290, to centrosomes. Interestingly, knockdown of mindbomb 1 (MIB1), a ubiquitin ligase that promotes PCM1 ubiquitylation and degradation, in USP9X-depleted cells largely restores PCM1 protein levels and corrects defects caused by the loss of USP9X. Overall, our study reveals that USP9X is a constituent of centriolar satellites and functions to maintain centriolar satellite integrity by stabilizing PCM1.

Monoubiquitination of survival motor neuron regulates its cellular localization and Cajal body integrity.

Low levels of the survival motor neuron (SMN) protein cause spinal muscular atrophy, the leading genetic disorder for infant mortality. SMN is ubiquitously expressed in various cell types and localizes in both the cytoplasm and the nucleus, where it concentrates in two subnuclear structures termed Cajal body (CB) and gems. In addition, SMN can also be detected in the nucleolus of neurons. Mechanisms that control SMN sorting in the cell remain largely unknown. Here, we report that the ubiquitin (Ub) ligase Itch directly interacts with and monoubiquitinates SMN. Monoubiquitination of SMN has a mild effect on promoting proteasomal degradation of SMN. We generated two SMN mutants, SMN(K0), in which all lysines are mutated to arginines and thereby abolishing SMN ubiquitination, and Ub-SMN(K0), in which a single Ub moiety is fused at the N-terminus of SMN(K0) and thereby mimicking SMN monoubiquitination. Immunostaining assays showed that SMN(K0) mainly localizes in the nucleus, whereas Ub-SMN(K0) localizes in both the cytoplasm and the nucleolus in neuronal SH-SY5Y cells. Interestingly, canonical CB foci and coilin/small nuclear ribonucleoprotein (snRNP) co-localization are significantly impaired in SH-SY5Y cells stably expressing SMN(K0) or Ub-SMN(K0). Thus, our studies discover that Itch monoubiquitinates SMN and monoubiquitination of SMN plays an important role in regulating its cellular localization. Moreover, mislocalization of SMN disrupts CB integrity and likely impairs snRNP maturation.

Ubiquitin-specific protease 9x deubiquitinates and stabilizes the spinal muscular atrophy protein-survival motor neuron.

Spinal muscular atrophy (SMA), the leading genetic disorder of infant mortality, is caused by low levels of survival motor neuron (SMN) protein. Currently it is not clear how the SMN protein levels are regulated at the post-transcriptional level. In this report, we find that Usp9x, a deubiquitinating enzyme, stably associates with the SMN complex via directly interacting with SMN. Usp9x deubiquitinates SMN that is mostly mono- and di-ubiquitinated. Knockdown of Usp9x promotes SMN degradation and reduces the protein levels of SMN and the SMN complex in cultured mammalian cells. Interestingly, Usp9x does not deubiquitinate nuclear SMNΔ7, the main protein product of the SMN2 gene, which is polyubiquitinated and rapidly degraded by the proteasome. Together, our results indicate that SMN and SMNΔ7 are differently ubiquitinated; Usp9x plays an important role in stabilizing SMN and the SMN complex, likely via antagonizing Ub-dependent SMN degradation.

The role of midbody-associated mRNAs in regulating abscission.

Midbodies function during telophase to regulate the abscission step of cytokinesis. Until recently, it was thought that abscission-regulating proteins, such as ESCRT-III complex subunits, accumulate at the MB by directly or indirectly binding to the MB resident protein, CEP55. However, recent studies have shown that depletion of CEP55 does not fully block ESCRT-III targeting the MB. Here, we show that MBs contain mRNAs and that these MB-associated mRNAs can be locally translated, resulting in the accumulation of abscission-regulating proteins. We demonstrate that localized MB-associated translation of CHMP4B is required for its targeting to the abscission site and that 3' UTR-dependent CHMP4B mRNA targeting to the MB is required for successful completion of cytokinesis. Finally, we identify regulatory cis-elements within RNAs that are necessary and sufficient for mRNA trafficking to the MB. We propose a novel method of regulating cytokinesis and abscission by MB-associated targeting and localized translation of selective mRNAs.

Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration.

Cell migration is a complex process that involves coordinated changes in membrane transport and actin cytoskeleton dynamics. Ras-like small monomeric GTPases, such as Rap2, play a key role in regulating actin cytoskeleton dynamics and cell adhesions. However, how Rap2 function, localization, and activation are regulated during cell migration is not fully understood. We previously identified the small GTPase Rab40b as a regulator of breast cancer cell migration. Rab40b contains a suppressor of cytokine signaling (SOCS) box, which facilitates binding to Cullin5, a known E3 ubiquitin ligase component responsible for protein ubiquitylation. In this study, we show that the Rab40b/Cullin5 complex ubiquitylates Rap2. Importantly, we demonstrate that ubiquitylation regulates Rap2 activation as well as recycling of Rap2 from the endolysosomal compartment to the lamellipodia of migrating breast cancer cells. Based on these data, we propose that Rab40b/Cullin5 ubiquitylates and regulates Rap2-dependent actin dynamics at the leading edge, a process that is required for breast cancer cell migration and invasion.

Rab40c regulates focal adhesions and PP6 activity by controlling ANKRD28 ubiquitylation.

Rab40c is a SOCS box-containing protein which binds Cullin5 to form a ubiquitin E3 ligase complex (Rab40c/CRL5) to regulate protein ubiquitylation. However, the exact functions of Rab40c remain to be determined, and what proteins are the targets of Rab40c-Cullin5-mediated ubiquitylation in mammalian cells are unknown. Here we showed that in migrating MDA-MB-231 cells Rab40c regulates focal adhesion's number, size, and distribution. Mechanistically, we found that Rab40c binds the protein phosphatase 6 (PP6) complex and ubiquitylates one of its subunits, ankyrin repeat domain 28 (ANKRD28), thus leading to its lysosomal degradation. Furthermore, we identified that phosphorylation of FAK and MOB1 is decreased in Rab40c knock-out cells, which may contribute to focal adhesion site regulation by Rab40c. Thus, we propose a model where Rab40c/CRL5 regulates ANKRD28 ubiquitylation and degradation, leading to a decrease in PP6 activity, which ultimately affects FAK and Hippo pathway signaling to alter focal adhesion dynamics.

Analysis of a TIR-less splice variant of TRIF reveals an unexpected mechanism of TLR3-mediated signaling.

Recognition of viral RNA by Toll-like receptor 3 (TLR3) triggers activation of the transcription factors NF-kappaB and IRF3 and induction of type I interferons. TRIF is a Toll-interleukin 1 receptor (TIR) domain-containing adapter protein critically involved in TLR3-mediated signaling. It has been shown that TRIF interacts with TLR3 through their respective TIR domains. In this study, we identified a splice variant of TRIF lacking the TIR domain, which is designated as TRIS. Overexpression of TRIS activates NF-kappaB, interferon-stimulated response element (ISRE), and the interferon-beta promoter, whereas knockdown of TRIS inhibited TLR3-mediated signaling, suggesting that TRIS is involved in TLR3-mediated signaling. Furthermore, we identified an N-terminal TBK1-binding motif of TRIS or TRIF that was important for its interaction with TBK1 and ability to activate ISRE. Activation of ISRE by TRIS also needs its dimerization or oligomerization mediated by its C-terminal RIP homotypic interaction motif. Finally, we demonstrated that TRIS was associated with TRIF upon TLR3 activation by poly(I-C). These findings reveal an unexpected mechanism of TLR3-mediated signaling.

Rab40-Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration.

Rab40b is a SOCS box-containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b-Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b-Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b-Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 s proteasome.

The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates.

Regulation of IRF2 transcriptional activity by its sumoylation.

IRFs constitute a family of transcription factors involved in IFN signaling and in the development and differentiation of the immune system. IRFs activities are regulated at transcriptional level (such as IRF1) and post-translational modifications (such as IRF3 and IRF7). Here we show that IRF2 interacts with the SUMO-E3 ligase PIASy and is sumoylated in vivo. Mutagenesis analysis suggests that IRF2 contains three sumoylation sites. Sumoylation of IRF2 has no significant effects on its nuclear localization and DNA-binding activity, but increases its ability to inhibit IRF1 transcriptional activity and decreases its ability to activate the ISRE and H4 promoters. Our findings suggest that sumoylation of IRF2 regulates its transcriptional activities.

[Interleukin-6 is involved in the enhanced proliferation of HEK293 cells by the tumor specific antigen of HCA520].

OBJECTIVE: To investigate the effect of tumor specific antigen of HCA520 on the proliferation of HEK293 cells and to obtain some functional implications for HCA520. METHODS: MTT assay was performed with HEK293 stable cell lines transfected with pcDNA3-HCA520-flag construct. Cytokines probably involved in HCA520 enhanced proliferation were screened by RT-PCR, and effect of these cytokines on the HEK293 cell proliferation was further confirmed by MTT assay. RESULTS: HCA520 significantly promoted the proliferation of HEK293 cells, which was at least partially attributed to the up-regulation of IL-6 in HEK293 cells by HCA520. CONCLUSION: HCA520 might accelerate tumorigenesis by promoting proliferation of cancerous cells.

PIASy represses TRIF-induced ISRE and NF-kappaB activation but not apoptosis.

The TIR domain-containing adapter protein TRIP is critically involved in TLR3-induced IFN-beta production through activation of NF-kappaB and ISRE. In addition, TRIF also induces apoptosis when overexpressed in 293 cells. In this report, we demonstrate that PIASy, a member of the PIAS SUMO-ligase family, interacts with TRIP, IRF-3 and IRF-7. In reporter gene assays, PIASy dramatically inhibits TRIF-induced NF-kappaB, ISRE and IFN-beta activation but not TRIF-induced apoptosis. Furthermore, PIASy also inhibits IRF-3, IRF-7 and Sendai virus-induced ISRE activation. Our results suggest that PIASy is an inhibitor of TRIF-induced ISRE and NF-kappaB activation but not apoptosis.

A20 is a potent inhibitor of TLR3- and Sendai virus-induced activation of NF-kappaB and ISRE and IFN-beta promoter.

Toll-like receptor 3 (TLR3) recognizes dsRNA generated during viral infection and activation of TLR3 results in induction of type I interferons (IFNs) and cellular anti-viral response. TLR3 is associated with a TIR domain-containing adapter protein TRIF, which activates distinct downstream pathways leading to activation of NF-kappaB and ISRE sites in the promoters of type I IFNs. We show here that A20, a NF-kappaB-inducible zinc finger protein that has been demonstrated to be an inhibitor of TNF-induced NF-kappaB activation and a physiological suppressor of inflammatory response, potently inhibited TLR3- and Sendai virus-mediated activation of ISRE and NF-kappaB and IFN-beta promoter in reporter gene assays. A20 also inhibited TRIF-, but not its downstream signaling components TBK1-, IKKbeta-, and IKKepsilon-mediated activation of ISRE and NF-kappaB and IFN-beta promoter. Moreover, A20 interacted with TRIF in co-immunoprecipitation experiments. Finally, expression of A20 could be induced at protein level by Sendai virus infection. These data suggest that A20 targets TRIF to inhibit TLR3-mediated induction of IFN-beta transcription and functions as a feedback negative regulator for TLR3 signaling and cellular anti-viral response.

CHP2 activates the calcineurin/nuclear factor of activated T cells signaling pathway and enhances the oncogenic potential of HEK293 cells.

CHP2 (calcineurin B homologous protein 2) was initially identified as a tumor-associated antigen highly expressed in hepatocellular carcinoma. Its biological function remains largely unknown except for a potential role in transmembrane Na(+)/H(+) exchange. In the present study, we observed that ectopic expression of CHP2 promoted the proliferation of HEK293 cells, whereas knockdown of endogenous CHP2 expression in HepG2 inhibited cell proliferation. When inoculated into nude mice, CHP2 transfected HEK293 cells displayed markedly increased oncogenic potential. In analysis of the underlying molecular mechanisms, we found that like calcineurin B, CHP2 was able to bind to and stimulate the phosphatase activity of calcineurin A. In accord with this, CHP2-transfected cells showed increased nuclear presence of NFATc3 (nuclear factor of activated T cells) and enhanced NFAT activity. Finally, both accelerated cell proliferation and NFAT activation following CHP2 transfection could be suppressed by the calcineurin inhibitor cyclosporine A, suggesting an intrinsic connection between these events. Taken together, our results highlighted a potential role of CHP2 in tumorigenesis and revealed a novel function of CHP2 as an activator of the calcineurin/NFAT signaling pathway.

Identification of RELT homologues that associate with RELT and are phosphorylated by OSR1.

RELL1 and RELL2 are two newly identified RELT homologues that bind to the TNF receptor family member RELT. The expression of RELL1 at the mRNA level is ubiquitous, whereas expression of RELL2 mRNA is more restricted to particular tissues. RELT, RELL1, and RELL2 co-localized with one another at the plasma membrane. The three proteins interacted with one another as demonstrated by in vitro co-immunoprecipitation experiments. We propose that RELL1 and RELL2 be considered RELT family members based on their similar amino acid sequences and on their ability to physically interact with one another. OSR1 was identified through a yeast two-hybrid screen utilizing the intracellular portion of RELL1 as bait, and OSR1 was shown to interact with the three RELT family members by in vitro co-immunoprecipitation experiments. Additionally, OSR1 phosphorylated the RELT family members in an in vitro kinase assay. These results report two novel homologues of RELT that interact with RELT and are phosphorylated by the OSR1 kinase.

The Ret finger protein inhibits signaling mediated by the noncanonical and canonical IkappaB kinase family members.

IFN regulatory factor-3 is a transcription factor that is required for the rapid induction of type I IFNs in the innate antiviral response. Two noncanonical IkappaB kinase (IKK) family members, IKKepsilon and TRAF family-associated NF-kappaB activator-binding kinase-1, have been shown to phosphorylate IFN regulatory factor-3 and are critically involved in virus-triggered and TLR3-mediated signaling leading to induction of type I IFNs. In yeast two-hybrid screens for potential IKKepsilon-interacting proteins, we identified Ret finger protein (RFP) as an IKKepsilon-interacting protein. Coimmunoprecipitation experiments indicated that RFP interacted with IKKepsilon and TRAF family-associated NF-kappaB activator-binding kinase-1 as well as the two canonical IKK family members, IKKbeta and IKKalpha. RFP inhibited activation of the IFN-stimulated response element and/or NF-kappaB mediated by the IKK family members and triggered by TNF, IL-1, polyinosinic-polycytidylic acid (ligand for TLR3), and viral infection. Moreover, knockdown of RFP expression by RNA interference-enhanced activation of IFN-stimulated response element and/or NF-kappaB triggered by polyinosinic-polycytidylic acid, TNF, and IL-1. Taken together, our findings suggest that RFP negatively regulates signaling involved in the antiviral response and inflammation by targeting the IKKs.

VISA is an adapter protein required for virus-triggered IFN-beta signaling.

Viral infection or stimulation of TLR3 triggers signaling cascades, leading to activation of the transcription factors IRF-3 and NF-kappaB, which collaborate to induce transcription of type I interferon (IFN) genes. In this study, we identified a protein termed VISA (for virus-induced signaling adaptor) as a critical component in the IFN-beta signaling pathways. VISA recruits IRF-3 to the cytoplasmic viral dsRNA sensor RIG-I. Depletion of VISA inhibits virus-triggered and RIG-I-mediated activation of IRF-3, NF-kappaB, and the IFN-beta promoter, suggesting that VISA plays a central role in virus-triggered TLR3-independent IFN-beta signaling. Our data also indicate that VISA interacts with TRIF and TRAF6 and mediates bifurcation of the TLR3-triggered NF-kappaB and IRF-3 activation pathways. These findings suggest that VISA is critically involved in both virus-triggered TLR3-independent and TLR3-mediated antiviral IFN signaling.

Identification of a ZU5 and death domain-containing inhibitor of NF-kappaB.

The transcription factor NF-kappaB plays important roles in inflammation and cell survival. NF-kappaB is composed of homodimeric and heterodimeric complexes of Rel/NF-kappaB family members, including p65 (RelA), c-Rel (Rel), RelB, NF-kappaB1/p50, and NF-kappaB2/p52. Here we report the identification and characterization of a novel ZU5 and death domain-containing protein designated ZUD. In reporter gene assays, overexpression of ZUD inhibited NF-kappaB-dependent transcription induced by both tumor necrosis factor (TNF) and interleukin-1 and their downstream signaling proteins. Gel shift assays indicated that the overexpression of ZUD inhibited binding of NF-kappaB to its target sequence. ZUD is a cytoplasmic protein, and coimmunoprecipitation assays indicated that ZUD interacted with the NF-kappaB subunit p105 and transactivator p65. Consistent with its role in inhibition of NF-kappaB-dependent transcription, ZUD sensitized cells to apoptosis induced by TNF and the TNF-related apoptosis-inducing ligand (TRAIL). Our findings suggest that ZUD is an inhibitor of NF-kappaB activation and that this protein may provide an alternative regulatory mechanism for NF-kappaB-mediated transcription.

[Hepatocellular carcinoma associated antigen HCA520, a novel Ca2+-binding protein].

AIM: To analyze the binding activity of the hepatocellular carcinoma associated antigen HCA520 to Ca2+. METHODS: The HCA520 gene was gained by PCR. Then, it was cloned into the prokaryotic expression vector pGEX-4T-3. The GST-HCA520 fusion gene was induced to express in E. coli and the expressed product was purified via GST-agarose affinity resin. The fusion protein was confirmed by Western blot analysis. The Ca2+ binding capability of the fusion protein was analyzed by dot blot. RESULTS: The GST-HCA520 fusion gene was constructed and the protein was successfully expressed and purified, which was identified by DNA sequencing and Western blot respectively. The fusion protein could bind to Ca2+ depend on dosages. CONCLUSION: HCA520, a novel hepatocellular carcinoma associated antigen, is a novel Ca2+ binding protein.

Mechanisms of the TRIF-induced interferon-stimulated response element and NF-kappaB activation and apoptosis pathways.

Toll-like receptor-3 is critically involved in host defense against viruses through induction of type I interferons (IFNs). Recent studies suggest that a Toll/interleukin-1 receptor domain-containing adapter protein (TRIF) and two protein kinases (TANK-binding kinase-1 (TBK1) and IkappaB kinase (IKK)-epsilon) are critically involved in Toll-like receptor-3-mediated IFN-beta production through activation of IFN regulatory factor (IRF)-3 and IRF-7. In this study, we demonstrate that TRIF interacts with both IRF-7 and IRF-3. In addition to TBK1 and IKKepsilon, our results indicate that IKKbeta can also phosphorylate IRF-3 and activate the IFN-stimulated response element. TRIF-induced IRF-3 and IRF-7 activation was mediated by TBK1 and its downstream kinases IKKbeta and IKKepsilon. TRIF induced NF-kappaB activation through an IKKbeta- and tumor necrosis factor receptor-associated factor-6-dependent (but not TBK1- and IKKepsilon-dependent) pathway. In addition, TRIF also induced apoptosis through a RIP/FADD/caspase-8-dependent and mitochondrion-independent pathway. Furthermore, our results suggest that the TRIF-induced IFN-stimulated response element and NF-kappaB activation and apoptosis pathways are uncoupled and provide a molecular explanation for the divergent effects induced by the adapter protein TRIF.