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At the neuron developmental stage, neuron-precursor cells can be differentiated into neuron or glia cells. However, precise molecular mechanism to determine the cell fate has not been clearly demonstrated. In this study, we reveal that Drosophila esgarcot and its mammalian homologue genes, Snail and Slug, play a key role in neuronal differentiation. In Drosophila model system, overexpression of Esg, like as Wingless, suppresses the bristle formation. In contrast, elimination of Esg though RNAi promotes double bristle phenotype. We can also observe the similar phenotype in Snail-overexpression system. In mammalian system, overexpression of Slug or Snail can induce neuronal differentiation. Esg and its mammalian homologue gene Slug directly interact with Daughtherless and its mammalian homologue HEB and eliminate them through siah-1 mediated protein degradation. Thus, overexpression of siah-1 can promote neuron cell differentiation, whereas si-siah-1 blocks the Slug-induced HEB suppression. In fact, Drosophila SINA, Siah-1 homologue, has been also known to be involved in bristle formation and Neuronal differentiation. In addition, it has been revealed that CK1 is involved in Esg or Snail stability and Neuronal differentiation. However, Snail is regulated only by CK1 but not by Siah. Considering the fact that Slug mutations have been found in human genetic disease, waardenberg syndrome, major symptoms of which is loss of hearing neuron and odd eye, our result implies that slug/Snail system is required for proper neuronal differentiation, like as Esg in Drosophila.

Polycomb Group (PcG) genes encode proteins that form large multimeric and chromatin-associated complexes implicated in the stable repression of developmentally essential genes. HPH2, the Homo sapiens polyhomeotic homologue 2, functions as one of the subunits of PcG complex 1. In our study, SIAH-1, an E3 ligase, could directly associate with HPH2 both in vitro and in vivo. Both the cysteine-rich region of SIAH-1 and the PxVxAxP motif of HPH2 were essential for the interaction. HPH2 was co-localized with SIAH-1 in nuclei. Furthermore, SIAH-1 was able to facilitate the ubiquitination and degradation of HPH2 via ubiquitin-proteasome pathway in vivo. The ubiquitination activity was severely impaired in the SIAH-1 mutant that either lost E3 ligase activity or had weakened binding ability with HPH2, strongly suggesting that SIAH-1 was the direct E3 ligase of HPH2. Thus, our results propose a novel role of SIAH-1 in regulating the expression level of HPH2 through the ubiquitin-proteasome pathway., (Copyright 2010 Elsevier Inc. All rights reserved.)

The molecular mechanisms by which hypoxic tumor cells escape radio- and chemotherapy are largely unclear. Homeodomain-interacting protein kinase 2 (HIPK2) drives the apoptotic program in response to DNA-damaging chemotherapeutic drug treatment by phosphorylating the tumor suppressor protein p53 at Ser46. HIPK2 is kept inactive in unstressed cells through ubiquitination and degradation facilitated by the ubiquitin ligases WSB1 and Siah1. Here, we demonstrate that HIPK2 is degraded during hypoxia in a proteasome-dependent and partially Siah1-dependent fashion. Concordantly, hypoxic tumor cells show an impaired p53 Ser46 phosphorylation in response to treatment with the chemotherapeutic Adriamycin. Remarkably, proteasome-inhibition rescues HIPK2 expression in hypoxic hepatoma cells and restores p53 Ser46 phosphorylation and caspase activity after Adriamycin treatment. Our findings suggest a molecular mechanism by which hypoxic cancer cells can escape chemotherapeutic drug treatment and suggest proteasome-inhibition as a promising approach to sensitise hypoxic cancer cells to therapy., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

Siah proteins are vertebrate homologs of the Drosophila 'seven in absentia' gene. In this study, we characterized two splicing forms, Siah1a and Siah1b, of the Xenopus seven in absentia homolog 1 gene (Siah1). Overexpression of xSiah1a led to severe suppression of embryo cleavage, while that of xSiah1b was not effective even at a high dose. Competition analysis demonstrated that co-expression of xSiah1a and 1b generated the same phenotype as overexpression of xSiah1a alone, suggesting that xSiah1b does not interfere with the function of xSiah1a. Since xSiah1b has an additional 31 amino acids in the N-terminus compared to xSiah1a, progressive truncation of xSiah1b from the N-terminus showed that inability of xSiah1b to affect embryo cleavage was associated with the length of the N-terminal extension of extra amino acids. The possible implication of this finding is discussed., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Seven in absentia homolog1 (SIAH1) was reported as a tumor suppressor and played an important role in regulating cell apoptosis. However, its effects on the breast carcinogenesis remain unclear. In this study, our aims were to examine the relationship between SIAH1 and Bcl-2-interacting mediator of cell death (Bim) and to explore the effects of SIAH1 on the breast carcinogenesis. Immunohistochemical analysis in 231 cases of breast tissues showed that the expression of SIAH1 and Bim were significantly decreased in the breast carcinogenesis. Moreover, SIAH1 expression was significantly correlated with Bim. Both SIAH1 and Bim expression were significantly higher in well to moderately differentiated and in early-stage breast cancer. Reverse transcription (RT)-polymerase chain reaction (PCR) and Western blot analysis in paired breast cancer tissues and breast cell lines found that the expression of SIAH1 was lower in the breast cancer tissues and cell lines. SIAH1 inducing apoptosis of the breast cancer cells was dependent on Bim. However, SIAH1 inhibiting invasion of the breast cancer cells was independent of Bim. The increase of the function of SIAH1 to upregulate the expression of Bim may play an important role in the progression of breast cancer. Restoration of the function of SIAH1 may be a new therapeutic target of human breast cancer., ((c) 2010 Wiley-Liss, Inc.)

Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.

We sought to determine the role of human papillomavirus (HPV) E6 and E7 oncogenes in nuclear beta-catenin accumulation, a hallmark of activated canonical Wnt signaling pathway. We used HPV16-positive oropharyngeal cancer cell lines 147T and 090, HPV-negative cell line 040T, and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) to measure the cytoplasmic and nuclear beta-catenin levels and the beta-catenin/Tcf transcriptional activity before and after E6/E7 gene silencing. Repression of HPV E6 and E7 genes induced a substantial reduction in nuclear beta-catenin levels. Luciferase assay showed that transcriptional activation of Tcf promoter by beta-catenin was lower after silencing. The protein levels of beta-catenin are tightly regulated by the ubiquitin/proteasome system. We therefore performed expression analysis of regulators of beta-catenin degradation and nuclear transport and showed that seven in absentia homologue (Siah-1) mRNA and protein levels were substantially upregulated after E6/E7 repression. Siah-1 protein promotes the degradation of beta-catenin through the ubiquitin/proteasome system. To determine whether Siah-1 is important for the proteasomal degradation of beta-catenin in HPV16-positive oropharyngeal cancer cells, we introduced a Siah-1 expression vector into 147T and 090 cells and found substantial reduction of endogenous beta-catenin in these cells. Thus, E6 and E7 are involved in beta-catenin nuclear accumulation and activation of Wnt signaling in HPV-induced cancers. In addition, we show the significance of the endogenous Siah-1-dependent ubiquitin/proteasome pathway for beta-catenin degradation and its regulation by E6/E7 viral oncoproteins in HPV16-positive oropharyngeal cancer cells.

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed.

The translocation and accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the nucleus has closely been associated with cell death induction. However, the mechanism of this process has not been completely understood. The E3 ubiquitin ligase siah-1 (seven in absentia homolog 1) has recently been identified as a potential shuttle protein to transport GAPDH from the cytosol to the nucleus. Previously, we have demonstrated that elevated glucose levels induce GAPDH nuclear accumulation in retinal Müller cells. Therefore, this study investigated the role of siah-1 in high glucose-induced GAPDH nuclear translocation and subsequent cell death in retinal Müller cells. High glucose significantly increased siah-1 expression within 12 h. Under hyperglycemic conditions, siah-1 formed a complex with GAPDH and was predominantly localized in the nucleus of Müller cells. siah-1 knockdown using 50 nm siah-1 small interfering RNA significantly decreased high glucose-induced GAPDH nuclear accumulation at 24 h by 43.8 +/- 4.0%. Further, knockdown of siah-1 prevented high glucose-induced cell death of Müller cells potentially by inhibiting p53 phosphorylation consistent with previous observations, indicating that nuclear GAPDH induces cell death via p53 activation. Therefore, inhibition of GAPDH nuclear translocation and accumulation by targeting siah-1 promotes Müller cell survival under hyperglycemic conditions.

Seven in absentia homolog 1 (SIAH1), a homologue of Drosophila seven in absentia (Sina), has emerged as a tumor suppressor and plays an important role in regulating cell apoptosis. To investigate the role and possible mechanism of SIAH1 in breast cancer cells, we up-regulated the expression of SIAH1 using pcDNA3-myc-SIAH1 and knocked down SIAH1 using SIAH1 siRNA. We found that the overexpression of SIAH1 induced cell apoptosis by up-regulating the level of Bim through the activation of the JNK signaling pathway, and the suppression of SIAH1 expression increased cell invasion via the activation of the ERK signaling pathway in breast cancer cells. All these results indicate that the JNK and ERK signaling pathways may play an important role in the SIAH1-dependent biological behavior of breast cancer, and it may be a good molecular therapeutic target to increase the expression level of SIAH1 through promoting cell apoptosis and inhibiting cell invasion in human breast cancer.

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 delta isoform (PP1cdelta) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.

Molecular lesions in Wnt/beta-catenin signaling and subsequent up-regulation of beta-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), and promoted the degradation of intracellular beta-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known beta-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer., (Copyright 2009 Elsevier Inc. All rights reserved.)

Background: During the development of the Drosophila eye, specific cell types differentiate from an initially equipotent group of uncommitted precursor cells. The lozenge (lz) gene, which is a member of the Runt family of transcriptional regulators, plays a pivotal role in mediating this process through regulating the expression of several fate-specifying transcription factors. However, the regulation of lz, and the control of lz expression levels in different cell types is not fully understood., Results: Here, we show a genetic interaction between Tramtrack69 (Ttk69) a key transcriptional repressor and an inhibitor of neuronal fate specification, and lz, the master patterning gene of cells posterior to the morphogenetic furrow in the Drosophila eye disc. Loss of Ttk69 expression causes the development of ectopic R7 cells in the third instar eye disc, with these cells being dependent upon Lz for their development. Using the binary UAS Gal4 system, we show that overexpression of Ttk69 causes the loss of lz-dependent differentiating cells, and a down-regulation of Lz expression in the developing eye. The loss of lz-dependent cells can be rescued by overexpressing lz via a GMR-lz transgene. We provide additional data showing that factors functioning upstream of Ttk69 in eye development regulate lz in a Ttk69-dependent manner., Conclusions: Our results lead us to conclude that Ttk69 can either directly or indirectly repress lz gene expression to prevent the premature development of R7 precursor cells in the developing eye of Drosophila. We therefore define a mechanism for the tight regulatory control of the master pre-patterning gene, lz, in early Drosophila eye development and provide insight into how differential levels of lz expression can be achieved to effect specific cell fate outcomes.

Cells induce the hypoxia responses to adapt to the environment when organisms are exposed to a low oxygen environment. The hypoxia response leads to the activation of multiple cellular signalling pathways involved in regulation of respiration, metabolism, cell survival and so forth. Hypoxia-Inducible-Factor (HIF) pathway plays a central role during the hypoxia response as its expression and activity are regulated in an oxygen-dependent manner and it also regulates the expression of multiple hypoxia responsive genes. The expression of HIF is regulated by proline hydroxylation, which is mediated by HIF prolyl-hydroxylase named PHD. The hydroxylated HIF-alpha subunit is degraded via the ubiquitin-proteasome pathway. The PHD activity needs to be strictly regulated to ensure the stabilization of HIF under hypoxic conditions, because PHD leads to HIF degradation. This review describes the regulatory mechanism of HIF stability and activity under normoxia and hypoxic conditions. Furthermore, the role of the HIF-independent pathways during the hypoxia response, which is as important as the HIF pathway, will also be described.

Laminin alpha2-deficient congenital muscular dystrophy, called MDC1A, is a rare, devastating genetic disease characterized by severe neonatal hypotonia ("floppy infant syndrome"), peripheral neuropathy, inability to stand or walk, respiratory distress, and premature death in early life. Transgenic overexpression of the apoptosis inhibitor protein BCL-2, or deletion of the proapoptotic Bax gene in a mouse model for MDC1A prolongs survival and mitigates pathology, indicating that apoptotic events are involved in the pathology. Here we demonstrate that the proapoptotic glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Siah1-CBP/p300-p53 pathway is activated in a mouse model for MDC1A. Moreover, we show that omigapil, which inhibits GAPDH-Siah1-mediated apoptosis, ameliorates several pathological hallmarks in the MDC1A mouse model. Specifically, we demonstrate that treatment with omigapil inhibits apoptosis in muscle, reduces body weight loss and skeletal deformation, increases locomotive activity, and protects from early mortality. These data qualify omigapil, which is in late phase of clinical development for human use, as a drug candidate for the treatment of MDC1A.

The Siah (seven in absentia homolog) family of RING-domain proteins are components of ubiquitin ligase complexes, targeting proteins for proteasomal degradation. Siah family members have been reported to function in Ras, estrogen, DNA-damage, and hypoxia response pathways. Although earlier reports implicated Siah proteins as tumor suppressors, recent studies in mouse models have shown that Siah inhibition impairs tumor growth and metastasis. Given their central role in oncogenic and angiogenic pathways, Siah proteins are attractive novel therapeutic targets in cancer.

Microtubule plus-end tracking proteins (+TIPs) control microtubule dynamics in fundamental processes such as cell cycle, intracellular transport, and cell motility, but how +TIPs are regulated during mitosis remains largely unclear. Here we show that the endogenous end-binding protein family EB3 is stable during mitosis, facilitates cell cycle progression at prometaphase, and then is down-regulated during the transition to G(1) phase. The ubiquitin-protein isopeptide ligase SIAH-1 facilitates EB3 polyubiquitination and subsequent proteasome-mediated degradation, whereas SIAH-1 knockdown increases EB3 stability and steady-state levels. Two mitotic kinases, Aurora-A and Aurora-B, phosphorylate endogenous EB3 at Ser-176, and the phosphorylation triggers disruption of the EB3-SIAH-1 complex, resulting in EB3 stabilization during mitosis. Our results provide new insight into a regulatory mechanism of +TIPs in cell cycle transition.

The different activities of the tumor suppressor p53 are tightly regulated by various negative and positive feedback loops, which allow accurate control of its function. Here we show that the p53-inducible ubiquitin E3 ligase Siah-1L can bind to the p53 phosphorylating kinase HIPK2 and thus allows its ubiquitination and proteasomal elimination. Siah-1L also eliminates the HIPK family member HIPK3, indicating that its activity is not restricted to one member of the HIPK family. The stimulating effect of HIPK2 on p53-triggered transcription is counteracted by Siah-1L, thus showing the occurrence of another negative feedback loop controlling the p53 response.

Aberrant accumulation of intracellular beta-catenin in intestinal epithelial cells is a frequent early event during the development of colon cancer. To identify small molecules that decrease the level of intracellular beta-catenin, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells to detect compounds that inhibit TOPFlash reporter activity, which was stimulated by Wnt3a-conditioned medium. We found that isoreserpine promoted the degradation of intracellular beta-catenin by up-regulation of Siah-1 in HEK293 and HCT116 colon cancer cells. Moreover, isoreserpine repressed the expression of beta-catenin/T-cell factor (TCF)-dependent genes, such as cyclin D1 and c-myc, resulting in the suppression of HCT116 cell proliferation. Our findings suggest that isoreserpine can potentially be used as a chemotherapeutic agent against colon cancer.

Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Its activity is strictly controlled by the pRB/E2F pathway. In the majority of cancer cells, however, this pathway is frequently found deregulated, and the underlying mechanism involving transcriptional control by E2F1 has not yet been fully elucidated. Here we report the identification of two putative E2F1-binding sites located upstream from Siah1 transcription start site (+1). Chromatin immunoprecipitation assay reveals that transcription factor E2F1 is capable of binding to the putative sites, and luciferase reporter assay shows that E2F1 can activate transcription from the Siah1 promoter. Ectopic expression of E2F1 elevates the Siah1 level, hence suppressing the beta-catenin/TCF activity. Consistently, knock-down of endogenous E2F1 by a shRNA strategy results in reduced expression of Siah1. Moreover, repression of beta-catenin/TCF activity by E2F1 can be attenuated by shRNA-based repression of endogenous Siah1, implying that Siah1 is a bona fide E2F1 target gene, which at least partly, mediates the suppression of beta-catenin/TCF signalling pathway.

We recently reported a cell death cascade whereby cellular stressors activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The nuclear GAPDH/Siah complex augments p300/CBP-associated acetylation of nuclear proteins, including p53, which mediate cell death. We report a 52 kDa cytosolic protein, GOSPEL, which physiologically binds GAPDH, in competition with Siah, retaining GAPDH in the cytosol and preventing its nuclear translocation. GOSPEL is neuroprotective, as its overexpression prevents NMDA-glutamate excitotoxicity while its depletion enhances death in primary neuron cultures. S-nitrosylation of GOSPEL at cysteine 47 enhances GAPDH-GOSPEL binding and the neuroprotective actions of GOSPEL. In intact mice, virally delivered GOSPEL selectively diminishes NMDA neurotoxicity. Thus, GOSPEL may physiologically regulate the viability of neurons and other cells.

Homeodomain-interacting protein kinase 2 (HIPK2) induces apoptosis and, thus, is maintained at a low level via ubiquitin-mediated proteolysis. In a yeast two-hybrid screen, we identified Siah1, a RING finger E3 ubiquitin ligase, as an interacting protein of HIPK2. Siah1 targeted HIPK2 for poly-ubiquitination-mediated proteasomal degradation. Degradation of HIPK2 by Siah1 was blocked by forced expression of either Mixed Lineage Kinase-3 or Epstein-Barr viral protein LMP-1, as well as by DNA damaging stimuli. These findings effectively illustrate the regulatory mechanisms underlying HIPK2 stabilization by escape from Siah1-mediated degradation, and that Siah1 is an integration target for several internal or external stimuli for HIPK2 stabilization.

Purpose: In our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance., Patients and Methods: SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression., Results: SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; P = 0.05) and progression-free survival (PFS) (HR = 0.79; P = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; P = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells (P = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF., Conclusion: In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF.

Oxygen deprivation (hypoxia) triggers a complex network of signaling pathways that result in changed gene expression patterns in order to cope with this challenge. Recent work has identified the serine/threonine kinase HIPK2 as a novel regulatory protein participating in hypoxic gene regulation. HIPK2 can affect apical as well as downstream events during the hypoxic response. Under normoxic conditions, HIPK2-mediated phosphorylation of the ubiquitin E3 ligase Siah2 weakens mutual binding and destabilizes the phosphorylated E3 ligase. Low oxygen levels result in strongly increased HIPK2/Siah2 interactions that lead to efficient polyubiquitylation and proteasomal degradation of the kinase. At the apical level, the Siah2 inhibiting phosphorylations are lost, thus allowing Siah2-dependent proteolysis of dioxygenases which in turn allows for activation of transcription factor HIF. Downstream events of the hypoxic response are affected by the proteasomal elimination of HIPK2 from gene repressing complexes, an event that allows for full induction of gene expression. Thus HIPK2 can regulate a subset of HIF-dependent and -independent genes during the hypoxic response.

Parkinson disease (PD) is characterized by the presence of ubiquitylated inclusions and the death of dopaminergic neurons. Seven in absentia homolog (SIAH) is a ubiquitin-ligase that ubiquitylates alpha-synuclein and synphilin-1 and is present in Lewy bodies of PD patients. Understanding the mechanisms that regulate the ubiquitylation of PD-related proteins might shed light on the events involved in the formation of Lewy bodies and death of neurons. We show in this study that the recently described synphilin-1 isoform, synphilin-1A, interacts in vitro and in vivo with the ubiquitin-protein isopeptide ligase SIAH and regulates its activity toward alpha-synuclein and synphilin-1. SIAH promotes limited ubiquitylation of synphilin-1A that does not lead to its degradation by the proteasome. SIAH also increases the formation of synphilin-1A inclusions in the presence of proteasome inhibitors, supporting the participation of ubiquitylated synphilin-1A in the formation of Lewy body-like inclusions. Synphilin-1A/SIAH inclusions recruit PD-related proteins, such as alpha-synuclein, synphilin-1, Parkin, PINK1, and UCH-L1. We found that synphilin-1A robustly increases the steady-state levels of SIAH by decreasing its auto-ubiquitylation and degradation. In addition, synphilin-1A blocks the ubiquitylation and degradation of the SIAH substrates synphilin-1 and deleted in colon cancer protein. Furthermore, synphilin-1A strongly decreases the monoubiquitylation of alpha-synuclein by SIAH and the formation of alpha-synuclein inclusions, supporting a role for monoubiquitylation in alpha-synuclein inclusion formation. Our results suggest a novel function for synphilin-1A as a regulator of SIAH activity and formation of Lewy body-like inclusions.

Many studies have shown that the activation of beta-catenin signaling can promote oncogenesis, and it is therefore of interest to find agents that modulate this pathway. Recent work has shown using B lymphoma cells that infection by Epstein-Barr virus (EBV) and expression of its latent membrane protein (LMP)-1, cause increases in the expression of beta-catenin and cellular transformation. Conversely, results from cell-based small molecule screening studies have shown that the antibiotic hexachlorophene can down-regulate beta-catenin in colon cancer cells. Here we report that hexachlorophene also counteracts the elevated beta-catenin levels in EBV-infected B lymphomas. This is associated with restoration in levels of Siah-1 (an E3 ubiquitin ligase that is active in beta-catenin regulation) which had been diminished by LMP-1. Our results suggest that Siah-1 is targeted by both LMP-1 and hexachlorophene with opposite effects. The hexachlorophene modulation of Siah-1 and beta-catenin is independent of p53 and results in reduced expression of cyclin-D1 and c-Myc (target genes of beta-catenin), leading to the growth arrest of B lymphoma cells. From these results we propose that hexachlorophene may provide a novel therapeutic strategy for EBV-infected B lymphoma cells by reducing beta-catenin levels via the restoration of Siah-1.

Growing evidence indicates that ubiquitin ligases play a critical role in the hypoxia response. Among them, Siah2, a RING finger ligase, is an important regulator of pathways activated under hypoxia. Siah2 regulates prolyl hydroxylases PHD3 and 1 under oxygen concentration of 2% to 5%, thereby allowing accumulation of hypoxia-inducible factor (HIF)-1alpha, a master regulator of the hypoxia response within the range of physiological normoxic to mild hypoxic conditions. Growing evidence also indicates an important function for Siah2 in tumor development and progression based on pancreatic cancer, mammary tumor, and melanoma mouse models. This review summarizes our current understanding of Siah2 regulation and function with emphasis on hypoxia and tumorigenesis.

How cells become malignant has preoccupied scientists for over a century. However, the converse question is also valid: are tumour cells capable of reverting from their malignant state? Askanazy's studies in 1907 indicated that teratoma cells could differentiate into normal somatic tissues and current evidence indicates that some tumour cells have acquired the molecular circuitry that results in the negation of chromosomal instability, translocations, oncogene activation and loss of tumour suppressor genes. Studying these extremely rare events of tumour reversion and deciphering these pathways, which involve SIAH1, presenilin 1, TSAP6 and translationally controlled tumour protein (TCTP), could lead to new avenues in cancer treatment.

Mutations in the gene encoding the E3 ubiquitin-protein ligase parkin have been shown to be a common genetic cause of familial early-onset Parkinson's disease (PD). In addition to its function in the ubiquitin-proteasome system (UPS), parkin has been ascribed general neuroprotective properties. Stress and mutation induced decreases in parkin solubility leading to compromised cytoprotection have recently been reported. We systematically investigated whether PD-related stresses including MG132 and epoxomicin (proteasomal impairment), tunicamycin (unfolded protein stress), and rotenone (mitochondrial dysfunction) resulted in expressional changes of parkin and other E3 ubiquitin ligases (dorfin, SIAH-1). Rotenone and tunicamycin did not change parkin mRNA levels, whereas proteasomal inhibition resulted in a reduction of parkin mRNA in PC12 cells as well as in SH-SY5Y cells. Therefore, surprisingly, cells did not react with a compensatory parkin upregulation under proteasomal inhibition, although, in parallel, parkin protein shifted to the insoluble fraction, reducing soluble parkin levels in the cytosol. Since the mRNA of the parkin-coregulated gene PACRG paralleled the parkin mRNA at least partly, we suspect a promoter-driven mechanism. Our study, therefore, shows a link between proteasomal impairment and parkin expression levels in cell culture, which is intriguing in the context of the described and debated proteasomal dysfunction in the substantia nigra of PD patients.

Homeodomain-interacting protein kinase 2 (HIPK2) is an emerging regulator of cell growth and apoptosis in various cell types, tissues and organisms. Previous work indicates that HIPK2 is a potential tumour suppressor and DNA damage-responsive kinase, which phosphorylation-dependently activates the apoptotic programme by engaging diverse downstream targets, including tumour suppressor p53 and the anti-apoptotic transcriptional corepressor C-terminal binding protein. The regulation of HIPK2, however, remained largely obscure. Recent studies show that HIPK2 activity is mainly controlled at the post-transcriptional level through targeted proteolysis. Caspase-dependent processing triggers HIPK2 hyperactivation, whereas the ubiquitin-proteasome system (UPS) keeps HIPK2 in check by targeting it for degradation. Both HIPK2 hyperactivation and HIPK2 degradation are under the control of transcription factor p53. Negative regulation of HIPK2 by the UPS is abolished in response to DNA damage, which facilitates HIPK2 stabilization and activation. Here we discuss these findings in the context of DNA damage signalling and tumour suppression.

Tumor hypoxia induces the upregulation of hypoxia-inducible factor 1alpha (Hif-1alpha), which in turn induces the expression of genes including VEGF to recruit new blood vessel outgrowth, enabling tumor growth and metastasis. Interference with the Hif-1 pathway and neoangiogenesis is an attractive antitumor target. The hydroxylation of Hif-1alpha by prolyl-hydroxylase (PHD) proteins during normoxia serves as a recognition motif for its proteasomal degradation. However, under hypoxic conditions, hydroxylation is inhibited and furthermore, PHD proteins are themselves polyubiquitylated and degraded by Siah ubiquitin ligases. Our data demonstrate for the first time that inhibition of the interaction between Siah and PHD proteins using a fragment derived from a Drosophila protein (phyllopod) interferes with the PHD degradation. Furthermore, cells stably expressing the phyllopod fragment display reduced upregulation of Hif-1alpha protein levels and Hif-1-mediated gene expression under hypoxia. In a syngeneic mouse model of breast cancer, the phyllopod fragment reduced tumor growth and neoangiogenesis and prolonged survival of the mice. In addition, levels of Hif-1alpha and its target Glut-1 are reduced in tumors expressing the phyllopod fragment. These data show, in a proof-of-principle study, that Siah protein, the most upstream component of the hypoxia pathway yet identified, is a viable drug target for antitumor therapies.

Oxygen deprivation (hypoxia) results in reprogrammed gene expression patterns that induce multifaceted cellular responses. Here we identify a regulated interaction between the serine/threonine kinase HIPK2 and the ubiquitin E3 ligase Siah2 as a mechanism controlling the hypoxic response. Under normoxic conditions, several mechanisms ensure HIPK2 stability: only a fraction of HIPK2 is found in association with Siah2, whereas HIPK2-mediated phosphorylation of this E3 ligase at positions 26, 28 and 68 weakens mutual binding and destabilizes its phosphorylated interaction partner. Hypoxic conditions allow a markedly increased HIPK2/Siah2 interaction and result in efficient polyubiquitylation and proteasomal degradation of the kinase. Accordingly, hypoxia-induced HIPK2 elimination is markedly reduced in Siah2-deficient cells. As HIPK2 has an important role as a negative regulator of gene expression, its elimination from promoter-associated repressor complexes allows the induction of a substantial fraction of hypoxia-induced genes.

Background: Hyperactivated epidermal growth factor receptor (EGFR) and/or RAS signaling drives cellular transformation and tumorigenesis in human lung cancers, but agents that block activated EGFR and RAS signaling have not yet been demonstrated to substantially extend patients' lives. The human homolog of Drosophila seven-in-absentia--SIAH-1 and SIAH-2--are ubiquitin E3 ligases and conserved downstream components of the RAS pathway that are required for mammalian RAS signal transduction. We examined whether inhibiting SIAH-2 function blocks lung cancer growth., Methods: The antiproliferative and antitumorigenic effects of lentiviral expression of anti-SIAH-2 molecules (ie, a dominant-negative protease-deficient mutant of SIAH-2 [SIAH-2(PD)] and short hairpin RNA [shRNA]-mediated gene knockdown against SIAH-2) were assayed in normal human lung epithelial BEAS-2B cells and in human lung cancer BZR, A549, H727, and UMC11 cells by measuring cell proliferation rates, by assessing MAPK and other activated downstream components of the RAS pathway by immunoblotting, assessing apoptosis by terminal deoxynucleotidyltransferase-mediated UTP end-labeling (TUNEL) assay, quantifying anchorage-independent cell growth in soft agar, and assessing A549 cell-derived tumor growth in athymic nude mice (groups of 10 mice, with two injections of 1 x 10(6) cells each at the dorsal left and right scapular areas). All statistical tests were two-sided., Results: SIAH-2 deficiency in human lung cancer cell lines reduced MAPK signaling and statistically significantly inhibited cell proliferation compared with those in SIAH-proficient cells (P < .001) and increased apoptosis (TUNEL-positive A549 cells 3 days after lentivirus infection: SIAH-2(PD) vs control, 30.1% vs 0.0%, difference = 30.1%, 95% confidence interval [CI] = 23.1% to 37.0%, P < .001; SIAH-2-shRNA#6 vs control shRNA, 27.9% vs 0.0%, difference = 27.9%, 95% CI = 23.1% to 32.6%, P < .001). SIAH-2 deficiency also reduced anchorage-independent growth of A549 cells in soft agar (mean number of colonies: SIAH-2(PD) vs control, 124.7 vs 57.3, difference = 67.3, 95% CI = 49.4 to 85.3, P < .001; shRNA-SIAH-2#6 vs shRNA control: 27.0 vs 119.7, difference = 92.7, 95% CI = 69.8 to 115.5, P < .001), and blocked the growth of A549 cell-derived tumors in nude mice (mean tumor volume on day 36 after A549 cell injection: SIAH-2(PD) infected vs uninfected, 191.0 vs 558.5 mm(3), difference = 367.5 mm(3), 95% CI = 237.6 to 497.4 mm(3), P < .001; SIAH-2(PD) infected vs control infected, 191.0 vs 418.3 mm(3), difference = 227.5 mm(3), 95% CI = 87.4 to 367.1 mm(3), P = .003; mean resected tumor weight: SIAH-2(PD) infected vs uninfected, 0.12 vs 0.48 g, difference = 0.36 g, 95% CI = 0.23 to 0.50 g, P < .001; SIAH-2(PD) infected vs control infected, 0.12 vs 0.29 g, difference = 0.17 g, 95% CI = 0.04 to 0.31 g, P = .016)., Conclusions: SIAH-2 may be a viable target for novel anti-RAS and anticancer agents aimed at inhibiting EGFR and/or RAS-mediated tumorigenesis.

Neurogenesis requires precise control of cell specification and division. In Drosophila, the timing of cell division of the sensory organ precursor (SOP) is under strict temporal control. But how the timing of mitotic entry is determined remains poorly understood. Here, we present evidence that the timing of the G2-M transition is determined by when proneural proteins are degraded from SOPs. This process requires the E3 ubiquitin ligase complex, including the RING protein Sina and the adaptor Phyl. In phyl mutants, proneural proteins accumulate, causing delay or arrest in the G2-M transition. The G2-M defect in phyl mutants is rescued by reducing the ac and sc gene doses. Misexpression of phyl downregulates proneural protein levels in a sina-dependent manner. Phyl directly associates with proneural proteins to act as a bridge between proneural proteins and Sina. As phyl is a direct transcriptional target of Ac and Sc, our data suggest that, in addition to mediating cell cycle arrest, proneural protein initiates a negative-feedback regulation to time the mitotic entry of neural precursors.

Protein ubiquitination is a posttranslational regulatory process essential for plant growth and interaction with the environment. E3 ligases, to which the seven in absentia (SINA) proteins belong, determine the specificity by selecting the target proteins for ubiquitination. SINA proteins are found in animals as well as in plants, and a small gene family with highly related members has been identified in the genome of rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), Medicago truncatula, and poplar (Populus trichocarpa). To acquire insight into the function of SINA proteins in nodulation, a dominant negative form of the Arabidopsis SINAT5 was ectopically expressed in the model legume M. truncatula. After rhizobial inoculation of the 35S:SINAT5DN transgenic plants, fewer nodules were formed than in control plants, and most nodules remained small and white, a sign of impaired symbiosis. Defects in rhizobial infection and symbiosome formation were observed by extensive microscopic analysis. Besides the nodulation phenotype, transgenic plants were affected in shoot growth, leaf size, and lateral root number. This work illustrates a function for SINA E3 ligases in a broad spectrum of plant developmental processes, including nodulation.

The tumour suppressor HIPK2 is an important regulator of cell death induced by DNA damage, but how its activity is regulated remains largely unclear. Here we demonstrate that HIPK2 is an unstable protein that colocalizes and interacts with the E3 ubiquitin ligase Siah-1 in unstressed cells. Siah-1 knockdown increases HIPK2 stability and steady-state levels, whereas Siah-1 expression facilitates HIPK2 polyubiquitination, degradation and thereby inactivation. During recovery from sublethal DNA damage, HIPK2, which is stabilized on DNA damage, is degraded through a Siah-1-dependent, p53-controlled pathway. Downregulation of Siah-1 inhibits HIPK2 degradation and recovery from damage, driving the cells into apoptosis. We have also demonstrated that DNA damage triggers disruption of the HIPK2-Siah-1 complex, resulting in HIPK2 stabilization and activation. Disruption of the HIPK2-Siah-1 complex is mediated by the ATM/ATR pathway and involves ATM/ATR-dependent phosphorylation of Siah-1 at Ser 19. Our results provide a molecular framework for HIPK2 regulation in unstressed and damaged cells.

We previously created a knock-in mutant mouse harboring a dominantly negative mutant thyroid hormone receptor beta (TRbeta(PV/PV) mouse) that spontaneously develops a follicular thyroid carcinoma similar to human thyroid cancer. We found that beta-catenin, which plays a critical role in oncogenesis, was highly elevated in thyroid tumors of TRbeta(PV/PV) mice. We sought to understand the molecular basis underlying aberrant accumulation of beta-catenin by mutations of TRbeta in vivo. Cell-based studies showed that thyroid hormone (T3) induced the degradation of beta-catenin in cells expressing TRbeta via proteasomal pathways. In contrast, no T3-induced degradation occurred in cells expressing the mutant receptor (TRbetaPV). In vitro binding studies and cell-based analyses revealed that beta-catenin physically associated with unliganded TRbeta or TRbetaPV. However, in the presence of T3, beta-catenin was dissociated from TRbeta-beta-catenin complexes but not from TRbetaPV-beta-catenin complexes. beta-Catenin signaling was repressed by T3 in TRbeta-expressing cells through decreasing beta-catenin-mediated transcription activity and target gene expression, whereas sustained beta-catenin signaling was observed in TRbetaPV-expressing cells. The stabilization of beta-catenin, via association with a mutated TRbeta, represents a novel activating mechanism of the oncogenic protein beta-catenin that could contribute to thyroid carcinogenesis in TRbeta(PV/PV) mice.

Tribbles 3 homolog (TRB3) is recently identified as a scaffold-like regulator of various signal transducers and has been implicated in several processes including insulin signaling, NF-kappaB signaling, lipid metabolism and BMP signaling. To further understand cellular mechanisms of TRB3 regulation, we performed a yeast two-hybrid screen to identify novel TRB3 interacting proteins and totally obtained ten in-frame fused preys. Candidate interactions were validated by co-immunoprecipitation assays in mammalian cells. We further characterized the identified proteins sorted by Gene Ontology Annotation. Its interaction with the E3 ubiquitin ligase SIAH1 was further investigated. SIAH1 could interact with TRB3 both in vitro and in vivo. Importantly, SIAH1 targeted TRB3 for proteasome-dependent degradation. Cotransfection of SIAH1 could withdraw up-regulation of TGF-beta signaling by TRB3, suggesting SIAH1-induced degradation of TRB3 represents a potential regulatory mechanism for TGF-beta signaling.

The chromosomal translocation products AML1-ETO and PML-RARalpha contribute to the pathogenesis of leukemias. Here, we demonstrate that both AML1-ETO and PML-RARalpha are degraded by the ubiquitin-proteasome system and that their turnover critically depends on the E2-conjugase UbcH8 and the E3-ligase SIAH-1. Contrary to its role in HDAC2 degradation, the E3-ligase RLIM does not target AML1-ETO and PML-RARalpha for ubiquitin-dependent elimination. RLIM rather is a substrate of SIAH-1, which indicates that these E3-ligases operate in a hierarchical order. Remarkably, proteasomal degradation of leukemia fusion proteins, in addition to the block of histone deacetylase (HDAC) enzymatic activity is a consequence of HDAC-inhibitor treatment. The former requires the induction of UbcH8 expression and each of these processes might be beneficial for leukemia treatment. Our observations shed light on the mechanism determining the interplay between E2-conjugases, E3-ligases, and their substrates and suggest a strategy for utilizing the ubiquitylation machinery in a therapeutic setting.

A-kinase anchor protein 121 (AKAP121) assembles a multivalent signalling complex on the outer mitochondrial membrane that controls persistence and amplitude of cAMP and src signalling to mitochondria, and plays an essential role in oxidative metabolism and cell survival. Here, we show that AKAP121 levels are regulated post-translationally by the ubiquitin/proteasome pathway. Seven In-Absentia Homolog 2 (Siah2), an E3-ubiquitin ligase whose expression is induced in hypoxic conditions, formed a complex and degraded AKAP121. In addition, we show that overexpression of Siah2 or oxygen and glucose deprivation (OGD) promotes Siah2-mediated ubiquitination and proteolysis of AKAP121. Upregulation of Siah2, by modulation of the cellular levels of AKAP121, significantly affects mitochondrial activity assessed as mitochondrial membrane potential and oxidative capacity. Also during cerebral ischaemia, AKAP121 is degraded in a Siah2-dependent manner. These findings reveal a novel mechanism of attenuation of cAMP/PKA signaling, which occurs at the distal sites of signal generation mediated by proteolysis of an AKAP scaffold protein. By regulating the stability of AKAP121-signalling complex at mitochondria, cells efficiently and rapidly adapt oxidative metabolism to fluctuations in oxygen availability.

The activity of hypoxia-inducible factors-1alpha (HIF-1alpha) is regulated by two types of hydroxylases, prolyl-hydroxylase (PHD) and aspargynyl-hydroxylase factor inhibiting HIF-1alpha (FIH). Hydroxylation of HIF-1alpha by PHD and FIH causes proteasomal degradation and transcriptional inhibition of HIF-1alpha, respectively. Siah ubiquitin ligases regulate the abundance of PHD via targeting for proteasomal degradation. The present study investigated the role of Siah-1 in the regulation of FIH abundance under normoxic conditions. Immunohistochemical analysis of the rat brains revealed that both Siah-1 and FIH were widely distributed in the central nervous system including the cerebral cortex, the hippocampus, the striatum, the olfactory bulb, the putamen, the thalamus, the celleberum, and the brain stem. In the hippocampus, both Siah-1 and FIH predominantly expressed in neurons. Siah-1 and FIH localized mostly in the cytoplasm. In mammalian cells, FIH expression levels were increased in the presence of a proteasomal inhibitor MG132, suggesting that FIH is degraded by the ubiquitin-proteasome system. Immunoprecipitation assay and ubiquitination assay revealed that Siah-1 interacted with, and ubiquitinated FIH. Under normoxic conditions, Siah-1 facilitated degradation of FIH. On the other hand, when endogenous Siah-1 expression was suppressed using siRNA, FIH expression levels were increased, as compared to control. These results suggest that Siah-1 might play a role as a regulator of FIH abundance under normoxic conditions.

Point mutations and gene multiplication of alpha-synuclein cause autosomal dominant familial Parkinson's disease (PD). Moreover, alpha-synuclein- and ubiquitin-positive inclusion bodies are the pathological hallmarks of PD and several other neurodegenerative diseases, such as dementia with Lewy bodies and multiple system atrophy. Despite the presence of ubiquitinated alpha-synuclein species in Lewy bodies, the regulation of alpha-synuclein ubiquitination and its role in Lewy body formation and neurodegeneration remain poorly understood. Here, we report that alpha-synuclein interacts and colocalizes with mammalian seven in absentia homologue-1 (Siah-1), a RING-type E3 ubiquitin-protein ligase. Siah-1 binds the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitates mono- and di-ubiquitination of alpha-synuclein in vivo. The ubiquitination of alpha-synuclein by Siah-1 is disrupted by the PD-linked A30P mutation but not by A53T mutation. We find that Siah-1-mediated ubiquitination does not target alpha-synuclein for degradation by the proteasome, but rather, it promotes alpha-synuclein aggregation and enhances alpha-synuclein toxicity. Our findings suggest that Siah-1-mediated alpha-synuclein ubiquitination may play a critical role in Lewy body formation and PD pathogenesis.

Seven-in-absentia homologue 1 (Siah1) is an E3 ubiquitin ligase that regulates the ubiquitination and proteasome-dependent degradation of a number of proteins. Here we report that Siah1 modulates multidrug resistance 1 (MDR1)/P-glycoprotein-mediated drug resistance in the cancer cell lines examined. Siah1, but not its ligase-dead mutant, down-regulates MDR1/P-glycoprotein and sensitizes the multidrug-resistant cells to chemotherapeutic agents. Mechanistically, Siah1 does not promote P-glycoprotein degradation but decreases its expression transcriptionally by promoting c-Jun transcription factor binding to the activator protein 1 (AP1) site in the MDR1 promoter. Moreover, Siah1 triggers c-Jun NH2-terminal kinase (JNK) activation, leading to enhanced phosphorylation of c-Jun, and the JNK/c-Jun signalling axis is critical for Siah1 to down-regulate MDR1/P-glycoprotein expression. These findings demonstrate a previously unidentified role for Siah1 in regulating MDR1/P-glycoprotein expression through the JNK/c-Jun pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection.

alpha-Synuclein plays a major role in Parkinson disease. Unraveling the mechanisms of alpha-synuclein aggregation is essential to understand the formation of Lewy bodies and their involvement in dopaminergic cell death. alpha-Synuclein is ubiquitylated in Lewy bodies, but the role of alpha-synuclein ubiquitylation has been mysterious. We now report that the ubiquitin-protein isopeptide ligase seven in absentia homolog (SIAH) directly interacts with and monoubiquitylates alpha-synuclein and promotes its aggregation in vitro and in vivo, which is toxic to cells. Mass spectrometry analysis demonstrates that SIAH monoubiquitylates alpha-synuclein at lysines 12, 21, and 23, which were previously shown to be ubiquitylated in Lewy bodies. SIAH ubiquitylates lysines 10, 34, 43, and 96 as well. Suppression of SIAH expression by short hairpin RNA to SIAH-1 and SIAH-2 abolished alpha-synuclein monoubiquitylation in dopaminergic cells, indicating that endogenous SIAH ubiquitylates alpha-synuclein. Moreover, SIAH co-immunoprecipitated with alpha-synuclein from brain extracts. Inhibition of proteasomal, lysosomal, and autophagic pathways, as well as overexpression of a ubiquitin mutant less prone to deubiquitylation, G76A, increased monoubiquitylation of alpha-synuclein by SIAH. Monoubiquitylation increased the aggregation of alpha-synuclein in vitro. At the electron microscopy level, monoubiquitylated alpha-synuclein promoted the formation of massive amounts of amorphous aggregates. Monoubiquitylation also increased alpha-synuclein aggregation in vivo as observed by increased formation of alpha-synuclein inclusion bodies within dopaminergic cells. These inclusions are toxic to cells, and their formation was prevented when endogenous SIAH expression was suppressed. Our data suggest that monoubiquitylation represents a possible trigger event for alpha-synuclein aggregation and Lewy body formation.

Phospholipase Cepsilon (PLCepsilon) is activated by various growth factors or G-protein-coupled receptor ligands via different activation mechanisms. The Ras association (RA) domain of PLCepsilon is known to be important for its ability to bind with Ras-family GTPase upon growth factor stimulation. In the present study, we identified Siah1 and Siah2 as novel binding partners of the PLCepsilon RA domain. Both Siah1 and Siah2 interacted with the RA2 domain of PLCepsilon, and the mutation of Lys-2186 of the PLCepsilon RA2 domain abolished this association. Moreover, Siah induced the ubiquitination and degradation of PLCepsilon upon epidermal growth factor (EGF) stimulation, and Siah proteins were phosphorylated on multiple tyrosine residues via an Src-dependent pathway upon EGF treatment. The Src inhibitor abolished the EGF-dependent ubiquitination of PLCepsilon, and the Siah1 phosphorylation-deficient mutant could not increase the EGF-dependent ubiquitination and degradation of PLCepsilon. The EGF-dependent degradation of PLCepsilon was blocked in mouse embryonic fibroblast (MEF) cells derived from Siah1a/Siah2 double knockout mice, and the extrinsic expression of wild-type Siah1 restored the degradation of PLCepsilon, whereas the phosphorylation-deficient mutant did not. Siah1 expression abolished PLCepsilon-dependent potentiation of EGF-dependent cell growth. In addition, the expression of wild-type Siah1 in Siah1a/Siah2-double knockout MEF cells inhibited EGF-dependent cell growth, and this inhibition was abolished by PLCepsilon knockdown. Our results suggest that the Siah-dependent degradation of PLCepsilon plays a role in the regulation of growth factor-dependent cell growth.

The Siah proteins, mammalian homologues of the Drosophila Sina protein, function as ubiquitin-protein isopeptide ligase enzymes to target a wide range of cellular proteins for degradation. We report here a novel Drosophila protein that is homologous to Sina, named Sina-Homologue (SinaH). We show that it can direct the degradation of the transcriptional repressor Tramtrack (Ttk) using two different mechanisms. One is similar to Sina and requires the adaptor Phyllopod, and the other is a novel mechanism of recognition. This novel mode of targeting for degradation is specific for the 69-kDa Ttk isoform, Ttk69. Ttk69 contains a region that is required for binding of SinaH and for SinaH-directed degradation. This region contains an AXVXP motif, which is the consensus sequence found in Siah substrate proteins. These results suggest that degradation directed by SinaH differs from that directed by Sina and is more similar to that found in vertebrates. We speculate that SinaH may be involved in regulating the levels of developmentally important transcription factors.

Constitutively active RAS small GTPases promote the genesis of human cancers. An important goal in cancer biology is to identify means of countervailing activated RAS signaling to reverse malignant transformation. Oncogenic K-RAS mutations are found in virtually all pancreatic adenocarcinomas, making the RAS pathway an ideal target for therapeutic intervention. How to best contravene hyperactivated RAS signaling has remained elusive in human pancreatic cancers. Guided by the Drosophila studies, we reasoned that a downstream mediator of RAS signals might be a suitable anti-RAS target. The E3 ubiquitin ligase seven in absentia (SINA) is an essential downstream component of the Drosophila RAS signal transduction pathway. Thus, we determined the roles of the conserved human homologues of SINA, SIAHs, in mammalian RAS signaling and RAS-mediated tumorigenesis. We report that similar to its Drosophila counterpart, human SIAH is also required for oncogenic RAS signaling in pancreatic cancer. Inhibiting SIAH-dependent proteolysis blocked RAS-mediated focus formation in fibroblasts and abolished the tumor growth of human pancreatic cancer cells in soft agar as well as in athymic nude mice. Given the high level of conservation of RAS and SIAH function, our study provides useful insights into altered proteolysis in the RAS pathway in tumor initiation, progression, and oncogenesis. By targeting SIAH, we have found a novel means to contravene oncogenic RAS signaling and block RAS-mediated transformation/tumorigenesis. Thus, SIAH may offer a novel therapeutic target to halt tumor growth and ameliorate RAS-mediated pancreatic cancer.

We report high expression of the maternally imprinted gene PEG10 in high-risk B-CLL defined by high LPL mRNA expression. Differential expression was initially identified by microarray analysis and confirmed by real time PCR in 42 B-CLL patients. mRNA expression ranged from 0.3- to 375.4-fold compared to normal peripheral blood mononuclear cells (PBMNC). Expression levels in CD19+ B-CLL cells were 100-fold higher than in B-cells from healthy donors. PEG10 expression levels in B-CLL patient samples remained stable over time even after chemotherapy. High PEG10 expression correlated with high LPL expression (p=0.001) and a positive Coombs' test (p=0.04). Interestingly, similar expression patterns were observed for the neighbouring imprinted gene sarcoglycan-epsilon (SGCE). Monoallelic expression and maintained imprinting of PEG10 were found by allele- or methylation-specific PCR. The intensity of intracellular staining of PEG10 protein corresponded to mRNA levels as confirmed by immunofluorescence staining. Short term knock-down of PEG10 in B-CLL cells and HepG2 cells was not associated with changes in cell survival but resulted in a significant change in the expression of 80 genes. However, long term inhibition of PEG10 led to induction of apoptosis in B-CLL cells. Our data indicate (i) a prognostic value of PEG10 in B-CLL patients; (ii) specific deregulation of the imprinted locus at 7q21 in high-risk B-CLL; (iii) a potential functional and biological role of PEG10 protein expression. Altogether, PEG10 represents a novel marker in B-CLL., (Copyright (c) 2007 Wiley-Liss, Inc.)

Human repp86 is a nuclear protein that is expressed in a tightly limited period of time during the cell cycle and plays an essential role in its progression. Manipulation of repp86 expression by reduction of endogenous repp86 or overexpression of exogenous repp86 results in cell cycle arrest. We found that repp86 interacts with human Siah2, which is a known mediator for proteasomal degradation. Siah2 failed to interact with repp86 lacking the first 67 N-terminal amino acids. Overexpression of Siah2 reduced endogenous and exogenous repp86 at the protein level without affecting its mRNA, as shown by cotransfection and RT-PCR experiments. Furthermore, MG-132--a specific inhibitor of the proteasome--blocked the degradation of repp86 in Siah2 overexpressing cells. Moreover, transiently transfected Siah2 abrogated the mitotic arrest in repp86 overexpressing cells. Our data show that Siah2 is an important mediator of repp86 protein degradation.