seven in absentia

EVOLUTIONARY HOMOLOGS

Members of the Siah family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. The crystal structure of the substrate-binding domain (SBD) of murine Siah1a has been determined to 2.6 Å resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded beta-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-alpha receptors and TNF-receptor associated death-domain (TRADD) proteins; however, the findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. The Siah1a SBD potentiates TNF-alpha-mediated NF-kappa B activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity (Polekhina, 2002).

Xenopus siah-2, a protein with 67% identity to Drosophila Sina (Seven in absentia) and 85% identity to the mouse and human siah-2 proteins, has been isolated. Sina is required in Drosophila for the R7 photoreceptor cell formation during eye development, because it down regulates proteins that inhibit R7 differentiation via the ubiquitin/proteasome pathway. Nothing is known about the developmental function of the siah protein in vertebrates. In Xenopus siah-2 is expressed maternally and is later restricted to the brain, spinal cord and the developing and mature eye. To demonstrate that the vertebrate factor participates in the process of eye formation, Xsiah-2 was overexpressed during Xenopus development; the formation of a small eye phenotype was observed. The vertebrate counterpart of a C-terminal loss of function sina mutant, that causes a deficiency of the R7 photoreceptor cells in Drosophila, also induces smaller eyes in Xenopus. The small eyes are characterized by a reduced size of the lens, the retina and the pigmented epithelium. Since this phenotype has been also described for flies expressing sina ectopically, the data demonstrate the functional and structural conservation of Xsiah-2 and sina in metazoan eye development (Bogdan, 2001).

The Siah family of mouse genes shows extensive homology to Drosophila sina: Siah genes fall into two main groups: Siah-1, consisting of four closely related members, two of which appear to be functional, and Siah-2, containing a single functional member. The SIAH proteins show a high degree of conservation with SINA over a majority of their lengths, diverging significantly only at their amino terminal ends. Siah-2 is expressed in olfactory epithelium, retina, forebrain and proliferating cartilage of developing bone. Siah-2 is expressed in germ cells of the mouse ovary and testis. The expression of Siah-2 in the testis is first detected in postmeiotic spermatids, and in primordial oocytes coincident with their recruitment from the pool of quiescent cells. Expression of Siah-2 appears to be an excellent marker for the inception of gonadotrophin-independent growth (Della, 1993).

HUMSIAH, a human homolog of SINA, shows 67% homology with SINA. HUMSIAH encodes a 282 amino acid C3HC4 RING zinc-finger motif. Recent structural studies of the Wilms tumor protein reveals an RNA recognition motif. HUMSIAH conserved domains resemble those described for WT1 RNA recognition. HUMSIAH is expressed in apoptotic cells of the epithelium of the small intestine. As the cells migrate from the lower part of the crypt up the villus toward the lumen, they differentiate and finally die by apoptosis at the tips of the villi. Human cancer-derived cells selected for suppression of their tumorigenic phenotype exhibit constitutively elevated levels of HUMSIAH mRNA. A similar pattern of expression is also displayed for p21waf1, a suppressor of cell proliferation. (See the Evolutionary Homologs section of E2F transcription factor). These results suggest that HUMSIAH is a target for activation by p53, and may play a role in apoptosis and tumor suppression (Nemani, 1996).

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the Zinc finger, C3HC4 type (RING finger) signature. ExPASy also gives information about the sequence and domains structure at ExPASy site for SINA.

To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A. Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods. BAG-1 is a widely expressed protein that was first discovered by virtue of its ability to bind to and collaborate with Bcl-2 in suppressing cell death. The murine and human BAG-1 proteins (also known as RAP46) contain a domain with strong sequence homology to ubiquitin, but otherwise do not share similarity with other known proteins. Since its initial discovery as a Bcl-2-binding protein, however, BAG-1 has been reported to interact with several steroid hormone receptors, the serine/threonine-specific protein kinase Raf-1 and some tyrosine kinase growth factor receptors [hepatocyte growth factor (HGF) receptor and platelet-derived growth factor (PDGF) receptor]. BAG-1 forms tight complexes with Hsp70/Hsc70-family proteins and modulates their chaperone activity. Thus, BAG-1 is probably a novel component of the chaperone system and presumably exerts its effects on various target proteins by recruiting Hsp70/Hsc70-family proteins to them. Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and co-immunoprecipitation studies. Siah-1A-induced growth arrest in 293 and GM701 cells is abolished by co-transfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A (Matsuzawa, 1998).

Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line. Over-expression of BAG-1 significantly inhibits p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG-1 also prevents growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21waf-1 expression. BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A. It is suggested that Siah-1A may be an important mediator of p53-dependent cell-cycle arrest and has been demonstrated that Siah-1A is directly inhibited by BAG-1 (Matsuzawa, 1998).

Repression of gene transcription is a fundamental property of nuclear hormone receptors. Cell-specific repression by nuclear receptors correlates with levels of nuclear receptor corepressor (N-CoR) protein. N-CoR protein levels are regulated by mSiah2, a mammalian homolog of Drosophila Seven in absentia that targets N-CoR for proteasomal degradation. mSiah2 expression is cell-type specific and differentially regulates the repressive activities of nuclear receptors. These findings establish targeted proteolysis of transcriptional coregulators as a mechanism for cell-specific regulation of gene transcription (Zhang, 1998).

The Drosophila seven in absentia (sina) gene was initially discovered because its inactivation leads to R7 photoreceptor defects. Recent data indicate that Sina binds to the Sevenless pathway protein Phyllopod, and together they mediate degradation of Tramtrack, a transcriptional repressor of R7 cell fate. Independent studies have shown that Sina and its highly related mammalian homologs Siah-1 and Siah-2 bind to the DCC (deleted in colorectal cancer) protein and promote its proteolysis via the ubiquitin-proteasome pathway. To determine the roles of mammalian Siahs in proteolysis and their interactions with target proteins, Siah-1 domains critical for regulation of DCC were determined. Mutant Siah-1 proteins, harboring missense mutations in the carboxy (C)-terminal domain analogous to those present in Drosophila sina loss-of-function alleles, fail to promote DCC degradation. Point mutations and deletion of the amino (N)-terminal RING finger domain of Siah-1 abrogates its ability to promote DCC proteolysis. In the course of defining Siah-1 sequences required for DCC degradation, it was found that Siah-1 is itself rapidly degraded via the proteasome pathway, and RING domain mutations stabilize the Siah-1 protein. Siah-1 oligomerizes with itself and other Sina and Siah proteins via C-terminal sequences. Finally, evidence that endogenous Siah-1 regulates DCC proteolysis in cells was obtained through studies of an apparent dominant negative mutant of Siah-1, as well as via an antisense approach. The data indicate that the Siah-1 N-terminal RING domain is required for its proteolysis function, while the C-terminal sequences regulate oligomerization and binding to target proteins, such as DCC (Hu, 1999).

By using H-1 parvovirus as a selective agent, cells with a strongly suppressed malignant phenotype (KS or US) were derived from malignant cell lines (K562 or U937). By using cDNA display on the K562/KS cells, 15 cDNAs have been isolated, corresponding to genes differentially regulated in tumor suppression. Of these, TSAP9 corresponds to a TCP-1 chaperonin, TSAP13 to a regulatory proteasome subunit, and TSAP21 to syntaxin 11, a vesicular trafficking molecule. A similar pattern of differential regulation is shared by activation of p53, p21Waf1, and the human homologue of Drosophila Seven in absentia, SIAH-1. BAG-1 is a potential negative modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to Hsp70 and Hsc70 and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. SIAH-1b is selectively up-regulated during the first hours of apoptosis induction by wild-type p53. SIAH-1 binds BAG-1, and this binding is responsible for the inhibition of the growth arrest effect of p53. BAG-1 targets Hsp70-Hsc70 to SIAH-1, inducing conformational changes that directly or indirectly abrogate SIAH-1 growth-regulatory function. SIAH also binds DCC (deleted in colorectal cancer) and regulates its degradation via the ubiquitin-proteasome pathway. SIAH's N-terminal RING-finger domain is required for proteolysis, whereas the C-terminal cystein-rich region is required for the binding to target proteins. Because SIAH-1 is differentially expressed in the various models, it was characterized at the protein and functional levels. The 32-kDa, mainly nuclear protein encoded by SIAH-1, can induce apoptosis and promote tumor suppression. These results suggest the existence of a common mechanism of tumor suppression and apoptosis shared by p53, p21Waf1, and SIAH-1 and involving regulation of the cellular machinery responsible for protein folding, unfolding, and trafficking (Roperch, 1999).

Transfection of U937 cells with SIAH-1 induces apoptosis and tumor suppression. This finding is in line with the strong induction of SIAH-1 expression in different models of tumor suppression and apoptosis, and with the fact that during differentiation of the intestinal epithelium, SIAH-1 is expressed only in cells that have reached the terminal stage and are dying by apoptosis. Furthermore, SIAH-1 induces growth arrest. The differential induction of either growth arrest or apoptosis is most probably due to the different cell types used. These experiments implicate once more SIAH-1 in the regulation of cell proliferation versus growth arrest and suggest that it is responsible for the modulation of important cell fate decisions. In contrast, the experiments with both the Drosophila Sina and the mammalian SIAH-1 suggest that these proteins may play a direct role in protein ubiquitination and degradation. Intriguingly, SIAH-1 binds BAG1 but does not promote its degradation as it does to DCC. Further identification of molecules interacting with SIAH might help elucidate its precise function in the cell. One clue might be the differential localization of SIAH in various cells. Most studies indicate that both Drosophila Sina and SIAH-1 are nuclear proteins. Nevertheless a small proportion of SIAH-1 is found in the cytoplasm. Such different localizations may allow SIAH to fulfill a different functions (Roperch, 1999).

Destruction of ß-catenin is regulated through phosphorylation-dependent interactions with the F box protein ß-TrCP. A novel pathway for ß-catenin degradation was discovered involving mammalian homologs of Drosophila Sina (Siah), which bind ubiquitin ß-conjugating enzymes, and Ebi, an F box protein that binds ß-catenin independent of the phosphorylation sites recognized by ß-TrCP. A series of protein interactions were identified in which Siah is physically linked to Ebi by association with a novel Sgt1 homolog SIP that binds Skp1, a central component of Skp1-Cullin-F box complexes. Expression of Siah is induced by p53, revealing a way of linking genotoxic injury to destruction of ß-catenin, thus reducing activity of Tcf/LEF transcription factors and contributing to cell cycle arrest (Matsuzawa, 2001).

A pathway linking the RING protein Siah-1 to the F box protein Ebi has been mapped and it has been shown that Ebi can bind ß-catenin. Unlike ß-TrCP, however, which requires GSK3ß-mediated phosphorylation of ß-catenin on serine 33 and serine 37, Ebi interacts with ß-catenin independently of these phosphorylation sites. Also, the Siah binding protein SIP associates with complexes containing Ebi but not ß-TrCP, suggesting differences compared to previously characterized E3 ubiquitin ligase complexes, where E2 enzymes are supplied via Cullin-mediated interactions with RING-containing proteins such as Rbx-1/Roc-1. Recent identification of interactions between Siah-1 and the ß-catenin binding protein APC suggest that this scaffold protein represents a point of common intersection of the Wnt and Siah-1 pathways for ß-catenin degradation (Matsuzawa, 2001).

Two alternative pathways for regulation of ß-catenin levels are presented, involving different F box proteins (Ebi versus ß-TrCP). One pathway is initiated by increases in the expression of Siah-family proteins, which can be induced, for example, by p53 in response to DNA damage, and involves sequential protein interactions with SIP, Skp1, and Ebi. Ebi binds ß-catenin, thus recruiting it to the Siah-1-SIP-Skp1 complex for polyubiquitination and subsequent proteosome-mediated degradation. Siah-1 binds the E2 UbcH5. The other pathway is regulated by Wnt signals (Dsh) and possibly PI3K/Akt. This pathway is phosphorylation dependent and involves GSK3ß-induced phosphorylation of Ser-33 and Ser-37 on ß-catenin, allowing ß-TrCP binding, resulting in recruitment of ß-catenin to Skp1-Cullin-1- ß-TrCP complexes (SCF). Cullin-1, in collaboration with other proteins, supplies this SCF complex with E2s, such as UbcH3. APC is required for both pathways as a scaffold protein, binding ß-catenin via one domain and also binding Siah-1 and GSK3ß (Matsuzawa, 2001).

In the fly, Sina recruits E2s to Phyllopod/Tramtrack complexes, targeting Tramtrack for ubiquitination. The ebi-gene product also binds Tramtrack and promotes its degradation in vitro and when expressed in insect cells in culture. Loss-of-function mutations of ebi cause Tramtrack accumulation and prevent R7 cell differentiation. Similar to ß-TrCP, the ebi gene of Drosophila encodes an F box/WD-40-repeat protein with sequence homology to Cdc4 (yeast), Sel-10 (C. elegans), and Slimb (Drosophila), suggesting that it provides a functional connection between a Sina-regulated pathway and SCF complexes. How this linkage between Sina and SCF complexes is achieved, however, has been unclear (Matsuzawa, 2001).

The finding that SIP functions as a molecular bridge between the human homologs of Sina and the SCF-component Skp1 provides evidence of a physical linkage between components of these two ubiquitin ligase systems, thus corroborating the genetic evidence from Drosophila that these two pathways for targeted protein degradation interact. The Drosophila ortholog of SIP is also capable of bridging the fly Skp1 and Sina proteins in three-hybrid experiments. Thus, an evolutionarily conserved network of protein interactions exists in which Siah-1 (Sina) binds to SIP, which in turn binds to Skp1, which binds Ebi (Matsuzawa, 2001).

p53 can induce expression of Siah-family genes in mammals, establishing p53 as one factor capable of invoking Siah-dependent pathways for protein degradation. Siah-family proteins are normally maintained at a relatively low level through ubiquitination-dependent protein turnover, where human Siah-1 and Siah-2 promote their own degradation through interactions of their RING domains with E2s. This therefore suggests that activation of p53 leads to a burst of Siah-1 mRNA and protein production, triggering the Siah/SIP/Skp1/Ebi pathway for ß-catenin degradation. In contrast to Siah-family proteins, it seems unlikely that SIP, Skp1, or Ebi are limiting components of this pathway, since overexpression of them has little effect on ß-catenin levels (Matsuzawa, 2001).

Though p53-mediated degradation of ß-catenin correlates with cell cycle arrest, it remains to be established whether these events are functionally linked. Activation of Tcf/LEF-family transcription factors by ß-catenin is known to induce expression of cyclin D1, c-myc, and other genes important for cell proliferation, making it plausible that ß-catenin degradation is linked to p53-mediated cell cycle arrest. However, given the role established for the cyclin-dependent kinase inhibitor p21^Waf1 in mediating G1 arrest induced by p53, it is unclear whether a parallel pathway for ß-catenin degradation would be required. Circumstances have been described where p53 fails to induce cell cycle arrest despite inducing p21^Waf1 expression, raising the question of whether p21^Waf1 is necessary but insufficient for p53-mediated G1 arrest. Recently, a genetic interaction between ebi and p21^Waf1 has been identified using an assay in Drosophila where flies are engineered to ectopically express human p21^Waf1 in the developing eye disc (Boulton, 2000). Specifically, mutant alleles of ebi abrogated inhibition of S phase entry by p21^Waf1, implying a need for Ebi in p21-mediated cell cycle arrest. Flies with mutant ebi also display ectopic S phases and overproliferation phenotypes (Boulton, 2000), further implying a role for ebi in growth suppression. Defects in cell cycle arrest in ebi mutants, however, do not necessarily implicate ß-catenin/Armadillo. For example, p53 can induce degradation of c-Myb through a proteosome-dependent mechanism partly mediated by Siah (Tanikawa, 2000). Thus, Ebi may have other targets in addition to ß-catenin that are relevant to mechanisms of p53-mediated cell cycle arrest. Future experiments should explore whether the fly homolog of p53 is linked to an ebi-dependent pathway for cell cycle arrest entailing degradation of Armadillo. In the M1 cell model, p53 induces both G1 arrest and apoptosis. Though Ebi(DeltaF)-expressing M1 cells may exhibit some delay in p53-induced apoptosis, this could result indirectly because of failed G1 arrest. Moreover, Siah-1 often fails to induce apoptosis when overexpressed in cells. However, links of Siah to apoptosis can occur under some circumstances, as demonstrated by the observation that coexpression of Siah-1 with a Siah binding protein Pw1/Peg3 causes apoptosis, whereas neither Siah-1 nor Pw1/Peg3 alone are sufficient. Mutations affecting components of the Wnt-signaling pathway are commonly observed in human cancers, resulting in aberrant accumulation of ß-catenin and activation of Tcf/LEF-target genes. Wnt-family ligands, frizzled-family receptors, and the signaling proteins downstream of these define one mechanism for regulating ß-catenin levels. However, additional inputs into pathways controlling ß-catenin turnover have recently been identified, including a mitogen-activated protein kinase pathway involving a Tak1 homolog and Nemo-like kinases in C. elegans and a cell adhesion-dependent pathway involving integrin-linked kinase. The findings reported here reveal yet another pathway for regulating ß-catenin levels that is linked at least in part to p53-dependent responses to genotoxic injury. It is speculated that loss of p53 or components of the Siah/SIP/Skp/Ebi pathway for ß-catenin destruction may contribute to aberrant ß-catenin accumulation in cancers (Matsuzawa, 2001).

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of ß-catenin by the proteasome. Siah-1, the human homolog of Drosophila Seven in absentia, is a p53-inducible mediator of cell cycle arrest, tumor suppression, and apoptosis. Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of ß-catenin in mammalian cells. The ability of Siahß-1 to downregulate ß-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of ß-catenin by Siah-1 is independent of GSK3ß-mediated phosphorylation and does not require the F box protein ß-TrCP. These results indicate that APC and Siahß-1 mediate a novel ß-catenin degradation pathway linking p53 activation to cell cycle control (Liu, 2001).

Induction of wild-type p53 in mouse fibroblasts causes cell cycle arrest at the G(1) phase, whereas coexpression of p53 and the protooncogene c-myc induces apoptosis. Although p53 transcriptional activity generally is required for both pathways, the molecular components mediating p53-dependent apoptosis are not well understood. To identify factors that could mediate p53-induced cell death, a comparative RNA differential display procedure was used. Pw1/Peg3 has been identified as a gene product induced during p53/c-myc-mediated apoptosis. Pw1/Peg3 is not induced during p53-mediated G(1) growth arrest nor by c-myc alone. Although it is not clear whether the induction of Pw1/Peg3 depends on p53 activity, it has been shown that Pw1/Peg3 interacts with a p53-inducible gene product Siah1a. Coexpression of Pw1/Peg3 with Siah1a induces apoptosis independently of p53, whereas expression of Pw1/Peg3 or Siah1a separately has no effect on cell death. These data suggest that Siah1a and Pw1/Peg3 cooperate in the p53-mediated cell death pathway. Furthermore, inhibiting Pw1/Peg3 activity blocks p53-induced apoptosis. The observation that Pw1/Peg3 is necessary for the p53 apoptotic response suggests a pivotal role for this gene in determining cell death versus survival (Relaix, 2000).

OBF-1 (also known as Bob1 or OCA-B) is a B-cell-specific transcription coactivator that binds to conserved octamer elements (ATGCAAAT or reverse) in the DNA together with the POU domain transcription factors Oct-1 or Oct-2. OBF-1 is critical for development of a normal immune response and the formation of germinal centers in secondary lymphoid organs. The RING finger protein Siah-1 interacts specifically with OBF-1. This interaction is mediated by the C-terminal part of Siah-1 and by residues in the N-terminus of OBF-1, partly distinct from the residues required for formation of a complex with the Oct POU domains and the DNA. Interaction between Siah-1 and OBF-1 leads to downregulation of OBF-1 protein level but not mRNA, and to a corresponding reduction in octamer site-dependent transcription activation. Inhibition of the ubiquitin-proteasome pathway in B cells leads to elevated levels of OBF-1 protein. Furthermore, in immunized mice, OBF-1 protein amounts are dramatically increased in primary activated B cells, without concomitant increase in OBF-1 mRNA. These data suggest that Siah-1 is part of a novel regulatory loop controlling the level of OBF-1 protein in B cells (Tiedt, 2001).

The BOB.1/OBF.1 coactivator is critically involved in mediating octamer dependent transcriptional activity in B lymphocytes. Mice lacking this coactivator show various defects in B-cell development; most notably, they completely lack germinal centers. Consistent with this phenotype, BOB.1/OBF.1 levels are massively upregulated in germinal center B cells as compared with resting B cells. The mechanism of upregulation has been addressed and it has been found found that only a minor part of this regulation can be attributed to increased levels of BOB.1/OBF.1-specific mRNA. Apparently, BOB.1/OBF.1 is also regulated at the protein level. In support of this suggestion two related BOB.1/OBF.1 interacting proteins, SIAH1 and SIAH2, have been identified in a yeast two-hybrid screen. SIAH1 and SIAH2 are known regulators of protein stability. Coexpression of SIAH results in a destabilization of BOB.1/OBF.1 protein without affecting mRNA levels. Further more, proteasome inhibitors block the degradation of BOB.1/OBF.1 protein. Finally, B-cell receptor cross-linking also results in the degradation of BOB.1/OBF.1 and consequently reduces transcriptional activation of BOB.1/OBF.1-dependent reporters (Boehm, 2001).

The Drosophila Seven in absentia (Sina) gene product originally was described as a protein that controls cell fate decisions during eye development. Its mammalian homolog, Siah-1, recently was found to be involved in p53-dependent and -independent pathways of apoptosis and G1 arrest. Siah-1 is shown to interact directly with and promote the degradation of the cell fate regulator Numb. Siah-1-mediated Numb degradation leads to redistribution of endogenous cell-surface Notch to the cytoplasm and nucleus and to augmented Notch-regulated transcriptional activity. These data imply that through its ability to target Numb for degradation, Siah-1 can act as a key regulator of Numb-related activities, including Notch signaling (Susini, 2001).

Interaction-mapping experiments have revealed that GST-Numb binds to a minimal region of Siah-1 (Delta10), composed of amino acids 180-211. The region of Delta10 overlaps with the binding sites of other known Siah-1 interactors, including DCC and the recently described beta-catenin-binding proteins. Interestingly, in the corresponding site, two allelic mutations have been identified in Drosophila Sina that affect R7 photoreceptor development. Within the Numb protein, residues 91-400 are sufficient to bind Siah-1. This region includes the C-terminal part of the PTB domain of Numb. PTB domains have been implicated in phosphorylation-dependent and phosphorylation-independent molecular interactions (Susini, 2001).

Numb physically interacts with and inhibits the signaling of Notch1, a cell-surface receptor that promotes cell fate decisions by activating downstream transcription factors of the CSL family. This is achieved by proteolytic cleavage within an intracellular site of Notch that results in the release and subsequent translocation of its cytosolic fragment (NICD) into the nucleus. The consequences of Siah-1 overexpression on Notch subcellular localization were investigated. Confocal microscopy analysis of Notch1 immunofluorescence in control U937 cells has revealed a rim-like staining pattern typical of cell-surface receptors. In striking contrast, Siah-1-overexpressing cells exhibited a redistribution of Notch1 immunofluorescence in the cytoplasm and in the nucleus. Confocal imaging within the Z plane of the nucleus confirms Notch1 nuclear localization. To show that the observed pattern of Notch expression in Siah-1-overpressing U937 cells resembles that of an activation state, U937 vector control cells were analyzed for Notch translocation after EDTA treatment, which mimics the effects of ligand-induced nuclear translocation of Notch. By 30 min after EDTA removal, Notch1 immunofluorescence was visualized in and around the nucleus. Together, these observations suggest further that Siah-1 overexpression promotes Notch1 activation. This conclusion was validated directly by monitoring endogenous NICD activity in MCF-7 cells stably transfected with vector control or pBK-RSV-Siah-1. A luciferase reporter construct whose activation is proportional to NICD translocation to the nucleus was transfected into these cells. The presence of endogenous Notch1 was verified by Western blot analysis. A consistent, >3-fold increase in endogenous NICD activity was observed in MCF-7 cells overexpressing Siah-1, and this increase was reduced by transient transfection of Numb (Susini, 2001).

TRAF2 serves as a central regulator of the cellular response to stress and cytokines through the regulation of key stress-signaling cascades. Wild-type Siah2, but not RING mutant, targets TRAF2 for ubiquitylation and degradation in vitro. Siah2 mediates equally efficient ubiquitylation of RING mutant TRAF2. In vivo, Siah2 primarily targets TRAF2 for degradation under stress conditions. Tumor necrosis factor-a and actinomycin D treatment results in accelerated TRAF2 degradation in wild-type mouse embryo fibroblasts (MEFs), as compared with Siah2-/- cells. Similarly, TRAF2 half-life is prolonged in Siah2-/- compared with wild-type MEFs subjected to stress stimuli. Siah2 efficiently decreases TNF-a-dependent induction of JNK activity and transcriptional activation of NFkappa-B. Apoptosis induced by TNF-a and actinomycin D treatment is increased upon expression of Siah2, or attenuated upon expression of TRAF2 or RING mutant Siah2. Identifying Siah2 as a regulator of TRAF2 stability reveals its role in the regulation of TRAF2 signaling following exposure to stress (Habelhah, 2002).

Hypoxia-inducible factor-1alpha (HIF1alpha: Drosophila homolog - Similiar) is a central regulator of the cellular response to hypoxia. Prolyl-hydroxylation of HIF1alpha by PHD enzymes is prerequisite for HIF1alpha degradation. The abundance of PHD1 and PHD3 are regulated via their targeting for proteasome-dependent degradation by the E3 ubiquitin ligases Siah1a/2, under hypoxia conditions. Siah2 null fibroblasts exhibit prolonged PHD3 half-life, resulting in lower levels of HIF1alpha expression during hypoxia. Significantly, hypoxia-induced HIF1alpha expression is completely inhibited in Siah1a/2 null cells, yet can be rescued upon inhibition of PHD3 by RNAi. Siah2 targeting of PHD3 for degradation increases upon exposure to even mild hypoxic conditions, which coincides with increased Siah2 transcription. Siah2 null mice subjected to hypoxia display an impaired hyperpneic respiratory response and reduced levels of hemoglobin. Thus, the control of PHD1/3 by Siah1a/2 constitutes another level of complexity in the regulation of HIF1alpha during hypoxia (Nakayama, 2004).

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