Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2

PHD1-3 (prolyl hydroxylases 1-3) catalyse the hydroxylation of HIF (hypoxia-inducible factor)-alpha subunit that triggers the substrate ubiquitination and subsequent degradation. The RING (really interesting new gene) finger E3 ligase Siah2 preferentially targets PHD3 for degradation. Here, we identify the requirements for such selective targeting. Firstly, PHD3 lacks an N-terminal extension found in PHD1 and PHD2; deletion of this domain from PHD1 and PHD2 renders them susceptible to degradation by Siah2. Secondly, PHD3 can homo- and hetero-multimerize with other PHDs. Consequently, PHD3 is found in high-molecular-mass fractions that were enriched in hypoxia. Interestingly, within the lower-molecular-mass complex, PHD3 exhibits higher specific activity towards hydroxylation of HIF-1alpha and co-localizes with Siah2, suggesting that Siah2 limits the availability of the more active form of PHD3. These findings provide new insight into the mechanism underlying the regulation of PHD3 availability and activity in hypoxia by the E3 ligase Siah2.     

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis

The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF‐κB signaling pathways. Both Ras/mitogen‐activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high‐throughput electro‐chemiluninescent‐based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self‐ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF‐1α and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah‐KO cells, pointing to its Siah‐dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS‐independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.     

Investigating the Molecular Basis of Siah1 and Siah2 E3 Ubiquitin Ligase Substrate Specificity

The Siah1 and Siah2 E3 ubiquitin ligases play an important role in diverse signaling pathways and have been shown to be deregulated in cancer. The human Siah1 and Siah2 isoforms share high sequence similarity but possess contrary roles in cancer, with Siah1 more often acting as a tumor suppressor while Siah2 functions as a proto-oncogene. The different function of Siah1 and Siah2 in cancer is likely due to the ubiquitination of distinct substrates. Hence, we decided to investigate the molecular basis of the substrate specificity, utilizing the well-characterized Siah2 substrate PHD3. Using chimeric and mutational approaches, we identified critical residues in Siah2 that promote substrate specificity. Thus, we have found that four residues in the N-terminal region of the Siah2 substrate binding domain (SBD) (Ser132, His150, Pro155, Tyr163) are critical for substrate specificity. In the C-terminal region of the SBD, a single residue, Leu250, was identified to promote the specific binding of Siah2 SBD to PHD3. Our study may help to overcome the challenges in the identification of Siah2 specific inhibitors.     

Normal p53 function in primary cells deficient for Siah genes

Overexpression studies have suggested that Siah1 proteins may act as effectors of p53-mediated cellular responses and as regulators of mitotic progression. We have tested these hypotheses using Siah gene knockout mice. Siah1a and Siah1b were not induced by activation of endogenous p53 in tissues, primary murine embryonic fibroblasts (MEFs) or thymocytes. Furthermore, primary MEFs lacking Siah1a, Siah1b, Siah2, or both Siah2 and Siah1a displayed normal cell cycle progression, proliferation, p53-mediated senescence, and G(1) phase cell cycle arrest. Primary thymocytes deficient for Siah1a, Siah2, or both Siah2 and Siah1a, E1A-transformed MEFs lacking Siah1a, Siah1b, or Siah2, and Siah1b-null ES cells all underwent normal p53-mediated apoptosis. Finally, inhibition of Siah1b expression in Siah2 Siah1a double-mutant cells failed to inhibit cell division, p53-mediated induction of p21 expression, or cell cycle arrest. Our loss-of-function experiments do not support a general role for Siah genes in p53-mediated responses or mitosis.     

Siah proteins induce the epidermal growth factor-dependent degradation of phospholipase Cepsilon

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 RING domain of Siaz, the zebrafish homologue of Drosophila seven in absentia, is essential for cellular growth arrest

Siah is a mammalian homologue of Drosophila seven in absentia (sina) that is required for R7 photoreceptor development. Both the SINA and Siah family interact with ubiquitin-conjugating enzymes via an N-terminal RING domain and the C-terminal domain of SINA/ Siahs interacts with proteins targeted for degradation. Siah induces cell growth arrest by promoting beta-catenin degradation in a phosphorylation-independent manner as a result of indirect binding to beta-catenin. We previously cloned a zebrafish homologue (Siaz) of Siah. Siaz shares high sequence homology with vertebrate Siah-2. We have now examined the role of Siaz in growth regulation using the trypan blue exclusion assay and flow cytometry and found that Siaz induces cellular growth arrest by inhibiting the G2/M transition. The C-terminal domain of Siaz that interacts with target proteins is not required for growth inhibition. We conclude that the N-terminal RING and central domain of Siaz are sufficient to block the G2/M phase transition.     

Regulation of the ring finger E3 ligase Siah2 by p38 MAPK

The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.     

Isolation of Xenopus HIF-prolyl 4-hydroxylase and rescue of a small-eye phenotype caused by Siah2 over-expression

Hypoxia is an important physiological condition during embryonic development. Hypoxia-inducible factor (HIF) is the mediator of hypoxic response of cells. The prolyl hydroxylase (PHD) of HIF plays a key role in stabilizing of HIF and the oxygen homeostasis of organisms. In this study, we isolated two PHD proteins, PHD45 and PHD28, and characterized them during the embryonic development of Xenopus laevis, which is an excellent model for embryonic development because of the ease of embryonic manipulation and the feasibility of transgenesis. Based on amino acid sequences, Xenopus PHD45 and PHD28 were homologous with human PHD2 and PHD3, respectively. In embryonic development, PHD45 expression was complementary to that of PHD28. xHIF-1alpha protein level was at a maximum around stage 20 when expression of PHD45 disappeared, while expression of PHD28 reached a maximum at stage 20, suggesting that PHD28 is inducible by HIF-1alpha. Recently, Siah2 was found to be an ubiquitin ligase of PHD proteins and to regulate degradation of PHD proteins. Over-expression of xSiah2 decreased PHD45 but not PHD28 and caused the small-eye phenotype of Xenopus. Additional over-expression of PHD47 rescued the abnormality caused by xSiah2, suggesting that the level of expression or activity of PHD proteins is important to the maintenance of homeostasis in embryonic development.     

Regulation of 2-oxoglutarate (alpha-ketoglutarate) dehydrogenase stability by the RING finger ubiquitin ligase Siah

The 2-oxoglutarate dehydrogenase complex (OGHDC) (also known as the alpha-ketoglutarate dehydrogenase complex) is a rate-limiting enzyme in the mitochondrial Krebs cycle. Here we report that the RING finger ubiquitin-protein isopeptide ligase Siah2 binds to and targets OGDHC-E2 for ubiquitination-dependent degradation. OGDHC-E2 expression and activity are elevated in Siah2(-/-) cells compared with Siah2(+)(/)(+) cells. Deletion of the mitochondrial targeting sequence of OGDHC-E2 results in its cytoplasmic localization and rapid proteasome-dependent degradation in Siah2(+)(/)(+) but not in Siah2(-/-) cells. Significantly, because of its overexpression or disruption of the mitochondrial membrane potential, the release of OGDHC-E2 from mitochondria to the cytoplasm also results in its concomitant degradation. The role of the Siah family of ligases in the regulation of OGDHC-E2 stability is expected to take place under pathological conditions in which the levels of OGDHC-E2 are altered.     

Generation and analysis of Siah2 mutant mice

Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.     

TIN2 stability is regulated by the E3 ligase Siah2

Telomeres are coated by shelterin, a six-subunit complex that is required for protection and replication of chromosome ends. The central subunit TIN2, with binding sites to three subunits (TRF1, TRF2, and TPP1), is essential for stability and function of the complex. Here we show that TIN2 stability is regulated by the E3 ligase Siah2. We demonstrate that TIN2 binds to Siah2 and is ubiquitylated in vivo. We show using purified proteins that Siah2 acts as an E3 ligase to directly ubiquitylate TIN2 in vitro. Depletion of Siah2 led to stabilization of TIN2 protein, indicating that Siah2 regulates TIN2 protein levels in vivo. Overexpression of Siah2 in human cells led to loss of TIN2 at telomeres that was dependent on the presence of the catalytic RING domain of Siah2. In contrast to RNAi-mediated depletion of TIN2 that led to loss of TRF1 and TRF2 at telomeres, Siah2-mediated depletion of TIN2 allowed TRF1 and TRF2 to remain on telomeres, indicating a different fate for shelterin subunits when TIN2 is depleted posttranslationally. TPP1 was lost from telomeres, although its protein level was not reduced. We speculate that Siah2-mediated removal of TIN2 may allow dynamic remodeling of the shelterin complex and its associated factors during the cell cycle.     

Homology modeling and in silico screening of inhibitors for the substrate binding domain of human Siah2: implications for hypoxia-induced cancers

The three-dimensional (3D) structure of the substrate binding domain (SBD) of human ubiquitin ligase Siah2 (seven in absentia homolog) was constructed based on the homology modeling approach using the Modeller 9v7 program. The molecular dynamics method was utilized to refine the model and it was further assessed by ProSA, three-dimensional structural superposition (3d-SS) and PROCHEK in order to analyze the quality and reliability of the generated model. Furthermore, we predicted the binding pocket of Siah2 and also validated it by both blind and normal docking using a known functional inhibitor, menadione. Using structure-based high-throughput virtual screening, we identified five lead drug-like molecules against the modeled SBD of Siah2 and analyzed its pharmacokinetic properties to identify the potential inhibitors for Siah2. The docking results for menadione and the lead molecules at the ligand binding site of SBD of Siah2 revealed that the residue Ser39 (corresponding to Ser167 in the full-length protein) is consistently involved in strong hydrogen bonding, and plays an important role in phosphorylation and the enhanced activity of Siah2.