Effective therapy for malignant melanoma, the leading cause of death from skin cancer, remains an area of significant unmet need in oncology. The elevated expression of PKCε in advanced metastatic melanoma results in the increased phosphorylation of the transcription factor ATF2 on threonine 52, which causes its nuclear localization and confers its oncogenic activities. The nuclear-to-mitochondrial translocation of ATF2 following genotoxic stress promotes apoptosis, a function that is largely lost in melanoma cells, due to its confined nuclear localization. Therefore, promoting the nuclear export of ATF2, which sensitizes melanoma cells to apoptosis, represents a novel therapeutic modality.
We conducted a pilot high-throughput screen of 3,800 compounds to identify small molecules that promote melanoma cell death by inducing the cytoplasmic localization of ATF2. The imaging-based ATF2 translocation assay was performed using UACC903 melanoma cells that stably express doxycycline-inducible GFP-ATF2.
We identified 2 compounds (SBI-0089410 and SBI-0087702) that promoted the cytoplasmic localization of ATF2, reduced cell viability, inhibited colony formation, cell motility, anchorage-free growth, and increased mitochondrial membrane permeability. SBI-0089410 inhibited the TPA-induced membrane tranlocation of PKC isoforms, whereas both compounds decreased ATF2 phosphorylation by PKCε and ATF2 transcriptional activity. Overexpression of either constitutively active PKCε or phosphomimic mutant ATF2T52E attenuated the cellular effects of the compounds.
The imaging-based high-throughput screen provides a proof-of-concept for the identification of small molecules that block the oncogenic addiction to PKCε signaling by promoting ATF2 nuclear export, resulting in mitochondrial membrane leakage and melanoma cell death.
ATF2; PKCε; melanoma; nuclear translocation; high content screen
Folding of newly synthesized polypeptides (NSPs) into functional proteins is a highly regulated process. Rigorous quality control ensures that NSPs attain their native fold during or shortly after completion of translation. Nonetheless, signaling pathways that govern the degradation of NSPs in mammals remain elusive. We demonstrate that the stress-induced c-Jun N-terminal kinase (JNK) is recruited to ribosomes by the receptor for activated protein C kinase 1 (RACK1). RACK1 is an integral component of the 40S ribosome and an adaptor for protein kinases. Ribosome-associated JNK phosphorylates the eukaryotic translation elongation factor 1A isoform 2 (eEF1A2) on serines 205 and 358 to promote degradation of NSPs by the proteasome. These findings establish a role for a RACK1/JNK/eEF1A2 complex in the quality control of NSPs in response to stress.
The receptor for activated C kinase 1 (RACK1) serves as an adaptor for a number of proteins along the MAPK, protein kinase C, and Src signaling pathways. The abundance and near ubiquitous expression of RACK1 reflect its role in coordinating signaling molecules for many critical biological processes, from mRNA translation to cell motility to cell survival and death. Complete deficiency of Rack1 is embryonic lethal, but the recent development of genetic Rack1 hypomorphic mice has highlighted the central role that RACK1 plays in cell movement and protein synthesis. This review focuses on the importance of RACK1 in these processes and places the recent work in the larger context of understanding RACK1 function.
RACK1; translation; ribosomes; stress response; cell migration; cancer
The transcription factor ATF2 elicits oncogenic activities in melanoma and tumor suppressor activities in non-malignant skin cancer. Here we identify that ATF2 tumor suppressor function is determined by its ability to localize at the mitochondria, where it alters membrane permeability following genotoxic stress. The ability of ATF2 to reach the mitochondria is determined by PKCε, which directs ATF2 nuclear localization. Genotoxic stress attenuates PKCε effect on ATF2, enables ATF2 nuclear export and localization at the mitochondria, where it perturbs the HK1-VDAC1 complex, increases mitochondrial permeability and promotes apoptosis. Significantly, high levels of PKCε, as seen in melanoma cells, block ATF2 nuclear export and function at the mitochondria, thereby attenuating apoptosis following exposure to genotoxic stress. In melanoma tumor samples, high PKCε levels associates with poor prognosis. Overall, our findings provide the framework for understanding how subcellular localization enables ATF2 oncogenic or tumor suppressor functions.
Sensitize melanomas to apoptosis and inhibit their growth and metastatic potential by compounds that mimic the activities of activating transcription factor 2 (ATF2)-driven peptides.
Small-molecule chemical library consisting of 3,280 compounds was screened to identify compounds that elicit properties identified for ATF2 peptide, including (a) sensitization of melanoma cells to apoptosis, (b) inhibition of ATF2 transcriptional activity, (c) activation of c-Jun NH2-terminal kinase (JNK) and c-Jun transcriptional activity, and (d) inhibition of melanoma growth and metastasis in mouse models.
Two compounds, celastrol (CSL) and acetyl isogambogic acid, could, within a low micromolar range, efficiently elicit cell death inmelanoma cells. Both compounds efficiently inhibit ATF2 transcriptional activities, activate JNK, and increase c-Jun transcriptional activities. Similar to the ATF2 peptide, both compounds require JNK activity for their ability to inhibit melanoma cell viability. Derivatives of CSL were identified as potent inducers of cell death in mouse and human melanomas. CSL and a derivative (CA19) could also efficiently inhibit growth of human and mouse melanoma tumors and reduce the number of lung metastases in syngeneic and xenograft mouse models.
These studies show for the first time the effect of CSL and acetyl isogambogic acid on melanoma. These compounds elicit activities that resemble the well-characterized ATF2 peptide and may therefore offer new approaches for the treatment of this tumor type.
Understanding regulatory pathways involved in melanoma development and progression has advanced significantly in recent years. It is now appreciated that melanoma is the result of complex changes in multiple signaling pathways that affect growth control, metabolism, motility and the ability to escape cell death programs. Here we review the major signaling pathways currently known to be deregulated in melanoma with an implication to its development and progression. Among these pathways are Ras, B-Raf, MEK, PTEN, phosphatidylinositol-3 kinase (PI3Ks) and Akt which are constitutively activated in a significant number of melanoma tumors, in most cases due to genomic change. Other pathways discussed in this review include the [Janus kinase/signal transducer and activator of transcription (JAK/STAT), transforming growth factor-β pathways which are also activated in melanoma, although the underlying mechanism is not yet clear. As a paradigm for remodeled signaling pathways, melanoma also offers a unique opportunity for targeted drug development.
The AKT/PKB pathway plays a central role in tumor development and progression and is often up-regulated in different tumor types, including melanomas. We have recently reported on the in silico approach to identify putative inhibitors for AKT/PKB. Of the reported hits, we selected BI-69A11, a compound which was shown to inhibit AKT activity in in vitro kinase assays. Analysis of BI-69A11 was performed in melanoma cells, a tumor type that commonly exhibits up-regulation of AKT. Treatment of the UACC903 human melanoma cells, har-boring the PTEN mutation, with BI-69A11 caused efficient inhibition of AKT S473 phosphorylation with concomitant inhibition of AKT phosphorylation of PRAS40. Treatment of melanoma cells with BI-69A11 also reduced AKT protein expression, which coincided with inhibition of AKT association with HSP-90. BI-69A11 treatment not only caused cell death of melanoma, but also prostate tumor cell lines. Notably, the effect of BI-69A11 on cell death was more pronounced in cells that express an active form of AKT. Significantly, intra-peritoneal injection of BI-69A11 caused effective regression of melanoma tumor xenografts, which coincided with elevated levels of cell death. These findings identify BI-69A11 as a potent inhibitor of AKT that is capable of eliciting effective regression of xenograft melanoma tumors.
AKT; melanoma; BI-69A11; HSP90; Pten; PI3K
Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.
The c-Jun N-terminal kinases (JNKs) are activated in response to stress, DNA damage, and cytokines by MKK4 and MKK7. We recently demonstrated that PKC can augment the degree of JNK activation by phosphorylating JNK, which requires the adaptor protein RACK1. Here we report on the conditions required for PKC-dependent JNK activation. In vitro kinase assays reveal that PKC phosphorylation of JNK is not sufficient for its activation but rather augments JNK activation by canonical JNK upstream kinases MKK4 or MKK7 alone or in combination. Further, to enhance JNK activity, PKC phosphorylation of JNK should precede its phosphorylation by MKK4/7. Inhibition of PKC phosphorylation of JNK affects both early and late phases of JNK activation following UV-irradiation and reduces the apoptotic response mediated by JNK. These data provide important insight into the requirements for PKC activation of JNK signaling.
JNK; MKK4; MKK7; PKC; RACK1
Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/β-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle–muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5.
UNC-95; RNF-5; LIM; RING; E3 ligase
Melanomas are among the aggressive tumor types because of their notorious resistance to treatment and their high capacity to metastasize. ATF2 is among transcription factors implicated in the progression of melanoma and its resistance to treatment. Here we demonstrate that the expression of a peptide spanning amino acids 50–100 of ATF2 (ATF250–100) reduces ATF2 transcriptional activities while increasing the expression and activity of c-Jun. Altering the balance of Jun/ATF2 transcriptional activities sensitized melanoma cells to apoptosis, an effect that could be attenuated by inhibiting c-Jun. Inhibition of ATF2 via RNA interference likewise increased c-Jun expression and primed melanoma cells to undergo apoptosis. Growth and metastasis of SW1 and B16F10 mouse melanomas were inhibited by ATF250–100 to varying degrees up to a complete regression, depending on the mode (inducible, constitutive, or adenoviral delivery) of its expression.
The endoplasmic reticulum (ER) responds to changes in intracellular homeostasis through activation of the unfolded protein response (UPR). Yet, it is not known how UPR-signaling coordinates adaptation versus cell death. Previous studies suggested that signaling through PERK/ATF4 is required for cell death. We show that high levels of ER stress (i.e., ischemia-like conditions) induce transcription of the ubiquitin ligases Siah1/2 through the UPR transducers PERK/ATF4 and IRE1/sXBP1. In turn, Siah1/2 attenuates proline hydroxylation of ATF4, resulting in its stabilization, thereby augmenting ER stress output. Conversely, ATF4 activation is reduced upon Siah1/2 KD in cultured cells, which attenuates ER stress-induced cell death. Notably, Siah1a+/−::Siah2−/− mice subjected to neuronal ischemia exhibited smaller infarct volume and were protected from ischemia-induced death, compared with the wild type (WT) mice. In all, Siah1/2 constitutes an obligatory fine-tuning mechanism that predisposes cells to death under severe ER stress conditions.
Maintaining a balanced level of stress (protein folding, reactive oxygen radicals) is important for keeping cellular homeostasis (the ability of a cell to maintain internal equilibrium by adjusting its physiological processes). The accumulation of stress (external or internal) will trigger a well-orchestrated machinery that attempts to restore homeostasis, namely, the unfolded protein response (UPR). The UPR either restores balance to the cells or induces a cell death program, which clears the damaged cell. How this machinery activates cell survival versus cell death is not entirely clear. Here we identify a new layer in the regulation of the UPR, which determines the magnitude of this response. We demonstrate the importance of this newly identified regulatory component for cell death commitments, in response to the more severe conditions (ischemia, lack of oxygen and nutrients). Our findings highlight an undisclosed mechanism that is important for the cell death decision following severe stress conditions, while pointing to the ability to fine tune cellular response to stress.
Understanding the mechanism underlying the regulation of the androgen receptor (AR), a central player in the development of castration-resistant prostate cancer (CRPC), holds promise for overcoming the challenge of treating CRPC. We demonstrate that the ubiquitin ligase Siah2 targets a select pool of NCOR1-bound, transcriptionally-inactive AR for ubiquitin-dependent degradation, thereby promoting expression of select AR target genes implicated in lipid metabolism, cell motility, and proliferation. Siah2 is required for prostate cancer cell growth under androgen-deprivation conditions in vitro and in vivo, and Siah2 inhibition promotes prostate cancer regression upon castration. Notably, Siah2 expression is markedly increased in human CRPCs. Collectively, we find that selective regulation of AR transcriptional activity by the ubiquitin ligase Siah2 is important for CRPC development.
The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to β-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of β-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.
The Wnt signaling pathway plays a crucial role during embryonic development and in the maintenance of stem cell populations in various organs and tissues. Aberrant activation of this pathway through different mechanisms participates in the onset and progression of several types of human cancers. In the presence of Wnt ligands, stabilized β-catenin acts as a transcriptional activator to induce the expression of target genes through binding to the TCF/LEF family of transcription factors. Using in vitro and in vivo models, we show that TCF/LEF proteins can be activated independently of β-catenin through cooperation with members of the ATF2 subfamily of transcription factors. This novel alternative mechanism of TCF/LEF activation is constitutively up-regulated in certain hematopoietic tumor cells, where it regulates the expression of TCF/LEF target genes and promotes cell growth.
Despite advances in developing specific inhibitors to BRAF mutant melanomas, to date there are no effective therapies for tumors bearing NRAS mutations, present in approximately 15–20% of human melanomas. Here, we extend earlier studies, demonstrating that the small molecule BI-69A11 inhibits the growth of melanoma cell lines in vitro and in vivo. Gene expression microarray analysis of BI-69A11-responsive melanoma cells revealed the induction of interferon- and cell death-related genes that were associated with responsiveness to BI-69A11. Strikingly, the administration of BI-69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras(Q61K)::Ink4a−/−). Biweekly administration of BI-69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100% and 36%, respectively). BI-69A11 treatment did not inhibit the development of histiocytic sarcomas, which comprise about 50% of the tumors in this model. Immunofluorescent staining analyses of CD45 revealed increased levels of immune cell infiltration in BI-69A11–treated tumors. Gene expression profiling of BI-69A11-resistant Nras(Q61K)::Ink4a−/− tumors revealed the upregulation of functional gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate development of NRAS mutant melanomas by BI-69A11 even when administered at a late stage of the tumor development, support its further development and clinical assessment.
The TCA cycle is the central hub of oxidative metabolism, running in the classic forward direction to provide carbon for biosynthesis and reducing agents for generation of ATP. Our metabolic tracer studies in melanoma cells showed that in hypoxic conditions the TCA cycle is largely disconnected from glycolysis. By studying the TCA branch point metabolites, acetyl CoA and citrate, as well as the metabolic endpoints glutamine and fatty acids, we developed a comprehensive picture of the rewiring of the TCA cycle that occurs in hypoxia. Hypoxic tumor cells maintain proliferation by running the TCA cycle in reverse. The source of carbon for acetyl CoA, citrate, and fatty acids switches from glucose in normoxia to glutamine in hypoxia. This hypoxic flux from glutamine into fatty acids is mediated by reductive carboxylation. This reductive carboxylation is catalyzed by two isocitrate dehydrogenases, IDH1 and IDH2. Their combined action is necessary and sufficient to effect the reverse TCA flux and maintain cellular viability.
Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2−/− mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.
Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5−/− MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5−/− using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5−/− mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5−/− macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.
Autophagy is an intracellular catabolic process by which a cell's own components are degraded through the lysosomal machinery. Autophagy is implicated in various cellular processes such as growth and development, cancer, and inflammation. Using biochemistry, cell biology, and genetic models, we identify a ubiquitin ligase that limits autophagy in the absence of an inducing stimulus (e.g. starvation). The control of basal autophagy is mediated by the ubiquitin ligase RNF5 through its regulation of the membrane-associated ATG4B protease. Using RNF5 mutant mice we demonstrate the implications of this regulation for host defense mechanisms that limit intracellular infection by bacterial pathogens.
Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.
In melanoma, the activation of pro-survival signaling pathways, such as the AKT and NF-κB pathways, are critical for tumor growth. We have recently reported that the AKT inhibitor BI-69A11 causes efficient inhibition of melanoma growth. Here, we show that in addition to its AKT inhibitory activity, BI-69A11 also targets the NF-κB pathway. In melanoma cell lines, BI-69A11 inhibited TNF-α-stimulated IKKα/β and IκB phosphorylation as well as NF-κB reporter gene expression. Furthermore, the effective inhibition of melanoma growth by BI-69A11 was attenuated upon NF-κB activation. Mechanistically, reduced NF-κB signaling by BI-69-A11 is mediated by the inhibition of sphingosine kinase 1, identified in a screen of 315 kinases. Significantly, we demonstrate that BI-69A11 is well-tolerated and orally active against UACC 903 and SW1 melanoma xenografts. Our results demonstrate that BI-69A11 inhibits both the AKT and NF-κB pathways and that the dual targeting of these pathways may be efficacious as a therapeutic strategy in melanoma.
Although B-RAF and MEK inhibitors have shown promise in the clinic against melanoma, the development of resistance to these singly targeted agents inevitably results. These observations underscore the plasticity of melanoma to chemotherapeutic agents and further emphasize the need to apply combinatorial targeting of signaling pathways as a strategy to maximize therapeutic response. The PI3K/AKT and NF-κB signaling pathways are altered in melanoma, presenting additional opportunities for target inhibition. Our studies demonstrate that the AKT inhibitor, BI-69A11, also inhibits the NF-κB pathway and that dual inhibition of both pathways is responsible for the anti-tumor efficacy of this molecule.
melanoma; AKT; NF-kB; targeted therapy
p27kip1 has been implicated in cell cycle regulation, functioning as an inhibitor of cyclin-dependent kinase activity. In addition, p27 was also shown to affect cell migration, with accumulation of cytoplasmic p27 associated with tumor invasiveness. However, the mechanism underlying p27 regulation as a cytoplasmic protein is poorly understood. Here we show that glucose starvation induces proteasome-dependent degradation of cytoplasmic p27, accompanied by a decrease in cell motility. We also show that the glucose limitation-induced p27 degradation is regulated through an ubiquitin E3 ligase complex involving Siah1 and SIP/CacyBP. SIP−/− embryonic fibroblasts have increased levels of cytosolic p27 and exhibit increased cell motility compared with wild-type cells. These observations suggest that the Siah1/SIP E3 ligase complex regulates cell motility through degradation of p27.
p27kip1; Siah1; SIP; glucose starvation; cell migration
The anaphase-promoting complex or cyclosome (APC/C) is a multi-subunit ubiquitin ligase that regulates exit from mitosis and G1 phase of the cell cycle. Although the regulation and function of APC/CCdh1 in the unperturbed cell cycle is well studied, little is known of its role in non-genotoxic stress responses. Here, we demonstrate the role of APC/CCdh1 (APC/C activated by Cdh1 protein) in cellular protection from endoplasmic reticulum (ER) stress. Activation of APC/CCdh1 under ER stress conditions is evidenced by Cdh1-dependent degradation of its substrates. Importantly, the activity of APC/CCdh1 maintains the ER stress checkpoint, as depletion of Cdh1 by RNAi impairs cell cycle arrest and accelerates cell death following ER stress. Our findings identify APC/CCdh1 as a regulator of cell cycle checkpoint and cell survival in response to proteotoxic insults.
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)-1α, 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.
Mutations in PARKIN, pten-induced putative kinase 1
(PINK1), and DJ-1 are individually linked to
autosomal recessive early-onset familial forms of Parkinson disease (PD). Although
mutations in these genes lead to the same disease state, the functional relationships
between them and how their respective disease-associated mutations cause PD are
largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed
PPD complex) to promote ubiquitination and degradation of Parkin
substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human
brain lysates. Genetic ablation of either Pink1 or
Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well
as decreased degradation and increased accumulation of aberrantly expressed Parkin
substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat
shock–induced misfolded protein. In contrast, PD-pathogenic Parkin and
PINK1 mutations showed reduced ability to promote degradation of Parkin substrates.
This study identified a functional ubiquitin E3 ligase complex consisting of
PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded
proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of
the complex, which may constitute a mechanism underlying PD pathogenesis.
The small GTP-binding protein Arf6 regulates membrane remodeling at cell peripheries and plays crucial roles in higher orders of cellular functions including tumor invasion. Here we show that Fbx8, an F-box protein bearing the Sec7 domain, mediates ubiquitination of Arf6. This ubiquitination did not appear to be linked to immediate proteasomal degradation of Arf6, whereas Fbx8 knockdown caused hyperactivation of Arf6. Expression of Fbx8 protein was substantially lost in several breast tumor cell lines, in which Arf6 activity is pivotal for their invasion. Forced expression of Fbx8 in these cells suppressed their Arf6 activities and invasive activities, in which the F-box and Sec7 domains of Fbx8 are required. Together with the possible mechanism as to how Fbx8-mediated ubiquitination interferes with the functions of Arf6, we propose that Fbx8 provides a novel suppressive control of Arf6 activity through noncanonical ubiquitination. Our results indicate that dysfunction of Fbx8 expression may contribute to the invasiveness of some breast cancer cells.