Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression.

Melanoma is a type of skin cancer that originates in melanin-producing melanocytes. It is considered a multifactorial disease caused by both genetic and environmental factors, such as UV radiation. Dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) phosphorylates many substrates involved in signaling pathways, cell survival, cell cycle control, differentiation, and neuronal development. However, little is known about the cellular function of DYRK3, one of the five members of the DYRK family. Interestingly, it was observed that the expression of DYRK3, as well as p62 (a multifunctional signaling protein), is highly enhanced in most melanoma cell lines. This study aimed to investigate whether DYRK3 interacts with p62, and how this affects melanoma progression, particularly in melanoma cell lines. We found that DYRK3 directly phosphorylates p62 at the Ser-207 and Thr-269 residue. Phosphorylation at Thr-269 of p62 by DYRK3 increased the interaction of p62 with tumor necrosis factor receptor-associated factor 6 (TRAF6), an already known activator of mammalian target of rapamycin complex 1 (mTORC1) in the mTOR-involved signaling pathways. Moreover, the phosphorylation of p62 at Thr-269 promoted the activation of mTORC1. We also found that DYRK3-mediated phosphorylation of p62 at Thr-269 enhanced the growth of melanoma cell lines and melanoma progression. Conversely, DYRK3 knockdown or blockade of p62-T269 phosphorylation inhibited melanoma growth, colony formation, and cell migration. In conclusion, we demonstrated that DYRK3 phosphorylates p62, positively modulating the p62-TRAF6-mTORC1 pathway in melanoma cells. This finding suggests that DYRK3 suppression may be a novel therapy for preventing melanoma progression by regulating the mTORC1 pathway.

USP7 attenuates endoplasmic reticulum stress-induced apoptotic cell death through deubiquitination and stabilization of FBXO7.

Parkinson's disease (PD) is a common neurodegenerative disease (NDD) characterized by the loss of dopaminergic neurons in the substantia nigra. Similar to other NDDs, the buildup of toxic protein aggregates in PD leads to progressive neuronal loss, culminating in neurodegeneration. Accumulating evidence indicates that alterations in subcellular organelles, particularly the endoplasmic reticulum (ER), are critically involved in pathological neurodegenerative events in NDDs, including PD. Mutations in the F-box only protein 7 (FBXO7 or PARK15) gene have been found to cause early onset autosomal recessive familiar PD. FBXO7 functions as an adaptor protein in the Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex, which promotes substrate ubiquitination. Although FBXO7 is involved in the ubiquitination of various target proteins, little is known about the upstream regulatory mechanism of FBXO7 and/or its modulator(s). Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme that regulates the balance between protein synthesis and degradation by removing ubiquitin from target substrates. The role of USP7 in various types of cancer is well-established; however, its role in NDDs has not been elucidated to date. In this study, we identified that USP7 acts as a novel regulator of FBXO7, positively regulating the stability of FBXO7 through Lys48-linked deubiquitination. Moreover, USP7 was found to mitigate ER stress-induced cytotoxicity and apoptosis by preventing the proteasomal degradation of FBXO7. Taken together, our study suggests that the functional relationship between FBXO7 and USP7 may play a crucial role in ER stress-induced apoptosis and the pathogenesis of PD.

DYRK3 phosphorylates SNAPIN to regulate axonal retrograde transport and neurotransmitter release.

Among the five members of the dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) family, the cellular functions of DYRK3 have not been fully elucidated. Some studies have indicated limited physiological roles and substrates of DYRK3, including promotion of glioblastoma, requirement in influenza virus replication, and coupling of stress granule condensation with mammalian target of rapamycin complex 1 signaling. Here, we demonstrate that serum deprivation causes a decrease in intracellular DYRK3 levels via the proteolytic autophagy pathway, as well as the suppression of DYRK3 gene expression. To further demonstrate how DYRK3 affects cell viability, especially in neurons, we used a yeast two-hybrid assay and identified multiple DYRK3-binding proteins, including SNAPIN, a SNARE-associated protein implicated in synaptic transmission. We also found that DYRK3 directly phosphorylates SNAPIN at the threonine (Thr) 14 residue, increasing the interaction of SNAPIN with other proteins such as dynein and synaptotagmin-1. In central nervous system neurons, SNAPIN is associated with and mediate the retrograde axonal transport of diverse cellular products from the distal axon terminal to the soma and the synaptic release of neurotransmitters, respectively. Moreover, phosphorylation of SNAPIN at Thr-14 was found to positively modulate mitochondrial retrograde transport in mouse cortical neurons and the recycling pool size of synaptic vesicles, contributing to neuronal viability. In conclusion, the present study demonstrates that DYRK3 phosphorylates SNAPIN, positively regulating the dynein-mediated retrograde transport of mitochondria and SNARE complex-mediated exocytosis of synaptic vesicles within the neurons. This finding further suggests that DYRK3 affects cell viability and provides a novel neuroprotective mechanism.

Covalent conjugation of ubiquitin-like ISG15 to apoptosis-inducing factor exacerbates toxic stimuli-induced apoptotic cell death.

Apoptosis-inducing factor (AIF) is a mitochondrion-localized flavoprotein with NADH oxidase activity. AIF normally acts as an oxidoreductase to catalyze the transfer of electrons between molecules, but it can also kill cells when exposed to certain stimuli. For example, intact AIF is cleaved upon exposure to DNA-damaging agents such as etoposide, and truncated AIF (tAIF) is released from the mitochondria to the cytoplasm and translocated to the nucleus where it induces apoptosis. Although the serial events during tAIF-mediated apoptosis and the transition of AIF function have been widely studied from various perspectives, their underlying regulatory mechanisms and the factors involved are not fully understood. Here, we demonstrated that tAIF is a target of the covalent conjugation of the ubiquitin-like moiety ISG15 (referred to as ISGylation), which is mediated by the ISG15 E3 ligase HERC5. In addition, ISGylation increases the stability of tAIF protein as well as its K6-linked polyubiquitination. Moreover, we found that ISGylation increases the nuclear translocation of tAIF upon cytotoxic etoposide treatment, subsequently causing apoptotic cell death in human lung A549 carcinoma cells. Collectively, these results suggest that HERC5-mediated ISG15 conjugation is a key factor in the positive regulation of tAIF-mediated apoptosis, highlighting a novel role of posttranslational ISG15 modification as a switch that allows cells to live or die under the stress that triggers tAIF release.

FBXO7 triggers caspase 8-mediated proteolysis of the transcription factor FOXO4 and exacerbates neuronal cytotoxicity.

Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopamine neurons in the substantia nigra. Mutations in the F-box only protein 7 gene (Fbxo7) have been reported to cause an autosomal recessive form of early-onset familial PD. FBXO7 is a part of the SKP1-Cullin1-F-box (SCF) E3 ubiquitin ligase complex, which mediates ubiquitination of numerous substrates. FBXO7 also regulates mitophagy, cell growth, and proteasome activity. A member of the FOXO family, the transcription factor FOXO4, is also known to modulate several cellular responses, including cell cycle progression and apoptosis; however, the relationship between FBXO7 and FOXO4 has not been investigated. In this study, we determined that FBXO7 binds to FOXO4 and negatively regulates intracellular FOXO4 levels. Interestingly, we also found that FBXO7-mediated degradation of FOXO4 did not occur through either of two major proteolysis systems, the ubiquitin-proteasome system or the lysosome-autophagy pathway, although it was blocked by a caspase 8-specific inhibitor and caspase 8-knockdown. Moreover, intracellular FOXO4 levels were greatly reduced in dopaminergic MN9D cells following treatment with neurotoxic 6-hydroxydopamine (6-OHDA), which was produced upon FBXO7-mediated and caspase 8-mediated proteolysis. Taken together, these results suggest that FOXO4 is negatively regulated in FBXO7-linked PD through caspase 8 activation, suppressing the cytoprotective effect of FOXO4 during 6-OHDA-induced neuronal cell death.

Death-associated Protein Kinase 1 Phosphorylates α-Synuclein at Ser129 and Exacerbates Rotenone-induced Toxic Aggregation of α-Synuclein in Dopaminergic SH-SY5Y Cells.

The formation of Lewy bodies (LBs), intracellular filamentous inclusions, is one of the hallmarks of Parkinson's disease (PD). α-Synuclein is the main component of LBs and its abnormal accumulation contributes to the pathogenesis of PD. Direct phosphorylation of α-synuclein at multiple Ser/Tyr residues is known to induce its aggregation, consequently promoting LB formation. Death-associated protein kinase 1 (DAPK1), originally identified as a positive mediator of γ-interferon-induced programmed cell death, possesses tumor-suppressive activity and mediates a wide range of cellular processes, including apoptosis and autophagy. Accumulating evidence suggests that DAPK1 is also associated with neuronal cell death and neurodegeneration. For example, DAPK1 phosphorylates tau and amyloid precursor protein, and induces tau aggregation and amyloid ß production, respectively, in Alzheimer's disease. DAPK1 is also accumulated to a larger extent in a mouse model of PD, causing synucleinopathy and dopaminergic neuron degeneration. In this study, we attempted to determine whether DAPK1 phosphorylates α-synuclein and affects cell viability in human dopaminergic neuroblastoma SH-SY5Y cells. We demonstrated that DAPK1 directly phosphorylates α-synuclein at Ser129, and induces the formation of insoluble α-synuclein aggregates. We also showed that DAPK1 enhances rotenone-induced aggregation of α-synuclein, potentiating neuronal cell death. Taken together, these findings suggest that DAPK1 acts as a novel regulator of toxic α-synuclein aggregation, possibly affecting and playing a role in the development of PD.

Gintonin Mitigates MPTP-Induced Loss of Nigrostriatal Dopaminergic Neurons and Accumulation of α-Synuclein via the Nrf2/HO-1 Pathway.

Gintonin, a ginseng-derived glycolipoprotein isolated from ginseng, has been shown to be neuroprotective in several neurological disorders such as Alzheimer's disease models and depressive-like behaviors. In this study, we sought to investigate the potential protective mechanisms of gintonin in an in vivo MPTP and in vitro MPP+-mediated Parkinson's disease (PD) model. We hypothesized that activation of nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1, potential therapeutic targets for neurodegeneration) with gintonin could abrogate PD-associated neurotoxicity by modulating the accumulation of α-synuclein, neuroinflammation, and apoptotic cell death in an MPTP/MPP+ models of PD. Our in vivo and in vitro findings suggest that the neuroprotective effects of gintonin were associated with the regulation of the Nrf2/HO-1 pathway, which regulated the expression of proinflammatory cytokines and nitric oxide synthase and apoptotic markers in the substantia nigra and striatum of the mice. Moreover, the neuroprotective effects of gintonin were also associated with a reduction in α-synuclein accumulation in the mouse substantia nigra and striatum. The neuroprotective effects of gintonin were further validated by analyzing the effects of gintonin on MPP+-treated SH-SY5Y cells, which confirmed the protective effects of gintonin. It remains for future basic and clinical research to determine the potential use of gintonin in Parkinson's disease. However, to the best of our knowledge, marked alterations in biochemical and morphological setup of midbrain dopaminergic pathways by gintonin in MPTP mice model have not been previously reported. We believe that gintonin might be explored as an important therapeutic agent in the treatment of PD.

Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon.

The carboxyl terminus of Hsp70-interacting protein (CHIP) acts as a ubiquitin E3 ligase and a link between the chaperones Hsp70/90 and the proteasome system, playing a vital role in maintaining protein homeostasis. CHIP regulates a number of proteins involved in a myriad of physiological and pathological processes, but the underlying mechanism of action via posttranslational modification has not been extensively explored. In this study, we investigated a novel modulatory mode of CHIP and its effect on CHIP enzymatic activity. ISG15, an ubiquitin-like modifier, is induced by type I interferon (IFN) stimulation and can be conjugated to target proteins (ISGylation). Here we demonstrated that CHIP may be a novel target of ISGylation in HEK293 cells stimulated with type I IFN. We also found that Lys143/144/145 and Lys287 residues in CHIP are important for and target residues of ISGylation. Moreover, ISGylation promotes the E3 ubiquitin ligase activity of CHIP, subsequently causing a decrease in levels of oncogenic c-Myc, one of its many ubiquitination targets, in A549 lung cancer cells and inhibiting A549 cell and tumor growth. In conclusion, the present study demonstrates that covalent ISG15 conjugation produces a novel CHIP regulatory mode that enhances the tumor-suppressive activity of CHIP, thereby contributing to the antitumor effect of type I IFN.

Deubiquitinating enzyme USP22 positively regulates c-Myc stability and tumorigenic activity in mammalian and breast cancer cells.

The proto-oncogene c-Myc has a pivotal function in growth control, differentiation, and apoptosis and is frequently affected in human cancer, including breast cancer. Ubiquitin-specific protease 22 (USP22), a member of the USP family of deubiquitinating enzymes (DUBs), mediates deubiquitination of target proteins, including histone H2B and H2A, telomeric repeat binding factor 1, and cyclin B1. USP22 is also a component of the mammalian SAGA transcriptional co-activating complex. In this study, we explored the functional role of USP22 in modulating c-Myc stability and its physiological relevance in breast cancer progression. We found that USP22 promotes deubiquitination of c-Myc in several breast cancer cell lines, resulting in increased levels of c-Myc. Consistent with this, USP22 knockdown reduces c-Myc levels. Furthermore, overexpression of USP22 stimulates breast cancer cell growth and colony formation, and increases c-Myc tumorigenic activity. In conclusion, the present study reveals that USP22 in breast cancer cell lines increases c-Myc stability through c-Myc deubiquitination, which is closely correlated with breast cancer progression.

Leucine-rich repeat kinase 2 exacerbates neuronal cytotoxicity through phosphorylation of histone deacetylase 3 and histone deacetylation.

Parkinson's disease (PD) is characterized by slow, progressive degeneration of dopaminergic neurons in the substantia nigra. The cause of neuronal death in PD is largely unknown, but several genetic loci, including leucine-rich repeat kinase 2 (LRRK2), have been identified. LRRK2 has guanosine triphosphatase (GTPase) and kinase activities, and mutations in LRRK2 are the major cause of autosomal-dominant familial PD. Histone deacetylases (HDACs) remove acetyl groups from lysine residues on histone tails, promoting transcriptional repression via condensation of chromatin. Here, we demonstrate that LRRK2 binds to and directly phosphorylates HDAC3 at Ser-424, thereby stimulating HDAC activity. Specifically, LRRK2 promoted the deacetylation of Lys-5 and Lys-12 on histone H4, causing repression of gene transcription. Moreover, LRRK2 stimulated nuclear translocation of HDAC3 via the phoshorylation of karyopherin subunit α2 and α6. HDAC3 phosphorylation and its nuclear translocation were increased in response to 6-hydroxydopamine (6-OHDA) treatment. LRRK2 also inhibited myocyte-specific enhancer factor 2D activity, which is required for neuronal survival. LRRK2 ultimately promoted 6-OHDA-induced cell death via positive modulation of HDAC3. These findings suggest that LRRK2 affects epigenetic histone modification and neuronal survival by facilitating HDAC3 activity and regulating its localization.

Human Telomerase Reverse Transcriptase (hTERT) Positively Regulates 26S Proteasome Activity.

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, an RNA-dependent DNA polymerase that elongates telomeric DNA. hTERT displays several extra-telomeric functions that are independent of its telomere-regulatory function, including tumor progression, and neuronal cell death regulation. In this study, we evaluated these additional hTERT non-telomeric functions. We determined that hTERT interacts with several 19S and 20S proteasome subunits. The 19S regulatory particle and 20S core particle are part of 26S proteasome complex, which plays a central role in ubiquitin-dependent proteolysis. In addition, hTERT positively regulated 26S proteasome activity independent of its enzymatic activity. Moreover, hTERT enhanced subunit interactions, which may underlie hTERT's ability of hTERT to stimulate the 26S proteasome. Furthermore, hTERT displayed cytoprotective effect against ER stress via the activation of 26S proteasome in acute myeloid leukemia cells. Our data suggest that hTERT acts as a novel chaperone to promote 26S proteasome assembly and maintenance. J. Cell. Physiol. 232: 2083-2093, 2017. © 2016 Wiley Periodicals, Inc.

Estradiol Synthesis in Gut-Associated Lymphoid Tissue: Leukocyte Regulation by a Sexually Monomorphic System.

17ß-estradiol is a potent sex hormone synthesized primarily by gonads in females and males that regulates development and function of the reproductive system. Recent studies show that 17ß-estradiol is locally synthesized in nonreproductive tissues and regulates a myriad of events, including local inflammatory responses. In this study, we report that mesenteric lymph nodes (mLNs) and Peyer's patches (Pps) are novel sites of de novo synthesis of 17ß-estradiol. These secondary lymphoid organs are located within or close to the gastrointestinal tract, contain leukocytes, and function at the forefront of immune surveillance. 17ß-estradiol synthesis was initially identified using a transgenic mouse with red fluorescent protein coexpressed in cells that express aromatase, the enzyme responsible for 17ß-estradiol synthesis. Subsequent immunohistochemistry and tissue culture experiments revealed that aromatase expression was localized to high endothelial venules of these lymphoid organs, and these high endothelial venule cells synthesized 17ß-estradiol when isolated and cultured in vitro. Both mLNs and Pps contained 17ß-estradiol with concentrations that were significantly higher than those of peripheral blood. Furthermore, the total amount of 17ß-estradiol in these organs exceeded that of the gonads. Mice lacking either aromatase or estrogen receptor-ß had hypertrophic Pps and mLNs with more leukocytes than their wild-type littermates, demonstrating a role for 17ß-estradiol in leukocyte regulation. Importantly, we did not observe any sex-dependent differences in aromatase expression, 17ß-estradiol content, or steroidogenic capacity in these lymphoid organs.

Leucine-Rich Repeat Kinase 2 (LRRK2) phosphorylates p53 and induces p21(WAF1/CIP1) expression.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) is a gene in which a mutation causes Parkinson's disease (PD), and p53 is a prototype tumor suppressor. In addition, activation of p53 in patient with PD has been reported by several studies. Because phosphorylation of p53 is critical for regulating its activity and LRRK2 is a kinase, we tested whether p53 is phosphorylated by LRRK2. RESULTS: LRRK2 phosphorylates threonine (Thr) at TXR sites in an in vitro kinase assay, and the T304 and T377 were identified as putative phosphorylated residues. An increase of phospho-Thr in the p53 TXR motif was confirmed in the cells overexpressing G2019S, and human induced pluripotent stem (iPS) cells of a G2019S carrier. Interactions between LRRK2 and p53 were confirmed by co-immunoprecipitation of lysates of differentiated SH-SY5Y cells. LRRK2 mediated p53 phosphorylation translocalizes p53 predominantly to nucleus and increases p21(WAF1/CIP1) expression in SH-SY5Y cells based on reverse transcription-polymerase chain reaction and Western blot assay results. The luciferase assay using the p21(WAF1/CIP1) promoter-reporter also confirmed that LRRK2 kinase activity increases p21 expression. Exogenous expression of G2019S and the phosphomimetic p53 T304/377D mutants increased expression of p21(WAF1/CIP1) and cleaved PARP, and cytotoxicity in the same cells. We also observed increase of p21 expression in rat primary neuron cells after transient expression of p53 T304/377D mutants and the mid-brain lysates of the G2019S transgenic mice. CONCLUSION: p53 is a LRRK2 kinase substrate. Phosphorylation of p53 by LRRK2 induces p21(WAF1/CIP1) expression and apoptosis in differentiated SH-SY5Y cells and rat primary neurons.

Dyrk1A phosphorylates parkin at Ser-131 and negatively regulates its ubiquitin E3 ligase activity.

Mutations of parkin are associated with the occurrence of autosomal recessive familial Parkinson's disease (PD). Parkin acts an E3 ubiquitin ligase, which ubiquitinates target proteins and subsequently regulates either their steady-state levels through the ubiquitin-proteasome system or biochemical properties. In this study, we identify a novel regulatory mechanism of parkin by searching for new regulatory factors. After screening human fetal brain using a yeast two hybrid assay, we found dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (Dyrk1A) as a novel binding partner of parkin. We also observed that parkin interacts and co-localizes with Dyrk1A in mammalian cells. In addition, Dyrk1A directly phosphorylated parkin at Ser-131, causing the inhibition of its E3 ubiquitin ligase activity. Moreover, Dyrk1A-mediated phosphorylation reduced the binding affinity of parkin to its ubiquitin-conjugating E2 enzyme and substrate, which could be the underlying inhibitory mechanism of parkin activity. Furthermore, Dyrk1A-mediated phosphorylation inhibited the neuroprotective action of parkin against 6-hydroxydopamine toxicity in dopaminergic SH-SY5Y cells. These findings suggest that Dyrk1A acts as a novel functional modulator of parkin. Parkin phosphorylation by Dyrk1A suppresses its E3 ubiquitin ligase activity potentially contributing to the pathogenesis of PD under PD-inducing pathological conditions. Mutations of parkin are linked to autosomal recessive forms of familial Parkinson's disease (PD). According to its functional relevance in abnormal protein aggregation and neuronal cell death, a number of post-translational modifications regulate the ubiquitin E3 ligase activity of parkin. Here we propose a novel inhibitory mechanism of parkin E3 ubiquitin ligase through dual-specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1A)-mediated phosphorylation as well as its neuroprotective action against 6-hydroxydopamine (6-OHDA)-induced cell death. The present work suggests that parkin phosphorylation by Dyrk1A may affect the pathogenesis of PD under PD-inducing pathological conditions.

Ubiquitin-specific protease 22 (USP22) positively regulates RCAN1 protein levels through RCAN1 de-ubiquitination.

Protein ubiquitination can be reversed by de-ubiquitinating enzymes (DUBs), which are classified into two main classes, cysteine proteases and metalloproteases. Cysteine proteases include ubiquitin-specific proteases (USPs) and ubiquitin C-terminal hydrolases. USP22 is a USP family member and a component of the mammalian Spt-Ada-Gcn5 acetyltransferase transcriptional coactivating complex. Regulator of calcineurin 1 (RCAN1; also known as DSCR1 or MCIP1) functions as an endogenous inhibitor of calcineurin signaling. In the present study, we have identified a novel interaction between USP22 and RCAN1 (RCAN1-1S) in the mammalian cells. In addition, the overexpression of USP22 caused the increase of RCAN1 protein stability. USP22 antagonized the actions of FBW7, NEDD4-2, and ß-TrCP E3 ligase on RCAN1 and promoted RCAN1 de-ubiquitination. Moreover, we found that RCAN1 was bound to USP22 in basal conditions, and interferon-α (IFN-α) treatment caused the dissociation of RCAN1 from USP22, which subsequently triggered RCAN1 ubiquitination and proteasome degradation. Taken together, these results suggest that USP22 positively regulates RCAN1 levels, which would consequently affect diverse RCAN1-linked cellular processes, such as the inflammatory process involving the release of IFN-α.

The F-box protein FBXO7 positively regulates bone morphogenetic protein-mediated signaling through Lys-63-specific ubiquitination of neurotrophin receptor-interacting MAGE (NRAGE).

Parkinson's disease (PD) is characterized by progressive midbrain dopaminergic neuron degeneration and the formation of intracellular protein aggregates, referred to as Lewy bodies. F-box only protein 7 (FBXO7) gene mutations are closely associated with progression of the autosomal recessive form of familial PD. FBXO7 encodes a component of Skp1, cullin, F-box ubiquitin ligase complexes; however, its cellular targets, including substrates and regulators, are not yet clarified. To identify potential substrates of FBXO7, we performed a yeast two-hybrid screen of a human fetal brain library and identified neurotrophin receptor-interacting MAGE protein (NRAGE) as a novel FBXO7-binding partner. We found that FBXO7 interacts with NRAGE and mediates Lys-63-linked poly-ubiquitination of NRAGE in mammalian cells. FBXO7 overexpression accelerates formation of NRAGE-TAK1-TAB1 complexes, whereas FBXO7 knockdown correspondingly decreases complex formation. In addition, BMP4 stimulation enhances NRAGE ubiquitination through FBXO7 and facilitates endogenous NRAGE-TAK1-TAB1 complex formation. Furthermore, FBXO7 positively regulates formation of the BMP receptor-NRAGE-TAK1-TAB1 complex, and up-regulates NF-κB activity. Taken together, our results suggest that FBXO7 affects BMP4-mediated signaling through proteasome-independent ubiquitination of NRAGE and augments formation of downstream signaling components.

Reactive oxygen species mediate IL-8 expression in Down syndrome candidate region-1-overexpressed cells.

Reactive oxygen species (ROS) have been considered to mediate inflammation in Down syndrome (DS). The present study is purposed to examine the mechanism of increased ROS levels and inflammatory cytokine IL-8 expression in Down syndrome candidate region-1 (DSCR1)-transfected cells, by determining ROS levels, IL-8 expression, NF-κB activation, and SOD1 levels in human embryonic kidney (HEK) 293 cells. The cells were treated with an antioxidant N-acetyl cysteine (NAC) or a calcium chelator BAPTA and stimulated with or without IL-1ß. As a result, basal levels of ROS, IL-8, and NF-κB-DNA binding activity were higher, and basal SOD1 levels were higher in DSCR1-transfected cells than pcDNA-transfected cells. BAPTA and NAC inhibited increase in ROS (intracellular and mitochondrial levels) in DSCR-1-transfected cells without treatment of IL-1ß. DSCR1 transfection-induced changes were increased by treatment with IL-1ß, which was suppressed by NAC and BAPTA. Transfection of SOD1 inhibited ROS levels in DSCR1-transfected cells. In conclusion, ROS activate NF-κB and IL-8 induction in DSCR1-transfected cells in a calcium-dependent manner, which is augmented by IL-1ß since IL-1ß increases calcium and ROS levels in the cells. Reducing ROS levels by treatment of antioxidants may be beneficial for preventing DS-associated inflammation by suppressing cytokine expression.

Zinc finger protein 131 inhibits estrogen signaling by suppressing estrogen receptor α homo-dimerization.

Steroid hormone estrogen elicits various physiological functions, many of which are mediated through two structurally and functionally distinct estrogen receptors, ERα and ERß. The functional role of zinc finger protein 131 (ZNF131) is poorly understood, but it is assumed to possess transcriptional regulation activity due to the presence of a DNA binding motif. A few recent reports, including ours, revealed that ZNF131 acts as a negative regulator of ERα and that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling. However, its molecular mechanism for ERα inhibition has not been elucidated in detail. Here, we demonstrate that ZNF131 directly interacts with ERα, which consequently inhibits ERα-mediated trans-activation by suppressing its homo-dimerization. Moreover, we show that the C-terminal region of ZNF131 containing the SUMOylation site is necessary for its inhibition of estrogen signaling. Taken together, these data suggest that ZNF131 inhibits estrogen signaling by acting as an ERα-co-repressor.

Small ubiquitin-like modifier (SUMO) modification of zinc finger protein 131 potentiates its negative effect on estrogen signaling.

Like ubiquitin, small ubiquitin-like modifier (SUMO) covalently attaches to specific target proteins and modulates their functional properties, including subcellular localization, protein dimerization, DNA binding, and transactivation of transcription factors. Diverse transcriptional co-regulator complexes regulate the ability of estrogen receptors to respond to positive and negative acting hormones. Zinc finger protein 131 (ZNF131) is poorly characterized but may act as a repressor of estrogen receptor α (ERα)-mediated trans-activation. Here, we identify ZNF131 as a target for SUMO modification and as a substrate for the SUMO E3 ligase human polycomb protein 2 (hPc2). We report that the SUMO-interacting motif 1 (SIM1) and the C-box of hPc2 are critical regions required for ZNF131 SUMOylation and define the ZNF131 SUMOylation site as lysine 567. We further show that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling and consequently attenuates estrogen-induced cell growth in a breast cancer cell line. Our findings suggest that SUMOylation is a novel regulator of ZNF131 action in estrogen signaling and breast cancer cell proliferation.

The transcription factor STAT2 enhances proteasomal degradation of RCAN1 through the ubiquitin E3 ligase FBW7.

Down syndrome is the most common genetic disorder and is characterized by three copies of chromosome 21. Regulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (distal part of chromosome 21), and its product functions as an endogenous inhibitor of calcineurin signaling. RCAN1 protein stability is regulated by several inflammatory signaling factors, though the underlying mechanisms remain incompletely understood. Here, we report that RCAN1 interacts with the inflammation-linked transcription factor, signal transducer and activator of transcription 2 (STAT2) in mammalian cells. STAT2 overexpression decreased levels of RCAN1 protein. Decreases in RCAN1 were blocked by a proteasome inhibitor, indicating that STAT2 regulates RCAN1 degradation via the ubiquitin-proteasome system. Co-immunoprecipitation/immunoblot analyses showed that STAT2 enhanced RCAN1 ubiquitination through the ubiquitin E3 ligase FBW7. This pathway appeared to be physiologically relevant, as treatment of cells with interferon-α reduced RCAN1 levels through the activation of STAT2 and FBW7. Together, these results suggest that STAT2 influences diverse cellular processes linked to RCAN1 by negatively affecting RCAN1 protein stability.