Chae Young Hwang

Tumor Suppressor VDUP1 Increases p27kip1 Stability by Inhibiting JAB1

Vitamin D3 up-regulated protein 1 (VDUP1) is a stress-response gene that is up-regulated by 1,25(OH)2D3 in many cells. It has been reported that VDUP1 expression is reduced in many tumor cells and the enforced expression of VDUP1 inhibits cell proliferation by arresting cell cycle progression. Here, we found that VDUP1-/- fibroblast cells proliferated more rapidly compared with wild-type cells with reduced expression of p27(kip1), a cyclin-dependent kinase inhibitor. JAB1 is known to interact with p27(kip1) and to decrease the stability of p27(kip1). VDUP1 interacted with JAB1 and restored JAB1-induced suppression of p27(kip1) stability. In this process, VDUP1 blocked the JAB1-mediated translocation of p27(kip1) from the nucleus to the cytoplasm. In addition, VDUP1 inhibited JAB1-mediated activator protein-1 activation and cell proliferation. Taken together, these results indicate that VDUP1 is a novel factor of p27(kip1) stability via regulating JAB1.

Crucial Role for Mst1 and Mst2 Kinases in Early Embryonic Development of the Mouse

ABSTRACT Mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2, respectively) are potent serine/threonine kinases that are involved in cell proliferation and cell death. To investigate the physiological functions of Mst1 and Mst2, we generated Mst1 and Mst2 mutant mice. Mst1−/− and Mst2−/− mice were viable and fertile and developed normally, suggesting possible functional overlaps between the two genes. A characterization of heterozygous and homozygous combinations of Mst1 and Mst2 mutant mice showed that mice containing a single copy of either gene underwent normal organ development; however, Mst1−/−; Mst2−/− mice lacking both Mst1 and Mst2 genes started dying in utero at approximately embryonic day 8.5. Mst1−/−; Mst2−/− mice exhibited severe growth retardation, failed placental development, impaired yolk sac/embryo vascular patterning and primitive hematopoiesis, increased apoptosis in placentas and embryos, and disorganized proliferating cells in the embryo proper. These findings indicate that both Mst1 and Mst2 kinases play essential roles in early mouse development, regulating placental development, vascular patterning, primitive hematopoiesis, and cell proliferation and survival.

Thioredoxin modulates activator protein 1 (AP-1) activity and p27Kip1 degradation through direct interaction with Jab1

Thioredoxin (Trx) is a cellular redox enzyme that plays multiple roles in regulating cell growth and apoptosis. Jun activation domain-binding protein 1 (Jab1) was originally identified as a coactivator of activator protein 1 (AP-1) transcription and was also shown to promote degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Recently, Jab1 expression was associated with the progression and poor prognosis of pituitary, epithelial ovarian, and breast cancers, suggesting that it plays a role in oncogenesis. Here, we report that Trx specifically interacts with and modulates the function of Jab1. Fluorescence resonance energy transfer and co-immunoprecipitation studies revealed that Trx and Jab1 colocalize and directly interact with each other. Further, Trx negatively regulates two important Jab1-controlled signaling pathways, activation of AP-1 transcription and degradation of p27Kip1, probably through a direct interaction between Trx and C-terminal of Jab1. The negative effect of Trx on AP-1 activity is Jab1-dependent, as it disappears when Jab1 levels are suppressed by an antisense approach. In addition, Trx competes with p27Kip1 for Jab1 binding. Taken together, our results suggest that Trx may regulate cell cycle and growth through a novel modulation of Jab1-mediated proliferation signals, further indicating that Trx may have the ability to control tumor progression.

Extracellular signal-regulated kinase 2-dependent phosphorylation induces cytoplasmic localization and degradation of p21Cip1.

ABSTRACT p21Cip1 is an inhibitor of cell cycle progression that promotes G1-phase arrest by direct binding to cyclin-dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21Cip1 downregulation at the posttranslational level. This downregulation requires the sustained activation of extracellular signal-regulated kinase 2 (ERK2), which directly interacts with and phosphorylates p21Cip1, promoting p21Cip1 nucleocytoplasmic translocation and ubiquitin-dependent degradation, thereby resulting in cell cycle progression. ERK1 is not likely involved in this process. Phosphopeptide analysis of in vitro ERK2-phosphorylated p21Cip1 revealed two phosphorylation sites, Thr57 and Ser130. Double mutation of these sites abolished ERK2-mediated p21Cip1 translocation and degradation, thereby impairing ERK2-dependent cell cycle progression at the G1/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by downregulating the cell cycle inhibitory protein p21Cip1.

TRIM32 Protein Sensitizes Cells to Tumor Necrosis Factor (TNFα)-induced Apoptosis via Its RING Domain-dependent E3 Ligase Activity against X-linked Inhibitor of Apoptosis (XIAP)

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

The tumour suppressor PTEN mediates a negative regulation of the E3 ubiquitin-protein ligase Nedd4

The tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) is a multifunctional protein deregulated in many types of cancer. It is suggested that a number of proteins that relate with PTEN functionally or physically have not yet been found. In order to search for PTEN-interacting proteins that might be crucial in the regulation of PTEN, we exploited a proteomics-based approach. PTEN-expressing NIH 3T3 cell lysates were used in affinity chromatography and then analysed by LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem MS). A total of 93 proteins were identified. Among the proteins identified, we concentrated on the E3 ubiquitin-protein ligase Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated gene 4), and performed subsequent validation experiments using HeLa cells. Nedd4 inhibited PTEN-induced apoptotic cell death and, conversely, the Nedd4 level was down-regulated by PTEN. The down-regulation effect was diminished by a mutation (C124S) in the catalytic site of PTEN. Nedd4 expression was also decreased by a PI3K (phosphoinositide 3-kinase) inhibitor, LY294002, suggesting that the regulation is dependent on the phosphatase-kinase activity of the PTEN-PI3K/Akt pathway. Semi-quantitative real-time PCR analysis revealed that Nedd4 was transcriptionally regulated by PTEN. Thus our results have important implications regarding the roles of PTEN upon the E3 ubquitin ligase Nedd4 as a negative feedback regulator as well as a substrate.

Quantitative proteome analysis of age‐related changes in mouse gastrocnemius muscle using mTRAQ

Aging is associated with a progressive loss of skeletal muscular function that often leads to progressive disability and loss of independence. Although muscle aging is well documented, the molecular mechanisms of this condition still remain unclear. To gain greater insight into the changes associated with aging of skeletal muscle, we performed quantitative proteomic analyses on young (6 months) and aged (27 months) mouse gastrocnemius muscles using mTRAQ stable isotope mass tags. We identified and quantified a total of 4585 peptides corresponding to 236 proteins (protein probability >0.9). Among them, 33 proteins were more than 1.5‐fold upregulated and 20 proteins were more than 1.5‐fold downregulated in aged muscle compared with young muscle. An ontological analysis revealed that differentially expressed proteins belonged to distinct functional groups, including ion homeostasis, energy metabolism, protein turnover, and Ca2+ signaling. Identified proteins included aralar1, β‐enolase, fatty acid‐binding protein 3, 3‐hydroxyacyl‐CoA dehydrogenase (Hadh), F‐box protein 22, F‐box, and leucine‐rich repeat protein 18, voltage‐dependent L‐type calcium channel subunit beta‐1, ryanodine receptor (RyR), and calsequestrin. Ectopic expression of calsequestrin in C2C12 myoblast resulted in decreased activity of nuclear factor of activated T‐cells and increased levels of atrogin‐1 and MuRF1 E3 ligase, suggesting that these differentially expressed proteins are involved in muscle aging.

Role of Junctin Protein Interactions in Cellular Dynamics of Calsequestrin Polymer upon Calcium Perturbation

Background: In vitro studies have reported reversible calsequestrin polymerization and depolymerization. Results: Live cell imaging analysis revealed Ca2+-dependent decondensation of calsequestrin speckles, consistent with in vitro microscopic data. Conclusion: Calsequestrin depolymerization by calcium depletion requires coexistence of junctin. Significance: The role of calsequestrin in intracellular calcium homeostasis was explored. Calsequestrin (CSQ), the major intrasarcoplasmic reticulum calcium storage protein, undergoes dynamic polymerization and depolymerization in a Ca2+-dependent manner. However, no direct evidence of CSQ depolymerization in vivo with physiological relevance has been obtained. In the present study, live cell imaging analysis facilitated characterization of the in vivo dynamics of the macromolecular CSQ structure. CSQ2 appeared as speckles in the presence of normal sarcoplasmic reticulum (SR) Ca2+ that were decondensed upon Ca2+ depletion. Moreover, CSQ2 decondensation occurred only in the stoichiometric presence of junctin (JNT). When expressed alone, CSQ2 speckles remained unchanged, even after Ca2+ depletion. FRET analysis revealed constant interactions between CSQ2 and JNT, regardless of the SR Ca2+ concentration, implying that JNT is an essential component of the CSQ scaffold. In vitro solubility assay, electron microscopy, and atomic force microscopy studies using purified recombinant proteins confirmed Ca2+ and JNT-dependent disassembly of the CSQ2 polymer. Accordingly, we conclude that reversible polymerization and depolymerization of CSQ are critical in SR Ca2+ homeostasis.

Hsp70 Interacts with the Retroviral Restriction Factor TRIM5α and Assists the Folding of TRIM5α

Tripartite motif (TRIM) protein TRIM5α has been shown to restrict human immunodeficiency virus, type 1 infection in Old World monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5α is not known. In this study, we showed that transiently overexpressed TRIM5α causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5α suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5α-binding partner. The interaction between Hsp70 and TRIM5α was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5α-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5α, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5α-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5α. Studies using a series of TRIM5α deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5α structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5α, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5α in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5α-induced cellular insults.

CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species.

Reactive oxygen species modulate cell fate in a context-dependent manner. Sublethal doses of H(2)O(2) decreased the level of proliferating cell nuclear antigen (PCNA) in normal cells (including primary human dermal fibroblasts and IMR-90 cells) without affecting cyclin-dependent kinase 2 (CDK2) activity, leading to cell cycle arrest and subsequent senescence. In contrast, exposure of cancer cells (such as HeLa and MCF7 cells) to H(2)O(2) increased CDK2 activity with no accompanying change in the PCNA level, leading to cell proliferation. A CDK2 inhibitor, CVT-313, prevented H(2)O(2)-induced cancer cell proliferation. These results support the notion that the cyclin/CDK2/p21(Cip1)/PCNA complex plays an important role as a regulator of cell fate decisions.