Seyun Kim

Skp2 regulates the antiproliferative function of the tumor suppressor RASSF1A via ubiquitin-mediated degradation at the G1-S transition.

The tumor suppressor RASSF1A is inactivated in many human cancers and is implicated in regulation of microtubule stability, cell cycle progression and apoptosis. However, the precise mechanisms of RASSF1A action and their regulation remain unclear. Here we show that Skp2, an oncogenic subunit of the Skp1–Cul1–F–box ubiquitin ligase complex, interacts with, ubiquitinates, and promotes the degradation of RASSF1A at the G1–S transition of the cell cycle. This Skp2-dependent destruction of RASSF1A requires phosphorylation of the latter on serine-203 by cyclin D–cyclin-dependent kinase 4. Interestingly, mutation of RASSF1A-phosphorylation site Ser203 to alanine results in a delay in cell cycle progression from G1 to S phase. Moreover, enforced expression of Skp2 abolishes the inhibitory effect of RASSF1A on cell proliferation. Finally, the delay in G1–S progression after Skp2 removal is normalized by depletion of RASSF1A. These findings suggest that the Skp2-mediated degradation of RASSF1A plays an important role in cell proliferation and survival.

Inositol polyphosphate multikinase signaling in the regulation of metabolism

Inositol phosphates (IPs) act as signaling messengers to regulate various cellular processes such as growth. Inositol polyphosphate multikinase (IPMK) generates inositol tetrakis‐ and pentakisphosphates (IP4 and IP5), acting as a key enzyme for inositol polyphosphate biosynthesis. IPMK was initially discovered as an essential subunit of the arginine‐sensing transcription complex in budding yeast. In mammals, IPMK is also known as a physiologically important phosphatidylinositol 3 kinase (PI3K) that forms phosphatidylinositol 3,4,5‐trisphosphate (PIP3), which activates Akt/PKB and stimulates its signaling. Acting in a catalytically independent fashion, IPMK mediates the activation of mammalian target of rapamycin (mTOR) in response to essential amino acids. In addition, IPMK binds and modulates AMP‐activated protein kinase (AMPK) signaling pathways, including those involved in hypothalamic control of food intake. These recent findings strongly suggest that IPMK is a versatile player in insulin‐, nutrient‐, and energy‐mediated metabolism signaling networks. Agents that control IPMK functions may provide novel therapeutics in metabolic syndromes such as obesity and diabetes.

A basal-like breast cancer-specific role for SRF–IL6 in YAP-induced cancer stemness

The switch between stem/progenitor cell expansion and differentiation is critical for organ homeostasis. The mammalian Hippo pathway effector and oncoprotein YAP expands undifferentiated stem/progenitor cells in various tissues. However, the YAP-associated transcription factors and downstream targets underlying this stemness-promoting activity are poorly understood. Here we show that the SRF–IL6 axis is the critical mediator of YAP-induced stemness in mammary epithelial cells and breast cancer. Specifically, serum response factor (SRF)-mediated binding and recruitment of YAP to mammary stem cell (MaSC) signature-gene promoters induce numerous MaSC signature genes, among which the target interleukin (IL)-6 is critical for YAP-induced stemness. High SRF–YAP/TAZ expression is correlated with IL6-enriched MaSC/basal-like breast cancer (BLBC). Finally, we show that this high SRF expression enables YAP to more efficiently induce IL6 and stemness in BLBC compared with luminal-type breast cancer. Collectively, our results establish the importance of SRF–YAP–IL6 signalling in promoting MaSC-like properties in a BLBC-specific manner.

Huntington’s disease: Neural dysfunction linked to inositol polyphosphate multikinase

Significance Huntington’s disease (HD) is a progressive neurodegenerative disorder affecting the striatum. The striatal-enriched transcription factor COUP-TF-interacting protein 2 (Ctip2) is depleted in HD and has been identified as a putative transcription factor for the enzyme inositol polyphosphate multikinase (IPMK). IPMK displays soluble inositol phosphate kinase activity, lipid kinase activity, and several noncatalytic activities including its role as a transcriptional coactivator. We describe severe depletion in IPMK protein in HD patients and several animal and cell models of the disease. IPMK overexpression rescues the metabolic impairments in a cell model of HD. Furthermore, delivery of IPMK in a transgenic HD model improves pathological changes and motor performance. The Ctip2–IPMK–Akt signaling pathway provides a previously unidentified therapeutic target for HD. Huntington’s disease (HD) is a progressive neurodegenerative disease caused by a glutamine repeat expansion in mutant huntingtin (mHtt). Despite the known genetic cause of HD, the pathophysiology of this disease remains to be elucidated. Inositol polyphosphate multikinase (IPMK) is an enzyme that displays soluble inositol phosphate kinase activity, lipid kinase activity, and various noncatalytic interactions. We report a severe loss of IPMK in the striatum of HD patients and in several cellular and animal models of the disease. This depletion reflects mHtt-induced impairment of COUP-TF-interacting protein 2 (Ctip2), a striatal-enriched transcription factor for IPMK, as well as alterations in IPMK protein stability. IPMK overexpression reverses the metabolic activity deficit in a cell model of HD. IPMK depletion appears to mediate neural dysfunction, because intrastriatal delivery of IPMK abates the progression of motor abnormalities and rescues striatal pathology in transgenic murine models of HD.

IPMK: A versatile regulator of nuclear signaling events

Inositol-derived metabolites (e.g., phosphoinositides and inositol polyphosphates) are key second messengers that are essential for controlling a wide range of cellular events. Inositol polyphosphate multikinase (IPMK) exhibits complex catalytic activities that eventually yield water-soluble inositol polyphosphates (e.g., IP4 and IP5) and lipid-bound phosphatidylinositol 3,4,5-trisphosphate. A series of recent studies have suggested that IPMK may be a multifunctional regulator in the nucleus of mammalian cells. In this review, we highlight the novel modes of action of IPMK in transcriptional and epigenetic regulation, and discuss its roles in physiology and disease.

Inositol polyphosphate multikinase is a coactivator for serum response factor-dependent induction of immediate early genes

Significance The inositol polyphosphate system is a major intracellular signaling modality. Inositol polyphosphate multikinase (IPMK) is essential for this system, converting inositol 1,4,5-trisphosphate (IP3) to IP4 and IP4 to IP5. IPMK also is a physiologic PI3-kinase. In a noncatalytic manner, IPMK stabilizes the mammalian target of rapamycin (mTOR) complex as well as tumor suppressor protein p53 and CREB-binding protein (CBP)/E1A binding protein p300. We now demonstrate that IPMK is a transcriptional coactivator for serum response factor (SRF) signaling, which is markedly reduced in IPMK-deleted mice and augmented by IPMK overexpression. IPMK binds directly to SRF, facilitating its interaction with serum response elements of multiple immediate early genes. Agents influencing IPMK interactions with SRF may be useful in regulating the SRF signaling system. Inositol polyphosphate multikinase (IPMK) is a notably pleiotropic protein. It displays both inositol phosphate kinase and phosphatidylinositol kinase catalytic activities. Noncatalytically, IPMK stabilizes the mammalian target of rapamycin complex 1 and acts as a transcriptional coactivator for CREB-binding protein/E1A binding protein p300 and tumor suppressor protein p53. Serum response factor (SRF) is a major transcription factor for a wide range of immediate early genes. We report that IPMK, in a noncatalytic role, is a transcriptional coactivator for SRF mediating the transcription of immediate early genes. Stimulation by serum of many immediate early genes is greatly reduced by IPMK deletion. IPMK stimulates expression of these genes, an influence also displayed by catalytically inactive IPMK. IPMK acts by binding directly to SRF and thereby enhancing interactions of SRF with the serum response element of diverse genes.

Mitochondrial respiratory defect causes dysfunctional lactate turnover via AMP-activated protein kinase activation in human induced pluripotent stem cell-derived hepatocytes

Background: Metabolic influences of defective mitochondrial respiration in hepatocytes remain elusive. Results: Mutation of mitochondrion-encoded NADH dehydrogenase 1 enhanced the activity of AMP-activated protein kinase (AMPK) and decreased lactate turnover in differentiated hepatocytes. Conclusion: Dysfunctional mitochondria in the hepatocytes may be responsible for lactic acidosis. Significance: This study provides new insight on the role of AMPK in lactic acidosis caused by mitochondrial dysfunction. A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T→C) in the mitochondrion-encoded NADH dehydrogenase 1 (MTND1) gene and that differentiated into hepatocytes (DMRC hepatocytes) in vitro. DMRC hepatocytes showed abnormalities in mitochondrial characteristics, the NAD+/NADH ratio, the glycogen storage level, the lactate turnover rate, and AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver.

Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Significance Inositol phosphates have long been considered to be negative regulators of synaptic exocytosis, but the function of diphosphoinositol pentakisphosphate (IP7) has remained elusive. We found that overexpression and depletion of inositol hexakisphosphate (IP6) kinase in PC12 cells or hippocampal neurons led to a reduction and increase in neurotransmitter release, respectively. Biophysical assays revealed that 5-IP7 inhibited Ca2+-induced synaptic membrane fusion at a concentration one order of magnitude lower than that required for IP6. We further elucidated the molecular mechanism responsible for 5-IP7 actions, demonstrating that 5-IP7 directly bound with high affinity to synaptotagmin 1 (Syt1), a Ca2+ sensor in cellular exocytosis, and suppressed its fusogenic activity. Thus, our data propose 5-IP7 as a potent inhibitor of Syt1 actions on Ca2+-mediated synaptic vesicle fusion. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6. Synaptotagmin 1 (Syt1), a Ca2+ sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7. Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6. In addition, 5-IP7–dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca2+ levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca2+. These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

Prediction of drug-induced immune-mediated hepatotoxicity using hepatocyte-like cells derived from human embryonic stem cells

Drug-induced liver injury (DILI) is a leading cause of liver disease and a key safety factor during drug development. In addition to the initiation events of drug-specific hepatotoxicity, dysregulated immune responses have been proposed as major pathological events of DILI. Thus, there is a need for a reliable cell culture model with which to assess drug-induced immune reactions to predict hepatotoxicity for drug development. To this end, stem cell-derived hepatocytes have shown great potentials. Here we report that hepatocyte-like cells derived from human embryonic stem cells (hES-HLCs) can be used to evaluate drug-induced hepatotoxic immunological events. Treatment with acetaminophen significantly elevated the levels of inflammatory cytokines by hES-HLCs. Moreover, three human immune cell lines, Jurkat, THP-1, and NK92MI, were activated when cultured in conditioned medium obtained from acetaminophen-treated hES-HLCs. To further validate, we tested thiazolidinedione (TZD) class, antidiabetic drugs, including troglitazone withdrawn from the market because of severe idiosyncratic drug hepatotoxicity. We found that TZD drug treatment to hES-HLCs resulted in the production of pro-inflammatory cytokines and eventually associated immune cell activation. In summary, our study demonstrates for the first time the potential of hES-HLCs as an in vitro model system for assessment of drug-induced as well as immune-mediated hepatotoxicity.

Inositol polyphosphate multikinase promotes Toll-like receptor–induced inflammation by stabilizing TRAF6

Inositol polyphosphate multikinase acts as a key determinant of inflammation by stabilizing the signaling adaptor TRAF6. Toll-like receptor (TLR) signaling is tightly controlled to protect hosts from microorganisms while simultaneously preventing uncontrolled immune responses. Tumor necrosis factor receptor–associated factor 6 (TRAF6) is a critical mediator of TLR signaling, but the precise mechanism of how TRAF6 protein stability is strictly controlled still remains obscure. We show that myeloid-specific deletion of inositol polyphosphate multikinase (IPMK), which has both inositol polyphosphate kinase activities and noncatalytic signaling functions, protects mice against polymicrobial sepsis and lipopolysaccharide-induced systemic inflammation. IPMK depletion in macrophages results in decreased levels of TRAF6 protein, thereby dampening TLR-induced signaling and proinflammatory cytokine production. Mechanistically, the regulatory role of IPMK is independent of its catalytic function, instead reflecting its direct binding to TRAF6. This interaction stabilizes TRAF6 by blocking its K48-linked ubiquitination and subsequent degradation by the proteasome. Thus, these findings identify IPMK as a key determinant of TRAF6 stability and elucidate the physiological function of IPMK in TLR-induced innate immunity.