Yousang Gwack | UCLA David Geffen School of Medicine (original) (raw)

Papers by Yousang Gwack

Small GTPases, Jan 2, 2017

More than 60 Rab GTPases exist in the human genome to regulate vesicle trafficking between organe... more More than 60 Rab GTPases exist in the human genome to regulate vesicle trafficking between organelles. Rab GTPases are members of the Ras GTPase superfamily that broadly control budding, uncoating, motility and fusion of vesicles in most cell types. Rab proteins interconvert between active, GTP-bound form and inactive, GDP-bound form. In their active conformation, they interact with various effector molecules to carry out diverse functions. Rab GTPases are usually small containing only a GTPase domain with a C-terminal prenylation site for membrane anchoring. Recently, we identified a large G protein, CRACR2A (CRAC channel regulator 2A), which uncovers novel functions of Rab GTPases. First, CRACR2A encodes a large Rab GTPase containing multiple functional domains contrary to small Rab GTPases. Second, CRACR2A plays an unexpected role in regulating intracellular signaling pathways important for T cell activation, unlike the canonical role of small Rab GTPases. Vesicles containing CRA...

Circulation research, Jan 6, 2017

Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear ... more Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. Primary endothelial cells from dermal blood and lymphatic vessels (BECs and LECs) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in BECs and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the VEGF-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium (CRAC) channel, was identified to induce the shear stress phen...

Oncotarget, 2016

Emerging evidence indicates that Orai1, a key calcium channel for store-operated Ca2+ entry, is a... more Emerging evidence indicates that Orai1, a key calcium channel for store-operated Ca2+ entry, is associated with human cancer. However, the underlying mechanism by which Orai1 regulates cancer progression remains unknown. Here we report that intracellular level of Orai1 is increased in a stepwise manner during oral/oropharyngeal carcinogenesis and highly expressed in cancer stem-like cell (CSC)-enriched populations of human oral/oropharyngeal squamous cell carcinoma (OSCC). Ectopic Orai1 expression converted non-tumorigenic immortalized oral epithelial cells to malignant cells that showed CSC properties, e.g., self-renewal capacity, increased ALDH1HIGH cell population, increased key stemness transcription factors, and enhanced mobility. Conversely, inhibition of Orai1 suppressed tumorigenicity and CSC phenotype of OSCC, indicating that Orai1 could be an important element for tumorigenicity and stemness of OSCC. Mechanistically, Orai1 activates its major downstream effector molecule, ...

The Journal of Immunology, May 1, 2014

Biochemical and biophysical research communications, Jan 14, 2016

Orai1 is a pore-subunit of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel that med... more Orai1 is a pore-subunit of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel that mediates Ca(2+) influx in most non-excitable cells via store-operated Ca(2+) entry (SOCE) mechanism. We previously demonstrated that Orai1 is involved in mediating osteogenic potential of mesenchymal stem cells (MSCs), but the underlying mechanism of this function remains unknown. Here, we report that Orai1 mediates osteogenic differentiation via bone morphogenic protein (BMP) signaling pathway in bone marrow MSCs (BMSCs). In osteogenic conditions, BMSCs derived from wild-type mice underwent osteoblastic differentiation and induced mineralization as demonstrated by increased alkaline phosphatase activity and alizarin red S staining, respectively. The expression of Runx2, a master regulator of osteoblast differentiation, and osteogenic differentiation markers were markedly increased in wild-type BMSCs under osteogenic conditions. In contrast, osteogenic conditions failed to induce such effec...

Science signaling, Jan 22, 2016

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human ... more More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed thatCRACR2A(Ca(2+)release-activated Ca(2+)channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca(2+)and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine...

Proceedings of the National Academy of Sciences, 2016

Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in im... more Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca(2+) sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER-PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER-PM junctions to regulate Ca(2+) signaling. Silencing or genetic manipulation of JP4 decreased ER Ca(2+) content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca(2+)-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate-JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca(2+) homeostasis and mediate SOCE in T cells.

Journal of Virology, Apr 1, 1999

ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 pr... more ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviviridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3-to-5 direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/ NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5 and 3 overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRT-GRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.

Journal of Virology, Dec 1, 1997

The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to ... more The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.

Journal of General Virology, Nov 1, 2000

Latency-associated nuclear antigen (LANA), encoded by ORF 73 of Kaposi's sarcoma-associated herpe... more Latency-associated nuclear antigen (LANA), encoded by ORF 73 of Kaposi's sarcoma-associated herpesvirus (KSHV ; human herpesvirus-8), may play an important role in the persistence of the viral episome by tethering it to host chromosomes during mitosis. It also has been suggested from its amino acid sequence features that LANA may have transcription-regulatory activity. Here, it is reported that LANA interacts with activating transcription factor (ATF) 4/cAMP response elementbinding protein (CREB) 2, a member of the ATF/CREB family of transcription factors, and represses the transcriptional activation activity of ATF4/CREB2. Repression by LANA is independent of the DNA-binding ability of ATF4/CREB2, since LANA also represses transactivation of ATF4/CREB2 fused to the GAL4 DNA-binding domain and does not affect the DNA-binding ability of ATF4/CREB2 in an electrophoretic mobility shift assay. The putative leucine zipper domain of LANA is required for binding to the relatively conserved basic region/leucine zipper domain (bZIP) of ATF4/CREB2, suggesting that the interaction may involve leucine zipper dimerization. 0001-7100 # 2000 SGM CGEF Downloaded from www.microbiologyresearch.org by

Molecules and Cells, Nov 1, 2002

ABSTRACT

Scientific reports, Jan 30, 2015

Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) d... more Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) rel...

The NFAT family of Ca 2+ -regulated transcription factors has a critical role in vertebrate devel... more The NFAT family of Ca 2+ -regulated transcription factors has a critical role in vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; upon stimulation they are dephosphorylated by the calmodulindependent phosphatase calcineurin and translocate to the nucleus. NFAT proteins are not represented in invertebrates, but the pathways regulating their subcellular localization --Ca 2+ homeostasis, Ca 2+ influx, calcineurin and NFAT kinases --are strongly conserved across species.

European journal of biochemistry / FEBS, Jan 15, 1997

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a known RNA helicase, an enzyme that unw... more Hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a known RNA helicase, an enzyme that unwinds RNA x DNA and RNA x RNA duplexes. We have now deciphered the biochemical characteristics of the HCV NS3 DNA helicase activity. Recombinant NS3 was expressed in Escherichia coli, purified to near homogeneity, and tested for DNA helicase activity. The optimal conditions for DNA unwinding (for example, the preferred pH and magnesium ion concentration) were similar to those for RNA unwinding. The DNA helicase activity was very sensitive to potassium ion concentration, while DNA binding and DNA-stimulated ATPase activities were not. The direction of DNA unwinding was determined to be 3' to 5'. All four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) and deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) could serve as energy sources, but GTP and dGTP were less efficient than the others. When nucleotide analog inhibitors were added to the DNA helicase reaction, the overall o...

The Journal of physiology, 2012

Store-operated Ca(2+) (SOC) entry is one of the major mechanisms to raise intracellular Ca(2+) co... more Store-operated Ca(2+) (SOC) entry is one of the major mechanisms to raise intracellular Ca(2+) concentration in non-excitable cells. Ca(2+)-release-activated Ca(2+) (CRAC) channels are a subtype of SOC channels that are extensively characterized in immune cells. Identification of STIM1 as an endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore subunit has dramatically advanced the molecular understanding of CRAC channels. Recent efforts have focused on understanding the physiological aspects of CRAC channels at an organism level using transgenic animal models and at a molecular level using electrophysiological and biochemical tools. In this review, we summarize our current understanding of the interacting partners of Orai and STIM proteins in the regulation of CRAC channel activity and other non-CRAC channel-related functions.

Methods in molecular biology (Clifton, N.J.), 2013

Measurement of intracellular Ca(2+) concentration ([Ca(2+)](i)) is useful to study the upstream a... more Measurement of intracellular Ca(2+) concentration ([Ca(2+)](i)) is useful to study the upstream and downstream events of Ca(2+) signaling. Ca(2+)-binding proteins including EF-hand-containing proteins are important downstream effector molecules after an increase of [Ca(2+)](i). Conversely, these proteins can also act as key modulators for regulation of [Ca(2+)](i) by sensing the Ca(2+) levels in the intracellular organelles and cytoplasm. Here we describe a single-cell Ca(2+) imaging technique that was used to measure the intracellular Ca(2+) levels to examine the function of Ca(2+)-binding proteins, STIM1 and Calcium release-activated Calcium channel regulator 2A (CRACR2A), using ratiometric Ca(2+) dye Fura-2 in adherent and non-adherent cells.

Current topics in membranes, 2013

Store-operated Ca(2+) entry (SOCE) is a fundamental mechanism ubiquitously employed by cells to e... more Store-operated Ca(2+) entry (SOCE) is a fundamental mechanism ubiquitously employed by cells to elevate intracellular Ca(2+) concentrations ([Ca(2+)]i). Increased intracellular Ca(2+) ions act as a second messenger that can stimulate a variety of downstream signaling pathways affecting proliferation, secretion, differentiation, and death of cells. In immune cells, immune receptor stimulation induces endoplasmic reticulum Ca(2+) store depletion that subsequently activates Ca(2+)-release-activated-Ca(2+) (CRAC) channels, a prototype of store-operated Ca(2+) (SOC) channels. Identification of Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tool to dissect the mechanism of activation and regulation of these channels. In this review, we discuss the recent advances in understanding the regulatory mechanisms and posttranslational modifications that regulate diverse aspects of CRAC channel function.

Methods in enzymology, 2014

Ca(2+) is a ubiquitous second messenger that is involved in regulation of various signaling pathw... more Ca(2+) is a ubiquitous second messenger that is involved in regulation of various signaling pathways. Cytoplasmic Ca(2+) is maintained at low concentrations (~100 nM) by many active mechanisms. Increases in intracellular Ca(2+) concentration ([Ca(2+)]i) indeed can initiate multiple signaling pathways, depending both on their pattern and subcellular localization. In T cells, the stimulation of T-cell receptor leads to an increase in [Ca(2+)]i upon the opening of Ca(2+) release-activated calcium (CRAC) channels. T cells can actually sustain high [Ca(2+)]i for several hours, resulting in the activation of transcriptional programs orchestrated by members of the nuclear factor of activated T-cell (NFAT) protein family. Here, we describe an imaging method widely employed to measure cytoplasmic [Ca(2+)] in naïve and effector T cells based on the ratiometric dye Fura-2. Furthermore, we discuss a pharmacological method relying on an inhibitor of CRAC channels, 2-aminoethyldiphenyl borate, to...