Nammi Park

A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes

TWIK-1 is a member of the two-pore domain K(+) (K2P) channel family that plays an essential part in the regulation of resting membrane potential and cellular excitability. The physiological role of TWIK-1 has remained enigmatic because functional expression of TWIK-1 channels is elusive. Here we report that native TWIK-1 forms a functional channel at the plasma membrane of astrocytes. A search for TWIK-1-binding proteins led to the identification of TREK-1, another member of the K2P family. The TWIK-1/TREK-1 heterodimeric channel is formed via a disulphide bridge between residue C69 in TWIK-1 and C93 in TREK-1. Gene silencing demonstrates that surface expression of TWIK-1 and TREK-1 are interdependent. TWIK-1/TREK-1 heterodimers mediate astrocytic passive conductance and cannabinoid-induced glutamate release from astrocytes. Our study sheds new light on the diversity of K2P channels.

Pn-AMPs, the hevein-like proteins from Pharbitis nil confers disease resistance against phytopathogenic fungi in tomato, Lycopersicum esculentum

The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMPs antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.

TRPM4b channel suppresses store-operated Ca2+ entry by a novel protein–protein interaction with the TRPC3 channel

We identified human TRPC3 protein by yeast two-hybrid screening of a human brain cDNA library with human TRPM4b as a bait. Immunoprecipitation and confocal microscopic analyses confirmed the protein-protein interaction between TRPM4b and TRPC3, and these two TRPs were found to be highly colocalized at the plasma membrane of HEK293T cells. Overexpression of TRPM4b suppressed TRPC3-mediated whole cell currents by more than 90% compared to those in TRPC3-expressed HEK293T cells. Furthermore, HEK293T cells stably overexpressing red fluorescent protein (RFP)-TRPM4b exhibited an almost complete abolition of UTP-induced store-operated Ca(2+) entry, which is known to take place via endogenous TRPC channels in HEK293T cells. This study is believed to provide the first clear evidence that TRPM4b interacts physically with TRPC3, a member of a different TRP subfamily, and regulates negatively the channel activity, in turn suppressing store-operated Ca(2+) entry through the TRPC3 channel.

Enhancement of TREK1 channel surface expression by protein–protein interaction with β-COP

TREK1 belongs to a family of two-pore-domain K(+) (K(2P)) channels and produce background currents that regulate cell excitability. In the present study, we identified a vesicle transport protein, beta-COP, as an interacting partner by yeast two-hybrid screening of a human brain cDNA library with N-terminal region of TREK1 (TREK1-N) as bait. Several in vitro and in vivo binding assays confirmed the protein-protein interaction between TREK1 and beta-COP. We also found that beta-COP was associated with TREK1 in native condition at the PC3 cells. When RFP-beta-COP was co-transfected with GFP-TREK1 into COS-7 cells, both proteins were found localized to the plasma membrane. In addition, the channel activity and surface expression of GFP-TREK1 increased dramatically by co-transfection with RFP-beta-COP. Surface expression of the TREK1 channel was also clearly reduced with the addition of beta-COP-specific shRNA. Collectively, these data suggest that beta-COP plays a critical role in the forward transport of TREK1 channel to the plasma membrane.

Copine1 enhances neuronal differentiation of the hippocampal progenitor HiB5 cells

Copine1 is a ubiquitously expressed protein found in various tissues including the brain, but little is known about the physiological function of this protein. Here, we showed that copine1 is involved in neuronal differentiation. Over-expression of copine1 clearly increased neurite outgrowth and expression of Tuj1, a neuronal marker protein, in HiB5 cells. In addition, endogenous copine1 was transiently increased at the early time during neuronal differentiation of HiB5 cells. When the expression of endogenous copine1 was knocked-down by its specific shRNA, PDGF-mediated neurite outgrowth was clearly decreased in HiB5 cells. Furthermore, over-expression of copine1 increased phosphorylation of Akt and copine1-specific shRNA decreased phosphorylation of Akt during neuronal differentiation of HiB5 cells. Interestingly, the phosphorylation level of PI3K, generally known as an upstream protein of Akt, was not changed by copine1 expression. These results suggest that copine1 enhances neuronal differentiation of HiB5 cells not through the PI3K-Akt pathway, but by using another Akt activated signal pathway.

Comparative analysis of the role of small G proteins in cell migration and cell death: cytoprotective and promigratory effects of RalA.

Small G protein superfamily consists of more than 150 members, and is classified into six families: the Ras, Rho, Rab, Arf, Ran, and RGK families. They regulate a wide variety of cell functions such as cell proliferation/differentiation, cytoskeletal reorganization, vesicle trafficking, nucleocytoplasmic transport and microtubule organization. The small G proteins have also been shown to regulate cell death/survival and cell shape. In this study, we compared the role of representative members of the six families of small G proteins in cell migration and cell death/survival, two cellular phenotypes that are associated with inflammation, tumorigenesis, and metastasis. Our results show that small G proteins of the six families differentially regulate cell death and cell cycle distribution. In particular, our results indicate that Rho family of small G proteins is antiapoptotic. Ras, Rho, and Ran families promoted cell migration. There was no significant correlation between the cell death- and cell migration-regulating activities of the small G proteins. Nevertheless, RalA was not only cytoprotective against multiple chemotherapeutic drugs, but also promigratory inducing stress fiber formation, which was accompanied by the activation of Akt and Erk pathways. Our study provides a framework for further systematic investigation of small G proteins in the perspectives of cell death/survival and motility in inflammation and cancer.

Resveratrol Induces Glioma Cell Apoptosis through Activation of Tristetraprolin

Tristetraprolin (TTP) is an AU-rich elements (AREs)-binding protein, which regulates the decay of AREs-containing mRNAs such as proto-oncogenes, anti-apoptotic genes and immune regulatory genes. Despite the low expression of TTP in various human cancers, the mechanism involving suppressed expression of TTP is not fully understood. Here, we demonstrate that Resveratrol (3,5,4′-trihydroxystilbene, Res), a naturally occurring compound, induces glioma cell apoptosis through activation of tristetraprolin (TTP). Res increased TTP expression in U87MG human glioma cells. Res-induced TTP destabilized the urokinase plasminogen activator and urokinase plasminogen activator receptor mRNAs by binding to the ARE regions containing the 3′ untranslated regions of their mRNAs. Furthermore, TTP induced by Res suppressed cell growth and induced apoptosis in the human glioma cells. Because of its regulation of TTP expression, these findings suggest that the bioactive dietary compound Res can be used as a novel anti-cancer agent for the treatment of human malignant gliomas.

Copine1 C2 domains have a critical calcium-independent role in the neuronal differentiation of hippocampal progenitor HiB5 cells.

Copine1 (CPNE1) has tandem C2 domains and an A domain and is known as a calcium-dependent membrane-binding protein that regulates signal transduction and membrane trafficking. We previously demonstrated that CPNE1 directly induces neuronal differentiation via Akt phosphorylation in the hippocampal progenitor cell line, HiB5. To determine which region of CPNE1 is related to HiB5 cell neurite outgrowth, we constructed several mutants. Our results show that over-expression of each C2 domain of CPNE1 increased neurite outgrowth and expression of the neuronal marker protein neurofilament (NF). Even though protein localization of the calcium binding-deficient mutant of CPNE1 was not affected by ionomycin, this mutant increased neurite outgrowth and NF expression in HiB5 cells. Furthermore, Akt phosphorylation was increased by over-expression of the calcium binding-deficient CPNE1 mutant. These results suggest that neither cellular calcium levels nor the localization of CPNE1 affect its function in neuronal differentiation. Collectively, our findings indicating that the C2 domains of CPNE1 play a calcium-independent role in regulating the neuronal differentiation of HiB5 cells.

Identification and characterization of a truncated isoform of NELL2

NELL2 is a neuron-specific secreted glycoprotein containing an N-terminal thrombospondin I-like domain (TSP-N). In this study, we describe NELL2-Tsp, a novel alternative splice variant of rat NELL2. NELL2-Tsp uses an alternate stop codon resulting in a C-terminal truncated form of NELL2, containing a signal peptide and a TSP-N domain. NELL2-Tsp is a glycosylated protein specifically expressed in brain tissue. NELL2-Tsp and NELL2 are secreted, likely due to the putative signal peptide. However, due to the truncation, the secreted portion of NELL2-Tsp is smaller than that of NELL2. Immunoprecipitation analysis confirmed that NELL2-Tsp was able to associate with NELL2 and with itself. In addition, expression of NELL2-Tsp notably reduced secretion of NELL2 and inhibited NELL2-mediated neurite outgrowth. These results suggest that NELL2-Tsp may act as a negative regulator of wild-type NELL2.

Direct binding of Copine3 with Jab1 activates downstream ErbB2 signaling and motility in SKBr3 breast cancer cells

Copine3, a known calcium-dependent membrane binding protein, contains two tandem C2 domains and an A domain. This protein has been shown to interact with receptor tyrosine kinase 2 (ErbB2), but little is known concerning the physiological function of Copine3. To better understand its cellular function, we carried out a yeast two-hybrid screen to find Copine3 binding partners. Among the identified proteins, Jun activation domain-binding protein 1 (Jab1) appears to directly interact with Copine3. This physical interaction between Copine3 and Jab1 as well as the specific binding regions of both proteins were confirmed in vitro and in vivo. Our results also demonstrate that binding of Copine3 to ErbB2 is increased when Jab1 is overexpressed in SKBr3 breast cancer cells. Furthermore, two ErbB2 downstream signaling proteins [phosphatidylinositol 3 (PI3) kinase and protein kinase B (AKT)] were also activated by Jab1 overexpression in these cells. These data suggest that binding of Copine3 and Jab1 regulates, at least to some extent, the ErbB2 signaling pathway. Moreover, overexpression of both Copine3 and Jab1 in SKBr3 cells effectively increased cellular migration. Collectively, our findings indicating that Jab1 enhances the ErbB2 binding ability of Copine3, further activating the ErbB2 signaling pathways involved in breast cancer cell pathogenesis.