Dongeun Park

Sequential Activation of Phosphatidylinositol 3-Kinase, βPix, Rac1, and Nox1 in Growth Factor-Induced Production of H2O2

ABSTRACT The generation of reactive oxygen species (ROS) in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the Rac protein. We report here that the COOH-terminal region of Nox1, a protein related to gp91phox (Nox2) of phagocytic cells, is constitutively associated with βPix, a guanine nucleotide exchange factor for Rac. Both growth factor-induced ROS production and Rac1 activation were completely blocked in cells depleted of βPix by RNA interference. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Moreover, the depletion of Nox1 by RNA interference inhibited growth factor-induced ROS generation. These results suggest that ROS production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, βPix, and Rac1, which then binds to Nox1 to stimulate its NADPH oxidase activity.

βPix-enhanced p38 Activation by Cdc42/Rac/PAK/MKK3/6-mediated Pathway IMPLICATION IN THE REGULATION OF MEMBRANE RUFFLING

βPix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor for Rho family small G protein Cdc42/Rac. The protein interacts with p21-activated protein kinase (PAK) through its SH3 domain. We examined the effect of βPix on MAP kinase signaling and cytoskeletal rearrangement in NIH3T3 fibroblast cells. Overexpression of βPix enhanced the activation of p38 in the absence of other stimuli and also induced translocation of p38 to the nucleus. This βPix-induced p38 activation was blocked by coexpression of dominant-negative Cdc42/Rac or kinase-inactive PAK, indicating that the effect of βPix on p38 is exerted through the Cdc42/Rac-PAK pathway and requires PAK kinase activity. The essential role of βPix in growth factor-stimulated p38 activation was evidenced by the blocking of platelet-derived growth factor-induced p38 activation in the cells expressing βPix SH3m (W43K) and βPix DHm (L238R,L239R). In addition, SB203580, a p38 inhibitor, and kinase-inactive p38 (T180A,Y182F) blocked membrane ruffling induced by βPix, suggesting that p38 might be involved in mediating βPix-induced membrane ruffling. The results in this study suggest that βPix might have a role in nuclear signaling, as well as in actin cytoskeleton regulation, and that some part of these cellular functions is possibly mediated by p38 MAP kinase.

Antagonistic regulation of myogenesis by two deubiquitinating enzymes, UBP45 and UBP69

Protein modification by ubiquitin is a dynamic and reversible process that is involved in the regulation of a variety of cellular processes. Here, we show that myogenic differentiation of embryonic muscle cells is antagonistically regulated by two deubiquitinating enzymes, UBP45 and UBP69, that are generated by alternative splicing. Both enzymes cleaved off ubiquitin from polyubiquitinated protein conjugates in vivo as well as from linear ubiquitin–protein fusions in vitro. In cultured myoblasts, the level of UBP69 mRNA markedly but transiently increased before membrane fusion, whereas that of UBP45 mRNA increased as the cells fused to form myotubes. Both myoblast fusion and accumulation of myosin heavy chain were dramatically stimulated by the stable expression of UBP69 but strongly attenuated by that of the catalytically inactive form of the protease, suggesting that the mutant enzyme acts dominant negatively on the function of the wild-type protease. In contrast, stable expression of UBP45 completely blocked both of the myogenic processes but that of inactive enzyme did not, indicating that the catalytic activity of the enzyme is essential for its inhibitory effects. These results indicate that differential expression of UBP45 and UBP69 is involved in the regulation of muscle cell differentiation.

Overexpression of βPix-a in human breast cancer tissues

Pak interacting exchange factor (βPix) is a recently cloned protein that contains a multidomain with many potential binding sites and is known to be involved in the regulation of Cdc42/Rac GTPases and Pak kinase activity. These domains of βPix appear to play a critical role in the regulation of the cytoskeletal organization. The overexpression of βPix enhances the activation of p38, which is thought to be an important downstream effector of the Rho GTPase family (Rac, Cdc42), which are involved in increased membrane ruffling and cell motility. This increase of cell mobility is an important feature of cancer invasion. We examined the expression of βPix-a in human breast cancer tissues and adjacent normal tissues obtained from 39 breast cancer patients. Immunoblot analysis and RT-PCR revealed that βPix-a expression was significantly increased in 37 of the 39 breast cancer tissues (94.9%) versus normal breast tissues. Immunohistochemical analysis showed that breast cancer tissues have consistently stronger immunoreactivity to βPix-a antibodies than normal tissues. βPix-a overexpression was inversely associated with extensive intraductal component (P<0.001). In conclusion, βPix-a expression was found to be higher in human breast cancer tissues than in normal breast tissues, which implies a role for βPix-a in human breast tumorigenesis. We suggest that βPix-a may be a useful marker of malignant disease in the breast.

Filamin B serves as a molecular scaffold for type I interferon-induced c-Jun NH2-terminal kinase signaling pathway.

Type I interferons (IFNs) activate Janus tyrosine kinase-signal transducer and activator of transcription pathway for exerting pleiotropic biological effects, including antiviral, antiproliferative, and immunomodulatory responses. Here, we demonstrate that filamin B functions as a scaffold that links between activated Rac1 and a c-Jun NH(2)-terminal kinase (JNK) cascade module for mediating type I IFN signaling. Filamin B interacted with Rac1, mitogen-activated protein kinase kinase kinase 1, mitogen-activated protein kinase kinase 4, and JNK. Filamin B markedly enhanced IFNalpha-dependent Rac1 activation and the sequential activation of the JNK cascade members. Complementation assays using M2 melanoma cells revealed that filamin B, but not filamin A, is required for IFNalpha-dependent activation of JNK. Furthermore, filamin B promoted IFNalpha-induced apoptosis, whereas short hairpin RNA-mediated knockdown of filamin B prevented it. These results establish a novel function of filamin B as a molecular scaffold in the JNK signaling pathway for type I IFN-induced apoptosis, thus providing the biological basis for antitumor and antiviral functions of type I IFNs.

Filamin is essential for shedding of the transmembrane serine protease, epithin

Epithin is a type II transmembrane serine protease that exists in a soluble and membrane‐bound form. Shedding is thought to be important in regulating its action, but little is known regarding the intracellular events that trigger such shedding. Here, we show that phorbol myristate acetate (PMA) causes the release of epithin. It also causes accumulation of the protein at the site of cell–cell contacts, and this accumulation is dependent on the formation of cortical actin. In addition, we have identified the actin‐binding protein, filamin, as the linker between epithin and the actin cytoskeleton. The interaction of epithin and filamin was enhanced by PMA, and epithin was not released from filamin‐deficient M2 cells. We also show that the release of epithin does not require its own activity and is blocked by a metalloprotease inhibitor, GM6001. These results show that filamin has an essential role in shedding by linking epithin to the as yet unidentified metalloprotease‐shedding enzyme(s).

GluA1 phosphorylation at serine 831 in the lateral amygdala is required for fear renewal.

Fear renewal, a widely pursued model of post-traumatic stress disorder and phobias, refers to the context-specific relapse of conditioned fear after extinction. However, its molecular mechanisms are largely unknown. We found that renewal-inducing stimuli, generally believed to be insufficient to induce synaptic plasticity, enhanced excitatory synaptic strength, activity of synaptic GluA2-lacking AMPA receptors and Ser831 phosphorylation of synaptic surface GluA1 in the lateral nucleus of the amygdala (LAn) of fear-extinguished rats. Consistently, the induction threshold for LAn synaptic potentiation was considerably lowered after extinction, and renewal occluded this low-threshold potentiation. The low-threshold potentiation (a potential cellular substrate for renewal), but not long-term potentiation, was attenuated by dialysis into LAn neurons of a GluA1-derived peptide that competes with Ser831-phosphorylated GluA1. Microinjections of the same peptide into the LAn attenuated fear renewal, but not fear learning. Our findings suggest that GluA1 phosphorylation constitutes a promising target for clinical treatment of aberrant fear-related disorders.

Matriptase/epithin participates in mammary epithelial cell growth and morphogenesis through HGF activation.

The epithelial-derived, type II transmembrane serine protease matriptase, the mouse homologue of which is epithin, has been shown to be involved in epidermal differentiation, hair formation, and thymus function. We show in this study that epithin/matriptase (Epi/MTP) plays a significant role in mammary epithelial cell growth and morphogenesis. Epi/MTP is expressed at low level in the mouse mammary epithelium of young animals and it accumulates at the terminal end-bud of the growing ducts. The level of Epi/MTP is elevated in the mammary glands at stages when epithelial proliferation and modeling occur. It is primarily present in the luminal epithelial cells of mouse mammary ducts and lobules. Using an ex vivo three-dimensional culture system for mammary epithelial functional assays, we show that mammary epithelial growth and morphogenesis in the presence of the latent form hepatocyte growth factor (pro-HGF) are blocked either by an inhibitor of the Epi/MTP protease activity or by siRNA knockdown of the Epi/MTP expression. These studies demonstrate that Epi/MTP participates in mammary epithelial growth and modeling through activation of pro-HGF. Our findings reveal an important pathway in normal mammary epithelial morphogenesis which may participate in breast cancer progression.

Crystal structure of the dimerization domain of human filamin A.

Crystal structure of the dimerization domain of human filamin A Min-Duk Seo,1y Seung-Hyeon Seok,1y Hookang Im, Ae-Ran Kwon, Sang Jae Lee, Hyung-Ryong Kim, Yongcheol Cho, Dongeun Park, and Bong-Jin Lee* 1 Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea 2 Promeditech Ltd., Seoul 151-010, Korea 3Department of Dental Pharmacology, School of Dentistry, Wonkwang University, Iksan, Chonbuk 570-749, Korea 4 School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

Cellular Localization of α3β1 Integrin Isoforms in Association with Myofibrillogenesis during Cardiac Myocyte Development in Culture

The cellular localization of α3β1 integrin isoforms was examined in cultured neonatal myocytes at selected times during development using double immunofluorescence assays. The distribution of α3A subunits began as diffuse and patternless, but as the cells matured, the distribution assumed a sarcomeric banding pattern, and α3A appeared to be localized in costameres - sarcolemmal regions adjacent to the Z-disks. α-actinin, a component of the Z-disk, was localized in the same intracellular regions. Temporal analysis of the incorporation of the α3A subunit and other myofibrillar proteins into sarcomeres revealed that α3A was integrated into sarcomeres following incorporation of α-actinin and myosin heavy chain (MHC) but prior to that of desmin. This suggests that α3A integrins are incorporated into a pre-existing myofibrillar structure, and it is unlikely that α3A integrins participate in the initial assembly of myofibrillar proteins. The α3B, β1A and β1D subunits were also localized in costameres, where they...