We have examined the function of a member of the vasodilator-stimulated phosphoprotein family of proteins (DdVASP) in Dictyostelium. Ddvasp null cells lack filopodia, whereas targeting DdVASP to the plasma membrane with a myristoyl tag results in a significant increase in filopodia. The proline-rich domain-Ena/ VASP homology 2 structure is required for both actin polymerization activity and filopodia formation. Ddvasp null cells exhibit a chemotaxis defect, which appears to be due to a defect in the ability of the cells to properly adhere to the substratum and to suppress lateral pseudopod extension. We demonstrate that during chemotaxis, the anterior ϳ50% of the cell lifts from the substratum and remains elevated for up to 1 min. These defects lead to a significant decrease in chemotaxis efficiency. DdVASP localizes to the leading edge in migrating cells and to the tips of filopodia. In addition, Ddvasp null cells have a defect in particle adhesion but internalize particles normally. Our results provide new insights into the function of DdVASP in controlling the actin cytoskeleton during chemotaxis and filopodia formation.
Vasodilator-stimulated phosphoprotein (VASP)1 was originally identified as a protein phosphorylated in response to an elevation of intracellular cAMP or cGMP levels by cAMP-and cGMP-dependent protein kinase in human platelets (1). VASP is a member of the Ena/VASP family of proteins that share a domain structure composed of three conserved domains: an N-terminal Ena/VASP homology 1 (EVH1) domain; a central proline-rich, Src homology 3-binding domain (PRD); and a Cterminal EVH2 domain. The EVH1 domain, which is also present in Wiskott-Aldrich syndrome protein (WASP) and N-WASP, binds to proteins containing the consensus sequence FPPPP, such as Listeria monocytogenes surface protein ActA, and interacts with the PRD of WASP (2-4). The PRD mediates VASP's interaction with the Src homology 3 domain on Src and F-actin-binding protein profilin (5-8). The EVH2 domain, which is unique to the Ena/VASP family, mediates VASP's tetramerization, F-actin binding, and actin bundling (9).Involvement of VASP in the regulation of actin assembly and cell motility was suggested by the results of studies on actinbased movement of Listeria. Listeria movement was abolished in VASP-depleted platelet extracts and could be restored by the addition of recombinant VASP (10). VASP and EVL (Ena/ VASP-like protein) induce polymerization of G-actin into Factin bundles in in vitro assays in the presence of low salt and stabilize F-actin in a phosphorylation-dependent manner (10). As expected from the ability of VASP to induce actin polymerization, in vitro reconstitution of Listeria movement using purified proteins demonstrated that the speed of bacterial movement is greatly enhanced if VASP is added to the reconstitution mixture (11). These findings suggested that VASP enhances actin polymerization at a site on the Listeria surface to generate propelling power. The mechanism of membrane protrusion through localized actin poly...
Epithelial-mesenchymal transition (EMT) is essential for increased invasion and metastasis during cancer progression. Among the candidate EMT-regulating microRNAs that we previously identified, miR-181b-3p was found to induce EMT in MCF7 breast cancer cells, as indicated by an EMT-characteristic morphological change, increased invasiveness, and altered expression of an EMT marker. Transfection with a miR-181b-3p inhibitor reduced the expression of mesenchymal markers and the migration and invasion of highly invasive breast cancer cells. miR-181b-3p induced the upregulation of Snail, a master EMT inducer and transcriptional repressor of E-cadherin, through protein stabilization. YWHAG was identified as a direct target of miR-181b-3p, downregulation of which induced Snail stabilization and EMT phenotypes. Ectopic expression of YWHAG abrogated the effect of miR-181b-3p, including Snail stabilization and the promotion of invasion. In situ hybridization and immunohistochemical analyses indicated that YWHAG expression was inversely correlated with the expression of miR-181b-3p and Snail in human breast cancer tissues. Furthermore, transfection with miR-181b-3p increased the frequency of metastatic nodule formation in the lungs of mice in experimental metastasis assays using MDA-MB-231 cells. Taken together, our data suggest that miR-181b-3p functions as a metastasis activator by promoting Snail-induced EMT, and may therefore be a therapeutic target in metastatic cancers.
Apoptotic and antiproliferative activities of small heterodimer partner (SHP) nuclear receptor ligand (E)-4-[3′-(1-adamantyl)-4′-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC), which was derived from 6-[3′-(1-adamantyl)-4′-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), and several carboxyl isosteric or hydrogen bond-accepting analogues were examined. 3-Cl-AHPC continued to be the most effective apoptotic agent, whereas tetrazole, thiazolidine-2,4-dione, methyldinitrile, hydroxamic acid, boronic acid, 2-oxoaldehyde, and ethyl phosphonic acid hydrogen bond-acceptor analogues were inactive or less efficient inducers of KG-1 acute myeloid leukemia and MDA-MB-231 breast, H292 lung, and DU-145 prostate cancer cell apoptosis. Similarly, 3-Cl-AHPC was the most potent inhibitor of cell proliferation. 4-[3′-(1-Adamantyl)-4′-hydroxyphenyl]-3chlorophenyltetrazole, (2E)-5-{2-[3′-(1-adamantyl)-2-chloro-4′-hydroxy-4-biphenyl]-ethenyl}-1H
MicroRNAs (miRNAs) play pivotal roles in tumorigenesis as either tumor suppressors or oncogenes. In the present study, we discovered and demonstrated the tumor suppressive function of a novel miRNA miR-5582-5p. miR-5582-5p induced apoptosis and cell cycle arrest in cancer cells, but not in normal cells. GAB1, SHC1, and CDK2 were identified as direct targets of miR-5582-5p. Knockdown of GAB1/SHC1 or CDK2 phenocopied the apoptotic or cell cycle arrest-inducing function of miR-5582-5p, respectively. The expression of miR-5582-5p was lower in tumor tissues than in adjacent normal tissues of colorectal cancer patients, while the expression of the target proteins exhibited patterns opposite to that of miR-5582-5p. Intratumoral injection of a miR-5582-5p mimic or induced expression of miR-5582-5p in tumor cells suppressed tumor growth in HCT116 xenografts. Collectively, our results suggest a novel tumor suppressive function for miR-5582-5p and its potential applicability for tumor control.
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