The human thyroid hormone receptor-associated protein (TRAP) complex, an earlier described coactivator for nuclear receptors, and an SRB- and MED-containing cofactor complex (SMCC) that mediates activation by Gal4-p53 are shown to be virtually the same with respect to specific polypeptide subunits, coactivator functions, and mechanisms of action (activator interactions). In parallel with ligand-dependent interactions of nuclear receptors with the TRAP220 subunit, p53 and VP16 activation domains interact directly with a newly cloned TRAP80 subunit. These results indicate novel pathways for the function of nuclear receptors and other activators (p53 and VP16) through a common coactivator complex that is likely to target RNA polymerase II. Identification of the TRAP230 subunit as a previously predicted gene product also suggests a coactivator-related transcription defect in certain disease states.
The AML1-ETO fusion protein, generated by the t(8;21) chromosomal translocation, is causally involved in nearly 15% of acute myeloid leukemia (AML) cases. This study shows that AML1-ETO, as well as ETO, inhibits transcriptional activation by E proteins through stable interactions that preclude recruitment of p300/CREB-binding protein (CBP) coactivators. These interactions are mediated by a conserved ETO TAF4 homology domain and a 17-amino acid p300/CBP and ETO target motif within AD1 activation domains of E proteins. In t(8;21) leukemic cells, very stable interactions between AML1-ETO and E proteins underlie a t(8;21) translocation-specific silencing of E protein function through an aberrant cofactor exchange mechanism. These studies identify E proteins as AML1-ETO targets whose dysregulation may be important for t(8;21) leukemogenesis, as well as an E protein silencing mechanism that is distinct from that associated with differentiation-inhibitory proteins.
Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide-or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e. buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding.
Purpose: Advanced metastatic prostate cancer (PCa) is a fatal disease, with only palliative therapeutic options. Though almost 80% of cases of metastatic PCa present bone metastasis, our current understanding of the molecular mechanisms that govern this metastatic dissemination remains fragmentary. The main objective of the present study was to identify microRNA (miRNA) genes that regulate metastatic PCa.Experimental Design: miRNA expression profiling was done in human prostate cell lines to identify dysregulated miRNA components of advanced PCa. miR-203 expression was assessed in prostate carcinoma cell lines and clinical specimens by real-time PCR and in situ hybridization. To assess the biological significance of miR-203, miR-203 was reexpressed in bone metastatic PCa cell lines followed by in vitro and in vivo functional assays.Results: miR-203 expression is specifically attenuated in bone metastatic PCa suggesting a fundamental antimetastatic role for this miRNA. Reintroduction of miR-203 in bone metastatic PCa cell lines suppresses metastasis via inhibition of several critical steps of the metastatic cascade including epithelial-mesenchymal transition, invasion, and motility. Ectopic miR-203 significantly attenuated the development of metastasis in a bone metastatic model of PCa. Importantly, miR-203 regulates a cohort of pro-metastatic genes including ZEB2, Bmi, survivin, and bone-specific effectors including Runx2, a master regulator of bone metastasis.Conclusions: miR-203 is an "antimetastatic" miRNA in PCa that acts at multiple steps of the PCa metastatic cascade via repression of a cohort of prometastatic targets. miR-203 may be an attractive target for therapeutic intervention in advanced PCa.
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