We have examined the structure and expression of the products associated with the t(2;13)(q35;q14) translocation associated with alveolar rhabdomyosarcoma. The chromosome 13 gene (FKHR) is identified as a member of the fork head domain family of transcription factors characterized by a conserved DNA binding motif. Polymerase chain reaction analysis demonstrates that a 5'PAX3-3' FKHR chimaeric transcript is expressed in all eight alveolar rhabdomyosarcomas investigated. Immunoprecipitation experiments detect the predicted fusion protein. These findings indicate that the t(2;13) generates a potentially tumorigenic fusion transcription factor consisting of intact PAX3 DNA binding domains, a truncated fork head DNA binding domain and C-terminal FKHR regions.

Tumor-specific translocations that result in altered expression of cellular genes occur frequently enough to be considered a general mechanism of oncogenesis (reviewed in references 18, 28, and 53). Chromosomal translocations may lead to oncogenesis via insertional activation in which one gene is placed under the control of an active expression element of another gene, typically an immunoglobulin or T-cell receptor locus, and thus inappropriately expressed. Translocation-mediated oncogenesis may also involve the recombination of portions of two genes to produce a chimeric gene encoding a fusion protein with aberrant functional properties. Several examples of tumor-specific translocations which generate chimeric transcription factors with altered regulatory function have been identified, and their oncogenic potentials have been demonstrated. It appears that functional modules from each factor collaborate in the fusion, e.g., a DNA-binding domain is fused to a transcriptional regulation or protein association domain, and the gain of the function results in neoplastic transformation.Many of the examples of translocation-generated fusion proteins have been found in the hematopoietic system (reviewed in references 28 and 66). A prototype is the BCR-ABL fusion associated with the t(9;22) Philadelphia chromosome of chronic myelocytic leukemia (7,22). In acute lymphoblastic leukemia, there are two different translocations of chromosome 19 involving the E2A gene. A t(1;19) translocation fuses the E2A activation domain to the DNA-binding domain of the homeobox gene PBX-1, thus creating an activator of PBX-1-regulated target genes (50,62,84). The product of the fusion of E2A and HLF genes, associated with the t(17;19) translocation, exhibits altered DNA-binding properties attributed to a restricted protein interaction potential (46, 47). In acute promyelocytic leukemia, the t(15;17) translocation fuses the retinoic acid receptor to the putative transcription factor PML and results in a functionally altered receptor (23,36,49).Only a few examples of translocation-generated fusion proteins in human solid tumors have been characterized. The Ewing's sarcoma EWS-FLI1 fusion protein associated with the t(11;22) translocation has greater transactivating and transforming capacity than its wild-type counterpart FLI1 (1, 57). In myxoid liposarcomas, the t(12;16) translocation fuses the NH 2 terminus of the TLS (FUS) protein with the COOH-terminal DNA-binding and leucine zipper dimerization regions of the CHOP protein (20,67). In clear-cell sarcoma, the t(12;22) translocation joins the NH 2 -terminal EWS transactivation domain to the ATF1 DNA-binding and protein interaction domain (88). For all of the above examples, it is significant that the fusion gene and/or protein(s) potentially represents unique tumor-specific targets for therapy. A common goal is to determine how these chimeric transcription factors alter normal gene expression.Alveolar rhabdomyosarcomas are pediatric malignant tumors that characteristically exhibit a t(2;13)...

Na ؉ /taurocholate (Na ؉ /TC) cotransport in hepatocytes is mediated primarily by Na ؉ /TC cotransporting polypeptide (Ntcp), and cyclic adenosine monophosphate (cAMP) stimulates Na ؉ /TC cotransport by inducing translocation of Ntcp to the plasma membrane. The aim of the present study was to determine if Ntcp is a phosphoprotein and if cAMP alters Ntcp phosphorylation. Freshly prepared hepatocytes from rat livers were incubated with carrier-free 32 PO 4 for 2 hours, followed by incubation with 10 mol/L 8-chlorophenylthio adenosin 3Ј:5Ј-cyclic monophosphate (CPT-cAMP) for 15 minutes. Subcellular fractions isolated from 32 P-labeled hepatocytes were subjected to immunoprecipitation using Ntcp antibody, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography to determine if Ntcp is phosphorylated. Ntcp immunoprecipitated from plasma membranes isolated from nonlabeled hepatocytes was subjected to immunoblot analysis using anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibody to determine whether Ntcp is a serine, threonine, or tyrosine phosphoprotein. Hepatocytes were loaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,NЈ,NЈ-tetraacetic acid (MAPTA), a Ca 2؉ buffering agent, and the effect of CPT-cAMP on TC uptake, cytosolic [Ca 2؉ ], and ntcp phosphorylation and translocation was determined. In addition, the effect of cAMP on protein phosphatases 1 and 2A (PP1/2A) was determined in homogenates and plasma membranes obtained from CPTcAMP-treated hepatocytes. Phosphorylation study showed that phosphorylated Ntcp is detectable in plasma membranes, and cAMP treatment resulted in dephosphorylation of Ntcp. Immunoblot analysis with phosphoamino antibodies revealed that Ntcp is a serine/threonine, and not a tyrosine, phosphoprotein, and cAMP inhibited both serine and threonine phosphorylation. In MAPTA-loaded hepatocytes, CPT-cAMP failed to stimulate TC uptake, failed to increase cytosolic [Ca 2؉ ], and failed to induce translocation and dephosphorylation of Ntcp. cAMP did not alter the activity of PP1/2A in either homogenates or in plasma membranes. Taken together, these results suggest that Ntcp is a serine/threonine phosphoprotein and is dephosphorylated by cAMP treatment. Activation of PP1/2A is not involved in cAMP-mediated dephosphorylation of Ntcp. Both translocation and dephosphorylation of Ntcp may be involved in the regulation of hepatic Na ؉ /TC cotransport.

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