Tvl-1, a 269 amino acid ankyrin repeat protein, was identified by screening a murine T-cell two hybrid cDNA library for proteins that associate with Raf-1. The gene encoding Tvl-1 maps to human chromosome 19p12, 0.7 kb upstream of MEF2B and is expressed in all tested tissues with highest levels in hematopoietic and lymphoid organs and testes. The Tvl-1 protein is localized in both the nucleus and the cytoplasm. The Raf-1/Tvl-1 association is responsible for a two-way interaction, with Raf-1 phosphorylating Tvl-1 and Tvl-1 enhancing the EGF or phosphorylation-induced activation of Raf-1. Screening an interaction mating library of approximately 500 proteins for Tvl-1 interactors, revealed that Tvl-1 also binds Mcl-1, Bcl-XL and Bad, as well as cyclins D1, D2 and D3. Moreover, Tvl-1 binds to and is phosphorylated by two additional protein kinases, Tpl-2 and Akt. Tvl-1 as well as Tpl-2 also binds Apaf-1, and the Apaf-1-associated procaspase-9, but they do not bind procaspase-3. Recently, the applicant showed that Tvl-1 and Tpl-2, when co-expressed, reciprocally enhance the interaction of each other with procaspase-9. More interesting, in the presence of wild type, but not kinase-dead Tpl-2, Tvl-1 binds procaspase-3 and juxtaposes it to caspase-9. The Tpl-2-mediated juxtaposition of procaspase-3 with caspase-9 is inhibited by Akt. These data suggest that Tvl-1, in combination with Tpl-2, promotes the assembly/stability of the Apaf-1 apoptosome while Akt either interferes with its assembly or promotes its dissociation. Tvl-1 and procaspase-9 associated with the complex assembled in the presence of the combination of Tvl-1 with Tpl-2 are both phosphorylated. As predicted from the preceding interactions, the caspase-9-dependent caspase-3 activation and apoptosis induced by Tpl-2 in immortalized non-transformed cell lines, was enhanced by Tvl-1. Ectopic expression of Tvl-1 also enhanced the TNFalpha-induced activation of caspase-3 and apoptosis in NIH3T3 cells. Independent studies by others revealed that Tvl-1 (RFXANK) contributes to the assembly/stability of a multimolecular transcriptional complex on the X site on class II MHC promoters. Mutations of the Tvl-1 gene silence these promoters, and they are responsible for complementation group B of the bare lymphocyte syndrome. The combination of the preceding data suggests that Tvl-1 is an adaptor protein that contributes to the assembly/stability of both cytoplasmic and nuclear multi-molecular complexes. This application addresses the role of Tvl-1 in the assembly and function of such complexes.
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