A Novel Strategy for Controlling the Metastic Phenotype: Targeting the SNAG Repression Domain in the SNAIL Zinc-Finger Protein
Abstract
Considerable progress has been made toward tasks aimed at reconstitution, mapping and determining the specificity of the SNAIL- SNAG domain/SNAP interaction in vitro and in vivo. We have obtained epitope-tagged full length cDNA clones of the SNAG domain zinc finger protein, SNAIL, and the SNAPs, Ajuba and LIMDI and verified stable protein expression in mammalian cells. We have demonstrated that Ajuba can interact with SNAIL by co-immunoprecipitation and have shown that wild type but not SNAG-domain-deleted SNAIL can augment SNAIL-mediated repression of transiently transfected E-Cadherin promoter Luciferase reporter plasmids. Immunocytochemical analysis has shown that in the absence of cotransfected SNAG domain proteins, both Ajuba and LlMDl exhibit predominantly cytoplasmic subcellular localization: however, Ajuba deleted for nuclear exclusion signals localizes in both the cytoplasm and nucleus. We have generated a panel of HEK293 stable cell lines expressing Ajuba and the LIMDl and examined differences in the biochemical complexes associated with Ajuba and LIMDl in cytoplasmic compared to nuclear fractions. We have generated and characterized extremely useful polyclonal antibodies capable of detecting and discriminating between Ajuba and LIMD1. Our current efforts are aimed at definition of potential dominant negative SNAG/SNAP interaction surfaces towards our goal of reactivating E-Cadherin to control the metastatic phenotype.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 01, 2003
- Accession Number
- ADA417783
Entities
People
- Frank J. Rauscher Iii
Organizations
- University of Pennsylvania