XXXV Reunião Anual da SBBqResumoID:8301


Tissue Distribution and Subcellular Localization of Endogenous FBXO25 Ubiquitin Ligase in Cultured Cells

Adriana O. Manfiolli; Ana Leticia G. Maragno; Munira M. A. Baqui; Sami Yokoo; Eduardo B. Oliveira; and Marcelo D. Gomes



Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP


Ubiquitin (Ub)-dependent proteolysis regulates protein abundance and serves a central regulatory function in many biological processes. The ubiquitination of the target protein is mediated by the ub-ligases (E3), which represent a diverse family of proteins and complexes. The Skp1/Cul1/F-box (SCF) complex is the largest family of E3 (human genome contains about 68 F-box proteins). Our data has been shown that FBXO25 protein is a component of a productive SCF with ub-ligase activity. The aim of this study is to characterize the tissue distribution and subcellular localization of endogenous FBXO25 in cultured cells. For this, we generated an affinity purified antibody against the N-terminal region of the protein, which was able to recognize specifically the endogenous protein in all major tissues of the adult mouse but not in striate muscle. In addition, immunofluorescence studies revealed that the FBXO25 is localized primarily to the nucleus of cultured cells (HeLa, B16-F10, and Neuro-2A) and excluded from the nucleoli. Striking, FBXO25 is found in prominent dot-like structures, which are generally adjacent to Cajal bodies and there is no overlapping with splicing speckles. The functional significance of this distribution is presently being studied. Finally, after Actinomycin D treatment, a transcription inhibitor, we observed in HeLa cells a dramatic reorganization of FBX025 from a pattern of large dots to a diffuse nuclear staining at the nucleus. This response contrasts with the behavior of splicing speckle proteins, which concentrate in enlarged speckles in the nucleoplasm. These data collectively suggest a possible role of FBXO25 on the transcriptional apparatus.
Supported by FAPESP and FAEPA.