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.
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