Alpha-fetoprotein (AFP) is one of the major serum proteins in the early life of mammals. We have previously identified a novel cis-acting element designated as DAS at the 5'-flanking region of the AFP gene and demonstrated that the DAS sequence can be specifically recognized by nuclear protein DAP-II in AFP-producing hepatoma cells and retinoic acid (RA)-induced AFP-producing F9 cells. In this study, we used DNA affinity chromatography to purify the DAP-II proteins from the nuclear extracts (NE) of RA-treated F9 cells. The purified DAP-II complex mainly contained five proteins, with molecular weights of 45, 42, 32, 30, and 20 kDa, respectively. The identification of these proteins was determined by MALDI-TOF mass spectrometric analysis and a database search. These proteins were found to belong to the AUF1 RNA-binding protein family. Protein (30 kDa), one of five proteins in an isolated DAP-II complex, was matched with amino acid sequence highly similar to muAUF1-3. The expression of this protein is inducible by RA, and the pattern of the protein expression is the same as DAP-II proteins in F9 cells after treatment with RA during differentiation. Our results suggest that the 30-kDa protein is a novel isoform of AUF1 family and is the main component of the DAP-II complex that binds to the DAS sequence.
(c) 2006 Wiley-Liss, Inc.