A RING finger protein prajal regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/necdin family protein, Dlxin-1

Aya Sasaki, Yoshiko Masuda, Kazuhiro Iwai, Kyoji Ikeda, Ken Watanabe

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Msx2 and Dlx5 are homeodomain proteins that play an important role in osteoblast differentiation and whose expression is induced by bone morphogenetic proteins. Recently we have identified a novel protein, Dlxin-1, that associates with these homeodomain proteins and regulates Dlx5-dependent transcriptional function (Masuda, Y., Sasaki, A., Shibuya, H., Ueno, N., Ikeda, K., and Watanabe, K. (2001) J. Biol. Chem. 276, 5331-5338). In an attempt to elucidate the molecular function of Dlxin-1, two closely related RING finger proteins, Prajal and Neurodap-1, were isolated by yeast two-hybrid screening using the C-terminal necdin homology domain of Dlxin-1 as bait. Glutathione S-transferase pull-down and immunoprecipitation/Western blotting assays following co-transfection of Dlxin-1 and Prajal revealed that Prajal binds to the C-terminal necdin homology domain of Dlxin-1 in vitro and in vivo, respectively. Overexpression of Praja1 caused a decrease in Dlxln-1 protein level, which was reversed when a proteasome inhibitor was added. Overexpression of Prajal with a mutation in the RING finger inhibited the decrease in Dlxin-1 protein, pointing to the importance of ubiquitin-protein isopeptide ligase (E3) activity associated with RING finger. Wild-type Prajal, but not its RING finger mutant, promoted ubiquitination of Dlxin-1 in vivo. Finally, expression of Prajal down-regulated Dlx5-dependent transcriptional activity in a GAL4-dependent assay. These results suggest that Prajal regulates the transcription function of the homeodomain protein Dlx5 by controlling the stability of Dlxin-1 via an ubiquitin-dependent degradation pathway.

Original languageEnglish
Pages (from-to)22541-22546
Number of pages6
JournalJournal of Biological Chemistry
Issue number25
Publication statusPublished - 2002 Jun 21


ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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