A RING finger protein Praja1 regulates Dlx5-dependent transcription through its ubiquitin ligase activity for the Dlx/Msx-interacting MAGE/Necdin family protein, Dlxin-1.
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, Praja1 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 Praja1 revealed that Praja1 binds to the C-terminal necdin homology domain of Dlxin-1 in vitro and in vivo, respectively. Overexpression of Praja1 caused a decrease in Dlxin-1 protein level, which was reversed when a proteasome inhibitor was added. Overexpression of Praja1 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 Praja1, but not its RING finger mutant, promoted ubiquitination of Dlxin-1 in vivo. Finally, expression of Praja1 down-regulated Dlx5-dependent transcriptional activity in a GAL4-dependent assay. These results suggest that Praja1 regulates the transcription function of the homeodomain protein Dlx5 by controlling the stability of Dlxin-1 via an ubiquitin-dependent degradation pathway.
Sasaki
Aya
A
Department of Geriatric Research, National Institute for Longevity Sciences, Obu, Aichi 474-8522, Japan.
Masuda
Yoshiko
Y
Iwai
Kazuhiro
K
Ikeda
Kyoji
K
Watanabe
Ken
K
D000951:Antigens, Neoplasm; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D009154:Mutation; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D000951:Antigens, Neoplasm; D002352:Carrier Proteins; C100596:DLX5 protein, human; D018076:DNA, Complementary; C088334:Distal-less homeobox proteins; C501120:Dlx5 protein, mouse; D018398:Homeodomain Proteins; C120897:MAGED1 protein, human; C486541:Maged1 protein, mouse; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D025801:Ubiquitin; C069766:necdin; C484661:PJA1 protein, human; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D008025:Ligases