Mammalian Numb is a target protein of Mdm2, ubiquitin ligase.
Drosophila Numb protein functions as an antagonist against Notch signal. The expression of this protein is asymmetrical in divided cells and thought to be involved in the neural cell differentiation and/or cell fate. Human homologue of Numb (hNumb) was cloned as Mdm2-binding protein by yeast two-hybrid screening. Since Mdm2 is an oncoprotein and has ubiquitin ligase activity toward tumor suppressor p53, we assessed to find out whether Mdm2 ubiquitinylates the hNumb protein. The recombinant hNumb expressed in Sf-9 cells using baculovirus protein expression system bound to Mdm2 in vitro. When hNumb was subjected to in vitro ubiquitinylation assay system, which contains E1, E2, or UbcH5c, and Mdm2, hNumb was ubiquitinylated as efficiently as the p53 protein. However, when the Ring-finger domain mutant of Mdm2 was used in place of wild-type Mdm2, hNumb was not ubiquitinylated. Furthermore, when U2OS cells were co-transfected with hNumb and Mdm2, the hNumb protein was ubiquitinylated and degraded. These data strongly suggest that Mdm2 functions as the ubiquitin ligase toward hNumb and that it induces its degradation in intact cells.
Yogosawa
Satomi
S
Division of Molecular Life Science, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Miyauchi
Yasuhiro
Y
Honda
Reiko
R
Tanaka
Hirofumi
H
Yasuda
Hideyo
H
D000818:Animals; D017930:Genes, Reporter; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C113026:NUMB protein, human; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2