Reiko Honda

Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53

The tumor suppressor p53 is degraded by the ubiquitin‐proteasome system. p53 was polyubiquitinated in the presence of E1, UbcH5 as E2 and MDM2 oncoprotein. A ubiquitin molecule bound MDM2 through sulfhydroxy bond which is characteristic of ubiquitin ligase (E3)‐ubiquitin binding. The cysteine residue in the carboxyl terminus of MDM2 was essential for the activity. These data suggest that the MDM2 protein, which is induced by p53, functions as a ubiquitin ligase, E3, in human papillomavirus‐uninfected cells which do not have E6 protein.

Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53.

We have demonstrated previously that the oncoprotein Mdm2 has a ubiquitin ligase activity for the tumor suppressor p53 protein. In the present study, we characterize this ubiquitin ligase activity of Mdm2. We first demonstrate the ubiquitination of several p53 point mutants and deletion mutants by Mdm2. The point mutants, which cannot bind to Mdm2, are not ubiquitinated by Mdm2. The ubiquitination of the C‐terminal deletion mutants, which contain so‐called Mdm2‐binding sites, is markedly decreased, compared with that of wild‐type p53. The binding of Mdm2 to p53 is essential for ubiquitination, but p53′s tertiary structure and/or C‐terminal region may also be important for this reaction. DNA‐dependent protein kinase is known to phosphorylate p53 on Mdm2‐binding sites, where DNA damage induces phosphorylation, and p53 phosphorylated by this kinase is not a good substrate for Mdm2. This suggests that DNA damage‐induced phosphorylation stabilizes p53 by inhibiting its ubiquitination by Mdm2. We further investigated whether the tumor suppressor p19ARF affects the ubiquitin ligase activity of Mdm2 for p53. The activity of p19ARF‐bound Mdm2 was found to be lower than that of free Mdm2, suggesting that p19ARF promotes the stabilization of p53 by inactivating Mdm2.

Sumoylation of Mdm2 by Protein Inhibitor of Activated STAT (PIAS) and RanBP2 Enzymes

Mdm2, a ubiquitin ligase that acts on p53, is regulated by sumoylation. In the current study, we identify the enzymes responsible for the sumoylation of Mdm2. When mammalian cells are co-transfected with cDNAs encoding Mdm2 and PIAS1 or PIASxβ (protein inhibitor of activatedSTAT) as sumoylation enzymes, Mdm2 is highly sumoylated. Mdm2 is also sumoylated in an in vitro system containing PIASxβ, PIAS1, and RanBP2. When several lysine residues of Mdm2 were sequentially mutated to arginine, the K182R mutant was not sumoylated in intact cells; however, in the in vitro system this mutant was sumoylated by PIAS1, PIASxβ, and RanBP2 as efficiently as the wild-type Mdm2 protein. Lysine residues 182 and 185 map within the nuclear localization signal of Mdm2. A K185R mutant of Mdm2 is sumoylated in intact cells, whereas a K182R protein is not. Only a Mdm2 protein bearing the K182R mutation is localized exclusively in the cytoplasm. Because RanBP2 is a nuclear pore protein and PIAS proteins are localized within the nucleus, our data suggest that Mdm2 is sumoylated during nuclear translocation by RanBP2 and then further sumoylated once in the nucleus by PIASxβ and PIAS1.

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.

Mouse p87 wee1 kinase is regulated by M-phase specific phosphorylation

We have cloned a mousewee1 kinase cDNA (mwee1). The clone is 2258 bp in length and its open reading frame corresponds to 646 amino acid residues. The molecular weight of this kinase is 87 kDa in SDS-PAGE, which is about 1.7-fold larger than the human p50wee1 kinase reported previously. In a cell cycle, the mousewee1 kinase is phosphorylated at M-phase, and anin vitro study using a mitotic extract revealed that phosphorylation occurs in the N-terminal domain, which is absent from the humanwee1 kinase, resulting in inactivation of the kinase activity. The N-terminal domain or entire molecule is extensively phosphorylated bycdc2-cyclin B kinase. Furthermore, the activity of thewee1 kinase was reduced by phosphorylation with the mitotic extract which containedcdc2-cyclin B kinase