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. 2018 Feb 5;217(2):745–762. doi: 10.1083/jcb.201709092

Figure 8.

Figure 8.

Role of Numb isoforms in the regulation of Mdm2 activity. (A) Numb inhibits Mdm2-dependent ubiquitination of p53 in the cytoplasm. Mdm2 and p53 shuttle between the nucleus and the cytoplasm (Roth et al., 1998; Tao and Levine, 1999), whereas the bulk of Numb is cytosolic (Santolini et al., 2000). However, p53 ubiquitination/degradation occurs both at the cytoplasmic and at the nuclear level (Joseph et al., 2003). We therefore overexpressed in H1299 cells two p53 variants with subcellular localizations restricted to the cytoplasm (p53-ΔNLS) or to the nucleus (p53-ΔNES). The p53-ΔNLS construct carries a nonfunctional p53 nuclear localization signal (with mutations of Lys305, Arg306, Lys319, Lys320, and Lys321 to Ala), and the p53-ΔNES construct carries a nonfunctional p53 nuclear export signal (with mutations of Met340, Leu344, Leu348, and Leu350 to Ala). Cells were also cotransfected with Mdm2 and Numb-1 to test the ability of the latter to inhibit in vivo the Mdm2-mediated ubiquitination of p53-WT or of its mutants. The ubiquitination pattern of the different p53 variants revealed that Numb-1 affects p53 ubiquitination only in the cytoplasm, whereas p53 ubiquitination at the nuclear level was unperturbed. Left: Mdm2-mediated p53 in vivo ubiquitination in H1299 cells transfected with the indicated plasmids and increasing concentrations of FLAG-tagged Numb isoform-1 (see also the Biochemical studies section of Materials and methods). GFP was transfected as an internal normalizer for efficiency of transfection. Lysates were subjected to IB as indicated. Right: the cellular localization of the p53 mutants was monitored by IF with anti-p53. Nuclei were counterstained with DAPI. Bar, 10 µM. (B) Mdm2-mediated p53 in vivo ubiquitination in H1299 cells transfected as indicated on the top (see also the Biochemical studies section of Materials and methods). GFP was transfected as an internal normalizer for efficiency of transfection. Lysates were immunoblotted as indicated. (C) p53 in vitro ubiquitination assay with the components and the Numb species indicated on top fused to a thioredoxin scaffold (Trx; see also the Biochemical studies section of Materials and methods). RG7112 is a small molecule belonging to the Nutlin family of Mdm2 inhibitors (designed to occupy the p53-binding pocket of Mdm2) used as control. Molecular masses are given in kilodaltons.