Mdm2 does not alter hNumb mRNA levels. 293 cells were transfected with the indicated plasmids. Total cellular RNA was prepared 70 h posttransfection, and 10 μg of each sample was subjected to Northern blot analysis. The blots were hybridized sequentially with probes corresponding to hNumb and to human GAPDH. The positions of 28S and 18S rRNA are indicated. Cont., control.

Mdm2 associates with hNumb in vivo. Mdm2 and FLAG-hNumb were overexpressed in 293 cells by transient transfection. (A) Cell extracts were precleared with preimmune serum, followed by immunoprecipitation with anti-Mdm2 polyclonal serum. Immunoprecipitates were subjected to Western blot (WB) analysis with anti-FLAG monoclonal antibodies. (B) Cell extracts were precleared with preimmune serum, followed by immunoprecipitation with anti-FLAG monoclonal antibodies. Immunoprecipitates (IP) were subjected to Western blot analysis with anti-Mdm2 polyclonal serum. Positions of protein molecular size markers are indicated on the left.

In vitro interaction of Mdm2 with hNumb deletion mutants. (A) Schematic representation of the hNumb deletion mutants; all mutants were constructed in pcDNA3FLAG. Indicated are the positions of the PTB domain, the zinc finger-like domain (Zn), and the eight putative SH3-binding motifs (vertical bars). (B) hNumb polypeptides described in panel A were translated in vitro in the presence of [³⁵S]methionine and analyzed for binding to either GST or GST-Mdm2 as described in the legend to Fig. 2. aa, amino acids.

Mdm2 affects the subcellular localization of hNumb. Saos-2 cells were transfected transiently with FLAG-hNumb expression plasmid DNA together with either vector control (A) or Mdm2 expression plasmid (B and C). Cells were fixed with methanol, stained with anti-FLAG monoclonal antibody (A and B), and visualized by indirect immunofluorescence. Arrowheads and long arrows identify cells with predominantly nuclear and predominantly cytoplasmic hNumb staining, respectively. (C) DAPI staining of the field shown in panel B. The photographs were taken at a magnification of ×400.

Protein sequence of hNumb. Sequence of the ORF encoded by the hNumb cDNA clone isolated from the ALL-1 library (see text). hNumb-S denotes the extra 11 residues present in the hNumb clone reported by Salcini et al. (54). The putative PTB domain is underlined. Eight putative SH3-binding motifs, Pro-X-X-Pro (X being any amino acid), are underlined by empty boxes. The most C-terminal box fits the extended consensus SH3-binding motif with Pro residues at positions 2, 7, and 10. A zinc finger-like domain is underlined by dashes.

hNumb binds specifically to Mdm2 in vitro. Full-length hNumb was produced by in vitro translation in a rabbit reticulocyte lysate in the presence of [³⁵S]methionine. Equal amounts of radiolabeled hNumb were incubated with the indicated GST fusion proteins or with GST alone. Bound proteins were eluted in protein sample buffer, resolved by SDS-PAGE, and visualized by exposure to X-ray film after fluorography. Positions and molecular masses of protein molecular size markers are indicated. (A and B) Results are from separate experiments.

Pulse-chase analysis of transfected hNumb in the absence or presence of excess Mdm2. 293 cells were transiently transfected with an hNumb expression plasmid, together with either Mdm2 expression plasmid (+Mdm2) or control vector (−Mdm2). Cells were metabolically labelled by incubation with [³⁵S]methionine for 15 min and then chased in nonradioactive medium for the numbers of hours indicated at the top. Portions of each extract, containing equal amounts of acid-insoluble radioactivity, were immunoprecipitated with anti-hNumb polyclonal serum. The immunoprecipitates were resolved by SDS-7.5% PAGE and visualized by exposure to X-ray film after fluorography. The positions of protein molecular size markers are indicated.

Effect of pervanadate on the in vivo association of Mdm2 and hNumb. 293 cells were transiently transfected with a hNumb expression plasmid, together with either Mdm2 expression plasmid (+) or control vector (−). The cells were pretreated with pervanadate (1 mM Na₃VO₄, 3 mM H₂O₂) for 0, 10, or 20 min prior to harvesting. Cell extracts were precleared with preimmune serum, followed by immunoprecipitation with anti-hNumb polyclonal serum. Immunoprecipitates were subjected to Western blot analysis with anti-Mdm2 polyclonal serum. The positions of protein molecular size markers are indicated on the left.

Mdm2 reduces the steady-state level of hNumb. (A and B) 1% of each cell extract employed for immunoprecipitation in Fig. 3 was subjected directly to Western blot analysis with either anti-FLAG monoclonal antibodies (A) or anti-Mdm2 polyclonal serum (B). hNumb and Mdm2 are indicated by arrows. Positions of protein molecular size markers are shown on the left. ECL exposure time is identical to that used in Fig. 3. (C) Expression plasmids encoding different forms of Mdm2 (see Fig. 5 and text) were transfected into Saos-2 cells, together with a FLAG-hNumb expression plasmid. Total cell extracts were subjected to Western blot (WB) analysis with anti-FLAG monoclonal antibodies. hNumb is indicated by an arrow. The positions of protein molecular size markers are shown on the left. Equal loading was confirmed by reprobing the same blot with an anti-α-tubulin antibody (bottom panel).

In vivo interaction of hNumb with Mdm2 mutants. Expression plasmids encoding different forms of Mdm2 were transfected into 293 cells together with an hNumb expression plasmid. Cell extracts were immunoprecipitated with anti-hNumb polyclonal serum. Immunoprecipitates (A) as well as 1% of each direct cell extract (B) were subjected to Western blot analysis with anti-Mdm2 polyclonal serum. X2 and KC encode full-length Mdm2 protein; D, B, and ΔXM encode Mdm2 polypeptides lacking the N-terminal portion of the protein; and 58A, 58D, 58V, and Δ58 encode Mdm2 proteins with alterations at amino acid position 58 (see text). The positions of protein molecular size markers are indicated on the left. The band present slightly above Mdm2 in panel A, particularly in lanes 1 to 5, corresponds to a nonspecific contaminant observable also when immunoprecipitation is performed with preimmune serum (data not shown). Lanes 1 to 5 and 6 to 10, respectively, are from separate experiments differing in nonspecific background.