The alternative translated MDMX^p60 isoform regulates MDM2 activity

Figures & data

Figure 1. The hMDMX^p60 isoform is initiated at the 7th in frame AUG codon. (A) Cartoon illustrating the hmdmx mRNA constructs and the mutated AUG sites (left). The 6 in frame AUG codons after the +1 AUG of the hmdmx coding sequence were substituted with alanine (GCG) (hmdmxΔ2–7AUG) to express only full length hMDMX (hMDMX^FL). The hmdmxΔ1–6AUG mRNA lacks the first 6 in frame AUG codons and only expresses hMDMX^p60 which is initiated from the 7th in frame AUG (+384). The hMDMX^p60 lacks the first 127 amino acids, including the p53 binding domain (right). (B) Western blot showing the expression of the two hMDMX isoforms from indicated constructs in H1299 cells. The hmdmx^wt mRNA expresses both isoforms. Actin is used as loading control. (C) The relative expression of the two hMDMX isoforms in different cell lines. H1299 and Saos-2 are p53 negative cell lines whereas HeLa expresses wild type p53 and MDA-MB-231 expresses a mutant p53(R280K). Western blots show one representative experiment out of 3.

Figure 2. (See previous page) hMDMX^p60 and hMDMX^FL are derived from cap-independent mRNA translation initiation. (A) Cartoon illustrating the bicistronic hmdmx mRNA inserted downstream of the GFP open reading frame and downstream of a hairpin structure preventing ribosomal read through from cap-dependent translation initiation of the GFP open reading frame. The location of primers (P1 to P5) is indicated. These were used for RT-PCR and RT-qPCR to ensure that the bicistronic mRNA remains intact in cells and that no cryptic promoter activity causes alternative RNA species. (B) The expression of the two hMDMX isoforms from the wild type mRNA and from the bicistronic construct in H1299 cells. The relative level of expression of the two hMDMX isoforms from wild type vs. bicistronic constructs is estimated (upper graph). (C) RT-PCR using indicated primer pairs repeated 4 times each for the bicistroninc hmdmx mRNA. (D) The relative amount of RT-PCR products from the bicistronic and the wild type hmdmx mRNAs as estimated using quantitative RT-PCR from indicated primers. The ratio of RT-PCR products derived from the primers covering the 5’UTR and the initiation site for hmdmx^FL (P2 and P3) and from a sequence covering the initiation site of hMDMX^p60 (P4 and P5) are similar between the wild type and the bicistronic hmdmx mRNAs. This shows that no cryptic promoter or alternative splicing was created by fusing the hmdmx mRNA to the bicistronic construct. No added RT enzyme (-RT) shows that the quantified PCR products are derived from mRNA. Actin mRNA serves as control. The graph shows data from 3 independent experiments plus SD. (E) The use of siRNA against the GFP results in a similar reduction in expression of GFP and hMDMX from the bicistronic hmdmx mRNA. There is no effect on hMDMX expression form the wild type mRNA (Fig. S1). (F) Western blot showing the effect of indicated UV doses 4 hours after treatment on cap-independent translation of the hmdmx mRNA as compared to cap-dependent translation of gfp (Fig. S2). Western blots show one representative experiment out of 3.

Figure 3. hMDMX^p60 shows higher affinity toward hMDM2 as compared to hMDMX^FL. (A) Immunoprecipitation (IP) against hMDM2 shows that hMDM2 interacts with both hMDMX isoforms (upper panel). The protein levels in whole lysates are shown below. (NS stands for non-specific). (B) Co-immunoprecipitation of hMDMX isoforms (left) or hMDM2 (right) using an HA-tagged hMDMX^p60. Hsp90 and actin serves as loading controls of total lysates. (C) ELISA using a fixed (500 ng) amount of recombinant purified hMDMX^p60 or hMDMX^FL and increasing amounts of hMDM2. The affinity of hMDMX^p60 to hMDM2 is higher as compared to hMDMX^FL. Both proteins bind hMDM2 in a biphasic fashion. The data shows the average of 7 independent experiments and SD. Western blots (A and B) show one representative experiment out of 3.

Figure 4. hMDMX^p60 stabilizes hMDMX^FL in the presence of hMDM2. (A) The levels of expression of hMDMX^FL in H1299 cells following increasing amounts of hMDM2 (left). A similar experiment but using a fixed amount of hMDMX^p60 and increasing levels of hMDM2 (right). Compare differences in the expression of hMDMX isoform in cells transfected with 50 ng hmdm2 cDNA. (B) hMDMX^FL is protected from hMDM2-mediated degradation in the presence of hMDMX^p60. (C) Increasing levels of hMDMX^p60 stabilizes hMDMXFL in cells expressing endogenous hMDM2. (D) Mutation of each, or all 6 together, of the lysine residues in the N-terminus of hMDMX upstream of the initiation site for hMDMX^p60 do not affect hMDM2-mediated degradation of hMDMX. The data are representative from 3 independent experiments (Fig. S3A and B).

Figure 5. Increasing levels of hMDMX^p60 induces oscillation in hMDM2 expression levels in H1299 cells. (A) Expression of a fixed amount of hmdm2 and increasing levels of either (a) hMDMX^p60 alone or (b) together with a fixed amount of hmdmx^fl, or (c) increasing levels of both isoforms. The hmdm2(C464A) mutant (d) is E3 ligase dead and is not affected by hMDMX^p60 (Fig. S4A and B). (B) In vitro autoubiquitination of recombinant hMDM2 in the presence of increasing levels of hMDMX^p60. Data shows one representative experiment out of 3 (Fig. S5A and B).