Dominant-Negative Effect of NPM-RAR on NPM Mediated Regulation of p53.

We have been studying the variant Acute Promyelocytic Leukemia (APL) translocations with a view towards identifying common molecular pathways underlying APL. The t(5;17) variant of APL fuses the N-terminal 117 amino acids of nucleophosmin (NPM) with the C-terminal 402 amino acids of the retinoic acid receptor alpha (RAR). NPM plays a role in several important pathways within cells beyond its originally described function in the transport of ribonucleoproteins. It serves a chaperone function in transport of proteins such as p53, Tat, Rb, YY1, IRF-1, and Arf. NPM binds to and becomes a target of CDK2/cyclin E to play a role in regulation of centrosomal duplication. The importance of NPM in myeloid leukemias has been highlighted by the recent finding that mutation of the NPM C-terminus is the most common genetic event in non-APL acute myeloid leukemia. We have previously shown that the RAR portion of NPM-RAR acts as a dominant-negative towards wild-type RAR. We now investigate the hypothesis that NPM-RAR serves as a dominant negative for NPM and partially contributes to the leukemic phenotype through disruption of NPM-dependent cellular functions. We have focused our studies on the effects of NPM-RAR on p53. It has previously been shown that NPM directly binds to p53 to increase p53 stability and p53-dependent transcription. The p53-interaction domain maps to a region of NPM that is not contained in NPM-RAR. We hypothesized that NPM-RAR might bind directly with NPM, disrupt NPM association with p53, and thereby indirectly affect p53 activity. We first used pull-down assays of Maltose Binding Protein (MBP)-NPM or MBP-NPM-RAR fusions incubated with 35S-NPM to determine that the N-terminal domain of NPM-RAR is sufficient to interact with NPM. To investigate whether NPM-RAR alters NPM interaction with p53, we transfected COS cells with expression vectors encoding NPM, p53 and either NPM-RAR or control plasmid (pBluescript). Complexes were precipitated with an anti-NPM antibody (that recognizes epitopes not contained in NPM-RAR) and immunoblotted with an anti-p53 antibody. We found a decrease in the p53 signal captured by NPM in the lysate expressing NPM-RAR, compared to the control lysate. To further investigate the effects of NPM-RAR on p53, we made use of a transcriptional assay utilizing a reporter gene under control of a p53 response element. We co-transfected COS cells with a mix containing NPM-RAR, p53, and NPM expression plasmids, along with a p53-luciferase reporter (containing a 20 base pair p53 response element derived from the p21 promoter), and a ß-gal transfection control plasmid, or a similar mix in which NPM-RAR was replaced by an equivalent amount of carrier DNA (pBluescript). We found that the co-expression of NPM-RAR led to a decrease in the luciferase activity. These results support our hypothesis that NPM-RAR binding to NPM interferes with NPM’s ability to regulate p53.