Essential role for the dimerization domain of NuMA-RARα in its oncogenic activities and localization to NuMA sites within the nucleus

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Abstract
Nuclear mitotic apparatus protein-retinoic acid receptor α (NuMA-RARα) is the fourth of five fusion proteins identified in acute promyelocytic leukemia (APL) patients. The molecular basis for its oncogenic activity has not been delineated. In gel-shift assays, NuMA-RARα bound to retinoic acid response elements (RAREs) both as a homodimer and as a heterodimer with RXRα. The binding profile of NuMA-RARα to a panel of RAREs was very similar to PML-RARα and PLZF-RARα. In transient transfection assays using HepG2 cells, NuMA-RARα inhibited wild-type RARα transcriptional activity, while it augmented STAT3 transcriptional activity. In GST-pull down experiments, NuMA-RARα formed a complex with the corepressor SMRT, was released from the NuMA-RARα/SMRT complexes by all-trans retinoic acid (ATRA) at 10−7–10−6 M and became associated with the coactivator TRAM-1 at 10−8 M ATRA. Studies comparing NuMA-RARα with NuMA-RARα(ΔCC) demonstrated that the dimerization or α-helical coiled-coil domain of NuMA was required for homodimer formation, transcriptional repression of wild-type RARα, transcriptional activation of STAT3, and stability of the NuMA-RARα/SMRT complex. Confocal fluorescent microscopy of HeLa cells was performed following transient expression of cyan fluorescent protein (CFP)-tagged proteins and incubation of cells with or without ATRA. Within the nucleus, CFP-NuMA-RARα exhibited a speckled pattern identical to that observed in cells transfected with CFP-NuMA. Furthermore, CFP-NuMA-RARα colocalized with yellow fluorescent protein-tagged (YFP)-NuMA. In contrast, CFP-NuMA-RARα(ΔCC) exhibited a diffuse granular pattern within the nucleus, similar to RARα. These results indicate that the dimerization domain of NuMA-RARα is critical for each of the known oncogenic activities of NuMA fusion proteins as well as its sequestration to nuclear sites normally occupied by NuMA and is distinct from RARα.