The Retinoic Acid Receptor (RAR ) Chimeric Proteins PML-, PLZF-, NPM-, and NuMA-RAR Have Distinct Intracellular Localization Patterns

Retinoic acid receptor (RAR ) gene rearrangement by reciprocal chromosome translocation is the molecular signature of acute promyelocytic leukemia (APL). Disruption of RAR function appears to be the likely cause of aberrant myelopoiesis observed in APL, because PML-RAR expression has been shown to deregulate the transcription of genes that control myelopoiesis. To target RAR chimeric proteins, we engineered epitope-tagged versions of PML-RAR , PLZF-RAR , NPM-RAR , and NuMA-RAR (X-RAR ) and generated a panel of stable COS cell lines expressing X-RAR . Protein fractionation and Western analysis of these COS lines reveal that X-RAR proteins localize to both the cytoplasm and nucleus. NPM-RAR is predominantly nuclear whereas NuMA-RAR is predominantly cytoplasmic. Confocal immunofluorescent microscopy reveals that PMLRAR and PLZF-RAR share a primarily diffuse nuclear pattern that excludes the nucleolus. NPM-RAR is also diffuse in the nucleus but, in contrast to PML-RAR and PLZF-RAR , is strongly associated with the nucleolus. Strikingly, NuMA-RAR predominantly localizes throughout the cytoplasm in a microspeckled pattern. We further demonstrate that NPM and NuMA interact with NPM-RAR and NuMA-RAR , respectively. The distinct intracellular localization patterns and the shared ability of X-RAR to interact with their respective RAR partner proteins (X) further support the hypothesis that deregulation of these partners may play a role in APL pathogenesis. Introduction APL is characterized by the accumulation of abnormal promyelocytes in the bone marrow, attributable to a block in the myeloid differentiation program. Nearly 100% of APL patients achieve remission after differentiation therapy with RA (1). This is the direct result of the abnormal promyelocytes reentering the differentiation program. APL is associated with a balanced t(15;17)(q22;q21) translocation (2) that fuses the RAR (17q21) gene (3, 4) to the PML (15q22) gene (5–8) and results in the expression of a PML-RAR chimeric protein. Several rare variant APL translocations have been identified involving the PLZF, (11q23; Ref. 9), NuMA (11q13; Refs. 10, 11), NPM (5q35; Refs. 12, 13), and STAT5b genes (17q21; Ref. 14) fused to the essential functional domains of RAR . RAR , a member of the steroid/hormone nuclear receptor family that specifically binds to 9-cis RA and ATRA (3, 15, 16), helps orchestrate myeloid development by acting as a transcription factor at specific DNA sequences called RAREs (17–22). Similar to other steroid/hormone nuclear receptors (TR, VD3R, and PPAR), RAR functions as a heterodimer with the RXR (19, 21, 23). Because RAREs have been identified in the upstream regions of several genes believed to control myelopoiesis (2, 20, 23) the disruption of RAR function is believed to be the likely cause of the aberrant myelopoiesis observed in APL. In addition, all APL fusion genes result in the expression of RAR chimeric proteins (X-RAR ), which have been directly implicated in APL pathogenesis. PML-RAR , PLZF-RAR , and NPM-RAR have been shown to possess altered transcriptional activity at RAREs, with a tendency to repress transcription in the presence of physiological levels of RA. Pharmacological levels of ATRA (10 6 M) can reestablish transcriptional activation at RAREs in cells expressing PML-RAR and NPM-RAR ; however, these levels have little effect on cells expressing PLZF-RAR (7, 24–28). Consistent with this, APL patients bearing the t(11;17)(q23;q21) respond poorly to ATRA treatment (29). Although the RAR partner proteins (X) appear to have different functions, each contains a multimerization domain that potentiates the formation of X-RAR homodimers. It has recently been determined by several groups that RAREs appear to be occupied by X-RAR homodimers in conditions where PML-RAR , PLZF-RAR , and NPM-RAR are overexpressed (30–35). In fact, increased repression at RAREs Received 11/15/01; revised 2/19/02; accepted 2/20/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the National Cancer Institute of Canada (to S. K-R.), the Canadian Institute of Health Research (to S. K-R.), and the Leukemia Research Fund of Canada (to S. K-R.), and by the Ontario Graduate Scholarship Program (to J. L. H.). 2 To whom requests for reprints should be addressed, at Princess Margaret Hospital, Ontario Cancer Institute, Room 9-622, 610 University Avenue, Toronto, Ontario, M5G 2M9. Phone: (416) 946-2806; E-mail: [email protected]. 3 The abbreviations used are: APL, acute PML; PML, promyelocytic leukemia; RA, retinoic acid; RAR , RA receptor ; PLZF, promyelocytic zinc finger; NPM, nucleophosmin; NuMA, nuclear mitotic apparatus; STAT5b, signal transducer and transcriptional activator 5b; ATRA, all-trans RA; RARE, RA response element; RXR , retinoid X receptor ; CML, chronic myeloid leukemia; GST, glutathione S-transferase; TNT, transcribed/ translated. 173 Vol. 13, 173–183, April 2002 Cell Growth & Differentiation