Arregi Igor, Falces Jorge, Olazabal-Herrero Anne, Alonso-Mariño Marián, Taneva Stefka G, Rodríguez José A, Urbaneja María A, Bañuelos Sonia
Unidad de Biofísica (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country, Leioa, Spain.
Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, Leioa, Spain.
PLoS One. 2015 Jun 19;10(6):e0130610. doi: 10.1371/journal.pone.0130610. eCollection 2015.
Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.
核磷蛋白（NPM）是一种穿梭于细胞核与细胞质之间的蛋白质，通常在核仁中富集，具有多种与细胞生长相关的活性。NPM突变是急性髓系白血病（AML）一种亚型的特征，其中突变的NPM似乎发挥致癌作用。与AML相关的NPM突变体表现出亚细胞运输改变，异常定位于原始白细胞的细胞质中。AML相关的NPM变体的过度输出由核输出受体CRM1介导，部分原因是通过突变获得了新的核输出信号（NES）。为了深入了解生理和病理条件下NPM运输的分子基础，我们评估了NPM在细胞中的输出效率，并提供了新的数据表明，在正常条件下，野生型NPM由CRM1微弱输出。另一方面，我们发现与AML相关的NPM突变体与CRM1HA（一种对NES具有更高亲和力的突变CRM1）有效形成复合物，并且我们使用荧光各向异性和等温滴定量热法对CRM1HA与这些突变体的NES基序的相互作用进行了定量分析。我们观察到CRM1HA对这些NES的亲和力相似，这可能有助于解释突变体的运输特性。我们还描述了NPM被输入机制识别的情况。我们结合细胞和生物物理研究进一步阐明了NPM运输的决定因素，以及它如何因AML相关突变而发生显著改变。
