Aukrust Ingvild, Rosenberg Linn Andersen, Ankerud Mia Madeleine, Bertelsen Vibeke, Hollås Hanne, Saraste Jaakko, Grindheim Ann Kari, Vedeler Anni
Department of Biomedicine University of Bergen Norway; Present address: Centre for Medical Genetics and Molecular Medicine Haukeland University Hospital Bergen Norway.
Department of Biomedicine University of Bergen Norway.
FEBS Open Bio. 2017 Jan 17;7(2):160-173. doi: 10.1002/2211-5463.12173. eCollection 2017 Feb.
Various post-translational modifications (PTMs) regulate the localisation and function of the multifunctional protein Annexin A2 (AnxA2). In addition to its various tasks as a cytoskeletal- and membrane-associated protein, AnxA2 can function as a -acting protein binding to -acting sequences of specific mRNAs. In the present study, we have examined the role of Ser25 phosphorylation in subcellular localisation of AnxA2 and its interaction with mRNP complexes. Subcellular fractionation and confocal microscopy of rat neuroendocrine PC12 cells showed that Ser25-phosphorylated AnxA2 (pSer25AnxA2) is absent from the nucleus and mainly localised to the perinuclear region, evidently associating with both membranes and cytoskeletal elements. Perinuclear targeting of AnxA2 was abolished by inhibition of protein kinase C activity, which resulted in cortical enrichment of the protein. Although oligo(dT)-affinity purification of mRNAs revealed that pSer25AnxA2 associates with nonpolysomal, translationally inactive mRNP complexes, it displayed only partial overlap with a marker of P-bodies. The phosphorylated protein is present as high-molecular-mass forms, indicating that it contains additional covalent PTMs, apparently triggered by its Ser25 phosphorylation. The subcellular distributions of these forms clearly differ from the main form of AnxA2 and are also distinct from that of Tyr23-phosphorylated AnxA2. Immunoprecipitation verified that these high-molecular-mass forms are due to ubiquitination and/or sumoylation. Moreover, these results indicate that Ser25 phosphorylation and ubiquitin/SUMO1 conjugation of AnxA2 promote its association with nonpolysomal mRNAs, providing evidence of a possible mechanism to sequester a subpopulation of mRNAs in a translationally inactive and transport competent form at a distinct subcellular localisation.
多种翻译后修饰（PTM）调节多功能蛋白膜联蛋白A2（AnxA2）的定位和功能。除了作为细胞骨架和膜相关蛋白承担的各种任务外，AnxA2还可作为一种作用蛋白，与特定mRNA的作用序列结合。在本研究中，我们研究了Ser25磷酸化在AnxA2亚细胞定位及其与mRNP复合物相互作用中的作用。大鼠神经内分泌PC12细胞的亚细胞分级分离和共聚焦显微镜检查显示，Ser25磷酸化的AnxA2（pSer25AnxA2）不存在于细胞核中，主要定位于核周区域，显然与膜和细胞骨架成分相关。抑制蛋白激酶C活性可消除AnxA2的核周靶向作用，导致该蛋白在皮质富集。尽管通过oligo(dT)亲和纯化mRNA显示pSer25AnxA2与非多聚体、翻译无活性的mRNP复合物相关，但它与P小体标记物仅部分重叠。磷酸化蛋白以高分子量形式存在，表明它含有额外的共价PTM，显然是由其Ser25磷酸化引发的。这些形式的亚细胞分布明显不同于AnxA2的主要形式，也不同于Tyr23磷酸化的AnxA2。免疫沉淀证实这些高分子量形式是由于泛素化和/或类泛素化。此外，这些结果表明，AnxA2的Ser25磷酸化和泛素/ SUMO1缀合促进了其与非多聚体mRNA的结合，为在不同亚细胞定位以翻译无活性和运输能力形式隔离一部分mRNA的可能机制提供了证据。
