The heterogeneous nuclear ribonucleoprotein hnRNP A1 is a nucleocytoplasmic-shuttling RNA-binding protein that plays an important role in nucleic acid metabolism and gene expression regulation. The function of hnRNP A1 is determined in part by its specific location within the cell. Although some work has been done to elucidate the signaling pathways that regulate the cellular localization of hnRNP A1, the precise mechanism(s), including physiological and pathophysiological conditions that alter hnRNP A1 localization, are not known. We previously conducted an unbiased RNAi-based kinome-wide screen to identify kinases that regulate hnRNP A1 localization during hypertonic stress. One of the hits from this screen is AMPK-related protein kinase 5 (ARK5). Here, we validate ARK5 as the kinase responsible for controlling hnRNP A1 subcellular localization in response to hypertonic stress. We find using immunoprecipitation and in vitro kinase assay methods that ARK5 directly interacts with and phosphorylates hnRNP A1 on serine residues within the F-peptide region. We further show that the M9 motif of hnRNP A1 is essential for the ARK5-hnRNP A1 interaction and subsequent phosphorylation. In addition, the silencing of ARK5 increases the expression of anti-apoptotic protein Bcl-xL and consequently delays caspase activation during hypertonic stress. Our results indicate that ARK5 phosphorylates hnRNP A1 and regulates its subcellular localization during hypertonic stress.

异质核核糖核蛋白hnRNP A1是一种核质穿梭RNA结合蛋白，在核酸代谢和基因表达调控中起重要作用。hnRNP A1 的功能部分取决于其在细胞内的特定位置。尽管已经进行了一些工作来阐明调节 hnRNP A1 细胞定位的信号通路，但确切的机制，包括改变 hnRNP A1 定位的生理和病理生理条件，尚不清楚。我们之前进行了无偏的基于 RNAi 的激酶组筛选，以识别在高渗应激期间调节 hnRNP A1 定位的激酶。此屏幕中的一个点击是 AMPK 相关蛋白激酶 5 (ARK5)。这里，我们验证 ARK5 是负责控制 hnRNP A1 亚细胞定位以响应高渗应激的激酶。我们发现使用免疫沉淀和体外激酶测定方法，ARK5 直接与 F 肽区域内的丝氨酸残基上的 hnRNP A1 相互作用并使其磷酸化。我们进一步表明hnRNP A1 的M9 基序对于ARK5-hnRNP A1 相互作用和随后的磷酸化是必不可少的。此外，ARK5 的沉默增加了抗凋亡蛋白 Bcl-xL 的表达，从而延迟了高渗应激期间的半胱天冬酶活化。我们的结果表明，ARK5 磷酸化 hnRNP A1 并在高渗应激期间调节其亚细胞定位。我们发现使用免疫沉淀和体外激酶测定方法，ARK5 直接与 F 肽区域内的丝氨酸残基上的 hnRNP A1 相互作用并使其磷酸化。我们进一步表明hnRNP A1 的M9 基序对于ARK5-hnRNP A1 相互作用和随后的磷酸化是必不可少的。此外，ARK5 的沉默增加了抗凋亡蛋白 Bcl-xL 的表达，从而延迟了高渗应激期间的半胱天冬酶活化。我们的结果表明，ARK5 磷酸化 hnRNP A1 并在高渗应激期间调节其亚细胞定位。我们发现使用免疫沉淀和体外激酶测定方法，ARK5 直接与 F 肽区域内的丝氨酸残基上的 hnRNP A1 相互作用并使其磷酸化。我们进一步表明hnRNP A1 的M9 基序对于ARK5-hnRNP A1 相互作用和随后的磷酸化是必不可少的。此外，ARK5 的沉默增加了抗凋亡蛋白 Bcl-xL 的表达，从而延迟了高渗应激期间的半胱天冬酶活化。我们的结果表明，ARK5 磷酸化 hnRNP A1 并在高渗应激期间调节其亚细胞定位。ARK5 的沉默增加了抗凋亡蛋白 Bcl-xL 的表达，从而延迟了高渗应激期间的半胱天冬酶活化。我们的结果表明，ARK5 磷酸化 hnRNP A1 并在高渗应激期间调节其亚细胞定位。ARK5 的沉默增加了抗凋亡蛋白 Bcl-xL 的表达，从而延迟了高渗应激期间的半胱天冬酶活化。我们的结果表明，ARK5 磷酸化 hnRNP A1 并在高渗应激期间调节其亚细胞定位。