Despite significant evidence that Rac1 is localized to the nucleus, little is known regarding the function and biological significance of nuclear Rac1. Here, we showed that in response to EGF Rac1 was translocated to nuclear speckles and co-localized with the nuclear speckle marker Serine/arginine-rich splicing factor 2 (SRSF2) in Cos-7 cells. We also showed that the nuclear speckle localization of Rac1 was dependent on its T108 phosphorylation and facilitated by Rac1 polybasic region (PBR) that contains a nuclear localization signal and Rac1 GTPase activity. To gain insight into the function of Rac1 in nuclear speckles, we searched for Rac1 binding proteins in the nucleus. We isolated nuclear fraction of HEK 293 cells and incubated with GST-Rac1 and the phosphomimetic GST-Rac1T108E. We identified 463 proteins that were associated with GST-Rac1T108E, but not with GST-Rac1 by LC-MS/MS. Three notable groups of these proteins are: the heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and SRSFs, all of which are involved in pre-mRNA splicing and associated with nuclear speckles. We further showed by co-immunoprecipitation that Rac1 interacts with SRSF2, hnRNPA1, and U2A' in response to EGF. The interaction is dependent on T108 phosphorylation and facilitated by Rac1 PBR and GTPase activity. We showed that hnRNPA1 translocated in and out of nucleus in response to EGF in a similar pattern to Rac1. Rac1 only partially colocalized with U2A' that localizes to the actual splicing sites adjacent to nuclear speckle. Finally, we showed that Rac1 regulated EGF-induced pre-mRNA splicing and this is mediated by T108 phosphorylation. We conclude that in response to EGF, T108 phosphorylated Rac1 is targeted to nuclear speckles, interacts with multiple groups of proteins involved in pre-mRNA splicing, and regulates EGF-induced pre-mRNA splicing.

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Rac1 核斑点定位和功能的调节

尽管有大量证据表明 Rac1 位于细胞核，但对核 Rac1 的功能和生物学意义知之甚少。在这里，我们发现响应于 EGF Rac1 易位到核斑点并与 Cos-7 细胞中的核斑点标记丝氨酸/富含精氨酸的剪接因子 2 (SRSF2) 共定位。我们还表明，Rac1 的核斑点定位取决于其 T108 磷酸化，并由包含核定位信号和 Rac1 GTPase 活性的 Rac1 多元区域（PBR）促进。为了深入了解 Rac1 在核斑点中的功能，我们搜索了细胞核中的 Rac1 结合蛋白。我们分离了 HEK 293 细胞的核部分，并与 GST-Rac1 和磷酸化 GST-Rac1T108E 一起孵育。我们通过 LC-MS/MS 鉴定了 463 种与 GST-Rac1T108E 相关的蛋白质，但与 GST-Rac1 无关。这些蛋白质的三个值得注意的组是：异质核核糖核蛋白 (hnRNPs)、小核核糖核蛋白 (snRNPs) 和 SRSFs，所有这些都参与前 mRNA 剪接并与核斑点相关。我们通过免疫共沉淀进一步表明，Rac1 与 SRSF2、hnRNPA1 和 U2A' 相互作用以响应 EGF。这种相互作用依赖于 T108 磷酸化，并由 Rac1 PBR 和 GTPase 活性促进。我们发现 hnRNPA1 以与 Rac1 类似的模式响应 EGF 易位进出细胞核。Rac1 仅与 U2A' 部分共定位，U2A' 定位到与核斑点相邻的实际剪接位点。最后，我们发现 Rac1 调节了 EGF 诱导的前 mRNA 剪接，这是由 T108 磷酸化介导的。我们得出结论，响应于 EGF，T108 磷酸化的 Rac1 靶向核斑点，与参与前 mRNA 剪接的多组蛋白质相互作用，并调节 EGF 诱导的前 mRNA 剪接。