Oct4 plays a crucial role in the regulation of self-renewal of embryonic stem cells (ESCs) and reprogramming of somatic cells to induced pluripotent stem cells. However, the molecular mechanisms underlying posttranslational regulation and protein stability of Oct4 remain unclear. Using affinity purification and mass spectrometry analysis, we identified Kap1 as an Oct4-binding protein. Silencing of Kap1 reduced the protein levels of Oct4 in ESCs, whereas the overexpression of Kap1 stimulated the levels of Oct4. In addition, Kap1 overexpression stimulated the self-renewal of ESCs and attenuated the spontaneous differentiation of ESCs in response to LIF withdrawal. Kap1 overexpression increased the stability of Oct4 by inhibiting the Itch-mediated ubiquitination of Oct4. Silencing of Kap1 augmented Itch-mediated ubiquitination and inhibited the stability of Oct4. We identified the lysine 133 (K133) residue in Oct4 as a ubiquitination site responsible for the Kap1-Itch-dependent regulation of Oct4 stability. Preventing ubiquitination at the lysine residue by mutation to arginine augmented the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. These results suggest that Kap1 plays a crucial role in the regulation of the pluripotency of ESCs and somatic cell reprogramming by preventing Itch-mediated ubiquitination and the subsequent degradation of Oct4.

Oct4 在调节胚胎干细胞 (ESC) 的自我更新和将体细胞重编程为诱导多能干细胞方面发挥着至关重要的作用。然而，Oct4 的翻译后调控和蛋白质稳定性的分子机制仍不清楚。使用亲和纯化和质谱分析，我们将 Kap1 鉴定为 Oct4 结合蛋白。Kap1 的沉默降低了 ESCs 中 Oct4 的蛋白质水平，而 Kap1 的过表达刺激了 Oct4 的水平。此外，Kap1 过表达刺激了 ESC 的自我更新，并减弱了 ESC 响应 LIF 退出的自发分化。Kap1 过表达通过抑制 Itch 介导的 Oct4 泛素化来增加 Oct4 的稳定性。Kap1 的沉默增强了 Itch 介导的泛素化并抑制了 Oct4 的稳定性。我们将 Oct4 中的赖氨酸 133 (K133) 残基鉴定为泛素化位点，负责对 Oct4 稳定性进行 Kap1-Itch 依赖性调节。通过突变为精氨酸来防止赖氨酸残基的泛素化增强了小鼠胚胎成纤维细胞向诱导多能干细胞的重编程。这些结果表明，Kap1 通过阻止 Itch 介导的泛素化和随后的 Oct4 降解，在调节 ESC 的多能性和体细胞重编程中起关键作用。通过突变为精氨酸来防止赖氨酸残基的泛素化增强了小鼠胚胎成纤维细胞向诱导多能干细胞的重编程。这些结果表明，Kap1 通过阻止 Itch 介导的泛素化和随后的 Oct4 降解，在调节 ESC 的多能性和体细胞重编程中起关键作用。通过突变为精氨酸来防止赖氨酸残基的泛素化增强了小鼠胚胎成纤维细胞向诱导多能干细胞的重编程。这些结果表明，Kap1 通过阻止 Itch 介导的泛素化和随后的 Oct4 降解，在调节 ESC 的多能性和体细胞重编程中起关键作用。