Department of Structural and Computational Biology, Max Perutz Labs, Vienna, Austria; Department of Medical Biochemistry, Medical University of Vienna, Vienna, Austria.
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.
J Biol Chem. 2021 Aug;297(2):100919. doi: 10.1016/j.jbc.2021.100919. Epub 2021 Jun 25.
Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.
血清和糖皮质激素调节激酶 3（Sgk3）是一种丝氨酸/苏氨酸蛋白激酶，通过生长因子信号传导下游的 I 类磷脂酰肌醇 3-激酶（PI3K）信号传导和内体上的 III 类 PI3K/Vps34 介导的 PI3P 产生被磷脂酰肌醇 3-磷酸（PI3P）激活。Sgk3 活性的上调最近与多种人类癌症有关；然而，Sgk3 的激活的确切机制尚不清楚。在这里，我们使用广泛的细胞生物学、生化和生物物理技术，包括氢氘交换质谱，来研究 PI3P 激活 Sgk3 的机制。我们表明，Sgk3 的调节是通过磷酸化和变构激活的组合来实现的。我们证明，Sgk3 通过其调节 PH 结构域（PX）与 PI3P 的结合诱导与 Sgk3 激活相关的大构象变化，并且 Sgk3 的 PX 结构域的 PI3P 结合口袋在其无活性构象中被隔离。最后，我们通过 Vps34 介导的在磷脂酰肌醇脂质体上的 PI3P 合成在体外重建 Sgk3 的激活。除了确定 PI3P 激活 Sgk3 的机制外，我们的发现还为变构抑制剂的开发开辟了潜在的治疗途径，以针对癌症中的 Sgk3。
