蛋白激酶B(AKT)在肿瘤发生和发展中的重要性已得到充分证实。但其通过己糖激酶(HK)同工酶的磷酸化对代谢重编程的潜在调节仍不清楚。有两名香港家庭成员(HK1/2)和三名AKT家庭成员(AKT1/2/3),AKT在不同的组织和细胞类型中表现出不同的功能。尽管已知AKT在苏氨酸473处磷酸化HK2,但尚未报道AKT介导的HK1磷酸化。我们检查了AKT1对HK1/2的直接结合和磷酸化,并使用共免疫沉淀鉴定了磷酸化修饰位点,谷胱甘肽下拉,西方印迹,和体外激酶测定。还检查了通过AKT1磷酸化对HK活性的调节。研究了2-[1,2-3H]-脱氧葡萄糖的摄取和乳酸的产生,以确定AKT1是否通过磷酸化HK1/2来调节葡萄糖代谢。功能测定,免疫组织化学,和在小鼠中进行肿瘤实验以研究AKT1介导的肿瘤发展调节是否依赖于其激酶活性和/或HK1/2的参与。AKT与HK1和HK2相互作用并磷酸化。丝氨酸磷酸化显著增加AKT激酶活性,从而增强糖酵解。机械上,AKT对丝氨酸178处HK1的磷酸化(S178)通过干扰HK1二聚体的形成而显着降低了Km并提高了Vmax。HK1或HK2的AKT磷酸化位点的突变显著消除了AKT对糖酵解的刺激特性,肿瘤发生,和细胞迁移,入侵,扩散,和转移。HK1-S178磷酸化水平与不同类型临床肿瘤的发生和转移显著相关。我们得出结论,AKT不仅通过直接磷酸化HK1和HK2调节肿瘤的糖代谢,而且在肿瘤的进展中发挥重要作用。扩散,和移民。
The importance of protein kinase B (AKT) in tumorigenesis and development is well established, but its potential regulation of metabolic reprogramming via phosphorylation of the hexokinase (HK) isozymes remains unclear. There are two HK family members (
HK1/2) and three AKT family members (AKT1/2/3), with varied distribution of AKTs exhibiting distinct functions in different tissues and cell types. Although AKT is known to phosphorylate HK2 at threonine 473, AKT-mediated phosphorylation of
HK1 has not been reported. We examined direct binding and phosphorylation of HK1/2 by AKT1 and identified the phosphorylation modification sites using coimmunoprecipitation, glutathione pull-down, western blotting, and in vitro kinase assays. Regulation of HK activity through phosphorylation by AKT1 was also examined. Uptake of 2-[1,2-3H]-deoxyglucose and production of lactate were investigated to determine whether AKT1 regulates glucose metabolism by phosphorylating
HK1/2. Functional assays, immunohistochemistry, and tumor experiments in mice were performed to investigate whether AKT1-mediated regulation of tumor development is dependent on its kinase activity and/or the involvement of HK1/2. AKT interacted with and phosphorylated
HK1 and HK2. Serine phosphorylation significantly increased AKT kinase activity, thereby enhancing glycolysis. Mechanistically, the phosphorylation of HK1 at serine 178 (S178) by AKT significantly decreased the Km and enhanced the Vmax by interfering with the formation of
HK1 dimers. Mutations in the AKT phosphorylation sites of HK1 or HK2 significantly abrogated the stimulatory characteristics of AKT on glycolysis, tumorigenesis, and cell migration, invasion, proliferation, and metastasis. HK1-S178 phosphorylation levels were significantly correlated with the occurrence and metastasis of different types of clinical tumors. We conclude that AKT not only regulates tumor glucose metabolism by directly phosphorylating HK1 and HK2, but also plays important roles in tumor progression, proliferation, and migration.