Matsuzaki Hitomi, Daitoku Hiroaki, Hatta Mitsutoki, Tanaka Keiji, Fukamizu Akiyoshi
Center for Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
Proc Natl Acad Sci U S A. 2003 Sep 30;100(20):11285-90. doi: 10.1073/pnas.1934283100. Epub 2003 Sep 17.
Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. It has been shown that FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. Here, we investigate the fate of FKHR after cells are stimulated by insulin. We show that insulin treatment decreases endogenous FKHR proteins in HepG2 cells, which is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and the mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation.
叉头转录因子FKHR（Foxo1）是葡萄糖稳态、细胞周期进程和细胞凋亡的关键调节因子。研究表明，FKHR通过胰岛素或生长因子信号级联反应发生磷酸化，导致其滞留在细胞质中并抑制靶基因表达。在此，我们研究细胞受胰岛素刺激后FKHR的命运。我们发现胰岛素处理可降低HepG2细胞中的内源性FKHR蛋白，而蛋白酶体抑制剂可抑制这种降低。FKHR在体内和体外均被泛素化，胰岛素可增强细胞中的泛素化作用。此外，胰岛素介导的FKHR降解信号由磷脂酰肌醇3激酶途径介导，并且FKHR在被磷脂酰肌醇3激酶的下游靶点蛋白激酶B磷酸化的丝氨酸或苏氨酸残基处发生突变，可在体内和体外抑制泛素化。最后，FKHR的有效泛素化需要细胞中的磷酸化和细胞质滞留。这些结果表明，胰岛素诱导的FKHR磷酸化导致多步骤负调控，不仅通过核排除，还通过泛素化介导的降解。
