AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins

Abstract

Edited by Wolfgang Peti Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1's transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr 24 and Ser 256 in FOXO1 to stimulate binding of 14-3-3 proteins, causing FOXO1 inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser 383 and Thr 649 in FOXO1. In this study, we identified Ser 22 as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser 22 phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser 22 phosphorylation impairs Thr 24 phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser 22 phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser 383 and Thr 649 complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity.

The crosstalk of NAD, ROS and autophagy in cellular health and ageing

“…Furthermore, FoxO1 acetylation permits access for upstream kinases to phosphorylate its Ser 253 residue that is otherwise shielded by FoxO1-DNA complex formation (Matsuzaki et al 2005). FoxO1 phosphorylation sites have since became known to act as docking or shielding sites for 14-3-3 protein binding, and the heterodimer exit into and retention within the cytoplasm (Brunet et al 1999;Saline et al 2019).…”

Persulfidation of transcription factor FOXO1 at cysteine 457: A novel mechanism by which H2S inhibits vascular smooth muscle cell proliferation

“…2 b), and Cys457 and Cys612 show particularly high conservation. A structural analysis indicated that Cys457 of FOXO1 is located in an important functional region [20] , which suggests that Cys457 is closely related to FOXO1 activity and might be persulfidated by H 2 S. As a result, we transfected WT and C457S-mutated FOXO1 plasmids into VSMCs and 293T cells and found that NaHS significantly persulfidated FOXO1 in the cells transfected with WT FOXO1 but could not persulfidate FOXO1 in the cells transfected with C457S FOXO1 ( Fig. 2 c and 2d).…”

“…To determine the exact site persulfidated by H 2 S, we first performed a sequence homology analysis of FOXO1 proteins from seven species and revealed that the cysteine residues were conserved in FOXO1 proteins from different species and that Cys457 and Cys612 were highly conserved among the seven species. Cys457 in FOXO1 is reportedly located in the transcriptional activation domain (aa 421–655) of FOXO1 [20] and in the functional region (aa 283–563) of FOXO1, which interacts with Nemo-like kinase (NLK), as described in the UniProt database. Previous studies have reported that NLK activates FOXO1 phosphorylation, FOXO1 nuclear exclusion to the cytoplasm and inhibits FOXO1 transcriptional activity [32] , which suggests that Cys457 is crucial for FOXO1 activity.…”

Trimetazidine improves hepatic lipogenesis and steatosis in non‑alcoholic fatty liver disease via AMPK‑ChREBP pathway

“…The carbohydrate-responsive element-binding protein (chreBP) has a crucial role in the regulation of hepatic lipogenesis, hepatic steatosis and insulin resistance (21,22). chreBP is a major mediator in glycolysis and lipogenesis via binding to the promoter region of lipogenic genes.…”