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

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 Thr²⁴ and Ser²⁵⁶ 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 Ser³⁸³ and Thr⁶⁴⁹ in FOXO1. In this study, we identified Ser²² as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser²² phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 σ at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser²² phosphorylation impairs Thr²⁴ phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser²² phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser³⁸³ and Thr⁶⁴⁹ 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.