Abstract
The FOXO family of forkhead transcription factors has a variety of important functions in stress response, metabolism, cell cycle, apoptosis, longevity, etc. The transcriptional activity and subcellular localization of FOXO are tightly regulated by post-translational modifications, including phosphorylation by various kinases. Here, we report that the transforming growth factor-beta-activated kinase (TAK1)-Nemo-like kinase (NLK) pathway negatively regulates FOXO1. We show that NLK binds and phosphorylates FOXO1 at Pro-directed Ser/Thr residues in the transactivation domain. The phosphorylation by TAK1-NLK pathway inhibits the transcriptional activity of FOXO1 and excludes FOXO1 from the nucleus, which is independent of phosphatidylinositol 3-kinase/Akt pathway. Consistently, knockdown of TAK1-NLK pathway dephosphorylates FOXO1 and decreases phospho-Ser-329 FOXO1 level. It also induces translocation of FOXO1 into the nucleus and leads to an increase in mRNA levels of FOXO target genes and poly(ADP-ribose) polymerase cleavage. In addition, we show the interaction between NLK and FOXO1 is evolutionarily conserved in Drosophila. Collectively, these findings provide the first evidence that TAK1-NLK pathway is a novel regulator of FOXO1.
Highlights
The best known example of the upstream signaling pathways of FOXO is the phosphatidylinositol 3-kinase (PI3K)3/Akt pathway that is activated by growth factors such as insulin and insulin-like growth factor-1 [2]
To investigate the relationship between Nemo-like kinase (NLK) and FOXO1, we tested whether NLK regulates the transcriptional activity of FOXO1, using a luciferase assay system based on the 8ϫFK1Tk
FOXO1 signal when the cells were transfected with NLK siRNA (Fig. 4D). These results strongly suggest that some or all of these eight Ser/Thr residues in the transactivation domain of FOXO1 are major phosphorylation sites for NLK
Summary
To investigate the relationship between NLK and FOXO1, we tested whether NLK regulates the transcriptional activity of FOXO1, using a luciferase assay system based on the 8ϫFK1Tk co-expressed with the NLK kinaseinactive mutant Among human, mouse, and rat (supplemental Fig. 3) To test whether these sites are phosphorylated by NLK, we replaced those Ser/Thr residues with Ala, one at a time, but the point mutants co-expressed with NLK WT did not produce a significant increase in mobility on SDS-PAGE (data not shown). These results strongly suggest that some or all of these eight Ser/Thr residues in the transactivation domain of FOXO1 are major phosphorylation sites for NLK. Erase activity of GAL4 DNA binding domain-FOXO1 WT was strongly inhibited by NLK (Fig. 5A) but that of FOXO1-8A was not significantly hindered (Fig. 5A) These results demonstrate that the FOXO1 phosphorylation by NLK impedes the activity of FOXO1 from activating transcriptional machinery
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