Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation - PubMed (original) (raw)

Comparative Study

. 2005 Aug 9;102(32):11278-83.

doi: 10.1073/pnas.0502738102. Epub 2005 Aug 2.

Affiliations

Comparative Study

Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation

Hitomi Matsuzaki et al. Proc Natl Acad Sci U S A. 2005.

Abstract

The FOXO family of forkhead transcription factors plays a key role in a variety of biological processes, including metabolism, cell proliferation, and oxidative stress response. We previously reported that Foxo1, a member of the FOXO family, is regulated through reversible acetylation catalyzed by histone acetyltransferase cAMP-response element-binding protein (CREB)-binding protein (CBP) and NAD-dependent histone deacetylase silent information regulator 2, and that the acetylation at Lys-242, Lys-245, and Lys-262 of Foxo1 attenuates its transcriptional activity. However, the molecular mechanism by which acetylation modulates Foxo1 activity remains unknown. Here, we show that the positive charge of these lysines in Foxo1 contributes to its DNA-binding, and acetylation at these residues by CBP attenuates its ability to bind cognate DNA sequence. Remarkably, we also show that acetylation of Foxo1 increases the levels of its phosphorylation at Ser-253 through the phosphatidylinositol 3-kinase-protein kinase B signaling pathway, and this effect was overridden on the acetylation-deficient Foxo1 mutant. Furthermore, in in vitro kinase reactions, the association of wild-type Foxo1 and its target DNA sequence inhibits the protein kinase B-dependent phosphorylation of Foxo1, whereas mutated Foxo1 proteins, which mimic constitutively acetylated states, are efficiently phosphorylated even in the presence of the DNA. These results suggest that acetylation regulates the function of Foxo1 through altering the affinity with the target DNA and the sensitivity for phosphorylation.

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Figures

Fig. 1.

Fig. 1.

Acetylation of Foxo1 attenuates DNA-binding and represses its transcriptional activity. (A) Schematic representation of Foxo1 point mutants. Lys-242, Lys-245, and Lys-262 are sites acetylated by CBP. Thr-24, Ser-253, and Ser-316 are sites phosphorylated by PKB. The gray box indicates the forkhead DNA-binding domain. (B) HepG2 cells were transiently transfected with 50 ng of p3×IRS-MLP-luc and 10 ng of wild-type or mutated Foxo1 expression plasmids, and the luciferase activity was measured. (C) EMSA was performed with bacterially expressed wild-type or mutated GST-fusion Foxo1 proteins (amino acids 157–268) and a 32P-labeled double-stranded oligonucleotide probe containing Foxo1-binding sequences from human G6Pase promoter. In the control lane, GST protein was used instead of GST-Foxo1 protein. The amounts of proteins used in the experiment were confirmed by Coomassie brilliant blue stain (Right). (D) The kinetics of binding of Foxo1 (wild-type or 3KQ mutant) to the probe DNA was measured by EMSA. Bound probe is quantified, and binding activity is shown. (E) HEK293T cells transfected with FLAG-Foxo1 and CBP-HA were treated with deacetylase inhibitors NIA and TSA. Whole-cell extracts were incubated with double-stranded 3×IRS immobilized on streptavidin agarose (avidin–biotin-conjugated DNA), and subsequently, the supernatants were recovered and immunoprecipitated with anti-FLAG antibody (IP). After washing the beads, proteins were detected by Western blotting using anti-acetylated-Foxo1 or anti-FLAG antibodies (Left). The amounts of FLAG-Foxo1 and CBP-HA in cell extracts were shown by Western blotting with anti-FLAG or anti-HA antibodies (Right). (F) Chromatin IP assays were performed in HepG2 cells treated with LY294002 and/or deacetylase inhibitors NIA and TSA by using normal rabbit IgG or anti-Foxo1 antibody. Immunoprecipitated (IP) DNA was analyzed by PCR using a primer set specific for G6Pase.

Fig. 2.

Fig. 2.

Deacetylase inhibitors promote phosphorylation of Foxo1. (A) HEK293T cells were transfected with FLAG-Foxo1 and CBP-HA and treated with histone deacetylase inhibitors NIA (N) and/or TSA (T). Whole-cell extracts were immunoprecipitated with anti-FLAG antibody, followed by Western blotting (WB). (B) HEK293T cells were transfected with FLAG-Foxo1 and CBP-HA and treated with NIA and TSA. FLAG-Foxo1 was immunopurified with anti-FLAG antibody and then incubated with alkaline phosphatase (AP) in the absence or presence of phosphatase inhibitor Na3VO4 in vitro. Reaction products and cell extracts were analyzed by Western blotting. (C) HEK293T cells were transfected with FLAG-Foxo1 and CBP-HA and treated with NIA, TSA, and LY294002. Whole-cell extracts were analyzed by Western blotting. (D) HEK293T cells were treated with NIA, TSA, and LY294002. Phosphorylated and total PKB in the whole-cell extracts were analyzed by Western blotting.

Fig. 3.

Fig. 3.

The mutations at acetylation sites affect the phosphorylation level of Foxo1. (A) HEK293T cells were transfected with FLAG-Foxo1 (wild-type or 3KR mutant) and CBP-HA, and treated with NIA and TSA. (B) HEK293T cells were transfected with FLAG-Foxo1 (wild-type or indicated mutants). Whole-cell extracts were analyzed by Western blotting. (C) HepG2 cells were transiently transfected with 50 ng of p3×IRS-MLP-luc together with 5 ng of the indicated Foxo1 expression plasmids, and the luciferase activity was measured. (D) H4IIE cells were treated with insulin and/or deacetylase inhibitors NIA and TSA. Total RNA was hybridized with 32P-labeled probes specific for G6Pase and β-actin genes.

Fig. 4.

Fig. 4.

The association of Foxo1 and its cognate DNA sequence inhibits the PKB-dependent phosphorylation of Foxo1 in vitro. (A) In vitro kinase assays were performed with 1 μg of GST-Foxo1 (amino acids 157–268), 10 ng of PKB, and 0.5 mM ATP. (B) One microgram of wild-type GST-Foxo1 (amino acids 157–268) was preincubated with the indicated double-stranded oligonucleotides (0.5 or 1 pmol) and phosphorylated by 10 ng of PKB and 0.5 mM ATP in vitro. (C) Wild-type or mutated GST-Foxo1 (1 μg) was phosphorylated by PKB (0, 2, or 10 ng). (D) Wild-type or mutated GST-Foxo1 (1 μg) was preincubated with double-stranded oligonucleotide (0, 0.5, or 1 pmol) and phosphorylated by 10 ng of PKB. All reaction products were analyzed by Western blotting using anti-phospho-Foxo1 antibody or silver stain.

Fig. 5.

Fig. 5.

A model for Foxo1 regulation through acetylation and phosphorylation.

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