Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1

Characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms

Xiangshan Zhao, Lixia Gan, Haiyun Pan, Donghui Kan, Michael Majeski, Stephen A. Adam, Terry G. Unterman

Research output: Contribution to journalArticle

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Abstract

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action.

Original languageEnglish (US)
Pages (from-to)839-849
Number of pages11
JournalBiochemical Journal
Volume378
Issue number3
DOIs
StatePublished - Mar 15 2004

Fingerprint

Phosphorylation
14-3-3 Proteins
Nuclear Export Signals
DNA
Leucine
Intercellular Signaling Peptides and Proteins
Insulin
Forkhead Transcription Factors
Amino Acids
Nuclear Localization Signals
Coprecipitation
Green Fluorescent Proteins
Proteins
Fusion reactions
leptomycin B

Keywords

  • 14-3-3 protein
  • Forkhead
  • Gene regulation
  • Growth factor
  • Insulin
  • Transcription

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1 : Characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms. / Zhao, Xiangshan; Gan, Lixia; Pan, Haiyun; Kan, Donghui; Majeski, Michael; Adam, Stephen A.; Unterman, Terry G.

In: Biochemical Journal, Vol. 378, No. 3, 15.03.2004, p. 839-849.

Research output: Contribution to journalArticle

Zhao, Xiangshan ; Gan, Lixia ; Pan, Haiyun ; Kan, Donghui ; Majeski, Michael ; Adam, Stephen A. ; Unterman, Terry G. / Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1 : Characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms. In: Biochemical Journal. 2004 ; Vol. 378, No. 3. pp. 839-849.
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