Both the N-terminal loop and wing W2 of the forkhead domain of transcription factor Foxo4 are important for DNA binding.
FoxO4 belongs to the "O" subset of forkhead transcription factors, which participate in various cellular processes. The forkhead DNA binding domain (DBD) consists of three-helix bundle resting on a small antiparallel beta-sheet from which two extended loops protrude and create two wing-like structures. The wing W2 of FoxO factors contains a 14-3-3 protein-binding motif that is phosphorylated by protein kinase B in response to insulin or growth factors. In this report, we investigated the role of the N-terminal loop (portion located upstream of first helix H1) and the C-terminal region (loop known as wing W2) of the forkhead domain of transcription factor FoxO4 in DNA binding. Although the deletion of either portion partly reduces the FoxO4-DBD binding to the DNA, the simultaneous deletion of both regions inhibits DNA binding significantly. Förster resonance energy transfer measurements and molecular dynamics simulations suggest that both studied N- and C-terminal regions of FoxO4-DBD directly interact with DNA. In the presence of the N-terminal loop the protein kinase B-induced phosphorylation of wing W2 by itself has negligible effect on DNA binding. On the other hand, in the absence of this loop the phosphorylation of wing W2 significantly inhibits the FoxO4-DBD binding to the DNA. The binding of the 14-3-3 protein efficiently reduces DNA-binding potential of phosphorylated FoxO4-DBD regardless of the presence of the N-terminal loop. Our results show that both N- and C-terminal regions of forkhead domain are important for stability of the FoxO4-DBD.DNA complex.
Boura
Evzen
E
Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic.
Silhan
Jan
J
Herman
Petr
P
Vecer
Jaroslav
J
Sulc
Miroslav
M
Teisinger
Jan
J
Obsilova
Veronika
V
Obsil
Tomas
T
D048948:14-3-3 Proteins / Q000737:chemistry; D000595:Amino Acid Sequence; D016880:Anisotropy; D018797:Cell Cycle Proteins; D004247:DNA / Q000737:chemistry*; D051858:Forkhead Transcription Factors; D017353:Gene Deletion; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; C108803:FOXO4 protein, human; D051858:Forkhead Transcription Factors; D014157:Transcription Factors; D004247:DNA; D051057:Proto-Oncogene Proteins c-akt