title: Two 14-3-3 binding motifs are required for stable association of Forkhead transcription factor FOXO4 with 14-3-3 proteins and inhibition of DNA binding.
abstract: The 14-3-3 proteins, a family of dimeric regulatory proteins, are involved in many biologically important processes. The common feature of 14-3-3 proteins is their ability to bind to other proteins in a phosphorylation-dependent manner. Through these binding interactions, 14-3-3 proteins work as molecular scaffolds, modulating the biological functions of their partners. 14-3-3 proteins recognize short motifs containing a phosphorylated serine or threonine residue. In this study, we have quantitatively characterized the in vitro interactions among 14-3-3, the Forkhead transcription factor FOXO4, and its target DNA, the insulin response element. Phosphorylation of FOXO4 (residues 11-213) by protein kinase B at Thr-28 and Ser-193 creates two 14-3-3 binding motifs. Analytical gel filtration and sedimentation equilibrium experiments indicate that doubly phosphorylated FOXO4 and 14-3-3zeta form a complex with 1:2 molar stoichiometry and a K(D) of less than 30 nM. In contrast, singly phosphorylated FOXO4 mutants bind 14-3-3zeta with significantly lower affinity while retaining the ability to bind DNA. An active role for 14-3-3 in the disassembly of the FOXO4/DNA complex is demonstrated by the fact that, in the presence of 14-3-3, two phosphorylated 14-3-3 binding motifs are needed for the complete inhibition of FOXO4 binding to its target DNA.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, 5 Center Drive, MSC 0560, Bethesda, Maryland 20892, USA.
authors.forename: Tomas
authors.initials: T
authors.lastname: Obsil
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.forename: D Eric
authors.initials: DE
authors.lastname: Anderson
authors.forename: Alison Burgess
authors.initials: AB
authors.lastname: Hickman
authors.forename: Fred
authors.initials: F
authors.lastname: Dyda
mesh_terms: D048948:14-3-3 Proteins; D020816:Amino Acid Motifs; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051858:Forkhead Transcription Factors; D006801:Humans; D007328:Insulin / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020218:Response Elements; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C108803:FOXO4 protein, human; D051858:Forkhead Transcription Factors; D007328:Insulin; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C075372:insulin response element binding protein, human; D013912:Threonine; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt