Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding.
14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.
Shen
Ying H
YH
Cardiovascular Research Institute, Division of Molecular Cardiology, The Texas A&M University System Health Science Center, College of Medicine, Temple, Texas 76504, USA.
Godlewski
Jakub
J
Bronisz
Agnieszka
A
Zhu
Jun
J
Comb
Michael J
MJ
Avruch
Joseph
J
Tzivion
Guri
G
D048948:14-3-3 Proteins; D000818:Animals; D019556:COS Cells; D029742:Caenorhabditis elegans Proteins; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D019281:Dimerization; D051858:Forkhead Transcription Factors; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D029742:Caenorhabditis elegans Proteins; D051858:Forkhead Transcription Factors; D014157:Transcription Factors; C108831:daf-16 protein, C elegans; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf