MOLECULAR MECHANISM OF 14-3-3 PROTEIN FUNCTION REGULATION

 

T. Obšil1,2

 

1Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague,

2Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague

 

The 14-3-3 protein family represents one of the most important group among proteins recognizing phosphorylated targets [1,2]. Many of 14-3-3 binding partners contain one of two consensus motifs, RSXpSXP, and RXY/FXpSXP, where pS denotes phosphoserine. 14-3-3 proteins function as molecular scaffolds modulating the conformation of their binding partners. As a result of this structural modulation they can: (i) affect the enzymatic properties of their partners [3]; (ii) interfere with the protein-protein interactions of their targets; or (iii) regulate the subcellular localization of their binding partners presumably by masking or obscuring a nearby targeting sequence, such as a nuclear localization sequence (NLS) or a nuclear export sequence (NES) [4]. The Cdc25 phosphatases, telomerase, histone deacetylase, or FoxO transcription factors have been suggested to be subject to such regulation.

All 14-3-3 proteins form very stable homo- and hetero-dimers with characteristic cup-like shape and a large, 40 Å-wide, deep central channel [2,3]. Each monomer consists of nine antiparallel a-helices and an amphipathic groove where the phosphorylated segment of the binding partner is bound. The primary structure of 14-3-3 proteins is highly conserved with completely conserved residues forming either the dimer interface or the walls of the ligand binding groove. Maximal sequence diversity occurs within the flexible C-terminal stretch and it has been suggested that this region is involved in the regulation of binding properties of 14-3-3 proteins [2,5]. In addition, the C-terminal stretch of two vertebrate 14-3-3 isoforms (tau and zeta) contains a casein kinase Ia phosphorylation site at position 232 [6]. Phosphorylation of 14-3-3 proteins has been suggested as an important regulatory mechanism of individual isoforms [1].

We have used various methods of fluorescence spectroscopy to study structural changes of the C-terminal segment induced by ligand binding and phosphorylation at T232, a casein kinase I phosphorylation site located within this region. We have showed that the phosphopeptide binding changes the conformation and increases the flexibility of the 14-3-3 C-terminal stretch. Phosphorylation of 14-3-3 at T232 resulted in inhibition of ligand binding and significant change of the C-terminal stretch conformation [7]. In order to fully understand the role of the C-terminal stretch in the regulation of 14-3-3 protein binding properties we have also investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose we have used Förster resonance energy transfer (FRET) measurements and molecular dynamics simulation. These studies demonstrated that in the absence of the ligand the C-terminal stretch occupies the ligand binding groove of the 14-3-3 protein. The phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove of the 14-3-3 protein molecule [8].

Our results are consistent with the hypothesis suggesting that the 14-3-3 C-terminal stretch functions as a suppressor of unspecific interactions between the 14-3-3 protein and inappropriate ligands. In the absence of the ligand the C-terminal stretch occupies ligand binding groove of the 14-3-3 molecule. Binding of the phosphopeptide with proper 14-3-3 binding motif displaces C-terminal stretch from the ligand binding groove. Phosphorylation of the T232, a casein kinase I phosphorylation site located within this region, likely modulates conformational changes of the C-terminal stretch and thus all its functions.

 

This work was supported by Grants 204/03/0714 and 309/02/1479 of the Grant Agency of the Czech Republic; by Grant B5011308 of the Grant Agency of the Czech Academy of Sciences; by Research Projects 1K03020, MSM 1131 00001, and 1132 00001 of the Ministry of Education, Youth and Sports of the Czech Republic, and by Research Project AVOZ 5011922.

 

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