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The 14-3-3 protein binds its target proteins with a common site located towards the C-terminus
1Department of Biochemistry, Faculty of Science, Niigata University, Japan.
Abstract:
The 14-3-3 protein family binds a variety of proteins in cell-signaling pathways, but the structural elements necessary for the ligand binding are poorly understood. Here we demonstrate that the 'box-1' region, which spans residues 171-213 in the eta-isoform and was previously identified as the binding site of 14-3-3 to the phosphorylated tryptophan hydroxylase, plays a critical role in the interaction with many target proteins. Using a series of truncated 14-3-3 mutants, we show that the mutant 167-213 carrying box-1 binds bacurovirus-expressed Raf-1 and Bcr protein kinases to the similar extent as the full-length 14-3-3 in a phosphorylation-dependent manner, while the mutants lacking this region abolish the binding activity. Furthermore, the box-1 region also appears essential for binding of 14-3-3 to more than 40 phosphoproteins found in the brainstem extract. These results suggest that the box-1 region, consisting of helices 7 and 8 in the tertiary structure, is a common structural element whereby the 14-3-3 protein binds many, if not all, target proteins.
Insights
The 14-3-3 protein's "box-1" region is crucial for binding numerous target proteins in cell signaling. This structural element, comprising helices 7 and 8, is essential for interactions with over 40 phosphoproteins.
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein Structure
Background:
- The 14-3-3 protein family regulates diverse cellular processes by interacting with numerous client proteins.
- The specific structural determinants governing 14-3-3 protein interactions with its diverse ligands remain incompletely characterized.
Purpose of the Study:
- To identify the critical structural elements within the 14-3-3 protein responsible for its broad ligand-binding capabilities.
- To elucidate the role of the 'box-1' region in mediating interactions with various phosphoproteins.
Main Methods:
- Utilized a series of truncated 14-3-3 eta-isoform mutants to probe the function of the 'box-1' region.
- Assessed binding affinities of 14-3-3 mutants to baculovirus-expressed Raf-1 and Bcr protein kinases.
- Investigated 14-3-3 binding to phosphoproteins in brainstem extracts.
Main Results:
- The 'box-1' region (residues 171-213) was found to be essential for 14-3-3 binding to Raf-1 and Bcr kinases in a phosphorylation-dependent manner.
- Mutants lacking the 'box-1' region exhibited abolished binding activity, while a mutant containing only this region retained significant binding.
- The 'box-1' region demonstrated essentiality for 14-3-3 interactions with over 40 distinct phosphoproteins from brainstem extracts.
Conclusions:
- The 'box-1' region, encompassing helices 7 and 8, serves as a common and critical structural motif for 14-3-3 protein interactions.
- This finding suggests a conserved mechanism for 14-3-3 protein engagement with a wide array of its target proteins.