A structural basis for 14-3-3sigma functional specificity.
The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.
Wilker
Erik W
EW
Center for Cancer Research, Department of Biology and Division of Biological Engineering, Massachsuetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Grant
Robert A
RA
Artim
Stephen C
SC
Yaffe
Michael B
MB
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism / Q000502:physiology*; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000737:chemistry; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D004249:DNA Damage; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D005092:Exonucleases / Q000378:metabolism / Q000502:physiology*; D005095:Exoribonucleases; D005822:Genetic Vectors; D006801:Humans; D047468:Immunoprecipitation; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism / Q000502:physiology*; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013379:Substrate Specificity; D014162:Transfection; D020687:cdc25 Phosphatases / Q000737:chemistry
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D018797:Cell Cycle Proteins; D008024:Ligands; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases