[show abstract][hide abstract] ABSTRACT: 14-3-3 proteins regulate cellular responses to stimuli by docking onto pairs of phosphorylated residues on target proteins. The present study shows that the human 14-3-3-binding phosphoproteome is highly enriched in 2R-ohnologues, which are proteins in families of two to four members that were generated by two rounds of whole genome duplication at the origin of the vertebrates. We identify 2R-ohnologue families whose members share a 'lynchpin', defined as a 14-3-3-binding phosphosite that is conserved across members of a given family, and aligns with a Ser/Thr residue in pro-orthologues from the invertebrate chordates. For example, the human receptor expression enhancing protein (REEP) 1-4 family has the commonest type of lynchpin motif in current datasets, with a phosphorylatable serine in the -2 position relative to the 14-3-3-binding phosphosite. In contrast, the second 14-3-3-binding sites of REEPs 1-4 differ and are phosphorylated by different kinases, and hence the REEPs display different affinities for 14-3-3 dimers. We suggest a conceptual model for intracellular regulation involving protein families whose evolution into signal multiplexing systems was facilitated by 14-3-3 dimer binding to lynchpins, which gave freedom for other regulatory sites to evolve. While increased signalling complexity was needed for vertebrate life, these systems also generate vulnerability to genetic disorders.
[show abstract][hide abstract] ABSTRACT: How does signalling via PI3K-PKB (AKT)-mTORC1-p70S6K and ERK-p90RSK mediate wide-ranging physiological responses to insulin? Quantitative proteomics and biochemical experiments are revealing that these signalling pathways induce the phosphorylation of large and overlapping sets of proteins, which are then captured by phosphoprotein-binding proteins named 14-3-3s. The 14-3-3s are dimers that dock onto dual-phosphorylated sites in a configuration with special signalling and mechanical properties. They interact with the Rab GTPase-activating proteins AS160 and TBC1D1 to regulate glucose uptake into target tissues in response to insulin and energy stress. Dynamic patterns in the 14-3-3-binding phosphoproteome are providing new insights into how insulin triggers coherent shifts in metabolism that are integrated with other cellular response systems.
Trends in Endocrinology and Metabolism 08/2011; 22(11):429-36. · 8.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease.