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1.  Visualization and Biochemical Analyses of the Emerging Mammalian 14-3-3-Phosphoproteome* 
Molecular & Cellular Proteomics : MCP  2011;10(10):M110.005751.
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 (, 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.
PMCID: PMC3205853  PMID: 21725060
2.  Bioinformatic and experimental survey of 14-3-3-binding sites 
Biochemical Journal  2010;427(Pt 1):69-78.
More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although the +2 proline residue occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different subtypes of 14-3-3-binding sites that overlap with the specificities of different basophilic AGC (protein kinase A/protein kinase G/protein kinase C family) and CaMK (Ca2+/calmodulin-dependent protein kinase) protein kinases, and a 14-3-3 dimer often engages with two tandem phosphorylated sites, which is a configuration with special signalling, mechanical and evolutionary properties. Thus 14-3-3 dimers can be digital logic gates that integrate more than one input to generate an action, and coincidence detectors when the two binding sites are phosphorylated by different protein kinases. Paired sites are generally located within disordered regions and/or straddle either side of functional domains, indicating how 14-3-3 dimers modulate the conformations and/or interactions of their targets. Finally, 14-3-3 proteins bind to members of several multi-protein families. Two 14-3-3-binding sites are conserved across the class IIa histone deacetylases, whereas other protein families display differential regulation by 14-3-3s. We speculate that 14-3-3 dimers may have contributed to the evolution of such families, tailoring regulatory inputs to different physiological demands.
PMCID: PMC2860806  PMID: 20141511
14-3-3 protein; AGC protein kinase; Ca2+/calmodulin-dependent protein kinase; disrupted-in-schizophrenia 1 (DISC1); evolution; AANAT, serotonin acetyltransferase; AGC, protein kinase A/protein kinase G/protein kinase C family kinase; AMPK, AMP-activated protein kinase; BAD, Bcl-XL/Bcl-2-associated death promoter; CaMK, Ca2+/calmodulin-dependent protein kinase; CDK5, cyclin-dependent kinase 5; DIG, digoxigenin; DISC1, disrupted-in-schizophrenia 1; DSTT, Division of Signal Transduction Therapy; EST, expressed sequence tag; FOXO, Forkhead box O; GLUT4, glucose transporter 4; GST, glutathione transferase; HA, haemagglutinin; HAP1A, Huntingtin-associated protein 1A; HDAC, histone deacetylase; HEK, human embryonic kidney; KLC, kinesin light chain; MARK, microtubule affinity-regulating kinase; PI4K, phosphoinositide 4-kinase; PKB, protein kinase B; PKC, protein kinase C; PP2A, protein phosphatase 2A; RSK, ribosomal S6 kinase; YAP1, yes-associated protein 1

Results 1-2 (2)