Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

BECN1/Beclin 1 is a critical protein in the initiation of autophagosome formation. Recent studies have shown that phosphorylation of BECN1 by STK4/MST1 at threonine 108 (T108) within its BH3 domain blocks macroautophagy/autophagy by increasing BECN1 affinity for its negative regulators, the anti-apoptotic proteins BCL2/Bcl-2 and BCL2L1/Bcl-x_L. It was proposed that this increased binding is due to formation of an electrostatic interaction with a conserved histidine residue on the anti-apoptotic molecules. Here, we performed biophysical studies which demonstrated that a peptide corresponding to the BECN1 BH3 domain in which T108 is phosphorylated (p-T108) does show increased affinity for anti-apoptotic proteins that is significant, though only minor (<2-fold). We also determined X-ray crystal structures of BCL2 and BCL2L1 with T108-modified BECN1 BH3 peptides, but only showed evidence of an interaction between the BH3 peptide and the conserved histidine residue when the histidine flexibility was restrained due to crystal contacts. These data, together with molecular dynamics studies, indicate that the histidine is highly flexible, even when complexed with BECN1 BH3. Binding studies also showed that detergent can increase the affinity of the interaction. Although this increase was similar for both the phosphorylated and non-phosphorylated peptides, it suggests factors such as membranes could impact on the interaction between BECN1 and BCL2 proteins, and therefore, on the regulation of autophagy. Hence, we propose that phosphorylation of BECN1 by STK4/MST1 can increase the affinity of the interaction between BECN1 and anti-apoptotic proteins and this interaction can be stabilized by local environmental factors. Abbreviations: asu: asymmetric unit; BH3: BCL2/Bcl-2 homology 3; DAPK: death associated protein kinase; MD: molecular dynamics; MST: microscale thermophoresis; NMR: nuclear magnetic resonance; PDB: protein data bank; p-T: phosphothreonine; SPR: surface plasmon resonance; STK4/MST1: serine/threonine kinase 4.
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PMID:Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1. 3062 84

¹⁹F NMR spectroscopy provides the ability to quantitatively analyze single species in complex solutions but is often limited by the modest sensitivity inherent to NMR. 4R- and 4S-Perfluoro-tert-buyl hydroxyproline contain 9 equivalent fluorines, in amino acids with strong conformational preferences. In order to test the ability to use these amino acids as sensitive probes of protein modifications, the perfluoro-tert-buyl hydroxyprolines were incorporated into substrate peptides of the protein kinases PKA and Akt. Peptides containing each diastereomeric proline were rapidly phosphorylated by each protein kinase and exhibited ¹⁹F chemical shift changes as a result of phosphorylation. The sensitivity of the perfluoro-tert-butyl group allowed quantitative analysis of the kinetics of phosphorylation over three half-lives at single-digit micromolar concentrations of each species. The distinct conformational preferences of these amino acids allowed the optimization of the substrate with a conformationally matched amino acid, in order to maximize the rate of phosphorylation. PKA preferred the 4R-amino acid at the -1 position, whereas the closely related AGC kinase Akt preferred the 4S-amino acid. These data, combined with analysis of structures of the Michaelis complexes of these kinases in the PDB, suggest that PKA recognizes the PPII conformation at the P-1 position relative to the phosphorylation site, while Akt/PKB recognizes an extended conformation at this position. These results suggest that conformational targeting may be employed to increase specificity in recognition by protein kinases. Perfluoro-tert-butyl hydroxyprolines were applied to the real-time detection and quantification of PKA activity and inhibition of PKA activity in HeLa cell extracts via ¹⁹F NMR spectroscopy. The coupling of proline ring pucker with main chain conformation suggests broad application of perfluoro-tert-butyl hydroxyprolines in molecular sensing and imaging.
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PMID:Perfluoro-tert-Butyl Hydroxyprolines as Sensitive, Conformationally Responsive Molecular Probes: Detection of Protein Kinase Activity by ¹⁹F NMR. 3212 21


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