Dataset Information

Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3? Protein.

ABSTRACT: Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3? to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3? protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615-630) in the presence of 14-3-3? protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3? protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3? protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3? binding at an atomic resolution.

SUBMITTER: Ghosh A

PROVIDER: S-EPMC4546627 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein.

Ghosh Anirban A Ratha Bhisma Narayan BN Gayen Nilanjan N Mroue Kamal H KH Kar Rajiv K RK Mandal Atin K AK Bhunia Anirban A

PloS one 20150821 8

Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possib ...[more]

PMID: 26295714

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Project description:ObjectiveMetabolic stress primes monocytes for accelerated chemokine-mediated adhesion, migration, and recruitment into vascular lesions by increasing actin remodeling. The mechanism linking metabolic stress to accelerated actin turnover and enhanced monocyte migration was not known. We tested the hypothesis that in metabolically primed monocytes, the acceleration of monocyte chemoattractant protein-1-induced chemotaxis is mediated by the hyperactivation of cofilin.Approach and resultsMetabolic priming was induced by exposing human THP-1 monocytes to diabetic conditions, that is, human native low-density lipoprotein plus high glucose concentrations. In healthy monocytes, monocyte chemoattractant protein-1 induced the phosphorylation and inactivation of cofilin. This response was completely blocked in metabolically primed monocytes but restored by overexpression of the thiol transferase, glutaredoxin 1. Cofilin kinase, LIM kinase 1, and cofilin phosphatase, Slingshot-1L, were not affected by metabolic stress. However, metabolic priming increased 3.8-fold the S-glutathionylation of the Slingshot-1L-binding protein 14-3-3ζ (zeta), resulting in its caspase-dependent degradation. Glutaredoxin 1 overexpression inhibited low-density lipoprotein plus high glucose-induced S-glutathionylation and degradation of 14-3-3ζ. The C25S mutant of 14-3-3ζ was resistant to both S-glutathionylation and degradation induced by low-density lipoprotein plus high glucose. Overexpression of the C25S mutant restored monocyte chemoattractant protein-1-induced cofilin phosphorylation and prevented accelerated migration of metabolically stressed monocytes, suggesting that loss of 14-3-3ζ increases the pool of free Slingshot-1L phosphatase, thereby preventing the phosphorylation and deactivation of cofilin in response to chemokine activation.ConclusionsBy preventing the inactivation of cofilin, metabolic stress-induced degradation of 14-3-3ζ promotes the conversion of blood monocytes into a hypermigratory, proatherogenic phenotype.

Dataset Information

Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3? Protein.

Publications

Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein.

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