Characterization of the interactions between inhibitor-1 and recombinant PP1 by NMR spectroscopy

Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activ...

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Bibliographic Details
Published in:Scientific reports 2018-01, Vol.8 (1), p.50, Article 50
Main Authors: Liang, Chu-Ting, Lin, Yu-Shan, Huang, Yi-Choang, Huang, Hsien-Lu, Yang, Jia-Qian, Wu, Tsung-Hsien, Chang, Chi-Fon, Huang, Shing-Jong, Huang, Hsien-Bin, Lin, Ta-Hsien
Format: Article
Language:English
Subjects:
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Cyclic AMP-Dependent Protein Kinases - chemistry
Cyclic AMP-Dependent Protein Kinases - metabolism
Humanities and Social Sciences
Kinases
Magnetic Resonance Spectroscopy
Models, Molecular
multidisciplinary
NMR
Nuclear magnetic resonance
Phosphoprotein phosphatase
Phosphorylation
Protein Binding
Protein Conformation
Protein kinase A
Protein phosphatase
Protein Phosphatase 1 - chemistry
Protein Phosphatase 1 - metabolism
Proteins - chemistry
Proteins - metabolism
Science
Science (multidisciplinary)
Site-directed mutagenesis
Spectrum analysis
Structure-Activity Relationship
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Summary:Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombinant PP1, resulting in PKA-thiophosphorylated form of inhibitor-1 displaying a different affinity to native and recombinant enzyme.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-017-18383-x