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Oligopeptide Competition Assay for Phosphorylation Site Determination

Title
Oligopeptide Competition Assay for Phosphorylation Site Determination
Author
임형신
Keywords
Biochemistry; Issue 123; In vitro kinase assay; peptide inhibitor; phosphorylation; AMPK; Nrf2; consensus motif; site-directed mutagenesis
Issue Date
2017-05
Publisher
Journal of Visualized Experiments
Citation
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, v. 2017, No. 123, Article no. e55708
Abstract
Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5' adenosine monophosphate-activated protein kinase (AMPK) has been established(1) and was validated using a positional scanning peptide library assay(2). Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.
URI
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608007/https://repository.hanyang.ac.kr/handle/20.500.11754/128446
ISSN
1940-087X
DOI
10.3791/55708
Appears in Collections:
COLLEGE OF PHARMACY[E](약학대학) > PHARMACY(약학과) > Articles
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