Oligopeptide Competition Assay for Phosphorylation Site Determination

Min Sung Joo; Ja Hyun Koo; Sol-Bi Shin; Hyungshin Yim; Sang Geon Kim

doi:10.3791/55708

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Method Article

Oligopeptide Competition Assay for Phosphorylation Site Determination

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DOI:

10.3791/55708

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May 18th, 2017

Min Sung Joo¹ , Ja Hyun Koo¹ , Sol-Bi Shin² , Hyungshin Yim² , Sang Geon Kim¹

¹College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, ²College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University

Summary

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

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 established¹ and was validated using a positional scanning peptide library assay². 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.

Introduction

Protein phosphorylation at a specific residue plays a significant role in a wide range of cellular processes. Thus, an understanding of signaling networks requires the identification of specific phosphorylation sites. In addition, the phosphorylation site determines the effect on protein function because individual domains within a protein possess different structures and functions. The activity of nuclear factor erythroid 2-related factor 2 (Nrf2), a key antioxidant transcription factor, is bi-directionally regulated through phosphorylation at different sites. Our studies have focused on the kinases that catalyze the phosphorylation of Nrf2. The stress response of Nr....