A simple method using 31P-NMR spectroscopy for the study of protein phosphorylation.

Nonradioactive 31P-NMR spectroscopy has previously been used for the study of protein phosphorylations. However, the procedures does not seem to be easy for non-experts of this field, hence, this approach has not been widely used. We introduce here a simple protocol with 31P-NMR spectroscopy to study in vitro phosphorylation in receptor proteins. The effectiveness of this method was verified using synthetic peptides and recombinant proteins of the C-terminus of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor, whose phosphorylations are considered to have important roles in synaptic plasticity. We show that a decrease in the pH of the sample solution after the phosphorylation reaction is critical for the separation of the phosphorylation signals. In the analysis of the C-terminal portion of the GluR2 AMPA receptor, the phosphorylation sites of which had not hitherto been well clarified, we found the presence of at least three protein kinase C (PKC) phosphorylation sites. Furthermore, this method allows prediction of the origins of each of the phosphorylation peaks. Thus, the techniques we described here is useful for examination of protein phosphorylation and permits us to safely conduct repetitive experiments.