Detection of Phosphate Ion and Protein Phosphorylation — Crystal Surfaces, Ionophore Monolayers, and Protein Interactions

Abstract

Several approaches for molecular recognition and detection of phosphate ions and of protein phosphorylation were reviewed herein. Crystal surfaces of a phosphate salts were formed to provide a remarkably selective molecular recognition site at which chemisorbed phosphate ions were detected by a underlying quartz crystal microbalance. Monolayers of phosphate receptors based on multitopic hydrogen bonds were found to function as a phosphate recognition site, where the phosphate ion is detected voltammetrically at their interface with aqueous phosphate solution, using an ion-channel mimetic sensing technique. Finally, a general method was described for genetically encoded fluorescent indicators to visualize the protein phosphorylation in living cells. The principle is phosphorylation-induced protein–protein interactions, which is converted to a signal of fluorescence resonance energy transfer.

Introduction

Several recent works from our research group on molecular recognition and detection of phosphate ions and of protein phosphorylation are reviewed herein. The site/domain or step for the molecular recognition and detection used respectively were crystal surfaces of phosphate salts,¹ phosphate-ionophore monolayers for phosphate detection,² and protein/protein interactions for protein phosphorylation.³

Molecular recognition and detection of inorganic anions and organic anions have been a topic of fundamental and practical importance.⁴ Molecular recognition of phosphate ions in aqueous solution has hardly been successful, because a highest hydrophilic nature of the phosphate ion makes it difficult for its direct binding with any synthetic receptors in aqueous solutions. In the human body, phosphate plays important roles not only in control of the pH in blood or lymph fluid, but also in the energy and nitrogen metabolism in cells. Moreover, the phosphate analysis in soil and wastewater are indexes of nutrition, environmental monitoring, and biomedical research. The analysis of orthophosphate ion is thus very important in clinical chemistry, environmental chemistry, biochemistry, pharmacology and others. Although many analytical methods for phosphate have been reported, most of inorganic and organic phosphorus compounds are quantified by spectrophotometric methods, using the formation of molybdenum blue. The reason for the fact that this technique is widely adopted is its excellent sensitivity to phosphate. However, on the other hand, the system is complicated and remains unsuitable for application to automated sensing and feedback control. Chemical sensing systems that can continuously and selectively monitor orthophosphate levels in aqueous solutions would therefore be extremely useful; thus, there is a clear need for well-functioning orthophosphate sensors in situ.

Protein phosphorylation by intracellular kinases plays one of the most pivotal roles in signaling pathways within cells (Fig. 1).⁵ The kinase proteins catalyze transfer of the γ-phosphate of ATP and phosphorylation of hydroxy groups of serines, threonines and/or tyrosines on the substrate proteins. Upon this phosphorylation, the substrate proteins are subject to conformational changes due to negative charges of the phosphates, which subsequently triggers their enzymatic activation and interaction with their respective target proteins. To reveal the biological issues related to the kinase proteins, electrophoresis, immunocytochemistry and in vitro kinase assay have been used. Recently, Bastiaens’ groups have improved an immunofluorescence staining method to detect protein phosphorylation in the cells and tissues.6, 7, 8 However, these conventional methods do not provide enough information about spatial and temporal dynamics of the signal transduction based on protein phosphorylation and dephosphorylation in living cells. In contrast to the kinase signaling, second messenger signaling, such as Ca²⁺,9, 10, 11 inositol 1,4,5-triphosphate,¹² diacylglycerol,¹³ cyclic AMP14, 15 and cyclic GMP,16, 17 have been visualized using fluorescent indicators in single living cells. The measurements based on those fluorescent indicators have been found to provide a high spatial and temporal resolution enough for dissecting the single cell events of the second messengers.¹⁸