Imaging protein phosphorylation by fluorescence in single living cells

Abstract

Protein phosphorylation by intracellular kinases plays one of the most pivotal roles in signaling pathways within cells. To reveal the biological issues related to the kinase proteins, electrophoresis, immunocytochemistry, and in vitro kinase assay have been used. 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. To overcome the limitation for investigating the kinase signaling, we developed genetically encoded fluorescent indicators for visualizing the protein phosphorylation in living cells. Using these indicators, we visualized under a fluorescence microscope when, where, and how the protein kinases are activated in single living cells.

Introduction

To visualize signal transduction based on protein phosphorylation in living cells, we have developed genetically encoded novel fluorescent indicators (Fig. 1) [1]. A substrate domain for a protein kinase of interest is fused with a phosphorylation recognition domain via a flexible linker sequence. The tandem fusion unit consisting of the substrate domain, linker sequence, and phosphorylation recognition domain is sandwiched with two different color fluorescent proteins, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), which serve as the donor and acceptor fluorophores for fluorescence resonance energy transfer (FRET). As a result of phosphorylation of the substrate domain and subsequent binding of the phosphorylated substrate domain with the adjacent phosphorylation recognition domain, FRET is induced between the two fluorescent units, which elicits the phosphorylation-dependent changes in fluorescence emission ratios of the donor and acceptor fluorophores. Upon activation of phosphatases, the phosphorylated substrate domain is dephosphorylated and the FRET signal is decreased. We named this indicator “phocus” (a fluorescent indicator for protein phosphorylation that can be custom-made). By using suitable substrate and phosphorylation recognition domains, we have developed a large number of phocuses for several key protein kinases including a receptor tyrosine kinase, insulin receptor [1], a serine/threonine protein kinase, Akt/PKB [2], and a non-receptor tyrosine kinase, c-Src [3] (Table 1). In addition, these phocuses have been further tailored to visualize local activity of the respective protein kinases in living cells by fusing appropriate localization sequences/domains with each phocus (Table 1).