Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity.

Shen D, Bai M, Tang R, Xu B, Ju X, Pestell RG, Achilefu S
Sci Rep. 2013 3: 1697

PMID: 23603888 · PMCID: PMC3631771 · DOI:10.1038/srep01697

Using a newly developed near-infrared (NIR) dye that fluoresces at two different wavelengths (dichromic fluorescence, DCF), we discovered a new fluorescent substrate for Akt, also known as protein kinase B, and a method to quantitatively report this enzyme's activity in real time. Upon insulin activation of cellular Akt, the enzyme multi-phosphorylated a single serine residue of a diserine DCF substrate in a time-dependent manner, culminating in monophospho- to triphospho-serine products. The NIR DCF probe was highly selective for the Akt1 isoform, which was demonstrated using Akt1 knockout cells derived from MMTV-ErbB2 transgenic mice. The DCF mechanism provides unparalleled potential to assess the stimulation, sustainability, and reversibility of Akt activation longitudinally. Importantly, NIR fluorescence provides a pathway to translate findings from cells to living organisms, a condition that could eventually facilitate the use of these probes in humans.

MeSH Terms (10)

3-Phosphoinositide-Dependent Protein Kinases Animals Enzyme Activation Fluorescent Dyes Gene Expression Profiling MCF-7 Cells Mice Microscopy, Fluorescence, Multiphoton Oncogene Protein v-akt Protein-Serine-Threonine Kinases

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