title: Dual fluorescent molecular substrates selectively report the activation, sustainability and reversibility of cellular PKB/Akt activity.
abstract: 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.
authors.affiliation: Department of Radiology, Washington University in St. Louis, Missouri 63110, USA.
authors.forename: Duanwen
authors.initials: D
authors.lastname: Shen
authors.forename: Mingfeng
authors.initials: M
authors.lastname: Bai
authors.forename: Rui
authors.initials: R
authors.lastname: Tang
authors.forename: Baogang
authors.initials: B
authors.lastname: Xu
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Ju
authors.forename: Richard G
authors.initials: RG
authors.lastname: Pestell
authors.forename: Samuel
authors.initials: S
authors.lastname: Achilefu
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling / Q000379:methods*; D061986:MCF-7 Cells; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D051058:Oncogene Protein v-akt / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D064413:3-Phosphoinositide-Dependent Protein Kinases; D051058:Oncogene Protein v-akt; D017346:Protein Serine-Threonine Kinases