Molecular imaging of Akt kinase activity

Memorial Sloan-Kettering Cancer Center, New York, New York, United States
Nature Medicine (Impact Factor: 27.36). 10/2007; 13(9):1114-9. DOI: 10.1038/nm1608
Source: PubMed


The serine/threonine kinase Akt mediates mitogenic and anti-apoptotic responses that result from activation of multiple signaling cascades. It is considered a key determinant of tumor aggressiveness and is a major target for anticancer drug development. Here, we describe a new reporter molecule whose bioluminescence activity within live cells and in mice can be used to measure Akt activity. Akt activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to activation or inhibition of receptor tyrosine kinase, inhibition of phosphoinositide 3-kinase, or direct inhibition of Akt. The results provide unique insights into the pharmacokinetics and pharmacodynamics of agents that modulate Akt activity, revealing the usefulness of this reporter for rapid dose and schedule optimization in the drug development process.

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    • "*Author for correspondence ( approaches have been presented, including a bioluminescent sensor employing a split-luciferase reporter (Zhang et al., 2007), but they offer few advantages over FRET-based reporter molecules. Here, we describe a robust fluorescent translocation sensor for measuring Akt activity. "
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    ABSTRACT: The protein kinase Akt is a key intracellular mediator of many biological processes, yet knowledge of Akt signaling dynamics is limited. Here we have constructed a fluorescent reporter molecule in a lentiviral delivery system to assess Akt kinase activity at the single cell level. The reporter, a fusion between a modified FoxO1 transcription factor and clover, a green fluorescent protein, rapidly translocates from the nucleus to the cytoplasm in response to Akt stimulation. Because of its long half-life and the intensity of clover fluorescence, the sensor provides a robust readout that can be tracked for days under a range of biological conditions. Using this reporter, we find that stimulation of Akt activity by IGF-I is encoded into stable and reproducible analog responses at the population level, but that single cell signaling outcomes are variable. This reporter, which provides a simple and dynamic measure of Akt activity, should be compatible with many cell types and experimental platforms, and thus opens the door to new insights into how Akt regulates its biological responses. © 2015. Published by The Company of Biologists Ltd.
    Journal of Cell Science 06/2015; 128(14). DOI:10.1242/jcs.168773 · 5.43 Impact Factor
    • "Consequently, the data suggest that MAPKAR undergoes a conformational change that causes an increase in luminescence when ERK1 dephosphorylation and inactivation occurs (Fig. 1). Thus, a decreased interaction between the two ERK1 moieties within MAPKAR enables structural and functional reassociation of the N- and C-terminal domains of click beetle luciferase to reconstitute bioluminescence activity, as has been proposed for the firefly split-luciferase reporter of Akt kinase activity (Zhang et al., 2007). In order to make the interpretation of traces more intuitive, note that the luminescent recordings are inverted so that the inactivation of ERK1 corresponds to a downward deflection in the trace. "
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    ABSTRACT: Egg activation at fertilization in mammals is initiated by prolonged Ca(2+) oscillations that trigger the completion of meiosis and formation of pronuclei. A late fall in MAPK activity is essential for pronuclear formation, but the precise timing and mechanism of decline are unknown. Here, we have measured the dynamics of MAPK inactivation in fertilizing mouse eggs using novel chemiluminescent MAPK activity reporters. This reveals that the MAPK activity decrease begins during the Ca(2+) oscillations, but MAPK does not completely inactivate until after pronuclear formation. MAPK in eggs consists of Mos, MEK and ERK1/2. Notably, the MAPK activity decline at fertilization is not explained by upstream destruction of Mos, because a decrease in Mos-luciferase signal is not associated with egg activation. Further, Mos over-expression does not affect the timing of MAPK inactivation or pronuclear formation. However, the late decrease in MAPK could be rapidly reversed by the protein phosphatase inhibitor, okadaic acid. These data suggest that the completion of meiosis in mouse zygotes is driven by an increased phosphatase activity and not by a decline in Mos levels, or MEK activity.
    Journal of Cell Science 04/2014; 127(12). DOI:10.1242/jcs.145045 · 5.43 Impact Factor
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    • "Although the treatments were selected on the basis of their current clinical relevance and their targeted effects on bone or tumor cells, this generalizable approach is anticipated to be useful in future studies identifying responses to experimental agents by obtaining a more complete understanding of the signaling pathways affected. These and other cancer cell lines have already been successfully engineered to express luciferase linked to cellular signals such as AKT, TGF-β, c-MET, epidermal growth factor receptor (EGFR), and others [26] [28] [29] [31] [32]. "
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    ABSTRACT: Cancer drug development generally performs in vivo evaluation of treatment effects that have traditionally relied on detection of morphologic changes. The emergence of new targeted therapies, which may not result in gross morphologic changes, has spurred investigation into more specific imaging methods to quantify response, such as targeted fluorescent probes and bioluminescent cells. The present study investigated tissue response to docetaxel or zoledronic acid (ZA) in a mouse model of bony metastasis. Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored throughout this study using several modalities: molecular resonance imaging (MRI) tumor volume and apparent diffusion coefficient (ADC), micro-computed tomography (µCT) bone volume, bioluminescence imaging (BLI) reporting cancer cell apoptosis, and fluorescence using Osteosense 800 and CatK 680-FAST. Docetaxel treatment resulted in tumor cell kill reflected by ADC and BLI increases and tumor volume reduction, with delayed bone recovery seen in µCT prefaced by increased osteoblastic activity (Osteosense 800). In contrast, the ZA treatment group produced similar values in MRI, BLI, and Osteosense 800 fluorescence imaging readouts when compared to controls. However, µCT bone volume increased significantly by the first week post-treatment and the CatK 680-FAST signal was slightly diminished by 4 weeks following ZA treatment. Multimodality imaging provides a more comprehensive tool for new drug evaluation and efficacy screening through identification of morphology as well as function and apoptotic signaling.
    Translational oncology 12/2012; 5(6):415-21. DOI:10.1593/tlo.12298 · 2.88 Impact Factor
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