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
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.
Available from: Peter Rotwein
- "*Author for correspondence ( email@example.com) 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.
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- "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.
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- "GFP-Akt-YFP Calleja et al., 2003 B-Raf Prin-BRaf Terai and Matsuda, 2006 C-Raf Prin-CRaf Terai and Matsuda, 2005 Death associated protein kinase 1 (DAPK1) DAPK1 sensor Pilji c et al., 2011 CaMKII Camui Takao et al., 2005; Kwok et al., 2008 Erk Miu2 Fujioka et al., 2006 MAPK-activated protein kinase 2 (MK2) GMB Neininger et al., 2001 Myosin light-chain kinase (MLCK) exMLCK Geguchadze et al., 2004 P21-activated kinase 1 (PAK1) Pakabi Parrini et al., 2009 PDK1 PARE Gao et al., 2011 Activity Probes Protein kinase A AKAR Zhang et al., 2001; Komatsu et al., 2011 Abl kinase Abl indicator Ting et al., 2001 Akt AktAR Gao and Zhang, 2008; Komatsu et al. 2011 Aktus Sasaki et al., 2003 BAR Zhang et al., 2007 BKAR Kunkel et al., 2005 AMPK AMPKAR Tsou et al., 2011 Aurora B kinase Aurora B sensor Chu et al., 2011 ATM kinase Atomic Johnson et al., 2007 Protein kinase C CKAR Violin et al., 2003; Komatsu et al., 2011; Wu-Zhang et al., 2012 Cyclin-dependent kinase 1 Cdk1 FRET sensor Gavet and Pines, 2010 Protein kinase D DKAR Fuchs et al., 2009; Eisler et al., 2012 EGFR EGFR indicator Ting et al., 2001 Erk Erkus Sato et al., 2007 EKAR Harvey et al. 2008; Komatsu et al., 2011 REV Xu et al., 2013 Focal adhesion kinase (FAK) FAK sensor Seong et al., 2011 Insulin receptor Phocus Sato and Umezawa, 2004 c-Jun N-terminal kinase (JNK) JNKAR Fosbrink et al., 2010; Komatsu et al., 2011 Microtubule affinity regulating kinase (MARK) MARK sensor Timm et al., 2011 Polo-like kinase 1 Plk sensor Mac urek et al., 2008 RSK Eevee-RSK Komatsu et al., 2011 S6K Eevee-S6K Komatsu et al., 2011 Stress-activated protein kinase kinase kinase SAP3K activity reporter Tomida et al., 2009 Src Src biosensor Ting et al., 2001; Wang et al., 2005; Ouyang et al., 2008 188 Chemistry & Biology 21, February 20, 2014 ª2014 Elsevier Ltd All rights reserved "
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ABSTRACT: Fluorescence-based, genetically encodable biosensors are widely used tools for real-time analysis of biological processes. Over the last few decades, the number of available genetically encodable biosensors and the types of processes they can monitor have increased rapidly. Here, we aim to introduce the reader to general principles and practices in biosensor development and highlight ways in which biosensors can be used to illuminate outstanding questions of biological function. Specifically, we focus on sensors developed for monitoring kinase activity and use them to illustrate some common considerations for biosensor design. We describe several uses to which kinase and second-messenger biosensors have been put, and conclude with considerations for the use of biosensors once they are developed. Overall, as fluorescence-based biosensors continue to diversify and improve, we expect them to continue to be widely used as reliable and fruitful tools for gaining deeper insights into cellular and organismal function.
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