Spatiotemporal mapping of PKA activity using biosensors

Abstract

Cyclic AMP-dependent protein kinases (PKA) are heterotetrameric enzymes whose activity is regulated by local concentrations of cAMP. cAMP/PKA signaling pathways regulate a variety of physiological processes, including calcium signaling, ion channel activity, exocytosis, intracellular transport mechanisms and cell cycle.1 The importance of the subcellular targeting of the cAMP/PKA pathway in the determination of its physiological output renders its study in the native context of a living cell, and with adequate spatio-temporal resolution, imperative. Classical methods of cell biology and biochemistry, which often rely on cell population, and usually involve cell fixation, cell lysis and pharmacological or genetical perturbation of the whole system, do not fulfill these requirements.2 In this context, genetically encoded sensors appear as a very attractive alternative as they allow direct visualization of signaling processes in living biological systems. As they are genetically encoded, these tools can also be introduced into specific cell populations in living organisms, or targeted to subcellular compartments. In recent years, genetically encoded sensors for kinase activity have been developed to monitor phosphorylation events. Among them are the A-kinase activity reporters (AKARs), composed of an A kinase-specific substrate peptide and a phosphoamino-acid-binding domain (PAABD) that are sandwiched between 2 fluorescent proteins that can undergo FRET. PAABD binds the substrate peptide once it is phosphorylated by PKA, resulting in a conformational modification, which causes a change in FRET.3 FRET can be detected by the fluorescent ratio acceptor/donor (ratiometric imaging), or the donor fluorescence lifetime (fluorescence lifetime imaging).4 Properties of the biosensor, such as pH sensitivity, dynamic range, and resistance to bleaching, can be adjusted by changing the fluorescent proteins or the linker between the 2 sensors moieties.5 One should remember that the substrate peptide in AKAR is also the target of phosphatases that will reverse the phosphorylation mediated by PKA. Consequently, AKARs are more reporters of the changes in kinase/phosphatase activity balance than a true reporter of PKA activity.3 In a recent issue of Cell Cycle, Vandame et al. used an AKAR-derived biosensor, AKAREV, to detect PKA activity during mitosis in HeLa cells.6 The spatiotemporal resolution of the sensor allowed them to demonstrate high levels of PKA activity near the chromosal plate during metaphase and anaphase. In the same way, differences in the local activity of PKA were demonstrated during anaphase with a strong activity detected near the chromatin and a lower one between the 2 bundles of chromosomes. Interestingly, the authors produced an inactive form of the sensor, AKAREV T>A, to discard any potential artifact. AKAREV T>A consists in a point mutant in which the phosphorylable threonine in the substrate is replaced by an alanine. The T>A replacement makes the phosphorylation of the sensor impossible, thus suppressing any variation of the FRET signal in response to changes in PKA activity, as demonstrated by the absence of response to the application of forskolin. The physiological question investigated in this article is a perfect example of how biosensors can bring new insights into well-studied processes. In this case, the data obtained are consistent with literature suggesting that PKA activity during mitosis is involved in chromosome positioning. In addition, the experimental design is almost a school case on how to collect and carefully analyze data using biosensors. Fluorescent sensors are extremely attractive tools for cell biologists as they allow direct, real time visualization of cell signaling. As the technique becomes more and more popular, researchers tend to forget that, as appealing as the sensor response can be, what you see is NOT what you get. Several parameters of the sensors, such as kinetics, dynamic range, and artifacts, such as pH or chloride sensitivity, need to be taken into account while interpreting the signal. The use of negative controls, as illustrated in Vandame et al.,6 should become a standard protocol as it is for other techniques such as immunocytochemistry or western blot. Genetically encoded sensors are shedding light on a variety of cellular processes in the context of living cells and organisms. Since this technique is generating new data that could not be obtained before, we have to be aware of its limitations to avoid misinterpretations. Enlightened use of these powerful tools will undoubtedly allow the dissection of disregarded signaling pathways.