FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2 +/PKA crosstalk
Introduction
Intracellular signal transduction requires precise physical interactions between specific signaling proteins within a receptor-directed signaling cascade. It is now clear that many of these protein–protein interactions are facilitated by scaffold proteins and not by random diffusion [1]. A-Kinase Anchoring Proteins (AKAPs) play an integral role in this by compartmentalizing cAMP-dependent protein kinase (PKA) and other enzymes to specific subcellular locations. AKAPs share a conserved amphipathic helical domain that binds the regulatory subunit PKA and a subcellular targeting domain that serves to anchor PKA and often additional kinases, phosphatases, and other regulatory enzymes to a diverse array of subcellular compartments [reviewed in 2]. Interestingly, some AKAPs are more than static “anchors”, but can traffic to alternative subcellular compartments in response to stimuli [3], [4], [5], [6].
Gravin (AKAP12), a 300 kDa AKAP with dramatic spatial targeting dynamics, anchors PKA and a host of other signaling enzymes to the plasma membrane through an N-myristoylation site and three polybasic domains (PB1-3). In response to either PKC activation or intracellular calcium ([Ca2 +]i) elevation, gravin is redistributed away from the membrane along with PKA that is bound to gravin. Gravin redistribution by PKC activation was shown by Yan et al. [7] to redirect gravin and PKA to a juxtanuclear vesicular compartment. In response to [Ca2 +]i elevation, Tao et al. [8] showed that gravin redistributes to the cytosol through a mechanism thought to involve Ca2 +/calmodulin binding to gravin's membrane-associated polybasic domains, PB1-3. A recent study from our laboratory further revealed that Ca2 +-mediated gravin redistribution triggers the relocalization of PKA away from the membrane, and a fourth putative calmodulin binding domain which we call CB4 may also be critical in this event [9]. Furthermore, we also showed that receptor-mediated signaling triggers gravin/PKA redistribution to the cytosol through a mechanism involving both calcium and PKC [9]. These findings raise the interesting possibility that gravin serves as a membrane-localized “switch” that can direct PKA away from the plasma membrane to alternative subcellular compartments in response to Ca2 +- and/or PKC signaling, thus facilitating crosstalk between these ubiquitous signaling pathways. However, gravin's impact on subcellular PKA activity, both basally and following Ca2 + mediated redistribution, is poorly understood. This could have important implications for disease contexts that utilize crosstalk between Ca2 +/PKC-dependent and PKA-dependent signaling pathways, such as cellular migration [10], [11], [12], cancer [reviewed in 13], learning and memory [14], cardiac function [15], and vascular biology [16], [17].
In the current study, we investigated the role of gravin in shaping subcellular PKA activity levels and in mediating crosstalk between Ca2 + and PKA-dependent signaling pathways. Gravin's role in targeting PKA to the plasma membrane suggests that gravin potentiates PKA signaling at the plasma membrane. This in turn implies that Ca2 + elevation may diminish plasma membrane PKA activity by triggering the redistribution of gravin/PKA into the cytosol. We hypothesize that through this mechanism of redistribution, gravin mediates cross-talk between calcium and PKA-dependent signaling pathways. We tested this hypothesis by targeting the genetically encoded FRET-based PKA biosensor AKAR3 to the plasma membrane and to the cytosol [18] and measuring the impact of exogenous gravin expression on compartmentalized PKA activity within these compartments. In addition, we tested the impact of calcium-mediated gravin redistribution on plasma membrane PKA activity.
Section snippets
Cell culture and transfection
AN3 CA cells (Manassas, VA, ATCC number: HTB-111), a human endometrial metastatic cancer cell line that does not express endogenous gravin, and HEC 1A cells (ATCC HTB-112) were maintained at 37 °C with 5% CO2 in low glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 100 units/ml penicillin and 100 μg/ml streptomycin. Growth medium was replaced three times each week and cells were split 1:25 upon reaching confluence.
Cells were plated at 25,000 cells per cm2
Forskolin treatment stimulates AKAR3 dynamics
Fluorescent micrographs of AN 3CA cells in Fig. 1A illustrate the dynamics of the AKAR3 biosensor following treatment with the agonist forskolin, which stimulates PKA activation through adenylyl cyclase-mediated elevation of cAMP. In response to forskolin treatment, AKAR3 undergoes a conformational change that positions CFP and Venus closer to one another (Fig. 1E), and this triggers a drop in CFP intensity and a corresponding increase in FRET intensity (Fig. 1A,B). These changes were measured
Discussion
In the current study, we investigated the role of gravin in shaping compartmentalized PKA activity and in mediating crosstalk between Ca2 + and PKA-dependent signaling pathways. We showed that upon stimulation with forskolin or isoproterenol, cells expressing gravin had elevated levels of plasma membrane (PM) PKA activity and reduced levels of cytosolic PKA activity. Gravin-PKA interaction at the PM was required for these alterations, as mutant gravin constructs lacking PKA interaction ([ΔPKA]
Conclusions
In conclusion, we report that gravin expression modulates both plasma membrane and cytosolic PKA activity levels and mediates a novel crosstalk mechanism between Ca2 + and PKA-dependent signaling pathways. This mechanism occurs through the Ca2 + mediated redistribution of gravin/PKA away from the plasma membrane, which in turn impacts PKA activity levels at the membrane. The current study lays important groundwork for future investigation of gravin-mediated Ca2 +/PKA crosstalk in physiological
Acknowledgments
This work was supported by NIH P30GM103329. We gratefully acknowledge Dr. Jin Zhang (Johns Hopkins University) for providing AKAR3-CAAX and AKAR3-NES constructs. In addition, the authors acknowledge use of the Edward C. Carlson Imaging and Image Analysis Core Facility which is also supported in part by NIH grant P30GM103329 and NIH Shared Instrumentation Grant 1S100D016250-01.
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Milestones in the development and implementation of FRET-based sensors of intracellular signals: A biological perspective of the history of FRET
2020, Cellular SignallingCitation Excerpt :The first approach is to target FRET probes to discrete cellular locations. These locations may be specific proteins (e.g., AKAPs [25,92–94]) or subcellular domains (e.g., plasma membrane and nucleus [95–97]). The advantages of this approach are several-fold:
Role of cyclic nucleotides and their downstream signaling cascades in memory function: Being at the right time at the right spot
2020, Neuroscience and Biobehavioral ReviewsCitation Excerpt :Along similar lines, a study from Schott et al. in which they targeted AKAR3 to the plasma membrane or the cytosol, reported that gravin (or AKAP12) impacts subcellular PKA activity levels through the spatial targeting of PKA in response to Ca2+ elevation. The above results support the hypothesis that gravin mediates crosstalk between Ca2+ and PKA-dependent signaling pathways (Schott et al., 2016). A modified version of AKAR4 that was targeted at lipid raft and non-raft regions of the plasma membrane gave first insight into the compartmentalization of PKA activity in the different microdomains in the membrane (Depry et al., 2011).
cAMP regulation of protein phosphatases PP1 and PP2A in brain
2019, Biochimica et Biophysica Acta - Molecular Cell ResearchCitation Excerpt :In the case of PKA, adaptor proteins that ensure the proximity of PKA to target substrates have long been recognized and appreciated for their role in regulating PKA activity [8,15,21–23]. For example, A-kinase anchoring proteins (AKAPs) create hubs of PKA regulation [21–25] by binding PKA itself as well as both the sources of cAMP production, ACs [26], and degradation, PDEs [27,28]. Thus, the regulation of PKA activity is a highly orchestrated process regulated by a variety of players in a spatiotemporally controlled manner.
Potential for therapeutic targeting of AKAP signaling complexes in nervous system disorders
2018, Pharmacology and TherapeuticsCitation Excerpt :These mechanisms include the rapid redistribution of Gravin-PKA to intracellular membrane compartments following stimulation of PKC (Lin et al., 1996; Wassler et al., 2001; Yan et al., 2009; Schott & Grove, 2013) or in response to increases in intracellular Ca2 + that activate Ca2 +-CaM. The interaction between Ca2 +-CaM and gravin occurs at the CB4 putative CaM binding site and is thought to play a key role in Ca2 +-CaM-dependent redistribution (Schott & Grove, 2013; Schott et al., 2016). Mechanisms such as these that direct the signaling complex to intracellular compartments may serve to limit PKA activity at the cell membrane.