The ability to both monitor and precisely manipulate levels of cAMP at different subcellular loci is important for fully understanding the role of compartmentalized cAMP signaling in cells. Towards this end, we describe a new method, SMICUS, for dictating the location, kinetics and magnitude of cAMP signal via manipulation of sAC using combined chemical and genetic controls. Using bicarbonate stimulation and washout, sAC-mediated cAMP production is under tight temporal control. Furthermore, we can also dictate where in the cell cAMP is being produced by using genetically encoded localization signals to target sACt
subcellularly. Second messenger generation in microdomains, such as mitochondria and vesicles, as well as nanodomains like membrane rafts, is made possible since the site of cAMP generation is dictated by molecular targeting rather than light as in the case of photolytic uncaging of cAMP analogs32
. Compared to the uncaging method, activation of sAC leads to more efficient generation of cAMP within the cell due to its catalytic nature. In addition, sAC has the potential for being expressed in tissues or in transgenic models for the study of cAMP signaling pathways in a multicellular context. More recently, light-sensitive adenylyl cyclases from Euglena gracilis
and the soil bacteria Beggiatoa
have been exploited to generate cAMP within the cell upon stimulation with light33,34
, although generation of cAMP in specific organelles has yet to be demonstrated by this method. These new approaches should allow for organelle-specific and efficient generation of cAMP under strict temporal control, thereby enabling mechanistic dissection of the spatiotemporal regulation of cAMP signaling in specific subcellular compartments.
On the computational modeling front, we adopted a unique strategy of assaying model structures via parameter estimation to infer the regulatory structure of a cell signaling network. Mechanistic models are often presumed to contain an appropriate structure and are used as surrogates for an experimental system35
. Here, we draw upon findings in information theory to directly infer and test different model structures when they are not fully known a priori22
. While traditional approaches in systems biology seek to quantify known relationships or integrate large data sets36
, our approach here illustrates a relatively under-utilized, but important application for computational models in complementing experimental studies: providing quantitative evidence for selecting from directly competing hypotheses.
In this study, we combined SMICUS with live-cell imaging and mathematical modeling to investigate how cAMP signals are translated into nuclear PKA activity. By showing that in addition to the translocated catalytic subunit of PKA, there exists a functional pool of PKA holoenzyme that contributes to nuclear PKA signaling, we revise the existing dogma of cAMP-PKA signaling in the nucleus1,5,9
. We provide the first set of quantitative evidence that this novel pool of nuclear PKA holoenzyme can be activated to produce fast responses in the nucleus. In addition, we form a new conceptual model for the regulation of nuclear PKA responses (Supplementary Fig. 13
): the presence of an AKAP-mediated signaling complex that localizes PDE4 in close proximity to nuclear PKA holoenzyme largely controls nuclear PKA activity. When cAMP is generated at the plasma membrane and diffuses into the nucleus, local cAMP concentration in this nuclear PKA signaling domain is kept low by AKAP-anchored PDE4s and is not capable of efficiently activating nuclear PKA holoenzyme. As a result, nuclear PKA responses are characterized by slow kinetics, rate-limited by the translocation of the catalytic domain of cytosolic PKA. However, when the local cAMP concentration is elevated above a threshold, for example via activation of cytoplasmic- or nuclear-targeted sACt
, activation of nuclear PKA holoenzyme can occur and generate fast kinetics of nuclear PKA responses. This nuclear PKA signaling domain assembled by AKAP therefore serves to manipulate the cAMP concentration threshold necessary to activate nuclear PKA holoenzyme in order to convert spatially distinct cAMP signals into temporal control of nuclear PKA activity. Future experiments will focus on identifying the responsible AKAP and characterizing the signaling complex. In addition, we will also evaluate the functional impact of such temporal control of nuclear PKA responses on processes such as transcription and RNA splicing as well as identify signaling sensing networks that are linked to this nuclear pool. A potential upstream component of nuclear PKA is endogenous sAC that has been suggested to exist in the nuclei of several cell types and produce cAMP in situ in response to changes in metabolic activities11
In summary, we combine targeted biochemical manipulation, real-time activity measurement and quantitative mechanistic modeling to provide evidence for the existence and distinct regulation of nuclear PKA holoenzyme. We propose a new model that localized negative regulators, in this case PDE4, help establish a local signaling threshold to convert spatial second messenger signals to temporal control of kinase activity. The quantitative native biochemistry approach17,37,38
utilized here should facilitate further testing of this model in this and other cell systems, leading to a better understanding of the mechanistic intricacies that underlie compartmentalized cAMP signaling.