cAMP compartmentalization provides a new conceptual framework to generate signaling specificity and transduction efficiency. The mechanisms involved in its establishment, maintenance and how cAMP effectors are targeted to it are not completely understood. Using thyroid cells we identified a new sub-membrane compartment, where the ERM protein radixin scaffolds both cAMP effectors Epac and PKA into a ternary complex. Maneuvers that disrupt this compartmentalization abrogate TSH/cAMP-mediated proliferation, and served as the basis for the development of new pathway-specific inhibitors. Interestingly, expression of constitutively active Rap1 but only in its phosphorylated form (G12V-S179D) rescues this inhibition, indicating the main role of this compartment is to position cAMP effectors in a local area of high cAMP concentration (i.e. a microdomain) to maximize effector activation. A sequential order of Epac-mediated activation followed by PKA-mediated phosphorylation was demonstrated. This sets an allosteric switch where pRap1 dissociates from its GEF promoting its association with new phospho-dependent binding partners, i.e. CAP1 (Cyclase- Associated Protein 1). Our preliminary studies are consistent with pRap1-CAP1 positively modulating the localized rate of cAMP synthesis. We advance here the hypothesis of a positive feedback loop as the mechanistic basis assuring that local cAMP levels in the microdomain are optimized for efficient effector activation. Defining the mechanisms involved in the pS179-dependent allosteric switch is therefore critical for the understanding of the phospho-dependent Rap1-CAP control of cAMP dynamics. We will accomplish this in two integrated aims.
In Aim #1 NMR approaches will be utilized to address the mechanism involved in the phospho-dependent allosteric communication aiming at the identification of the residues that are allosterically coupled, the population of the states and their exchange dynamics.
In Aim #2 a combination of biochemical and live cell imaging techniques will be used to characterize the phospho-dependent Rap1-CAP1 interaction and its ability to positively modulate compartmentalized cAMP synthesis. The long-term goal of this proposal is to understand the spatial and temporal regulation of the cAMP-dependent signaling events, and the role of Rap1 and its phosphorylation state as a signal integration unit. Understanding the mechanisms responsible for cAMP compartmentalization will eventually provide insights into the rational design of new specific inhibitors with effector pathway selectivity.

Public Health Relevance

cAMP is a universal second messenger used by several hormones in every cell type. It is involved in multiple aspects of normal physiology and disease. Particularly in endocrine systems, it is involved in normal cell proliferation, differentiation and tumorigenesis. Mutations rendering a constitutively active cAMP pathway are linked to hyperproliferative human disorders underscoring the biological significance of this pathway. However, the mechanisms involved are still ill-defined. Our studies demonstrated the need for both cAMP effectors, Epac and PKA, in cell proliferation. Their action was spatially restricted to a particular compartment, and signaling was integrated at the level of Rap1 activation and phosphorylation. The studies proposed should provide new insights into the spatial and temporal regulation of the cAMP- dependent signaling events. Unraveling the mechanisms underlying these new interactions might provide insights into the rational design of new specific inhibitors with effector pathway selectivity that might have implications in the development of new therapies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Dunsmore, Sarah
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University of Pittsburgh
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Zhang, Xuefeng; Cao, Shufen; Barila, Guillermo et al. (2018) Cyclase-associated protein 1 (CAP1) is a prenyl-binding partner of Rap1 GTPase. J Biol Chem 293:7659-7673
Li, ZhenLu; Cao, Shufen; Buck, Matthias (2016) K-Ras at Anionic Membranes: Orientation, Orientation…Orientation. Recent Simulations and Experiments. Biophys J 110:1033-5