From microdomains to organelles: Delving into spatiotemporally compartmentalized PKA signaling

Extracellular stimuli elicit specific cellular responses via intracellular signaling pathways. To achieve specific responses, these pathways are tightly controlled in space and time and this is frequently attained by regulating second messenger concentrations and kinase activity dynamics. The 3'-5'-cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase (PKA) pathway is spatiotemporally compartmentalized at various levels in order to regulate its control over diverse cellular activities. At the second messenger level, cAMP concentration gradients are maintained by actively controlling its accumulation and degradation. PKA activity is modulated in space and time by the spatiotemporal compartmentalization of upstream components, including cAMP gradients, and is itself spatially localized to specific subcellular compartments, contributing to highly specific substrate recognition. In order to study cAMP and PKA in their native cellular milieu, we used fluorescence resonance energy transfer (FRET)-based biosensors to capture the endogenous signaling dynamics of these ubiquitous molecules.;This dissertation is composed of three different studies, which each investigate spatiotemporally compartmentalized PKA signaling. In the first study, using FRET-based PICA biosensors targeted to distinct plasma membrane microdomains we uncovered that PICA is differentially regulated between membrane rafts and non-raft regions. Specifically, membrane rafts appear to negatively regulate stimulated PICA activity and possess high basal PKA activity levels. In the second study we build upon recently generated bimolecular FRET-based biosensors used to monitor kinase activity, we further examined the versatility and adaptable nature of complex bimolecular reporters in studying various kinase activity dynamics. Using these reporters we reveal that membrane raft-localized basal PKA dampens AMP-activated protein kinase (AMPK) activity. In the third study, we investigated the underlying mechanisms that contribute to heightened cardiac function in patients suffering from dyssynchronous heart failure after receiving cardiac resynchronization therapy (CRT). Using biochemical techniques in combination with plasma membrane- and sarcoplasmic reticulum-localized FRET-based biosensors we revealed that CRT alters the compartmentalization of beta2-adrenegic receptor stimulated PICA activity to enhance myocyte function.;Taken together, the studies presented herein highlight various modes and roles of spatiotemporally compartmentalized cAMP/PKA signaling within discrete subcellular compartments. These studies enhance our understanding of the cAMP/PKA pathway and should contribute to our understanding of how cells communicate.