Abstract
Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.
Highlights
Autophagy is a degradative process triggered by starvation to help cells compensate for the lack of nutrients [1]
To clarify the mechanisms underpinning the effects of cyclic AMP (cAMP) on autophagy, we performed a preliminary screening on different cell lines
B, treatment with FSK alone for 24 h had no significant effect on YFP-LC3 puncta compared to the vehicle control in both cell types, while treatment with FSK/IBMX increased the number of YFPLC3 vesicles in HT-29 but not in HeLa cells. Both lines responded with an increase in the number of YFP-LC3 puncta when treated with ChQ, a drug that inhibits the fusion between autophagosomes and lysosomes [38] and has no effect on the cAMP/protein kinase A (PKA) axis (Fig. 1A, B)
Summary
Autophagy is a degradative process triggered by starvation to help cells compensate for the lack of nutrients [1]. MTOR interacts with regulatoryassociated protein of mTOR (RAPTOR) [12] and other proteins to form mTORC1, a complex that directly suppresses autophagy in response to the nutrient status of the cells [13, 14]. Another kinase that regulates autophagy, independently from mTOR, is the cyclic AMP (cAMP)activated protein kinase A (PKA) [15]. AKAPs act as molecular platforms where functional signalosomes called microdomains containing PKA, its substrates, and signaling molecules of the cAMP pathway (phosphatases, phosphodiesterases (PDEs)) [16, 22] are generated. We found that only displacement of PKA-II reversed the effects on autophagy, providing a link between cAMP/PKA microdomains and this process
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
