PKA compartmentalization links cAMP signaling and autophagy

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.

Fig. 1. Cyclic AMP elevating agents increase the autophagic flux in HT-29 but not HeLa cells.

A Confocal photomicrographs of HT-29 and HeLa cells expressing YFP-LC3. Treatment with 5 µM forskolin (F) did not alter the number of YFP-LC3-positive structures, while 20 µM F combined to 500 µM IBMX (F/I) increased YFP-LC3 puncta in HT-29 but not in HeLa cells. Chloroquine (ChQ) (100 µM) was used as control. B Summary of the effects on YFP-LC3 puncta of each treatment normalized to vehicle control (DMSO). Average of HT-29 cells: DMSO: 282, F 5 µM: 77, F/I: 276, F/I/ChQ: 31, ChQ: 38 in at least six independent experiments. Average of HeLa cells: DMSO: 98, F 5 µM: 58, F/I: 80, F/I/ChQ: 69, ChQ: 67 in at least four independent experiments. (*p < 0,04; **p < 0,01). C Schematic representation of how the sensor mCherry-GFP-LC3 works (created with BioRender.com). D Confocal photomicrographs of HT-29 cells expressing mCherry-GFP-LC3. Treatment with 5 µM forskolin (F) did not alter either the number or the balance between autophagosomes (mCherry⁺/GFP⁺) and autolysosomes (mCherry⁺/GFP^–). Treatment with 20 µM F combined to 500 µM IBMX (F/I) for 6 or 24 h increased the number of autolysosomes and decreased the number of autophagosomes similarly to starvation (HBSS). Treatment with ChQ alone or in combination to F/I drastically decreased the number of autolysosomes and increased the number of autophagosomes. E Summary of the effects on mCherry-GFP-LC3 puncta number and type of each treatment. Average of HT-29 cells: DMSO: 83, F 5 µM: 38, F/I/6 h: 31, F/I/24 h: 37, F/I/6 h/ChQ: 12, F/I/24 h/ChQ: 15, ChQ: 40, HBSS: 22 in at least three independent experiments (*p < 0,01) (Scale bar 20 µm).

Fig. 2. HT-29 cells display low PKA-dependent phosphorylation in response to high cAMP levels.

A HeLa (averaged traces ± SD of six cells from one experiment) or B HT-29 cells (averaged traces ± SD of seven cells from one experiment) expressing the cAMP-sensitive FRET-based sensor H187 were challenged with increasing doses of forskolin (FSK) followed by FSK (20 µM) together with IBMX 500 µM (FSK/IBMX) to saturate the sensor. C HeLa cells expressing the PKA-dependent phosphorylation sensor AKAR4 marginally responded to FSK alone; however, addition of IBMX 500 µM produced a near-saturation response. Saturation was achieved by FSK/IBMX (averaged traces ± SD of 20 cells from four independent experiments). D In HT-29 cells AKAR4 gave extremely low responses independent of the treatment (averaged traces ± SD of 37 cells from 15 independent experiments).

Fig. 3. HT-29 and HeLa cells have comparable PKA levels but differ in PKA-dependent phosphorylation.

A Western blotting of PKA components in total cell lysates of HT-29 and HeLa cells. Both cell lines expressed similar levels of PKA catalytic subunit (PKA Cα) as well as PKA regulatory subunits I and II (PKA RII and PKA RI). GAPDH was used as loading control. Representative data of three independent experiments. B Western blotting of phospho-bands assessed by a phospho-PKA substrate antibody, RRX(S/T)^P of total cell lysates (40 µg) of HT-29 and HeLa cells treated with FSK alone (5 µM), FSK (20 µM) combined with IBMX (500 µM), 8CPT-cAMP (5 µM), or the PKA inhibitor H89 (30 µM). C Intensities of phospho-bands, normalized to GAPDH, for HT-29 and HeLa (D). Averages ± SD from at least three independent experiments.

Fig. 4. PKA-dependent phosphorylation remains compartmentalized in HT-29 but not in HeLa cells at high cAMP levels.

HT-29 cells expressing AKAR4 constructs targeted to A the outer mitochondrial membrane (OMM-AKAR4), B the endoplasmic reticulum facing the cytosol (ER-AKAR4), and C the plasma membrane (PM-AKAR4). All constructs responded to treatment with FSK 5 µM, while addition of FSK 20 µM combined to IBMX 500 µM did not further affect the FRET ratio, indicating that all sensors were saturated. Averaged traces ± SD of 15 cells in four independent experiments for OMM-AKAR4; 13 cells in four independent experiments for ER-AKAR4 and 4 cells in three independent experiments for PM-AKAR4. D HeLa cells expressing OMM-AKAR4, E ER-AKAR4, and F PM-AKAR4. Only PM-AKAR4 responded to treatment with FSK 5 µM, while all constructs saturated with FSK 20 µM combined to IBMX 500 µM. Averaged traces ± SD of 13 cells in three independent experiments of OMM-AKAR4; 10 cells in three independent experiments of ER-AKAR4, and 10 cells in three independent experiments for PM-AKAR4.

Fig. 5. Soluble phosphatases shape PKA-dependent phosphorylation in HT-29 cells.

A Western blotting of PKA components and phosphatases in cytosolic/soluble (cyto), mitochondria-enriched (Mito), and microsomal-enriched (Msms) fractions from HT-29 cells. An antibody mixture against ATP synthase subunit alpha (V-ATP5A) and GAPDH assessed mitochondrial enrichment and cytosol, respectively, while calreticulin (CALR) was used to determine ER enrichment. B Total cell lysates (40 µg) of HT-29 were treated with FSK (5 µM), Calyculin A 100 nM (CalA), or both combined and the phosphorylation status of endogenous PKA substrates was assessed by the phospho-PKA substrate-specific antibody, RRX(S/T)^P. Combination of CalA and FSK produced a dramatically greater responses than those of the individual drugs alone, indicating an additive effect as summarized in the quantification histogram (inset). β-actin was used as loading control. Experiments were repeated at least three times. C HT-29 cells expressing AKAR4 (black), OMM-AKAR4 (orange), or ER-AKAR4 (green) challenged with FSK (5 µM) followed by CalA (100 nM) to block phosphatases. Averaged traces ± SD of 12 cells for AKAR4, 15 cells for OMM-AKAR4, and 12 cells for ER-AKAR4, from three independent experiments for each sensor.

Fig. 6. Localized PKA RII is responsible for the effects of cAMP on autophagy.

A HT-29 cells were challenged with FSK (5 µM) or FSK (20 µM) combined to IBMX (500 µM) and total cell lysates were prepared at different timepoints. The phosphorylation status of endogenous PKA substrates was assessed by a phospho-PKA substrate-specific antibody, RRX(S/T)^P and is summarized in the inset (bar graphs: average ± SD of three independent experiments). B Summary of the effects on YFP-LC3 puncta of FSK 5 µM or FSK 20 µM combined to IBMX (500 µM) treatments, normalized to vehicle control (DMSO), measured after 4–6 h after treatment. Average of at least three independent experiments. C Summary of the effects of super AKAP-IS-mCherry (sAKAP-IS), RIAD-mCherry (RIAD), and scramble-mCherry (scr) on the number of YFP-LC3 puncta generated by FSK (20 µM)/IBMX (500 µM). Data presented are average of four independent experiments for sAKAP-IS, RIAD, and scramble, and ten experiments for control (15–25 cells for each treatment).