The inner and outer compartments of mitochondria are sites of distinct cAMP/PKA signaling dynamics

Abstract

Cyclic AMP (cAMP)-dependent phosphorylation has been reported to exert biological effects in both the mitochondrial matrix and outer mitochondrial membrane (OMM). However, the kinetics, targets, and effectors of the cAMP cascade in these organellar domains remain largely undefined. Here we used sensitive FRET-based sensors to monitor cAMP and protein kinase A (PKA) activity in different mitochondrial compartments in real time. We found that cytosolic cAMP did not enter the matrix, except during mitochondrial permeability transition. Bicarbonate treatment (expected to activate matrix-bound soluble adenylyl cyclase) increased intramitochondrial cAMP, but along with membrane-permeant cAMP analogues, failed to induce measureable matrix PKA activity. In contrast, the OMM proved to be a domain of exceptionally persistent cAMP-dependent PKA activity. Although cAMP signaling events measured on the OMM mirrored those of the cytosol, PKA phosphorylation at the OMM endured longer as a consequence of diminished control by local phosphatases. Our findings demonstrate that mitochondria host segregated cAMP cascades with distinct functional and kinetic signatures.

Figure 1.

Cyclic AMP produced in the cytosol does not reach the mitochondrial matrix. (A) Confocal images of HeLa cells expressing mito-EpacH90 and loaded with MitoTracker red suggesting proper localization of mito-EpacH90. (B) cAMP measurements in intact cells expressing cytosolic EpacH90 (black trace) or mito-EpacH90 (red trace; mean of five cells); typical of n = 9 experiments; 34 mito-H90, 19 Epac-H90 cells. (C) Digitonin-permeabilized HeLa cells expressing mito-D3cpv (black traces) or mito-EpacH90 (red trace; n = 6 experiments; 10 mito-D3cpv cells, 13 mito-EpacH90 cells; red trace indicates the mean of three cells). (D) Mixed populations of permeabilized HeLa cells expressing mito-D3cpv (black trace) or mito-EpacH90 (red traces; n = 3 experiments; 5 mito-D3cpv cells, 8 mito-EpacH90 cells). After Ca²⁺ pulses, mitochondria changed morphology and failed to retain Ca²⁺, (hallmarks of MPT), coincident with responses of mito-EpacH90 to exogenous cAMP. (E) Permeabilized HeLa cells expressing mito-EpacH90 (n = 5 experiments; 20 cells) subjected to increasing [Ca²⁺] in the presence of cAMP (10 µM). 100 µM Ca²⁺ induced a dramatic increase in cAMP measured by mito-EpacH90. (F) Cells expressing mito-EpacH90 bathed in Hepes-buffered normal Ringer’s solution (continuous perfusion) then switched to CO₂/HCO₃⁻-buffered Krebs-Ringer’s, inducing an apparent cAMP rise (presumably due to matrix sAC activation). Data from two representative cells are shown (n = 6 repeats; 16 mito-EpacH90 cells). Error bars indicate mean ± SD.

Figure 2.

PKA activity in the mitochondrial matrix measured using targeted FRET-based reporters. (A) Confocal images of HeLa cells expressing mito-AKAR4 loaded with MitoTracker red. (B) Mixed populations of HeLa cells transfected with mito-EpacH90 (black trace; mean of three cells) or mito-AKAR4 (red trace; mean of four cells) were initially bathed in Hepes-buffered solution and then switched to CO₂/HCO₃⁻-buffered solution (n = 5 experiments; 10 mito-AKAR4 cells, 12 mito-EpacH90 cells). (C) HeLa expressing mito-AKAR3 (two representative cells; red traces) or OMM-AKAR3 (two representative cells; black traces; n = 8; 20 OMM-AKAR3 cells, 20 mito-AKAR3 cells). (D) HeLa cells expressing OMM-AKAR3 (two representative cells; black traces) compared with neighboring mito-AKAR3 positive cells (two representative cells; red traces; n = 10 experiments; 24 OMM-AKAR3 cells, 28 mito-AKAR3 cells). (E) Cells expressing mito-AKAR3 (red trace; mean of three cells) or mito-EpacH90 plus mCherry (black traces; mean of six cells; typical of n = 4 experiments; 10 mito-AKAR3 cells, 16 mito-EpacH90 cells). (F) HEK cells transfected with mito-AKAR4 (red trace) or OMM-AKAR4 together with mCherry (black trace; mean of four cells); typical of n = 3 experiments; 6 OMM-AKAR4 cells, 8 mito-AKAR4 cells). Error bars indicate mean ± SD.

Figure 3.

Overexpression of PKA in the matrix is detected by AKAR sensors, and induces specific phosphorylation patterns. (A) Summary of the starting mito-AKAR4 ratios in the presence of mito-PKACat-mCherry, mito-PKI-mCherry, and cyto-PKI-mCherry (***, P < 0.0002 with respect to control). Error bars indicate mean ± SD. (B) Phosphorylation status of cytosolic and mitochondria-enriched fractions of HEK cells treated with cAMP-generating agonists or cell-permeant cAMP analogues, or transfected with mito-PKACat-mCherry. A phospho-(Ser/Thr) PKA substrate antibody unveiled mito-PKACat-mCherry–dependent mitochondria-specific phosphorylation bands (arrows). The purity of mitochondria was tested using an antibody cocktail against the human OXPHOS subunits, whereas GAPDH was used to detect any cytosolic contamination (typical of n = 3 independent experiments).

Figure 4.

Comparison of cAMP and PKA signals at the OMM and cytosol. (A) Mixed populations of HEK cells expressing OMM-AKAR4 (two representative cells; red traces) or soluble AKAR4 (two representative cells; black traces). Both sensors responded to 10 nM isoproterenol (ISO); however, the OMM-AKAR4 signal reversed with significant delay compared with AKAR4 upon ISO removal (n = 4 experiments; 14 AKAR4 cells, 16 OMM-AKAR4 cells). (B) cAMP responses to ISO measured by OMM-EpacH90 (red trace; mean of four cells) or cytosolic EpacH90 (black trace) in HEK 293 cells (n = 3 experiments; 9 EpacH90 cells, 12 OMM-EpacH90 cells). (C) cAMP kinetics measured by OMM-EpacH90 (red trace; mean of three representative cells) or cytosolic EpacH90 (black trace; mean of four representative cells) in HeLa cells (n = 3 experiments; 10 EpacH90 cells, 19 OMM-EpacH90 cells). (D) Comparison of AKAR4 and OMM-AKAR4. Termination of PKA activity by agonist removal or H89 resulted in slower FRET reversal specifically at the OMM; two representative cells are depicted (n = 3 repeats; 12 AKAR4 cells, 14 OMM-AKAR4 cells).

Figure 5.

Phosphatase-dependent termination of PKA signals at the OMM and cytosol. (A) Acute addition of 20 nM calyculin A to mixed populations of cells expressing OMM-AKAR4 (one representative cell; red trace) or AKAR4 (one representative cell; black trace; n = 6 experiments; 14 AKAR4 cells, 14 OMM-AKAR4 cells). (B) HEK cells expressing OMM-AKAR4 or AKAR4. (B, right) Mean of the slope of the responses to ISO (an estimate of PKA activity; n = 9 experiments; 40 AKAR4 cells, 22 OMM-AKAR4 cells) or 10 µM H89 on top of FSK/IBMX (an estimate of phosphatase activity). AKAR4, 39 cells; OMM-AKAR4, 25 cells; n = 12 experiments (***, P < 0.00015). (C) Mixed populations of HeLa cells expressing cyto-PKACat-mCherry together with OMM-AKAR4 (one representative cell; red trace) or with nontargeted AKAR4 (one representative cell; black trace; n = 11 repeats; 9 AKAR4 cells; 16 OMM-AKAR4 cells). The high starting ratios reversed with different kinetics upon addition of 10 µM H89 (P < 0.0002). (C, right) Mean slope of the responses to H89 across all experiments (***, P < 0.0002). Error bars indicate mean ± SD.