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• W2090848174 abstract "Mitochondria constantly respond to changes in substrate availability and energy utilization to maintain cellular ATP supplies, and at the same time control reactive oxygen radical (ROS) production. Reversible phosphorylation of mitochondrial proteins has been proposed to play a fundamental role in metabolic homeostasis, but very little is known about the signaling pathways involved. We show here that protein kinase A (PKA) regulates ATP production by phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase. The cyclic AMP (cAMP), which activates mitochondrial PKA, does not originate from cytoplasmic sources but is generated within mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase (sAC) in response to metabolically generated carbon dioxide. We demonstrate for the first time the existence of a CO2-HCO3−-sAC-cAMP-PKA (mito-sAC) signaling cascade wholly contained within mitochondria, which serves as a metabolic sensor modulating ATP generation and ROS production in response to nutrient availability. Mitochondria constantly respond to changes in substrate availability and energy utilization to maintain cellular ATP supplies, and at the same time control reactive oxygen radical (ROS) production. Reversible phosphorylation of mitochondrial proteins has been proposed to play a fundamental role in metabolic homeostasis, but very little is known about the signaling pathways involved. We show here that protein kinase A (PKA) regulates ATP production by phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase. The cyclic AMP (cAMP), which activates mitochondrial PKA, does not originate from cytoplasmic sources but is generated within mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase (sAC) in response to metabolically generated carbon dioxide. We demonstrate for the first time the existence of a CO2-HCO3−-sAC-cAMP-PKA (mito-sAC) signaling cascade wholly contained within mitochondria, which serves as a metabolic sensor modulating ATP generation and ROS production in response to nutrient availability. The Krebs Cycle (TCA cycle) produces the electron donors, which drive mitochondrial production of ATP via oxidative phosphorylation (OXPHOS). OXPHOS is subject to complex regulation, including short-term modulations essential for responding to transient changes in nutritional availability, environmental conditions, and energy requirements. If the reducing equivalents generated by the TCA cycle are not efficiently utilized by the OXPHOS machinery, reactive oxygen species (ROS) production may increase, and oxidative damage may ensue. It has been proposed that dynamic protein phosphorylation plays a major role in these rapid modulations (Hopper et al., 2006Hopper R.K. Carroll S. Aponte A.M. Johnson D.T. French S. Shen R.F. Witzmann F.A. Harris R.A. Balaban R.S. Mitochondrial matrix phosphoproteome: effect of extra mitochondrial calcium.Biochemistry. 2006; 45: 2524-2536Crossref PubMed Scopus (204) Google Scholar). Evidence has emerged suggesting that cyclic AMP (cAMP)-mediated phosphorylation of mitochondrial enzymes plays a role in OXPHOS regulation. Consistent with this hypothesis, both protein kinase A (PKA) (reviewed in Pagliarini and Dixon, 2006Pagliarini D.J. Dixon J.E. Mitochondrial modulation: reversible phosphorylation takes center stage?.Trends Biochem. Sci. 2006; 31: 26-34Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, Thomson, 2002Thomson M. Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria.Cell. Mol. Life Sci. 2002; 59: 213-219Crossref Scopus (68) Google Scholar) and A kinase-anchoring proteins (AKAPs) have been identified in mammalian mitochondria (Feliciello et al., 2005Feliciello A. Gottesman M.E. Avvedimento E.V. cAMP-PKA signaling to the mitochondria: protein scaffolds, mRNA and phosphatases.Cell. Signal. 2005; 17: 279-287Crossref Scopus (95) Google Scholar, Lewitt et al., 2001Lewitt M.S. Brismar K. Wang J. Wivall-Helleryd I.L. Sindelar P. Gonzalez F.J. Bergman T. Bobek G.A. Responses of insulin-like growth factor (IGF)-I and IGF-binding proteins to nutritional status in peroxisome proliferator-activated receptor-alpha knockout mice.Growth Horm. IGF Res. 2001; 11: 303-313Abstract Full Text PDF Scopus (16) Google Scholar). In particular, PKA in the mitochondrial matrix has been demonstrated by several independent groups using biochemical, pharmacological, and immunological methods, including immunoelectron microscopy (Livigni et al., 2006Livigni A. Scorziello A. Agnese S. Adornetto A. Carlucci A. Garbi C. Castaldo I. Annunziato L. Avvedimento E.V. Feliciello A. Mitochondrial AKAP121 links cAMP and src signaling to oxidative metabolism.Mol. Biol. Cell. 2006; 17: 263-271Crossref Scopus (119) Google Scholar, Prabu et al., 2006Prabu S.K. Anandatheerthavarada H.K. Raza H. Srinivasan S. Spear J.F. Avadhani N.G. Protein kinase A-mediated phosphorylation modulates cytochrome c oxidase function and augments hypoxia and myocardial ischemia-related injury.J. Biol. Chem. 2006; 281: 2061-2070Crossref PubMed Scopus (151) Google Scholar, Ryu et al., 2005Ryu H. Lee J. Impey S. Ratan R.R. Ferrante R.J. Antioxidants modulate mitochondrial PKA and increase CREB binding to D-loop DNA of the mitochondrial genome in neurons.Proc. Natl. Acad. Sci. USA. 2005; 102: 13915-13920Crossref Scopus (128) Google Scholar, Schwoch et al., 1990Schwoch G. Trinczek B. Bode C. Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.Biochem. J. 1990; 270: 181-188Crossref PubMed Scopus (63) Google Scholar). However, if PKA plays a role in phosphorylating mitochondrial proteins, it remains unclear how the cAMP that activates PKA is modulated. Specifically, cAMP does not diffuse far from its source (Bornfeldt, 2006Bornfeldt K.E. A single second messenger: several possible cellular responses depending on distinct subcellular pools.Circ. Res. 2006; 99: 790-792Crossref Scopus (11) Google Scholar, Zaccolo and Pozzan, 2002Zaccolo M. Pozzan T. Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes.Science. 2002; 295: 1711-1715Crossref PubMed Scopus (653) Google Scholar), and as we show here, it does not enter mitochondria. Papa et al. postulated that a source of this second messenger might reside inside mitochondria (Papa et al., 1999Papa S. Sardanelli A.M. Scacco S. Technikova-Dobrova Z. cAMP-dependent protein kinase and phosphoproteins in mammalian mitochondria. An extension of the cAMP-mediated intracellular signal transduction.FEBS Lett. 1999; 444: 245-249Abstract Full Text Full Text PDF Scopus (86) Google Scholar), but an intramitochondrial adenylyl cyclase had not been demonstrated so far. In mammalian cells, cAMP can be produced by a family of plasma membrane-bound forms of adenylyl cyclase (tmAC), or by a “soluble” adenylyl cyclase (sAC) (Buck et al., 1999Buck J. Sinclair M.L. Schapal L. Cann M.J. Levin L.R. Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals.Proc. Natl. Acad. Sci. USA. 1999; 96: 79-84Crossref PubMed Scopus (404) Google Scholar). We previously showed that sAC resides at multiple subcellular organelles, including mitochondria (Zippin et al., 2003Zippin J.H. Chen Y. Nahirney P. Kamenetsky M. Wuttke M.S. Fischman D.A. Levin L.R. Buck J. Compartmentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains.FASEB J. 2003; 17: 82-84Crossref Scopus (225) Google Scholar). Unlike tmACs, sAC is insensitive to heterotrimeric G protein regulation or forskolin; instead, it is stimulated by bicarbonate (Chen et al., 2000Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor.Science. 2000; 289: 625-628Crossref PubMed Scopus (640) Google Scholar) and sensitive to ATP (Litvin et al., 2003Litvin T.N. Kamenetsky M. Zarifyan A. Buck J. Levin L.R. Kinetic properties of “soluble” adenylyl cyclase. Synergism between calcium and bicarbonate.J. Biol. Chem. 2003; 278: 15922-15926Crossref PubMed Scopus (252) Google Scholar) and calcium levels (Jaiswal and Conti, 2003Jaiswal B.S. Conti M. Calcium regulation of the soluble adenylyl cyclase expressed in mammalian spermatozoa.Proc. Natl. Acad. Sci. USA. 2003; 100: 10676-10681Crossref PubMed Scopus (200) Google Scholar, Litvin et al., 2003Litvin T.N. Kamenetsky M. Zarifyan A. Buck J. Levin L.R. Kinetic properties of “soluble” adenylyl cyclase. Synergism between calcium and bicarbonate.J. Biol. Chem. 2003; 278: 15922-15926Crossref PubMed Scopus (252) Google Scholar). Bicarbonate stimulates sAC activity by facilitating active site closure, while calcium promotes activity by increasing the affinity for ATP (Litvin et al., 2003Litvin T.N. Kamenetsky M. Zarifyan A. Buck J. Levin L.R. Kinetic properties of “soluble” adenylyl cyclase. Synergism between calcium and bicarbonate.J. Biol. Chem. 2003; 278: 15922-15926Crossref PubMed Scopus (252) Google Scholar, Steegborn et al., 2005Steegborn C. Litvin T.N. Levin L.R. Buck J. Wu H. Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment.Nat. Struct. Mol. Biol. 2005; 12: 32-37Crossref PubMed Scopus (131) Google Scholar). In physiological systems, including mitochondria (Dodgson et al., 1980Dodgson S.J. Forster 2nd, R.E. Storey B.T. Mela L. Mitochondrial carbonic anhydrase.Proc. Natl. Acad. Sci. USA. 1980; 77: 5562-5566Crossref Scopus (101) Google Scholar), carbonic anhydrases (CA) convert CO2 into bicarbonate. While generating electron donors for OXPHOS, the TCA cycle generates CO2 and therefore bicarbonate. Thus, sAC represents an excellent candidate OXPHOS regulator, which ensures that respiration can keep pace with changes in nutritional availability and prevent ROS accumulation. Here we show that PKA modulation of OXPHOS activity is regulated by cAMP generated inside mitochondria by sAC in response to metabolically generated CO2. This study provides a functional understanding of the modulation of OXPHOS in direct response to nutrient metabolism by the mito-sAC signaling pathway. To test whether mitochondrial OXPHOS can be modulated by PKA, we stimulated HeLa cells with membrane-permeant 8Br-cAMP, which activates all cAMP-dependent kinases. We measured oxygen consumption as an indicator of mitochondrial respiratory chain function. 8Br-cAMP (1 mM for 30 min) resulted in a 25% (p < 0.0001) increase in oxygen consumption, as compared to untreated cells (Figure 1A). Then, we uncoupled oxygen consumption from ATP synthesis by inclusion of carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP). Under these conditions, where respiratory chain activity is independent from ATP synthesis by the F1F0 ATPase, 8Br-cAMP still increased oxygen consumption. Note that the residual ATP content in mitochondria treated with FCCP for 5 min was approximately 60% of the pre-FCCP content (2.5 ± 0.3 and 4.1 ± 0.2 nmol/mg protein, respectively). Thus, there still was sufficient ATP for phosphorylation of PKA target proteins. The exchange protein activated by cAMP-selective agonist, 8CPT methyl-cAMP, did not change coupled or uncoupled respiration. Conversely, H89 at 1 μM, a concentration that selectively blocks PKA, resulted in a 50% decrease in coupled and uncoupled respiration. RpcAMP (25 μM), which inhibits PKA by a different mechanism, also caused a significant decrease (20%, p < 0.001) in oxygen consumption (data not shown). The effects of PKA agonists and antagonists were replicated in 143B human osteosarcoma and 293T-HEK (data not shown), except that the increase in respiration induced by FCCP was higher (i.e., approximately 100% in 143B and 293T compared to 20% in HeLa cells), reflecting different coupling between oxygen consumption and ATP synthesis. Stimulation of tmAC with forskolin in combination with the phosphodiesterase (PDE) inhibitor 3-Isobutyl-1-methylxanthine (IBMX) did not affect mitochondrial respiration, despite an 8- to 10-fold increase in cytoplasmic cAMP (Figure 1B). Therefore, mitochondrial respiration is enhanced through PKA activation by membrane-permeant cAMP analogs, but not by cytoplasmic cAMP, suggesting that the PKA that modulates respiration is inside mitochondria. It was suggested that tmAC-generated cAMP enters the mitochondrial matrix. However, our data demonstrate that cytoplasmic cAMP does not have access to the intramitochondrial PKA pool. DiPilato and colleagues used a reporter protein targeted to mitochondria, but the mitochondrial import was partial, with a portion of the protein remaining on the cytosolic surface or in the mitochondrial intermembrane space (DiPilato et al., 2004DiPilato L.M. Cheng X. Zhang J. Fluorescent indicators of cAMP and Epac activation reveal differential dynamics of cAMP signaling within discrete subcellular compartments.Proc. Natl. Acad. Sci. USA. 2004; 101: 16513-16518Crossref PubMed Scopus (362) Google Scholar), where it remained accessible to cytosolic cAMP. To further explore the role of intramitochondrial PKA in modulating OXPHOS, we examined isolated mitochondria from mouse liver. First we measured state III (phosphorylating) respiration driven by different substrates: glutamate/malate (G + M) specific for complex I (Figure 1C), succinate for complex II (Figure 1D), and TMPD + ascorbate for complex IV (Figure 1E). Similar to intact cells, 8Br-cAMP produced a small but significant increase in respiration with glutamate/malate (12%, p < 0.05) and TMPD/ascorbate (13%, p < 0.05). Succinate-dependent respiration was unchanged, indicating that it cannot be upregulated by cAMP. As expected, addition of exogenous, membrane-impermeant cAMP, or 8CPT methyl-cAMP, or forskolin + IBMX had no effect on oxygen consumption (Figures 1C–1E). Similar to whole cells, the PKA inhibitor, H89, decreased oxygen consumption driven by all complexes (44% for glutamate/malate, p < 0.0001; 50% for succinate, p < 0.0001; 30% for TMPD/ascorbate, p < 0.0001). Another PKA-specific inhibitor, myristoylated PKI 14-22, also inhibited respiration (20% for glutamate/malate, p < 0.001; 36% for succinate, p < 0.001; 25% for TMPD/ascorbate, p < 0.0001). 8Br-cAMP was inert in the presence of PKI 14-22, confirming the role of PKA in modulating respiration. Second, we showed that ATP synthesis (Figure 1F) was also enhanced by 8Br-cAMP (75%, p < 0.0001), inhibited by H89 and PKI 14-22 (63% and 74%, respectively, p < 0.0001), and unchanged by membrane impermeant cAMP. Third, the capacity to generate mitochondrial membrane potential (ΔΨm) was measured fluorimetrically under nonphosphorylating conditions. Figure 1G shows representative fluorescence traces. 8Br-cAMP increased membrane potential driven by glutamate/malate or succinate (21% ± 8% and 29 ± 6%, respectively; p < 0.01), whereas H89 decreased it (37% ± 8% and 51% ± 10%, respectively; p < 0.0001). Because PKA inhibitors decreased oxygen consumption independent of which electron transfer complex was stimulated (Figures 1C–1E), the modulating effect of PKA likely targets COX, the terminal component of the respiratory chain. Consistently, COX activity (rate of oxidation of reduced cytochrome c) was stimulated by 8Br-cAMP (25%, p < 0.0001; Figure 1H), but not by cAMP, 8CPT methyl-cAMP, or forskolin + IBMX, and was inhibited by H89 and PKI 14-22 (22%% and 18%, respectively; p < 0.0001). The stimulation by 8Br-cAMP was blocked by PKI 14-22. There were no changes in steady-state protein levels of COX subunits I and IV after modulation of PKA (Figure S1), suggesting that the activity changes depend on posttranslational modification of the enzyme kinetics. Consistently, it was previously proposed that COX is regulated via protein phosphorylation (Bender and Kadenbach, 2000Bender E. Kadenbach B. The allosteric ATP-inhibition of cytochrome c oxidase activity is reversibly switched on by cAMP-dependent phosphorylation.FEBS Lett. 2000; 466: 130-134Abstract Full Text Full Text PDF Scopus (143) Google Scholar, Lee et al., 2005Lee I. Salomon A.R. Ficarro S. Mathes I. Lottspeich F. Grossman L.I. Huttemann M. cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity.J. Biol. Chem. 2005; 280: 6094-6100Crossref PubMed Scopus (175) Google Scholar, Miyazaki et al., 2003Miyazaki T. Neff L. Tanaka S. Horne W.C. Baron R. Regulation of cytochrome c oxidase activity by c-Src in osteoclasts.J. Cell Biol. 2003; 160: 709-718Crossref Scopus (172) Google Scholar). As expected for a short-term adaptation mechanism, the cAMP-induced changes in PKA modulation of COX activity were transient and readily reversible upon washout of the agonist (Figure S2). Membrane-impermeant cAMP had no effect on OXPHOS response, suggesting that a source of cAMP must reside within mitochondria. We previously showed that sAC immunoreactivity colocalizes and sAC activity copurifies with mitochondria (Zippin et al., 2003Zippin J.H. Chen Y. Nahirney P. Kamenetsky M. Wuttke M.S. Fischman D.A. Levin L.R. Buck J. Compartmentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains.FASEB J. 2003; 17: 82-84Crossref Scopus (225) Google Scholar). We now show that western blotting using the R21 monoclonal anti-sAC antibody (Zippin et al., 2003Zippin J.H. Chen Y. Nahirney P. Kamenetsky M. Wuttke M.S. Fischman D.A. Levin L.R. Buck J. Compartmentalization of bicarbonate-sensitive adenylyl cyclase in distinct signaling microdomains.FASEB J. 2003; 17: 82-84Crossref Scopus (225) Google Scholar) identifies multiple bands in the liver homogenate (Figure 2A), consistent with multiple sAC splice isoforms (Buck et al., 1999Buck J. Sinclair M.L. Schapal L. Cann M.J. Levin L.R. Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals.Proc. Natl. Acad. Sci. USA. 1999; 96: 79-84Crossref PubMed Scopus (404) Google Scholar, Farrell et al., 2008Farrell J. Ramos L. Tresguerres M. Kamenetsky M. Levin L.R. Buck J. Somatic ‘soluble’ adenylyl cyclase isoforms are unaffected in Sacy tm1Lex/Sacy tm1Lex ‘knockout’ mice.PLoS ONE. 2008; 3: e3251Crossref PubMed Scopus (45) Google Scholar, Geng et al., 2005Geng W. Wang Z. Zhang J. Reed B.Y. Pak C.Y. Moe O.W. Cloning and characterization of the human soluble adenylyl cyclase.Am. J. Physiol. Cell Physiol. 2005; 288: C1305-C1316Crossref PubMed Scopus (100) Google Scholar, Jaiswal and Conti, 2003Jaiswal B.S. Conti M. Calcium regulation of the soluble adenylyl cyclase expressed in mammalian spermatozoa.Proc. Natl. Acad. Sci. USA. 2003; 100: 10676-10681Crossref PubMed Scopus (200) Google Scholar). However, purified mitochondria contained only one sAC isoform of approximately 48kDa (Figure 2A, lane 4). This band was confirmed in mitochondria highly purified by a second gradient step (Figure 2A, lanes 4.4). Instead, ER-rich fractions contained a distinct sAC isoform migrating at approximately 35kDa (Figure 2A, lanes 2 and 3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was absent from the mitochondrial and ER fractions excluding detectable contamination with cytoplasmic proteins. A highly active isoform of rat sAC (sACt) (Chaloupka et al., 2006Chaloupka J.A. Bullock S.A. Iourgenko V. Levin L.R. Buck J. Autoinhibitory regulation of soluble adenylyl cyclase.Mol. Reprod. Dev. 2006; 73: 361-368Crossref PubMed Scopus (41) Google Scholar) containing an N-terminal HA tag was expressed in COS cells. In cell homogenates (Figure 2B, lane 1) sACt was detected both by the R21 and the HA antibodies. The crude mitochondrial fraction contained the same immunoreactive sACt bands (Figure 2B, lane 2). Likewise, endogenous PKA was detected in the mitochondrial fraction (Figure 2B, lane 2). A proteinase K protection assay performed on the mitochondrial fraction showed that portions of sACt and endogenous PKA were resistant to digestion, indicating that they resided in a protected mitochondrial compartment (Figure 2B, lane 3). Detergent solubilization of mitochondria allowed for complete digestion of sACt, PKA, as well as hsp60 and Tim23, localized in the mitochondrial matrix and inner membrane, respectively (Figure 2B, lane 4). sAC is stimulated by bicarbonate (Chen et al., 2000Chen Y. Cann M.J. Litvin T.N. Iourgenko V. Sinclair M.L. Levin L.R. Buck J. Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor.Science. 2000; 289: 625-628Crossref PubMed Scopus (640) Google Scholar), and adenylyl cyclase activity in mouse liver mitochondria demonstrated a significant bicarbonate stimulation (Figure 2C, p < 0.0001) and inhibition by the sAC-specific inhibitor, KH7 (Hess et al., 2005Hess K.C. Jones B.H. Marquez B. Chen Y. Ord T.S. Kamenetsky M. Miyamoto C. Zippin J.H. Kopf G.S. Suarez S.S. et al.The “soluble” adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.Dev. Cell. 2005; 9: 249-259Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), indicating that the mitochondrial component of sAC is enzymatically active. The reversibility of the cAMP signal predicts that phosphodiesterase (PDE) should also be contained inside mitochondria. To test this hypothesis we isolated mitoplasts (i.e., mitochondria stripped of their outer membrane) from mouse liver. Mitoplasts contained inner membrane and matrix proteins (COX I, and Hsp60, respectively), but no intermembrane space or outer membrane proteins (Cyt c and Tom40, respectively), whereas the postmitoplasts supernatants only contained Cyt c and Tom 40 (Figure 2D). After the contents of the matrix were made accessible by sonication, they degraded exogenous cAMP (Figure 2E). This cAMP catabolic activity was fully inhibited by IBMX, confirming the presence of intramitochondrial PDE activity. We used two distinct methods of increasing intramitochondrial sAC-generated cAMP to demonstrate a functional role for sAC in modulating OXPHOS activity. First, sACt was stably overexpressed in 293T-HEK cells, and was found in both the whole cell homogenate and in isolated mitochondria (Figure 3A, lanes hom and 4, respectively). sACt overexpression increased respiration by approximately 25% (Figure 3B, p < 0.0001), as compared to untransfected cells. Consistently, COX activity (Figure 3C) and ATP synthesis (Figure 3D) were also increased (by 28% and 87%, respectively; p < 0.0001). These effects were antagonized by the sAC-specific inhibitor, KH7. Second, we stimulated endogenous mouse liver sAC in isolated mitochondria with bicarbonate. We found that maximum stimulation of COX activity occurred at 30 mM bicarbonate (Figure 4A), which is consistent with the physiological intramitochondrial bicarbonate concentration (ranges between 10 and 40 mM) (Simpson and Hager, 1979Simpson D.P. Hager S.R. pH and bicarbonate effects on mitochondrial anion accumulation. Proposed mechanism for changes in renal metabolite levels in acute acid-base disturbances.J. Clin. Invest. 1979; 63: 704-712Crossref PubMed Scopus (29) Google Scholar). As predicted, bicarbonate enhanced ATP synthesis by 32% (Figure 4B, p < 0.001), and bicarbonate-dependent stimulation of COX activity at least partially accounted for this effect (18%) (Figure 4C, p < 0.001). To confirm that bicarbonate stimulates OXPHOS, isolated mitochondria were exposed to the CO2-generating combination of α-ketoglutarate dehydrogenase complex (KGDHC), its substrates ketoglutaric acid + NAD+, and its cofactors coenzyme-A and cocarboxylase. KGDHC stimulated COX-driven respiration by 32%, using TMPD/ascorbate as substrates (Figures 4D and 4E, p < 0.0001). Addition of the carbonic anhydrase inhibitor (CAI) acetazolamide diminished COX driven respiration by 10% (p < 0.01), indicating that CO2 diffusing through the mitochondrial membranes is converted into bicarbonate that activates sAC and stimulates COX. We note that, since OXPHOS activity is increased by both bicarbonate and by exogenously generated CO2, in a carbonic anhydrase-dependent manner, these effects cannot be due to pH changes (or ionic strength), because bicarbonate addition increases pH while CO2 addition decreases it. To further demonstrate the role of sAC in modulating OXPHOS activity, we used three independent methods of blocking intramitochondrial sAC. First, KH7, but not an inactive congener, KH7.15 (Wu et al., 2006Wu K.Y. Zippin J.H. Huron D.R. Kamenetsky M. Hengst U. Buck J. Levin L.R. Jaffrey S.R. Soluble adenylyl cyclase is required for netrin-1 signaling in nerve growth cones.Nat. Neurosci. 2006; 9: 1257-1264Crossref Scopus (80) Google Scholar), inhibited state III respiration driven by complexes I, II, and IV by 25%, 22%, and 35%, respectively (Figure 1C–1E, p < 0.0001). Inhibition of sAC by KH7 also markedly decreased ATP synthesis (80%) (Figure 4B, p < 0.0001). KH7, but not KH7.15, inhibited COX activity by 30% (Figure 4C, p < 0.0001). In each case, KH7 inhibition was rescued by membrane permeable 8Br-cAMP. Finally, KH7 decreased ΔΨm in isolated mitochondria by 46% ± 6% with glutamate/malate and by 62% ± 11% with succinate (Figure 1G, p < 0.0001). The effects of sAC inhibition on mitochondrial respiration were confirmed in whole cells, where KH7 induced a 30% decrease, which was rescued by 8Br-cAMP but not by cytosolic cAMP induced by forskolin + IBMX (Figure 4F, p < 0.0001). As a second independent method of blocking sAC activity, we used the anti-sAC monoclonal R21antibody, which has inhibitory properties on enzymatic activity (Figure S3). Neither R21 nor nonspecific isotype-matched IgG caused COX inhibition in intact mitochondria. However, R21, but not the IgG control, blocked the bicarbonate-induced increase in COX activity when sAC was made accessible to the antibody by mitochondrial sonication (Figure 4G, p < 0.01). Third, we measured COX activity in mitochondria treated with bicarbonate in the presence or absence of PDE. In intact mitochondria, bicarbonate stimulated COX activity was unaffected by PDE (Figure 4H). Thus, cAMP was produced in a compartment isolated from external PDE. After sonication, PDE degraded intramitochondrial cAMP (Figure 4I, p < 0.001), and the bicarbonate-induced COX stimulation was abolished (Figure 4H), in an IBMX sensitive manner. Taken together, this evidence indicates that the sAC-cAMP-PKA signaling pathway is wholly contained within mitochondria and that it modulates OXPHOS in response to physiologically relevant concentrations of bicarbonate. We investigated the pattern of PKA-dependent mitochondrial protein phosphorylation in isolated mitochondria. Proteins were resolved by isoelectric focusing two-dimensional electrophoresis and detected with a PKA substrate-specific anti-phospho Ser/Thr antibody (Bruce et al., 2002Bruce J.I. Shuttleworth T.J. Giovannucci D.R. Yule D.I. Phosphorylation of inositol 1,4,5-trisphosphate receptors in parotid acinar cells. A mechanism for the synergistic effects of cAMP on Ca2+ signaling.J. Biol. Chem. 2002; 277: 1340-1348Crossref Scopus (124) Google Scholar, Schmitt and Stork, 2002Schmitt J.M. Stork P.J. PKA phosphorylation of Src mediates cAMP's inhibition of cell growth via Rap1.Mol. Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF Scopus (207) Google Scholar). Several of the PKA-phosphorylated proteins detectable in untreated mitochondria (indicated by arrows in Figure S4A, upper panel) disappeared or were markedly reduced in the KH7-treated samples (Figure S4A, lower panel). Many hydrophobic subunits of the respiratory chain are not amenable to isoelectric focusing (unpublished data). Therefore, to examine respiratory chain complexes, we employed 2D-blue-native gel electrophoresis (Schagger and Pfeiffer, 2000Schagger H. Pfeiffer K. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (935) Google Scholar, Schagger and von Jagow, 1991Schagger H. von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1838) Google Scholar). Phosphoproteins were detected using either anti-phospho Ser/Thr antibody (Figure S5A) or the PKA substrate-specific antibody (Figure S5B). Replicate samples were treated with calf-intestinal phosphatase, which abolished most immunoreactive spots on the membrane probed with anti-phospho Ser/Thr antibody, demonstrating the specificity of the antibody (Figure S6). Both anti-phospho Ser/Thr and the PKA substrate-specific antibodies revealed a marked decrease of several phosphoproteins after KH7 or H89. Membranes were reprobed with antibodies against COX subunits, which revealed two of the phosphorylated proteins (denoted by asterisks in the upper panel of Figure S5A) to be COX I and COX IV type II (COX IV-2). The amounts of phosphorylated COX I and COX IV-2 were respectively reduced to 30% and 20% with H89, and to 25% and 5% with KH7. These results confirm that phosphorylation of several mitochondrial proteins responds to modulation of the mito-sAC signaling pathway and suggest that certain COX subunits are candidates for the regulation of OXPHOS activity. We hypothesized that the physiological role of mitochondrial sAC is to respond to CO2 metabolically generated by the TCA cycle. To test this hypothesis, we measured COX activity in isolated mitochondria “fed” with pyruvate and malate, which fuel the TCA cycle to stimulate CO2 generation. Under these conditions, CAI diminished COX activity by 37% (Figure 5A, p < 0.001), suggesting that the CO2 had to be converted to bicarbonate to sustain COX activity. The inhibitory effect of CAI was reversed by exogenous bicarbonate, which directly stimulates sAC, or by the addition of 8Br-cAMP, which bypasses sAC stimulation. Diminishing CO2 production by retrograde inhibiti" @default.
• W2090848174 created "2016-06-24" @default.
• W2090848174 creator A5008828417 @default.
• W2090848174 creator A5012834359 @default.
• W2090848174 creator A5017781653 @default.
• W2090848174 creator A5024790869 @default.
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• W2090848174 date "2009-03-01" @default.
• W2090848174 modified "2023-10-17" @default.
• W2090848174 title "Cyclic AMP Produced inside Mitochondria Regulates Oxidative Phosphorylation" @default.
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