Abstract
Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses, but the role of mitochondria in such responses is poorly understood. To define such roles, we used quantitative proteomic analysis of mitochondria-enriched fractions and performed functional and morphologic studies of wild-type (WT) and kin(-) (PKA-null) murine S49 lymphoma cells. Basally, 75 proteins significantly differed in abundance between WT and kin(-) S49 cells. WT, but not kin(-), S49 cells incubated with the cAMP analog 8-(4-chlorophenylthio)adenosine cAMP (CPT-cAMP) for 16 h have (a) increased expression of mitochondria-related genes and proteins, including ones in pathways of branched-chain amino acid and fatty acid metabolism and (b) increased maximal capacity of respiration on branched-chain keto acids and fatty acids. CPT-cAMP also regulates the cellular rate of ATP-utilization, as the rates of both ATP-linked respiration and proton efflux are decreased in WT but not kin(-) cells. CPT-cAMP protected WT S49 cells from glucose or glutamine deprivation, In contrast, CPT-cAMP did not protect kin(-) cells or WT cells treated with the PKA inhibitor H89 from glutamine deprivation. Under basal conditions, the mitochondrial structure of WT and kin(-) S49 cells is similar. Treatment with CPT-cAMP produced apoptotic changes (i.e. decreased mitochondrial density and size and loss of cristae) in WT, but not kin(-) cells. Together, these findings show that cAMP acts via PKA to regulate multiple aspects of mitochondrial function and structure. Mitochondrial perturbation thus likely contributes to cAMP/PKA-mediated cellular responses.
Highlights
S49 kinϪ cells, which lack active protein kinase A (PKA), can identify roles of PKA, including at the mitochondria, in which such roles are not well defined
Known or predicted mitochondrial localization was determined using the COMPARTMENTS database [30]; 1871 of the proteins we detected were listed in COMPARTMENTS, 1305 of which had known or predicted mitochondrial localization based on text mining or protein sequence
We studied S49 lymphoma cells and utilized multiple complementary approaches to determine if Cyclic AMP (cAMP)/PKA regulates the mitochondrial proteome and influences the structure and function of mitochondria
Summary
S49 kinϪ cells, which lack active protein kinase A (PKA), can identify roles of PKA, including at the mitochondria, in which such roles are not well defined. Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses, but the role of mitochondria in such responses is poorly understood To define such roles, we used quantitative proteomic analysis of mitochondria-enriched fractions and performed functional and morphologic studies of wildtype (WT) and kin؊ (PKA-null) murine S49 lymphoma cells. Treatment with CPT-cAMP produced apoptotic changes (i.e. decreased mitochondrial density and size and loss of cristae) in WT, but not kin؊ cells Together, these findings show that cAMP acts via PKA to regulate multiple aspects of mitochondrial function and structure. Due to the role of mitochondria in cAMP/PKA-mediated death of S49 cells [6, 7], here we used proteomic analysis to identify cAMP/PKA-mediated changes in protein expression in a mitochondria-enriched fraction of S49 cells and conducted additional studies to assess cAMP/ PKA-regulated control of mitochondrial proteins and mitochondrial function
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