Insight into the Interactome of Intramitochondrial PKA Using Biotinylation-Proximity Labeling

Abstract

Mitochondria are fully integrated in cell signaling. Reversible phosphorylation is involved in adjusting mitochondrial physiology to the cellular needs. Protein kinase A (PKA) phosphorylates several substrates present at the external surface of mitochondria to maintain cellular homeostasis. However, few targets of PKA located inside the organelle are known. The aim of this work was to characterize the impact and the interactome of PKA located inside mitochondria. Our results show that the overexpression of intramitochondrial PKA decreases cellular respiration and increases superoxide levels. Using proximity-dependent biotinylation, followed by LC-MS/MS analysis and in silico phospho-site prediction, we identified 21 mitochondrial proteins potentially targeted by PKA. We confirmed the interaction of PKA with TIM44 using coimmunoprecipitation and observed that TIM44-S80 is a key residue for the interaction between the protein and the kinase. These findings provide insights into the interactome of intramitochondrial PKA and suggest new potential mechanisms in the regulation of mitochondrial functions.

Regulation of Mitochondrial Homeostasis by sAC-Derived cAMP Pool: Basic and Translational Aspects

“…PKA and EPAC, two important cAMP effectors, are also present in the mitochondrial matrix, the IMS, and the OMM [24,25]. Several reports suggested there are a large number of PKA phosphorylation targets in mitochondria [26,27]. Similarly, EPAC targets localized in mitochondria have also been reported [28,29].…”

“…Cells meet their energy needs by glycolysis or by substrate oxidation via mitochondrial OXPHOS. Multiple signalling pathways precisely regulate both processes, and emerging data have revealed the critical role of sAC-generated cAMP under physiological and pathological conditions [16,24,27,92]. There is a tight balance between these processes, and both work in a coordinated manner to meet the energy demands of the cells.…”

Editorial of Special Issue “Protein Post-Translational Modifications in Signal Transduction and Diseases”

“…Even though PTMs in mitochondria have been often overlooked in the past, it has now been recognized that modifications of mitochondrial proteins represent a key mechanism in regulating organelle functions, including mitochondrial metabolic pathways, transport across the inner and outer membranes, fission, fusion, and degradation [ 9 , 10 ]. In their research article, Amer and Hebert-Chatelain, using a proximity-labelling technique, identified 21 proteins constituting the PKA intramitochondrial interactome and shed light on new potential functions exerted by the kinase in mitochondria [ 11 ]. Guerra-Castellano et al in their review describe the PTMs regulating cytochrome c, a double-face protein–an essential component of the mitochondrial electron transport chain–and, at the same time, responsible for triggering apoptosis when released into the cytosol [ 12 ].…”