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

• Published in: International journal of molecular sciences Vol. 21; no. 21; p. 8283
• Main Authors: Ould Amer, Yasmine, Hebert-Chatelain, Etienne
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI 05.11.2020; MDPI AG
• Subjects: BioID2; Biotinylation - methods; Cell Respiration - physiology; Cross-Linking Reagents - chemistry; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - metabolism; HEK293 Cells; HeLa Cells; Humans; Immunoprecipitation - methods; Microscopy, Fluorescence; mitochondria; Mitochondria - chemistry; Mitochondria - metabolism; Mitochondrial Proteins - chemistry; Mitochondrial Proteins - metabolism; Protein Binding; Protein Interaction Mapping - methods; protein kinase A; Protein Processing, Post-Translational; proteomics; serine/threonine phosphoprediction; Signal Transduction; Staining and Labeling - methods; TIM44; TIM44; serine/threonine phosphoprediction; BioID2; proteomics; protein kinase A; mitochondria
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21218283

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.