Protein Kinase D Modulates the Mitochondrial Permeability Transition Pore

Introduction: The mitochondrial permeability transition pore (mPTP) is a large, high-conductance channel formed under stress conditions, which promotes the release of Ca 2+ , reactive oxygen species (ROS), and proapoptotic proteins from the mitochondria, which initiates mitochondrial membrane depolarization and cell death signaling. The mPTP opening is a crucial mechanism of cell death under various pathological conditions, such as ischemia/reperfusion injury. Recent studies suggest that the c-subunit of F 1 F 0 ATP synthase, encoded by ATP5G1/2/3, is a key component of the mPTP, offering new insights into its regulation through cellular signaling. We previously reported that a stress-responsive serine/threonine kinase, protein kinase D (PKD), exists in the mitochondria, and PKD activation partially, but significantly, opens the mPTP during G q protein-coupled receptor (G q PCR) stimulation (Jhun BS, et al., J Physiol. 2018). This G q PCR-mediated mPTP opening is inhibited by treatment with a potent PKD inhibitor, suggesting a potential link between mPTP function and PKD signaling. Hypothesis: PKD regulates the mPTP functions Objective: To test whether PKD can interact with mPTP, and/or change the phosphorylation levels of the structural proteins, which ultimately modulates mPTP activity. Methods: HEK293T cells were transfected with GFP-tagged PKD1 (GFP-PKD), a main isoform of the PKD family, a dominant-negative mutant of PKD1 (GFP-PKD-K612W, termed GFP-PKD-DN), a constitutively active mutant of PKD1 (GFP-PKD-S738/742E), or its control vector. Cells were harvested 48-72 hours after transfection, and the whole cell lysates were used for biochemical assays. All Western blotting experiments were repeated at least three times using independent sample sets. Computational analyses were performed to predict PKD-specific phosphorylation sites on the c-subunit using the Group-based Prediction System with the light gradient boosting machine (GPS 6.0, Chen M et al., Nucleic Acids Res. 2023). Results: The mPTP pore-forming protein, c-subunit, and its accessory subunits (i.e., OSCP subunit of ATP synthase, adenine nucleotide translocator, and cyclophilin D) were not detected by co-immunoprecipitation with GFP-PKD1 using anti-GFP antibody followed by Western blotting. However, immunoprecipitation of the endogenous c-subunit with an anti-ATP5G1/2/3 antibody showed a trend of increasing serine/threonine phosphorylation levels in the c-subunit after overexpression of PKD1, further increased by overexpression of constitutively active phospho-mimetic PKD1. Similar results were observed using a phosphate-affinity electrophoresis technique (Phos-tag SDS-PAGE) with Zn 2+ as a divalent metal ion. The computational analysis predicted a serine residue of the c-subunit at S102 within the ATP5G1 structure as a potential PKD-specific phosphorylation site, residing between the two transmembrane domains and exposed to the mitochondrial matrix. Conclusion: PKD signaling could modulate mPTP function through phosphorylation of its pore subunit. Future studies will aim to investigate whether c-subunit is a direct substrate of PKD and further assess the impact of c-subunit phosphorylation on ATP synthase and mPTP functions. NIH/NHLBI R01HL160699 (to B.S.J.), R01HL171710 (to J.O.-U. and B.S.J.), and R01HL136757 (to J.O.-U.) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.