Role of tyrosine phosphorylation of Mfn2 in endoplasmic reticulum-mitochondria coupling

Introduction: A mitochondrial fusion protein, Mitofusin 2 (Mfn2) serves as one of the major components for tethering the mitochondria and endoplasmic reticulum (ER), which regulates the exchange of lipids, Ca2+, and reactive oxygen species (ROS) across the two organelles. Several post-translational modifications (PTMs) of Mfn2 have been identified via mass spectroscopy, such as tyrosine phosphorylation (P-Tyr). However, little information is available whether any PTMs of Mfn2 can regulate mitochondrial and ER tethering function under physiological and pathological conditions. Hypothesis: P-Tyr of Mfn2 regulates Mfn2 function such as ER-mitochondrial tethering. Methods: To achieve stable activation of c-Src, we stably overexpressed c-Src or an shRNA targeting the shRNA targeted to C-terminal Src kinase (CSK), a specific negative regulator of c-Src, in HEK293T cells. Stable cell lines were used for biochemical, cell biological, and physiological assays. Results: Two computational program for phosphorylation site prediction, NetPhos and Group-Based Prediction System, predicted at least five c-Src-specific P-Tyr sites within the Mfn2 structure. Using immunoprecipitation and a phosphate-affinity electrophoresis technique (Phos-tag SDS-PAGE), we found that overexpression of c-Src, but not other Src family members, leads to P-Tyr of Mfn2. Moreover, dephospho-mimetic mutations (replacing Tyr with Phe) in Mfn2 significantly decreased the c-Src-dependent P-Tyr in Mfn2. Stable c-Src activation decreased the distance between the ER and outer mitochondrial membrane (OMM) as assessed by transmission electron microscopy and Förster resonance energy transfer between an OMM-targeted cyan fluorescent protein and an ER membrane-targeted yellow fluorescent protein the ER membrane measured by confocal microscopy. Mitochondrial Ca 2+ (mtCa 2+ ) uptake in response to cytosolic Ca 2+ elevation increased with faster kinetics under c-Src activation, followed by increased mitochondrial ROS, and decreased oxygen consumption rate. Knocking down of endogenous Mfn2 by stable expression of shRNA targeting 3’ untranslated region of Mfn2 increased the distance between ER and OMM, and decreased mtCa 2+ uptake. The reintroduction of wild-type Mfn2 in Mfn2-knockdown cells reversed these changes to the levels observed in cells expressing control shRNA. However, re-expression of a dephspho-mimetic mutant of Mfn2 in Mfn2-knockdown cells failed to rescue the changes in mtCa 2+ uptake profile. Conclusion: P-Tyr of Mfn2 by c-Src modulates the distance between the OMM and ER, resulting in mtCa 2+ elevation and oxidative stress. NIH/NHLBI R01HL136757 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.