Phosphorylation of Mitofusin 2 Regulates Endoplasmic Reticulum‐Mitochondrial Calcium Coupling

Abstract

Introduction Contact sites between the endoplasmic reticulum (ER) and mitochondria (i.e., mitochondria‐associated membranes: MAMs) have important roles for the exchange of lipids, Ca2+, and reactive oxygen species (ROS), and greatly influence mitochondrial bioenergetics and cell fate. Mitofusin 2 (Mfn2), a mitochondrial fusion protein, is critical for MAM formation by tethering two organelles together to initiate contact. Although several post‐translational modifications (PTMs) of Mfn2 have been identified, including serine/threonine phosphorylation and ubiquitination, it remains unclear whether the PTMs of Mfn2 regulate its tethering function. In addition, while basal tyrosine phosphorylation (P‐Tyr) of Mfn2 was reported from mass spectroscopy data, the signaling pathways that regulate P‐Tyr levels of Mfn2 are completely unknown. Objective To determine whether P‐Tyr of Mfn2 modulates MAM functions. Methods Biochemical (mitochondrial fractionation), cell biological (Foster resonance energy transfer [FRET] efficiency between the outer mitochondrial membrane (OMM)‐targeted cyan fluorescent protein and ER membrane‐targeted yellow fluorescent protein), and physiological (imaging of mitochondrial Ca2+ [mtCa2+], ROS, and membrane potential [Δψm] in live cells) assays were performed in HEK293T cells. Results Endogenous expression of several tyrosine kinases, including proto‐oncogene tyrosine protein kinase (Src), C‐Terminal Src Kinase (CSK), and proline‐rich tyrosine kinase 2 (Pyk2), was found in the cytosolic and mitochondrial fractions of HEK293T cells. Overexpression of these proteins increased P‐Tyr levels of Mfn2, as detected by a general P‐Tyr antibody. Next, we found that CSK knockdown by shRNA in HEK293T cells enhances the physical coupling between the OMM and ER membrane compared to control cells, as determined by biochemical and live‐cell FRET assays. We also found that CSK knockdown induces mild, but significant, Δψm depolarization and increases basal mitochondrial ROS levels, which were quantified by a Δψm‐sensitive dye TMRE, and a mitochondria‐targeted H2O2 biosensor mt‐RoGFP2‐Orp1, respectively. Lastly, we observed mtCa2+ uptake in response to ER Ca2+ release induced by Gq protein‐coupled receptor stimulation, using a mitochondria‐targeted Ca2+ biosensor mt‐RCamp1h. Importantly, CSK‐knockdown enhanced mtCa2+ uptake in cells compared to control despite the mild Δψm depolarization. Conclusion Mfn2 has potential to be phosphorylated by tyrosine kinases in situ. P‐Tyr levels of Mfn2 may modulate the physical coupling and Ca2+ transport between organelles, which promotes mtCa2+‐dependent Δψm depolarization and mitochondrial ROS generation. Support or Funding Information A part of this research was supported by American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.