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Phosphorylation of Mitofusin 2 Regulates Endoplasmic Reticulum‐Mitochondrial Calcium Coupling
Author(s) -
Sung Jae Hwi,
Ford Kara A.,
Moeller Jacob,
Suzuki Yuta,
Cypress Michael W.,
Jhun Bong Sook,
O-Uchi Jin,
Zhang Peng
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.08911
Subject(s) - microbiology and biotechnology , mfn2 , endoplasmic reticulum , tyrosine phosphorylation , xbp1 , hek 293 cells , phosphorylation , mitochondrion , biology , mitochondrial fusion , chemistry , biochemistry , receptor , mitochondrial dna , rna , rna splicing , gene
Introduction Contact sites between the endoplasmic reticulum (ER) and mitochondria (i.e., mitochondria‐associated membranes: MAMs) have important roles for the exchange of lipids, Ca 2+ , 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 Ca 2+ [mtCa 2+ ], ROS, and membrane potential [ Δψ m ] 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, Δψ m depolarization and increases basal mitochondrial ROS levels, which were quantified by a Δψ m ‐sensitive dye TMRE, and a mitochondria‐targeted H 2 O 2 biosensor mt‐RoGFP2‐Orp1, respectively. Lastly, we observed mtCa 2+ uptake in response to ER Ca 2+ release induced by G q protein‐coupled receptor stimulation, using a mitochondria‐targeted Ca 2+ biosensor mt‐RCamp1h. Importantly, CSK‐knockdown enhanced mtCa 2+ uptake in cells compared to control despite the mild Δψ m depolarization. Conclusion Mfn2 has potential to be phosphorylated by tyrosine kinases in situ . P‐Tyr levels of Mfn2 may modulate the physical coupling and Ca 2+ transport between organelles, which promotes mtCa 2+ ‐dependent Δψ m 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.