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Role of Src‐Dependent Phosphorylation of Mitofusin 2 in Endoplasmic Reticulum‐Mitochondria Tethering
Author(s) -
Zhou Hana,
Polina Iuliia,
Cypress Michael,
Jhun Bong Sook,
Zhang Peng,
OUchi Jin
Publication year - 2021
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.2021.35.s1.02460
Subject(s) - mfn2 , microbiology and biotechnology , proto oncogene tyrosine protein kinase src , endoplasmic reticulum , mitochondrion , mitochondrial fusion , tyrosine phosphorylation , chemistry , mitophagy , biology , phosphorylation , biochemistry , autophagy , apoptosis , mitochondrial dna , gene
: Contact sites between the mitochondria and endoplasmic reticulum (ER) are one of the key structures involved in regulating lipid, Ca 2+ and reactive oxygen species exchange across the two organelles which is critical for the maintenance of mitochondrial bioenergetics. Recent studies have reported that a major component of the tethering structures between the two organelles is mitofusin 2 (Mfn2), a GTPase associated with fusion of the outer mitochondrial membrane (OMM) as well as the activities of mitophagy and ER stress. While several post‐translational modifications (PTMs) that influence the fusion and mitophagy functions of Mfn2 have been identified, it remains unclear whether there are PTMs that can regulate its tethering function. Data from mass spectroscopy reveals that there is basal tyrosine phosphorylation (P‐Tyr) of Mfn2, but it is unknown whether Mfn2 is a substrate of mitochondria‐localized tyrosine kinases (proline rich tyrosine kinase 2 [Pyk2] and proto‐oncogene Src kinase [Src]). Objective : To determine whether P‐Tyr of Mfn2 regulates Mfn2 functions. Methods HEK293T cells stably overexpressing shRNA targeted to a specific negative regulator of Src, C‐terminal Src kinase (CSK) or a control vector were subjected to several biochemical (Fluorescent western blotting), cell biological (Transmission electron microscopy [TEM], Förster Resonance Energy Transfer [FRET] between cyan‐fluorescent protein targeted to the OMM and yellow‐fluorescent protein targeted to the ER membrane observed under confocal microscopy) and physiological (mitochondrial calcium [mtCa 2+ ] uptake measurements using a mitochondria‐targeted Ca 2+ biosensor, mt‐RCaMp1h) assays. Results Overexpression of Pyk2 and Src lead to P‐Tyr of Mfn2 with Src having greater P‐Tyr levels of Mfn2. Generation of a HEK293T cell line with stable knockdown of CSK reveals that CSK knockdown promotes Src activation in the mitochondria and increases P‐Tyr of Mfn2 without changing expression levels of Src and Mfn2. CSK knockdown (CSK‐KD) cells showed decreased distance between the ER and OMM as assessed by FRET measurement under confocal microscopy. This change was abolished by pretreatment with a Src‐specific inhibitor, PP2. TEM images also showed that CSK knockdown decreases the distance between OMM and ER without changing the length of interface. To assess the impact of changes in the physical interaction between ER and mitochondria, mtCa 2+ uptake profiles in CSK‐KD cells were observed. In CSK‐KD cells, mtCa 2+ uptake in response to cytosolic Ca 2+ elevation induce by G q ‐protein coupled receptor stimulation was enhanced with faster kinetics compared to that in control cells. While physical coupling between ER and mitochondria increased, we found that hallmarks of ER stress and mitophagy were not altered in CSK‐KD cells compared to control cells. Conclusion : Src‐dependent phosphorylation of Mfn2 decreased the distance between the OMM and ER and lead to increased Ca 2+ exchange between ER and mitochondria.