Introduction: The entry of Ca 2+ into mitochondria drives a multitude of cell functions, including bioenergetics, lipid handling, and reactive oxygen species (ROS) generation. Cytosolic Ca 2+ elevation sufficient for triggering mitochondrial Ca 2+ (mtCa 2+ ) uptake specifically occurs at the endoplasmic reticulum (ER)-mitochondria (ER-mito) microdomains since the major Ca 2+ -releasing sites at ERs face outer mitochondrial membranes (OMM). Although mitofusin 2 (Mfn2) is established as one of the components of ER-mito tethering, the mechanism regulating the tethering function of Mfn2 by cellular signaling and its impact on Ca 2+ handling at ER-mito contact sites remain unclear. Previous studies by our group indicate that tyrosine phosphorylation (P-Tyr) of Mfn2, c-Src activation, and decreased ER-mito microdomains co-exist in activated cardiac fibroblasts (CFs) in an animal model of pulmonary arterial hypertension (PAH) and right ventricular (RV) hypertrophy. We also found that activation of c-Src in the primary human CFs induced P-Tyr of Mfn2, decreased ER-mito microdomains, and activated proliferative signaling. Hypothesis: Mfn2 is a substrate of c-Src and c-Src-dependent P-Tyr of Mfn2 alters ER-mito tethering function. Methods: In vitro kinase assays were performed using GST-fused recombinant proteins. Dephospho-mimetic mutants of Mfn2 were generated by replacing Tyr (Y) residue with phenylalanine (F). Mutants were introduced into HEK239T cells and stable cell lines were produced. Phosphorylation of Mfn2 was detected by immunoprecipitation (IP) or Phos-tag SDS-PAGE. The size of ER-mito microdomains was assessed by live-cell imaging using a Förster resonance energy transfer (FRET)-based assay with OMM-targeted monomeric CFP (mt-CFP) and ER membrane-targeted monomeric YFP (ER-YFP) as a donor and an acceptor, respectively. The changes in Ca 2+ concentration at the mitochondrial matrix in response to cytosolic Ca 2+ elevation were measured by the mitochondrial matrix-targeted Ca 2+ -sensitive biosensor mtRCamp1h. Results: Introduction of wild-type c-Src (c-Src-WT), but not its kinase-inactive dominant-negative mutant c-Src-K295R (c-Src-DN) in HEK293T cells significantly increased P-Tyr of Mfn2. Moreover, recombinant c-Src increased P-Tyr of recombinant Mfn2 in vitro, indicating that c-Src is capable of directly phosphorylating Mfn2. Phosphorylation prediction programs (NetPhos 3.1 and GPS 5.0) indicated four Tyr residues in human Mfn2 predicted to be phosphorylated by protein Tyr kinases and/or c-Src (Y49, Y61, Y269, Y752), all of which reside in the cytoplasmic face of the Mfn2 termini. Among these dephospho-mimetic mutants, Mfn2-Y752F, in which the mutation is located at the C-terminal tail of Mfn2, did not show increased P-Tyr of Mfn2 after transient transfection of constitutive active mutant of Mfn2 (c-Src-CA). Additionally, Mfn2-Y752F failed to form dimers with WT-Mfn2 as assessed by Co-IP. Reintroduction of Mfn2-WT, but not Mfn2-Y752F, into Mfn2-knockdown (Mfn2-KD) HEK293T cells normalized ER-mito distance and mtCa 2+ uptake to the levels observed in the control HEK293T cells. Conclusion: c-Src phosphorylates the C-terminal tail of Mfn2 to modulate ER-mito tethering. Funding: The work was partially supported by NIH/NHLBI R01HL160699 (to B.S.J.), NIH/NHLBI R01HL171710 (B.S.J. and J.O.-U.), and NIH/NHLBI R01HL136757 (to J.O.-U.). This abstract was presented at the American Physiology Summit 2026 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.
Slotabec et al. (Fri,) studied this question.