Phosphorylations in the Termini of Mitochondrial Calcium Uniporter Regulate Mitochondrial Calcium Uptake

Mitochondrial Ca2+ (mtCa2+) uptake via the mtCa2+ uniporter (MCU) mediates cell survival and death. Our group previously showed that activation and mitochondrial translocation of proline‐rich tyrosine kinase 2 (Pyk2), tyrosine phosphorylation (P‐Tyr) of MCU, and enhancement of mtCa2+ uptake concomitantly occur in cardiomyocytes under Gq protein–coupled receptor (GqPCR) stimulation, which activates apoptotic myocyte death. However, it is still unclear how Pyk2 access to MCU structure, phosphorylate MCU, and modulate its channel function as well as mitochondrial Ca2+ uptake profile. Here, we determined Pyk2‐specific P‐Tyr site(s) in MCU and investigated the detailed molecular mechanism underling the regulation of mtCa2+ uptake via P‐Tyr of MCU. Through computational prediction programs for phosphorylation sites, we identified three Tyr residues (Y158, Y289, and Y317) in human MCU as candidates for general PTK phosphorylation sites, which are highly conserved across all eukaryotic species. P‐Tyr levels at these candidate sites were biochemically determined before and after the stimulation of a GqPCR in HEK293T cells stably overexpressing wild type (WT)‐ or dephosphomimetic mutants of MCU (MCU‐YFs). Only two Tyr sites (Y517F at the N‐ and Y317 C‐termini of MCU) exhibited increased P‐Tyr levels in response to GqPCR stimulation. Overexpression of MCU‐Y317F failed to increase mtCa2+ uptake in response to cytosolic Ca2+ elevation, although the overexpression of WT‐ and the other two MCU‐YFs significantly augmented mtCa2+ uptake compared to non‐transfected cells. Furthermore, we also found that MCU‐Y317F‐flag exhibits less association with WT‐MCU‐GFP compared to those observed in WT‐MCU‐flag, indicating reduced subunit oligomerization by this point mutation. Finally, we demonstrated that increased in P‐Tyr of MCU levels occurs in the ventricle tissues from transgenic (TG) mice with cardiac‐specific overexpression of a constitutively active Gαq protein (Gαq‐Q209L TG mice) compared to those from WT, concomitant with higher Pyk2 activation and apoptotic signaling. In summary, we conclude that MCU contains Pyk2‐specific phosphorylation site(s) and Pyk2‐dependent P‐Tyr of MCU at these site(s) increases mitochondrial Ca2+ uptake possibly via enhanced subunit oligomerization.Support or Funding InformationJ.O.‐U. was supported by NIH/NHLBI R01HL136757, AHA 16SDG27260248, Rhode Island Foundation #20164376 Medical Research Grant, and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award. J.L.C. and S.M.A were supported by Undergraduate Teaching & Research Award (UTRA) from Brown University. B.S.J was supported by 18CDA34110091. S.S.S was supported by NIH/NHLBI R01HL093671 and R01HL122124.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.