Proline‐rich Tyrosine Kinase 2 Phosphorylates Mitochondrial Calcium Uniporter and Regulates Mitochondrial Calcium Uptake
Authors: Cao Jessica L. , Adaniya Stephanie M. , Yang Dongqin , King Michelle E. , Jhun Bong Sook , Mende Ulrike , Sheu Shey‐Shing , O‐Uchi Jin 
Published: June 2021

Mitochondrial Ca2+ (mtCa2+) uptake via the mtCa2+ uniporter (MCU) complex is a critical factor in determining cell survival or death. We previously reported that the activation of a Ca2+‐ and reactive oxygen species (ROS)‐sensitive protein tyrosine kinase (PTK), proline‐rich tyrosine kinase 2 (Pyk2), under Gq protein–coupled receptor (GqPCR) stimulation increases tyrosine phosphorylation (P‐Y) of MCU and mtCa2+ uptake, followed by the activation of mitochondrial permeability transition pore, and apoptotic cell death in cardiomyocytes. However, further investigation is required to determine 1) the identity of Pyk2‐specific phosphorylation sites within the MCU structure and 2) the functional relevance of P‐Y to MCU channel function as well as mitochondrial functions both in situ and in vivo. In this project, we determined specific Pyk2 phosphorylation site(s) in MCU and tested whether P‐Y at these site(s) modulates MCU channel function using cell lines stably expressing wild‐type (WT)‐ or dephosphomimetic mutants of MCU (MCU‐YFs). Furthermore, we investigated the role of MCU P‐Y in vivo using transgenic (TG) mice with cardiac‐specific overexpression of constitutively active Gαq protein (Gαq TG mice). Through phosphorylation prediction programs, we identified three tyrosine residues as candidate PTK phosphorylation sites, which are conserved across all eukaryotic species. In vitro kinase assays showed that purified active Pyk2 phosphorylated purified full‐length MCU. Next, P‐Y levels at candidate sites were biochemically detected after GqPCR stimulation using cell lines stably expressing Flag‐tagged WT‐ or MCU‐YFs. This in situ assay revealed that only two tyrosine sites increased P‐Y levels in response to GqPCR stimulation. We further assessed mtCa2+ uptake profiles in response to cytosolic Ca2+ elevation in cells stably expressing WT‐ and MCU‐YFs via live cell imaging using mitochondria‐targeted Ca2+‐sensitive biosensors. Using GFP‐tagged MCU‐YFs, we confirmed that all mutant MCUs, like WT‐MCU, were exclusively expressed in mitochondria. Overexpression of one MCU‐YF failed to increase mtCa2+ uptake in response to cytosolic Ca2+ elevation, although the overexpression of WT‐ and the other two MCU‐YFs significantly accelerated mtCa2+ uptake compared to non‐transfected cells. Finally, we demonstrated that P‐Y of MCU occurs in Gαq TG mouse hearts, concomitant with higher Pyk2 activation and apoptotic signaling. In summary, MCU contains Pyk2‐specific phosphorylation site(s) and Pyk2‐dependent P‐Y of MCU increase mtCa2+ uptake via the MCU complex. Moreover, GqPCR‐Pyk2 signaling may induce P‐Y of MCU and cardiomyocyte death in vivo. These findings suggest that inhibition of GqPCR‐Pyk2‐MCU signaling may be a novel therapeutic target to prevent mitochondrial Ca2+ overload, oxidative stress, and cardiomyocyte death during pathophysiological conditions such as heart failure, in which chronic GqPCR stimulation occurs.

Support or Funding Information

B.S.J was supported by NIH/NIGMS U54GM115677 and P30GM1114750. U.M. was supported by NIH/NHLBI R01HL114784. S.S.S was supported by NIH/NHLBI R01HL093671 and R01HL122124. J.O.‐U. was supported by NIH/NHLBI R01HL136757, NIH/NIGMS P30GM1114750, American Heart Association (AHA) 4BGIA18830032, AHA 16SDG27260248, Rhode Island Foundation #20164376 Medical Research Grant, and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.