Role of Tyrosine Phosphorylation of Mitochondrial Calcium Uniporter in Regulating Mitochondrial Calcium Homeostasis

Mitochondrial Ca2+ (mtCa2+) uptake via the mtCa2+ uniporter (MCU) complex is a critical factor in determining cell survival or death. Basal tyrosine phosphorylation (P-Y) of the pore forming subunit, MCU has been reported via mass spectroscopy, and our lab reported that the activation of a Ca2+- and reactive oxygen species (ROS)-sensitive protein tyrosine kinase (PTK), proline-rich tyrosine kinase 2 (Pyk2), increases P-Y of MCU and mtCa2+ uptake. However, further research 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. 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. Only three tyrosine residues were identified as potential phosphorylation candidate sites for PTKs using phosphorylation prediction programs, and they 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 Gq protein–coupled receptor (GqPCR) stimulation using cell lines stably expressing wild-type (WT)- or dephosphomimetic mutants of MCUs (MCU-YFs). This in-situ assay revealed that only two tyrosine sites increased P-Y levels in response to GqPCR stimulation. Finally, we assessed mtCa2+ uptake profiles in cells stably expressing WT- and MCU-YFs in response to cytosolic Ca2+ elevation via live cell imaging using mitochondria-targeted Ca2+-sensitive biosensors. Although the overexpression of WT- and two of the MCU-YFs significantly accelerated mtCa2+ uptake compared to non-transfected cells, overexpression of one of the MCU-YFs failed to increase mtCa2+ uptake in response to cytosolic Ca2+ elevation. In summary, MCU contains Pyk2-specific phosphorylation site(s) and Pyk2-dependent P-Y of MCU increases mtCa2+ uptake via the MCU complex.