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Role of Src homology/collagen adaptor protein p66Shc in Pyk2 translocation into mitochondria under G q protein–coupled receptor stimulation
Author(s) -
Dileepan Gayathri,
Polina Iuliia,
Cypress Michael,
Suzuki Yuta,
Adhikari Neeta,
Jhun Bong Sook,
O-Uchi Jin
Publication year - 2020
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.2020.34.s1.07016
Subject(s) - signal transducing adaptor protein , proto oncogene tyrosine protein kinase src , microbiology and biotechnology , immunoprecipitation , biology , cytosol , tyrosine , tyrosine kinase , hek 293 cells , signal transduction , tyrosine phosphorylation , chemistry , receptor , biochemistry , gene , enzyme
Introduction Mitochondrial Ca 2+ (mtCa 2+ ) uptake via a main mtCa 2+ channel, mtCa 2+ uniporter (MCU), mediates cell survival and death via its contribution of mitochondrial ATP synthesis and apoptotic signaling activation, respectively. Our group previously showed that G q protein–coupled receptor (G q PCR) stimulation promotes activation of a tyrosine kinase named proline‐rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to the mitochondrial matrix, which leads to the tyrosine phosphorylation of MCU, and mtCa 2+ overload, and activation of apoptotic signaling in the cardiomyocytes. However, it is still unclear how Pyk2 access from cytosol to mitochondrial matrix under G q PCR stimulation. The Src homology/collagen (Shc) adaptor protein, p66Shc, is also known to be capable to translocate to the mitochondria where it promotes increased ROS production and apoptosis. Importantly, p66Shc can bind to another tyrosine kinase Src, but its association to Pyk2 has not been well investigated. Hypothesis p66Shc serves as an important chaperon for Pyk2 translocation into the mitochondrial matrix. Aim to test whether p66Shc can associate with Pyk2. Methods Mammalian expression plasmids containing p66Shc or Pyk2 were transformed in to bacteria and purified. Overexpression of p66Shc and/or Pyk2 in HEK293T cells were performed by transient plasmid transfection. Whole cell lysates were collected and used for biochemical assay including Western blotting and co‐immunoprecipitation assay. Results We confirmed that transient transfections of the plasmids containing His‐tagged p66Shc and GFP‐tagged Pyk2 were successfully yield overexpression of these proteins in HEK293Tcells. Co‐immunoprecipitation assay using GFP antibody showed that p66Shc and Pyk2 forms complex in the whole cell lysates from cells co‐transfected with two plasmids. Conclusion p66Shc can bind Pyk2 and may contribute to the mechanism for Pyk2 translocation to mitochondria. Pur next plan is to test whether the removal (overexpression) of p66Shc blocks (Pyk2 transportation and prevent excessive calcium uptake by the MCU under G q PCR stimulation both in heterologous expression system and primary cardiomyocytes. Support or Funding Information Apart of this research was supported by the Lillehei Heart Institute Summer Research Fellowship (to G.D.), American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.