Gayathri Dileepan

Mitochondrial Ca 2+ (mtCa 2+) homeostasis is critical for cell survival and death by regulating mitochondrial ATP synthesis and apoptotic signaling. We previously showed that 1) an important heart failure signaling, Gq protein–coupled receptor (GqPCR) stimulation, promotes activation of an oxidation-sensitive tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to mitochondrial matrix; 2) Pyk2 phosphorylates a main mtCa2+-uptake channel, mtCa2+ uniporter (MCU), which promotes mtCa2+ overload, and cardiomyocyte apoptosis. However, the detailed molecular mechanism of Pyk2 translocation under GqPCR stimulation is still unknown. The Src homology/collagen (Shc) adaptor protein, p66Shc, known as an inducer of cellular oxidation, can bind another tyrosine kinase Src and translocate to the mitochondria under stress signaling. Therefore, we hypothesize that p66Shc serves as an important chaperone for Pyk2 translocation into mitochondria. In this study, we tested whether p66Shc can associate with Pyk2. Co-transfections of the plasmids containing tagged p66Shc and Pyk2 successfully yielded overexpression of both proteins in HEK293T cells. Co-immunoprecipitation assays using tag antibodies showed the association of p66Shc and Pyk2. These results suggest that p66Shc and pPyk2 are capable of forming a complex that may contribute to Pyk2 translocation into mitochondria. Our next plan is to test the genetic knockdown of p66Shc for blocking Pyk2 transportation and MCU-mediated mtCa2+ overload under GqPCR stimulation in primary cardiomyocytes. The outcome of this study may provide a new therapeutic approach for heart failure where mtCa2+ overload plays an important role for disease formation and progression.