. Author manuscript; available in PMC: 2021 Apr 1.

Published in final edited form as: Int J Biochem Cell Biol. 2020 Feb 2;121:105704. doi: 10.1016/j.biocel.2020.105704

Table 1.

Summary of functionally characterized phosphorylation sites of Cytc

Phosphorylation site	Low-throughput identification	Experimental models	Functional and structural characterization
Threonine 28	Electrospray ionization-mass spectrometry (bovine kidney) (Mahapatra et al., 2017).	In vivo phosphorylated protein purified from bovine kidney, phosphomimetic T28E recombinant protein, mouse lung fibroblast cells expressing T28E Cytc (Mahapatra et al., 2017); phosphomimetic T28D recombinant protein (Guerra-Castellano et al., 2016).	Lower Cytc-COX activity, lower redox potential, higher rate of reduction. Lower respiration, membrane potential and ROS production in intact cells. Phosphorylated by AMPK (Mahapatra et al., 2017). Lower binding affinity with Cytc₁ in bc₁ complex (Guerra-Castellano et al., 2016).
Serine 47	Electrospray ionization-mass spectrometry (porcine brain tissue) (Kalpage et al., 2019b).	In vivo phosphorylated protein purified from porcine brain, phosphomimetic S47E recombinant protein (Kalpage et al., 2019b); phosphomimetic S47D recombinant protein (Guerra-Castellano et al., 2016).	Lower Cytc-COX activity, lower caspase-3 activity, lower cardiolipin peroxidase activity, reduced heme degradation (Kalpage et al., 2019b).
Tyrosine 48	Electrospray ionization-mass spectrometry (bovine liver) (Yu et al., 2008).	In vivo phosphorylated protein purified from bovine liver (Yu et al., 2008), Phosphomimetic Y48E recombinant protein (Garcia-Heredia et al., 2011; Pecina et al., 2010), Y48pCMF phosphomimetic Cytc (Moreno-Beltran et al., 2017).	Lower Cytc-COX activity (Pecina et al., 2010; Yu et al., 2008), abolished caspase-3 activity, reduced cardiolipin peroxidase activity, reduced cardiolipin binding, lower redox potential (Pecina et al., 2010). Lower caspase-9 activity (Moreno-Beltran et al., 2017).
Threonine 58 (replaced with isoleucine in humans)	Electrospray ionization-mass spectrometry (rat kidney) (Wan et al., 2019).	Phosphomimetic T58E recombinant protein, mouse lung fibroblasts expressing T58E Cytc (Wan et al., 2019).	Lower Cytc-COX activity, lower caspase-3 activity, lower redox potential, lower rate of oxidation, lower cardiolipin peroxidase activity, higher rate of reduction, lower respiration, membrane potential and ROS production in intact cells. Decreased cell death when treated with H₂O₂ and staurosporine (Wan et al., 2019).
Tyrosine 97	Electrospray ionization-mass spectrometry (bovine heart, insulin-treated porcine brain).	In vivo phosphorylated protein purified from bovine heart (Lee et al., 2006), phosphomimetic Y97E recombinant Cytc (Garcia-Heredia et al., 2011); Y97pCMF phosphomimetic Cytc (Guerra-Castellano et al., 2018); insulin-treated rat model of global brain ischemia (Sanderson et al., 2013a).	Lower Cytc-COX activity, shift of the characteristic 695 nm peak to 687 nm (change in heme environment) (Lee et al., 2006), less thermally stable (Garcia-Heredia et al., 2011), lower caspase-3 activity (Guerra-Castellano et al., 2018), lower Cytc release from mitochondria, decreased neuronal cell death (Sanderson et al., 2013a).