Figure 2. Cys323 is the critical cysteine residue that is S-nitrosylated in parkin.
(a) A schematic representation of the structure of the RING1 and IBR domains in parkin. Yellow and red circles indicate cysteines involved in zinc coordination and not involved in zinc coordination, respectively. Blue circle indicates histidine involved in zinc coordination. (b) Cell lysates from SH-SY5Y cells transfected with the FLAG-tagged wild type, C323A or C323S parkin expression plasmid were analysed by SNO-RAC as above (upper panel). The quantity of S-nitrosylated parkin, as measured by scanning densitometry, is expressed as a percentage of control, normalized with respect to total parkin. Data shown are mean ± SE (n = 4); **p < 0.01 versus wild type (lower panel). SH-SY5Y cells transfected with the FLAG-tagged wild type, C323A or C323S parkin expression plasmid were incubated for 3 hr with 50 μM GSNO (c), 8 μM A23187 (d), 1 μM rotenone (e), or 10 μM CCCP (f), and then the cell lysates were analysed by SNO-RAC (upper panels). The quantity of S-nitrosylated parkin, as measured by scanning densitometry, is expressed as a percentage of control, normalized with respect to total parkin. Data shown are mean ± SE (n = 3); **p < 0.01 (lower panels). Full scans of the blots in b, c, d, e and f are available in Supplementary Information, Fig.S10.