PINK1 Defect Causes Mitochondrial Dysfunction, Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease
Figure 2
PINK1 auto-phosphorylation is impaired by L347P and E417G mutations.
PINK1 protein is auto-phosphorylated in the cells expressing wild type PINK1 and the auto-phosphorylation was decreased in cells expressing L347P- or E417G-PINK1. Lysates from the SH-SY5Y cells expressing wild type-PINK1-Flag (1st and 2nd panels), L347P-PINK1-Flag (3rd and 4th panels) or E417G- PINK1-Flag (5th and 6th panels) were subjected to 2-dimensional gel electrophoresis, followed by Western analyses using anti-Flag Ab. Of the same protein, the more phosphorylated ones migrated to the more acidic end (left hand) of the isoelectric focusing (IEF) gel, while less or non-phosphorylated ones migrated to the more basic end (right hand) of IEF. 1st panel: Wild type PINK1 along the pH gradient of the IEF. Spot 1, 2 (64 kD), and 3 (50 kD) migrated to the acidic end, and several others (open arrows) to the basic end. 2nd panel: A control of wild type PINK1 proteins treated with alkaline phosphotase (AP). While the spots on the basic end still remained in the same region of the gel as in the 1st panel, spot 1, 2, and 3 were missing. This indicated that these 3 spots were phospho-PINK1 in 1st panel, and were de-phosphorylated in the 2nd panel. The difference between spot 1 and 2 reflected the degree of phosphorylation on PINK1. In cells expressing L347P or E417G-PINK1, spot 1, 2 and 3 were greatly reduced, indicating reduced auto-phosphorylation on L347P- or E417G-PINK1 (3rd and 5th panels). The 4th and 6th panels are controls (treated with AP) for the 3rd and 5th panels respectively, similar to the 2nd panel.