Figure 5.
Mechanisms of kinase upregulation by mitochondrial superoxide. A, Inhibition of protein phosphatases by mitochondrial O2- promotes CaMKII activation. Superoxide-enhanced CaMKII autophosphorylation activated by high-frequency stimulation (100 Hz/18 sec) was not affected by inhibiting PP1 (high OA, 1 μm) or calcineurin (FK506) and was only weakly elevated by inhibiting PP2A (low OA, 20 nm) (left bar group). The low level of OA-sensitive PP2A activity observed here indicates that mitochondrial O2- suppressed but did not abolish phosphatase activity and is consistent with the idea that the effect of O2- is mainly modulatory. All three blockers not only reversed the suppression of CaMKII autophosphorylation normally observed in the presence of rotenone- oligomycin (compare white bars, gray bars; see also Fig. 3D) but even enhanced autophosphorylation compared with 100 Hz/18 sec stimulation without drugs (middle bar group); blocking PP1 was particularly effective. In neurons activated by weak stimulation (5 Hz/180 sec), pharmacological block of any of these phosphatases leads to enhancement of CaMKII phosphorylation (right bar group) because, under these conditions, protein phosphatases are not already suppressed by mitochondrially produced superoxide. Symbols indicating statistical significance are as defined in Figure 2. B, None of the three protein phosphatase inhibitors had an effect on pPKCα activation, regardless of stimulation protocol or the presence of respiratory inhibitors. Therefore, mitochondrial O2- must activate pPKCα (see Fig. 3D) by a mechanism(s) other than phosphatase inhibition.