Phosphorylation of Cyclophilin-D is Not Required for Regulation of The Mitochondrial Permeability Transition Pore by GSK3β

Genetic inhibition of cyclophilin D (CypD) delays the opening of the mitochondrial permeability transition pore (MPTP) and therefore reduces necrotic cell death. Elucidation of factors that impact CypD activity is therefore key to understanding the regulation of MPTP opening. Glycogen synthase kinase-3β (GSK3β) is a serine/threonine kinase that has been shown to modulate MPTP and cell death, potentially through phosphorylation of CypD. Therefore, we hypothesized that the mitochondrial fraction of GSK3β directly phosphorylates CypD and promotes opening of MPTP. Overexpression of full length GSK3β in mouse embryonic fibroblasts sensitized the MPTP and exacerbated oxidative stress-induced necrosis. In contrast, genetic inhibition of GSK3β protected against oxidant-induced cytotoxicity but did not affect the MPTP. Recombinant GSK3β could directly bind to and phosphorylate recombinant CypD. Mass spectrometry revealed several putative GSK3β phosphorylation sites on CypD. However, mutation of these sites did not affect the peptidyl prolyl isomerase activity of CypD and reconstitution of these phosphomutants in CypD-deficient cells increased MPTP sensitivity and oxidative-induced cell death to the same extent as wild-type CypD. Further, targeted overexpression of either wild-type or kinase-inactive GSK3β in the mitochondrial matrix did not impact MPTP or cell death. Moreover, while proteinase-K digestion of cardiac mitochondria showed a significant amount of GSK3β in the mitochondria, it was not localized to the matrix. Finally, overexpression of GSK3β was still able to increase MPTP sensitivity and oxidative stress-induced death in CypD-null cells. Taken together, these data indicate that, while GSK3β can modulate MPTP, this appears to be independent of GSK3β's interaction with, or phosphorylation of CypD.