VDAC Phosphorylation in Control of Mitochondrial Respiration

Abstract

Tubulin has been recently identified as a powerful regulator of voltage dependent anion channel (VDAC). Dimeric tubulin induces reversible blockage of VDAC reconstituted into planar lipid membranes and blocks the flux of ATP/ADP across the mitochondrial outer membrane. Here we show that phosphorylation of VDAC by cytosolic kinases protein kinase A (PKA) and glycogen synthase kinase-3β (GSK3β) increases the on-rate of tubulin binding by orders of magnitude, but only for tubulin at the cis (or cytosolic) side of the membrane. This and the fact that the basic properties of VDAC, such as single-channel conductance and selectivity, remained unaltered by phosphorylation allowed us to suggest the phosphorylation regions positioned on the cytosolic loops of VDAC and establish channel orientation in our reconstitution experiments. Experiments on human hepatoma cells HepG2 are in agreement with our findings that phosphorylation state of VDAC determines its blockage by tubulin. When HepG2 cells were treated with the microtubule-destabilizing agent colchicine that is known to increase free tubulin, mitochondrial potential, as measured by tetramethylrhodamine methyester (TMRM) uptake, decreased. Inhibition of PKA activity with H89 increased mitochondrial potential and, furthermore, reversed mitochondrial depolarization induced by colchicine. To further explore a role VDAC-tubulin interaction in cell proliferation and survival, we studied the effect of erastin, the selective anti-tumour agent involved in the RAS-RAF-MEK signalling pathway, on VDAC blockage by tubulin. We found that erastin reverses tubulin blockage of VDAC isolated from HepG2 cells, promoting the unblocked state of the channel. Our findings suggest a novel functional link between serine/threonine kinase signalling pathways, mitochondrial respiration, and highly dynamic microtubule network which is characteristic of carcinogenesis and cell proliferation.