VDAC Phosphorylation Regulates Interaction with Tubulin

Abstract

Mitochondria and mitochondrial bioenergetics are believed to be involved in glycogen synthase kinase (GSK3β)-related cardioprotection. Recently it was suggested that cardioprotection could be achieved through the preservation of mitochondrial binding of hexokinase II (HXKII), or/and through GSK3β phosphorylation of voltage dependent anion channel (VDAC) (Pastorino et al., Cancer Res., 2005; Das et al., Circ. Res., 2008). VDAC, the most abundant channel in the mitochondria outer membrane (MOM), is known to be responsible for most of the metabolite and ATP/ADP fluxes across MOM. Recently we have found that dimeric αβ-tubulin regulates mitochondrial respiration by directly blocking VDAC and hence, permeability of MOM for ATP/ADP (Rostovtseva et al., PNAS, 2008). Here, using mammalian VDAC reconstituted into planar lipid membrane, we show that tubulin-VDAC interaction appears to be very sensitive to the state of VDAC phosphorylation. When VDAC is phosphorylated in vitro by either GSK3β or protein kinase A (PKA), the on-rate of tubulin binding increases up to 100 times compared with untreated VDAC. Importantly, the basic properties of VDAC, such as single-channel conductance, selectivity, and voltage gating, remain almost unaltered after phosphorylation. Nonspecific alkaline phosphatase and tyrosine kinase inhibitor PP2A dephosphorylate VDAC, which results in decreased tubulin binding. Gel analysis and subsequent phospho-staining confirm that VDAC contains motifs recognized by both GSK3β and PKA. Phosphorylation causes a pronounced asymmetry of tubulin binding to VDAC. These findings allow us to point to the tentative GSK3β and PKA serine/threonine phosphorylation sites positioned on the cytosolic loops of VDAC. The results show that VDAC phosphorylation enhances tubulin-induced VDAC closure and thus could reduce MOM permeability and mitochondria respiration. We suggest that GSK3β cardioprotective effect is more complex that was initially thought because along with HXKII it involves tubulin as a potent regulator of VDAC and hence, cellular respiration.