Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin.

Kely L Sheldon,John J Lemasters,Sergey M Bezrukov,Tatiana K Rostovtseva,Eduardo N Maldonado,Philipp J Kahle

doi:10.1371/journal.pone.0025539

Abstract

Tubulin was recently found to be a uniquely potent regulator of the voltage-dependent anion channel (VDAC), the most abundant channel of the mitochondrial outer membrane, which constitutes a major pathway for ATP/ADP and other metabolites across this membrane. Dimeric tubulin induces reversible blockage of VDAC reconstituted into a planar lipid membrane and dramatically reduces respiration of isolated mitochondria. Here we show that VDAC phosphorylation is an important determinant of its interaction with dimeric tubulin. We demonstrate that in vitro phosphorylation of VDAC by either glycogen synthase kinase-3β (GSK3β) or cAMP-dependent protein kinase A (PKA), increases the on-rate of tubulin binding to the reconstituted channel by orders of magnitude, but only for tubulin at the cis side of the membrane. This and the fact 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 support our conjecture that VDAC permeability for the mitochondrial respiratory substrates is regulated by dimeric tubulin and channel phosphorylation. Treatment of HepG2 cells with colchicine prevents microtubule polymerization, thus increasing dimeric tubulin availability in the cytosol. Accordingly, this leads to a decrease of mitochondrial potential measured by assessing mitochondrial tetramethylrhodamine methyester uptake with confocal microscopy. Inhibition of PKA activity blocks and reverses mitochondrial depolarization induced by colchicine. Our findings suggest a novel functional link between serine/threonine kinase signaling pathways, mitochondrial respiration, and the highly dynamic microtubule network which is characteristic of cancerogenesis and cell proliferation.

Highlights

The role of mitochondria in energy production, calcium signaling, and promoting apoptotic signals is well established
To study effects of phosphorylation, voltage dependent anion channel (VDAC) isolated from mouse liver mitochondria was incubated with glycogen synthase kinase-3b (GSK3b) or protein kinase A (PKA) in MgATP cocktail for 1 hour at room temperature to ensure maximum phosphorylation and reconstituted into planar lipid membranes and tested for its interaction with tubulin
The increased sensitivity of VDAC to tubulin is manifested by the events of simultaneous blockage of the two phosphorylated channels that is seen as the transient current reduction to the level designated as c2

Summary

Introduction

The role of mitochondria in energy production, calcium signaling, and promoting apoptotic signals is well established. A significant portion of the MOM control functions is realized through the voltage dependent anion channel (VDAC) that constitutes a major pathway for ATP/ADP and other mitochondrial metabolites across MOM [4,5,6,7,8]. Any imbalance in this exchange leads to an essential disturbance of cell metabolism, especially in the processes of development and apoptosis that require extensive mitochondria participation. The tubulin-blocked state is virtually impermeable for ATP [12]

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Conclusion