Phosphorylation of Voltage-Dependent Anion Channel by Serine/Threonine Kinases Governs Its Interaction with Tubulin

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.

近期研究发现，微管蛋白（Tubulin）是电压依赖性阴离子通道（Voltage-dependent anion channel, VDAC）的强效特异性调控因子；VDAC是线粒体外膜中丰度最高的通道蛋白，亦是ATP、ADP及其他代谢物跨线粒体外膜转运的主要通路。二聚体微管蛋白可使重构至平面脂质膜中的VDAC发生可逆性阻断，并显著降低分离线粒体的呼吸活性。本研究证实，VDAC的磷酸化修饰是其与二聚体微管蛋白相互作用的重要调控因素。我们证实，通过糖原合酶激酶3β（GSK3β）或环腺苷酸依赖的蛋白激酶A（PKA）对VDAC进行体外磷酸化修饰，可使微管蛋白与重构通道的结合速率提升数个数量级，但仅当微管蛋白处于膜的顺式侧时才会产生该效应。结合VDAC的单通道电导、离子选择性等基本特性不受磷酸化修饰影响这一实验结果，我们可推定VDAC的磷酸化位点位于其胞质环区域，并明确了本研究重构实验中VDAC的通道取向。针对人肝癌细胞HepG2的实验结果支持了我们的推测：VDAC对线粒体呼吸底物的通透性受二聚体微管蛋白及通道磷酸化修饰共同调控。用秋水仙碱处理HepG2细胞可抑制微管聚合，进而提升胞质中二聚体微管蛋白的可利用水平。相应地，通过共聚焦显微镜检测线粒体对四甲基罗丹明甲酯的摄取量可发现，该处理会导致线粒体膜电位降低。抑制PKA的活性可阻断并逆转秋水仙碱诱导的线粒体去极化现象。本研究结果揭示了丝氨酸/苏氨酸激酶信号通路、线粒体呼吸与高度动态的微管网络之间的新型功能关联，而后者正是癌变与细胞增殖的典型特征。