Lin S X, Ferro K L, Collins C A
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611-3008.
J Cell Biol. 1994 Nov;127(4):1009-19. doi: 10.1083/jcb.127.4.1009.
Cytoplasmic dynein is a microtubule-binding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.
胞质动力蛋白是一种微管结合蛋白，被认为是逆行细胞器运动的动力。在培养的成纤维细胞中，胞质动力蛋白主要定位于溶酶体，溶酶体是一种膜性细胞器，其在细胞质中的运动和分布已被证明依赖于微管细胞骨架的完整性。我们最近发现了一些条件，这些条件会导致动力蛋白在体内明显从溶酶体解离，这表明膜结合的改变可能参与了逆行细胞器运动的调节（Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579 - 588）。短暂的血清撤除和低细胞外钙水平都会诱导这种改变，并且在添加血清或额外的钙后这种效应会逆转。在这里，我们证明了动力蛋白分子的磷酸化状态与其在正常大鼠肾成纤维细胞内分布的变化相关。在血清饥饿期间，动力蛋白重链的磷酸化水平升高，随后添加血清后又降至对照水平。我们发现，冈田酸，一种磷酸蛋白磷酸酶抑制剂，模拟了血清饥饿对磷酸化以及动力蛋白从膜相关池到更易溶池的细胞内重新分布的影响，两种现象具有相似的剂量依赖性。通过不同去污剂提取进行细胞分级分离显示，血清饥饿后可溶性池中存在的动力蛋白比例高于对照细胞的相应分级分离部分。我们的数据表明，胞质动力蛋白在体内被磷酸化，磷酸化状态的变化可能参与了影响该蛋白在细胞内区室间分布的调节机制。
