Abstract. Cytoplasmic dynein is a microtubulebinding 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.CUMULATING evidence indicates that retrograde and anterograde organelle transport along the microtubule network are mediated by the actions of microtubule-dependent motor molecules, cytoplasmic dynein, and kinesin (for review see Vallee and Shpetner, 1990;Walker and Sheetz, 1993). In contrast to the plethora of kinesin and kinesin-like molecules, only some of which may be anterograde organelle motors (for review see Endow, 1991;Goldstein, 1991;Walker and Sheetz, 1993), cytoplasmic dynein has been found in only one form in each cell type and species examined (Koonce et al., 1992;Paschal et al., 1992;Eshel et al., 1993;Li et al., 1993;Mikami et al., 1993;Zhang et al., 1993;Gibbons et al., 1994;Rasmusson et al., 1994). However, this retrograde translocator may carry out multiple cellular functions, as dynein has been localized to both membranous organdies and the mitotic apparatus in a number of cell types (Pfarr et al., 1990; Steuer et al., 1990;Lin and Collins, 1992 In our previous work cytoplasmic dynein in cultured fibroblasts was shown to be concentrated on large membrane-bounded organelles, identified as lysosomes (Lin and Collins, 1992). The association of dynein with these...
In this study we report that phospholipid vesicles activate ATP hydrolysis by cytoplasmic dynein but not kinesin, consistent with reported differences in the organelle/vesicle binding of these motor proteins. Dynein activation by phospholipids was comparable with that seen in the presence of microtubules but was not sensitive to moderate salt concentrations and was independent of the net charge of the phospholipid, suggesting that the means of interaction between dynein and the lipid vesicle was not strictly ionic in nature. Based on this result, previous data that show that the interaction between dynein and vesicles is not ATP sensitive, and the concentration dependence observed for lipid activation of cytoplasmic dynein, it is likely that the binding interaction between dynein and liposomes is a stable one. In contrast to a previous report, microtubules increased the hydrolysis rate of all naturally occurring nucleotides tested, whereas only ATPase activity was stimulated by phospholipids. As ATP is the physiologically relevant substrate and is the only nucleotide to promote motility, the activation of only the ATPase by phospholipids may represent a means of discriminating between coupled and uncoupled nucleotide hydrolysis in vitro.
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