Cytoplasmic dynein undergoes intracellular redistribution concomitant with phosphorylation of the heavy chain in response to serum starvation and okadaic acid.

Abstract

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...

Phosphorylation by cdc2-CyclinB1 Kinase Releases Cytoplasmic Dynein from Membranes

“…One interesting question is whether the regulatory mechanism we have characterized operates during mitosis in somatic cells (rather than through the embryonic cell cycle, as assessed here and previously), as there are conflicting reports of whether CD remains associated with its membranous cargoes (43,44).…”

Characterization and localization of the cytoplasmic dynein heavy chain in Aspergillus nidulans.

“…In mammalian cells the localization of CDHC to the membranous organelles is regulated by phosphorylation (20). Another large protein complex, the Glued/dynactin complex (21,22), is required for dynein to move vesicles in vitro (23) and may be required to couple the CDHC to the vesicles.…”

SPD-3 Is Required for Spindle Alignment in Caenorhabditis elegans Embryos and Localizes to Mitochondria

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