Cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation

Cytoplasmic dynein is a microtubule-associated motor that utilizes ATP hydr olysis to conduct minus-end directed transport of various organelles. Dynac tin is a multisubunit complex that has been proposed to both link dynein wi th cargo and activate dynein motor function. The mechanisms by which dynact in regulates dynein activity are not clear. In this study, we examine the r ole of dynactin in regulating dynein ATPase activity. We show that dynein-m icrotubule binding and ATP-dependent release of dynein from microtubules ar e reduced in dynactin null mutants, Delta ro-3 (p150(Glued)) and Delta ro-4 (Arp1), relative to wild-type. The dynein-microtubule binding activity, bu t not the ATP-dependent release of dynein from microtubules, is restored by in vitro mixing of extracts from dynein and dynactin mutants. Dynein produ ced in a Delta ro-3 mutant has similar to 8-fold reduced ATPase activity re lative to dynein isolated from wildtype. However, dynein ATPase activity fr om wild-type is not reduced when dynactin is separated from dynein, suggest ing that dynein produced in a dynactin mutant is inactivated. Treatment of dynein isolated from the Delta ro-3 mutant with lambda protein phosphatase restores the ATPase activity to near wild-type levels. The reduced dynein A TPase activity observed in dynactin null mutants is mainly due to altered a ffinity for ATP. Radiolabeling experiments revealed that low molecular mass proteins, particularly 20- and 8-kDa proteins, that immunoprecipitate with dynein heavy chain are hyperphosphorylated in the dynactin mutant and deph osphorylated upon A protein phosphatase treatment. The results suggest that cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phos phorylation of dynein light chains.