Cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation.
Cytoplasmic dynein is a microtubule-associated motor that utilizes ATP hydrolysis to conduct minus-end directed transport of various organelles. Dynactin is a multisubunit complex that has been proposed to both link dynein with cargo and activate dynein motor function. The mechanisms by which dynactin regulates dynein activity are not clear. In this study, we examine the role of dynactin in regulating dynein ATPase activity. We show that dynein-microtubule binding and ATP-dependent release of dynein from microtubules are reduced in dynactin null mutants, Deltaro-3 (p150(Glued)) and Deltaro-4 (Arp1), relative to wild-type. The dynein-microtubule binding activity, but not the ATP-dependent release of dynein from microtubules, is restored by in vitro mixing of extracts from dynein and dynactin mutants. Dynein produced in a Deltaro-3 mutant has approximately 8-fold reduced ATPase activity relative to dynein isolated from wild-type. However, dynein ATPase activity from wild-type is not reduced when dynactin is separated from dynein, suggesting that dynein produced in a dynactin mutant is inactivated. Treatment of dynein isolated from the Deltaro-3 mutant with lambda protein phosphatase restores the ATPase activity to near wild-type levels. The reduced dynein ATPase activity observed in dynactin null mutants is mainly due to altered affinity 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 dephosphorylated upon lambda protein phosphatase treatment. The results suggest that cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation of dynein light chains.
Kumar
S
S
School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499, USA.
Lee
I H
IH
Plamann
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001921:Brain; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000201:enzymology* / Q000378:metabolism; D000072159:Dynactin Complex; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2
D000072159:Dynactin Complex; D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; D000255:Adenosine Triphosphate; D017027:Protein Tyrosine Phosphatases; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D004398:Dyneins