Regulation of dynein-driven microtubule sliding by the axonemal protein kinase CK1 in Chlamydomonas flagella.
Experimental analysis of isolated ciliary/flagellar axonemes has implicated the protein kinase casein kinase I (CK1) in regulation of dynein. To test this hypothesis, we developed a novel in vitro reconstitution approach using purified recombinant Chlamydomonas reinhardtii CK1, together with CK1-depleted axonemes from the paralyzed flagellar mutant pf17, which is defective in radial spokes and impaired in dynein-driven microtubule sliding. The CK1 inhibitors (DRB and CK1-7) and solubilization of CK1 restored microtubule sliding in pf17 axonemes, which is consistent with an inhibitory role for CK1. The phosphatase inhibitor microcystin-LR blocked rescue of microtubule sliding, indicating that the axonemal phosphatases, required for rescue, were retained in the CK1-depleted axonemes. Reconstitution of depleted axonemes with purified, recombinant CK1 restored inhibition of microtubule sliding in a DRB- and CK1-7-sensitive manner. In contrast, a purified "kinase-dead" CK1 failed to restore inhibition. These results firmly establish that an axonemal CK1 regulates dynein activity and flagellar motility.
Gokhale
Avanti
A
Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Wirschell
Maureen
M
Sale
Winfield S
WS
D000818:Animals; D054468:Axoneme / Q000187:drug effects / Q000201:enzymology*; D047389:Casein Kinase I / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004398:Dyneins / Q000378:metabolism*; D005407:Flagella / Q000187:drug effects / Q000201:enzymology*; D007546:Isoquinolines / Q000494:pharmacology; D008387:Marine Toxins; D052998:Microcystins / Q000494:pharmacology; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism
D007546:Isoquinolines; D008387:Marine Toxins; D052998:Microcystins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; D004004:Dichlororibofuranosylbenzimidazole; D047389:Casein Kinase I; D010749:Phosphoprotein Phosphatases; D004398:Dyneins; C057862:cyanoginosin LR