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Regulation of dynein-driven microtubule sliding by the axonemal protein kinase CK1 in Chlamydomonas flagella

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322

Correspondence to Winfield S. Sale: win{at}cellbio.emory.edu

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.

Abbreviations used in this paper: CK1, casein kinase I; CK1-7, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; CP/RS, central pair–radial spoke; DRB, 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole; DRC, dynein regulatory complex; rCK1, recombinant CK1; rCK1-KD, kinase-dead rCK1.

© 2009 Gokhale et al.
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