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MBC in Press, published online ahead of print June 11, 2004
Mol. Biol. Cell 10.1091/mbc.E04-04-0352

A more recent version of this article appeared on August 1, 2004
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Submitted on April 29, 2004
Revised on May 21, 2004
Accepted on June 1, 2004

Flagellar Radial Spokes Contain a Ca2+-Stimulated Nucleoside Diphosphate Kinase

Ramila S. Patel-King, Oksana Gorbatyuk, Sachiko Takebe, and Stephen M. King*

Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3305

Monitoring Editor: Paul Matsudaira

The radial spokes are required for Ca2+-initiated intraflagellar signaling resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-sepharose beads in a Ca2+-independent manner, whereas IQ2 and IQ3 show Ca2+-sensitive associations. Wildtype axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca2+. This Ca2+-responsive enzyme, which accounts for ~45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca2+-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.

*Corresponding author. E-mail: steve{at}king2.uchc.edu






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