Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Aug;15(8):3891-902.
doi: 10.1091/mbc.e04-04-0352. Epub 2004 Jun 11.

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

Affiliations
Free PMC article

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

Ramila S Patel-King et al. Mol Biol Cell. .
Free PMC article

Abstract

The radial spokes are required for Ca(2+)-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 Ca(2+)-independent manner, whereas IQ2 and IQ3 show Ca(2+)-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca(2+). This Ca(2+)-responsive enzyme, which accounts for approximately 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 Ca(2+)-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.

Figures

Figure 1.
Figure 1.
NDK phylogeny and Northern/Southern blot analysis of p61. (a) Chlamydomonas ESTs AV645300 (encoding p61) and BG843793, the NDKs identified in scaffolds #154 and 279 of the Chlamydomonas genome, the NDK modules from the IC1 polypeptide of sea urchin dynein (Anthocidaris crassispina), human and murine NDK5, rat NDK7, human Sptrx-2, human Txl-2, and the cytosolic and chloroplast NDKs from spinach, pea, and Arabidopsis were aligned using ClustalW. Residues conserved in ≥30% of the sequences were shaded using BOXSHADE. (b) ClustalW alignment was used as the input to generate the neighbor-joining unrooted tree shown. The BG843793 EST and NDK encoded on scaffold 279 clearly group with the NDKs of higher plants. In contrast, AV645300 (p61) and the scaffold #154–9 NDK (p40) are more closely related to the testis-specific mammalian enzymes. (c) Southern blot analysis of Chlamydomonas genomic DNA restricted with SmaI, PvuII, PstI, and BamHI and probed with the 5′ region of the AV645300 EST clone. Single bands were detected in the SmaI-, PstI-, and BamHI-digested samples. (d) Northern analysis of Chlamydomonas RNA obtained from non-deflagellated cells (NDF) and from cells that had undergone flagella excision and been allowed to regenerate new flagella for 30 min (30′postDF). A 2.54-kb mRNA that is highly up-regulated after deflagellation was detected by the AV645300 probe.
Figure 2.
Figure 2.
p61 cDNA sequence. The entire 2.3-kb cDNA clone (AV645300) encoding full-length p61 was sequenced. The p61 protein consists of 586 residues and has a calculated mass of 61,394 Da and a pI of 4.48. The 5′-UTR contains a single in-frame stop codon and the putative polyadenylation signal in the 3′-UTR is underlined. The clone terminates in a polyA tract. This annotated sequence is available under GenBank/European Molecular Biology Laboratory/DNA Database of Japan accession no. AY452667.
Figure 3.
Figure 3.
Domain structure of p61. A map of p61 illustrating the location of the NDK domain, IQ motifs, and regions rich in specific amino acids is shown. A Kyte and Doolittle hydropathy plot is inserted above the map. Almost the entire C-terminal region is hydrophilic in nature. Bottom, dot plot comparison of p61 to illustrate the repetitive structure containing the three IQ motifs present within the C-terminal region of the protein. The inset at lower right is a molecular model for the p61 NDK domain (residues 2–142) built using SWISSMODEL. Indicated on the model are the residues known to be required for the catalytic activity of other NDKs (Tepper et al., 1994). All these active site residues are completely conserved in p61.
Figure 4.
Figure 4.
p61 migrates anomalously in SDS-polyacrylamide gels. (a) Twenty micrograms of the MBP-p61(7-586) fusion protein was electrophoresed in a 5–15% acrylamide gradient gel, both before and after incubation with factor Xa, and stained with Coomassie Blue. The intact fusion protein has a calculated mass of 103,130 Da but migrated with Mr ∼135,000. The minor bands derive from cleavage of MBP-p61(7-586) by endogenous bacterial proteases. After factor Xa cleavage of the fusion protein, MBP migrated at ∼Mr 40,000 as observed previously, whereas the p61(7-586) segment (calculated mass 60,662 Da) had Mr ∼100,000. (b) Wild-type axonemes (∼100 μg) prepared in the presence of a protease inhibitor cocktail were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie Blue (left) or blotted and probed with the CT220 antibody (right). Two prominent bands of Mr102,000 and 40,000 were obtained. The top band corresponds to p61, which migrates anomalously. The diffuse band located above tubulin is a proteolytic fragment of p61.
Figure 5.
Figure 5.
NDKs are integral axonemal components. Chlamydomonas flagella were treated with a nonionic detergent to remove the flagellar membrane and soluble matrix components, yielding microtubule-based axonemes that were subsequently extracted with 0.6 M NaCl. Equivalent samples of each fraction were separated in a 5–15% acrylamide gradient gel and either stained with Coomassie Blue (top) or blotted to nitrocellulose and probed with CT220 antibody (bottom). Both p61 and the Mr 40,000 band are integral axonemal components that are almost completely resistant to extraction by high ionic strength buffers containing 0.6 M NaCl.
Figure 6.
Figure 6.
p61 is missing in mutant axonemes lacking radial spokes. (a) Axonemes were obtained from wild-type Chlamydomonas (WT) and from mutants lacking outer (oda1) and inner (ida1 and ida4) dynein arms, radial spokes (pf14), and the central pair microtubule complex (pf18). After electrophoresis, samples were stained with Coomassie Blue (top) or examined for CT220 immunoreactivity (bottom). The Mr 40,000 protein (p40) was detected in all samples, whereas p61 was absent only in pf14 axonemes. (b) Axonemes from the pf14 mutant were prepared in the presence of a comprehensive protease inhibitor cocktail. The sample was electrophoresed and stained with Coomassie Blue (left) or blotted and probed with CT220 (right). These axonemes contain the Mr 40,000 band. However, p61 and its breakdown product migrating just above tubulin (see Figure 4b) were missing. (c) To assess the location of p61 within flagellar radial spokes, axonemes were prepared from wild type, pf14, pf17 (lack the spoke head), and pf24 (no spoke head and truncated stalk) axonemes. The p61 protein was present in pf17, but levels were drastically reduced in pf24 (upon prolonged exposure, a very weak p61 band was detected in this mutant). This suggests that p61 is located within the distal portion of the radial spoke stalk.
Figure 7.
Figure 7.
p61 copurifies with radial spoke protein 3.(a) Coomassie Blue-stained lane of isolated radial spokes (from the sucrose gradient shown in b). The p61 protein and several RSP components were identified by immunoblot analysis and/or by comparison with the electrophoretic analysis performed by Yang et al. (2001). (b) Axonemes from the mutant oda1 were treated with 0.6 M NaCl to remove inner arm dyneins, and the radial spokes were subsequently extracted with 0.5 M KI. This extract was then sedimented in a 5–20% sucrose density gradient. Fractions were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie Blue (top) or blotted to nitrocellulose and probed with CT220 (middle) and the RSP3 antibody (bottom). Most of the p61 and RSP3 proteins were found together in fractions 1–3. In contrast, the Mr 40,000 band detected by antibody CT220 sedimented near the top of the gradient in fractions 11–14. NDKase activity of each fraction is shown in the lower panel, and precisely copurified with p61 near the bottom of the gradient and also with p40 toward the top of the gradient. (c) Radial spokes lacking the spoke head were extracted from pf17 axonemes and sedimented in a 5–20% sucrose gradient. Both p61 and RSP3 were present nearer to the top of the gradient in fractions 1–7; sedimentation of the Mr 40,000 band was unaffected by the pf17 mutation. NDKase activity also was shifted up the gradient and again followed the p61 profile.
Figure 8.
Figure 8.
p61 binds calmodulin through both Ca2+-dependent and Ca2+-independent mechanisms. Equimolar amounts of the full-length MBP-p61(7-586), the truncated MBP-p61(7-199), MBP-p61(7-424), MBP-p61(7-494) fusion proteins, and a MBP protein to which only the IQ2 region (p61 residues 424–494) was attached, were incubated with calmodulin-Sepharose beads in the presence of 2 mM Ca2+. Protein that did not bind was recovered after a brief centrifugation (lane marked flow through). After several washes, protein bound in a Ca2+-dependent manner was eluted with buffer containing 2 mM EGTA (EGTA eluate). Any protein that remained associated through a Ca2+-independent interaction was obtained by incubating the beads in gel sample buffer (lane marked beads). Samples were electrophoresed in an 8% acrylamide gel and stained with Coomassie Blue. Maps illustrating the domain structure of the various constructs are shown at right.
Figure 9.
Figure 9.
Model for Ca2+-dependent GTP signaling through the radial spokes. The diagram illustrates the location of structures associated with a single flagellar doublet microtubule. Within the radial spoke stalk, RSP3 is located at the base and is thought to anchor cAMP-dependent protein kinase (PKA), resulting in phosphorylation of inner dynein arm proteins. The p61 protein and associated calmodulin (CaM) are also present in the stalk. Ca2+ binding results in activation of the p61 NDK module to generate GTP (or some other NTP), which subsequently could be used either by a GTP-binding switch protein, a specific GTPase or a guanylate cyclase. Intriguingly, the calmodulin-associated RSP2 protein, which contains a GAF domain that might potentially bind cGMP and act as a downstream target for p61-generated GTP, is also a component of the radial spoke (Yang et al., 2004) and is apparently located close to p61. The dynein regulatory complex (DRC) is located near the base of the radial spokes and inner arms.

Similar articles

See all similar articles

Cited by 41 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback