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Flagellar radial spoke protein 3 is an A-kinase anchoring protein (AKAP).

Gaillard AR, Diener DR, Rosenbaum JL, Sale WS - J. Cell Biol. (2001)

Bottom Line: By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk.Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding.RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

ABSTRACT
Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144-180. Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

Show MeSH
Conservation of the RSP3 RII-binding domain in orthologs of RSP3. Bold type indicates identical or conservatively substituted residues. Asterisks indicate proposed conserved positions of the RII-binding motif. aa, amino acid.
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Figure 5: Conservation of the RSP3 RII-binding domain in orthologs of RSP3. Bold type indicates identical or conservatively substituted residues. Asterisks indicate proposed conserved positions of the RII-binding motif. aa, amino acid.

Mentions: Orthologs to RSP3 have been identified in human, mouse, and Drosophila databases. Alignment of the RII-binding site of RSP3 with the corresponding residues in these orthologs reveals that the RII-binding site is well conserved (Fig. 5). The locations of the orthologs in their respective organisms is not known, but since the axoneme is a highly conserved structure, they most likely will be found in the radial spokes of flagellar and perhaps ciliary axonemes. It is important to test whether any of the orthologs is also an AKAP. If so, it suggests that the AKAP is an important component of the axoneme, and therefore may also be important in the regulation of motility. There is some evidence that AKAPs indeed play an important role in the control of flagellar motility. For example, cell-permeable analogues of Ht31 have been shown to inhibit sperm motility (Vijayaraghavan et al. 1997).


Flagellar radial spoke protein 3 is an A-kinase anchoring protein (AKAP).

Gaillard AR, Diener DR, Rosenbaum JL, Sale WS - J. Cell Biol. (2001)

Conservation of the RSP3 RII-binding domain in orthologs of RSP3. Bold type indicates identical or conservatively substituted residues. Asterisks indicate proposed conserved positions of the RII-binding motif. aa, amino acid.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169452&req=5

Figure 5: Conservation of the RSP3 RII-binding domain in orthologs of RSP3. Bold type indicates identical or conservatively substituted residues. Asterisks indicate proposed conserved positions of the RII-binding motif. aa, amino acid.
Mentions: Orthologs to RSP3 have been identified in human, mouse, and Drosophila databases. Alignment of the RII-binding site of RSP3 with the corresponding residues in these orthologs reveals that the RII-binding site is well conserved (Fig. 5). The locations of the orthologs in their respective organisms is not known, but since the axoneme is a highly conserved structure, they most likely will be found in the radial spokes of flagellar and perhaps ciliary axonemes. It is important to test whether any of the orthologs is also an AKAP. If so, it suggests that the AKAP is an important component of the axoneme, and therefore may also be important in the regulation of motility. There is some evidence that AKAPs indeed play an important role in the control of flagellar motility. For example, cell-permeable analogues of Ht31 have been shown to inhibit sperm motility (Vijayaraghavan et al. 1997).

Bottom Line: By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk.Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding.RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

ABSTRACT
Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144-180. Amino acids 161-180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.

Show MeSH