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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.

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Mapping of the RII-binding domain in RSP3. (A) Full-length (1–516) and various truncations of RSP3 were expressed as GST fusion proteins in bacteria and tested for RII binding by RII overlays. Overlays were followed by probing with anti-GST to verify protein expression. GST only was expressed as a negative control. (B) Alignment of RSP3 residues 161–178 with the RII-binding domains of other AKAPs. Bold type indicates identical or conservatively substituted residues. Asterisks identify proposed conserved positions of the RII-binding motif (adapted from Vijayaraghavan et al. 1999). aa, amino acid. (C) Helical wheel projection of RSP3 161–178 demonstrating an amphipathic helix. (D) Site-directed mutagenesis of leucine 170 to proline and of valine 169 and leucine 170 to alanines was performed as indicated. RSP3 mutants were expressed as GST fusion proteins and tested for RII binding by RII overlays. Overlays were followed by probing with anti-GST to verify protein expression.
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Figure 4: Mapping of the RII-binding domain in RSP3. (A) Full-length (1–516) and various truncations of RSP3 were expressed as GST fusion proteins in bacteria and tested for RII binding by RII overlays. Overlays were followed by probing with anti-GST to verify protein expression. GST only was expressed as a negative control. (B) Alignment of RSP3 residues 161–178 with the RII-binding domains of other AKAPs. Bold type indicates identical or conservatively substituted residues. Asterisks identify proposed conserved positions of the RII-binding motif (adapted from Vijayaraghavan et al. 1999). aa, amino acid. (C) Helical wheel projection of RSP3 161–178 demonstrating an amphipathic helix. (D) Site-directed mutagenesis of leucine 170 to proline and of valine 169 and leucine 170 to alanines was performed as indicated. RSP3 mutants were expressed as GST fusion proteins and tested for RII binding by RII overlays. Overlays were followed by probing with anti-GST to verify protein expression.

Mentions: To confirm that AKAP97 is RSP3, we expressed recombinant RSP3 as a GST fusion protein in E. coli and tested for RII binding using an RII blot overlay. The overlay demonstrated that recombinant RSP3 binds to RII (Fig. 4 A). RII binding was specifically inhibited by addition of the Ht31 peptide (data not shown). These results provided verification that RSP3 is an AKAP.


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

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

Mapping of the RII-binding domain in RSP3. (A) Full-length (1–516) and various truncations of RSP3 were expressed as GST fusion proteins in bacteria and tested for RII binding by RII overlays. Overlays were followed by probing with anti-GST to verify protein expression. GST only was expressed as a negative control. (B) Alignment of RSP3 residues 161–178 with the RII-binding domains of other AKAPs. Bold type indicates identical or conservatively substituted residues. Asterisks identify proposed conserved positions of the RII-binding motif (adapted from Vijayaraghavan et al. 1999). aa, amino acid. (C) Helical wheel projection of RSP3 161–178 demonstrating an amphipathic helix. (D) Site-directed mutagenesis of leucine 170 to proline and of valine 169 and leucine 170 to alanines was performed as indicated. RSP3 mutants were expressed as GST fusion proteins and tested for RII binding by RII overlays. Overlays were followed by probing with anti-GST to verify protein expression.
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Related In: Results  -  Collection

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Figure 4: Mapping of the RII-binding domain in RSP3. (A) Full-length (1–516) and various truncations of RSP3 were expressed as GST fusion proteins in bacteria and tested for RII binding by RII overlays. Overlays were followed by probing with anti-GST to verify protein expression. GST only was expressed as a negative control. (B) Alignment of RSP3 residues 161–178 with the RII-binding domains of other AKAPs. Bold type indicates identical or conservatively substituted residues. Asterisks identify proposed conserved positions of the RII-binding motif (adapted from Vijayaraghavan et al. 1999). aa, amino acid. (C) Helical wheel projection of RSP3 161–178 demonstrating an amphipathic helix. (D) Site-directed mutagenesis of leucine 170 to proline and of valine 169 and leucine 170 to alanines was performed as indicated. RSP3 mutants were expressed as GST fusion proteins and tested for RII binding by RII overlays. Overlays were followed by probing with anti-GST to verify protein expression.
Mentions: To confirm that AKAP97 is RSP3, we expressed recombinant RSP3 as a GST fusion protein in E. coli and tested for RII binding using an RII blot overlay. The overlay demonstrated that recombinant RSP3 binds to RII (Fig. 4 A). RII binding was specifically inhibited by addition of the Ht31 peptide (data not shown). These results provided verification that RSP3 is an AKAP.

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.

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