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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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Deficiency of AKAPs in selected motility mutants. (A) Diagram of axoneme cross section indicating the major structural components and the corresponding mutants (adapted from Porter and Sale 2000). (B) RII overlay of various motility mutants. wt, wild-type. (C) RII overlay of mutants defective in various structures of the central pair apparatus.
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Figure 2: Deficiency of AKAPs in selected motility mutants. (A) Diagram of axoneme cross section indicating the major structural components and the corresponding mutants (adapted from Porter and Sale 2000). (B) RII overlay of various motility mutants. wt, wild-type. (C) RII overlay of mutants defective in various structures of the central pair apparatus.

Mentions: To localize AKAP240 and AKAP97 within the axoneme, we performed RII overlays on axonemal protein from four different classes of Chlamydomonas motility mutants that are defective for specific axonemal structures (Fig. 2 A). By doing so, we localized the AKAPs within the axoneme based on whether or not the AKAPs are present in the structural mutants. The analysis revealed that AKAP240 is absent in axonemes from a mutant defective for the central pair apparatus (pf18), and that AKAP97 is absent in axonemes from a mutant lacking the radial spokes (pf14) (Fig. 2 B). Both AKAPs are present in axonemes from a mutant lacking the outer dynein arms and the I1 inner dynein arm (pf28pf30) as well as in axonemes from a mutant defective for the dynein regulatory complex (pf2) (Fig. 2 B). Coomassie blue staining of a corresponding SDS-PAGE gel was performed to verify equivalent protein load. RII binding to the AKAPs was inhibited by addition of the Ht31 peptide (data not shown). The results of the mutant analysis suggest a model in which AKAP240 is associated with the central pair apparatus and AKAP97 is a component of the radial spoke.


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

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

Deficiency of AKAPs in selected motility mutants. (A) Diagram of axoneme cross section indicating the major structural components and the corresponding mutants (adapted from Porter and Sale 2000). (B) RII overlay of various motility mutants. wt, wild-type. (C) RII overlay of mutants defective in various structures of the central pair apparatus.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Deficiency of AKAPs in selected motility mutants. (A) Diagram of axoneme cross section indicating the major structural components and the corresponding mutants (adapted from Porter and Sale 2000). (B) RII overlay of various motility mutants. wt, wild-type. (C) RII overlay of mutants defective in various structures of the central pair apparatus.
Mentions: To localize AKAP240 and AKAP97 within the axoneme, we performed RII overlays on axonemal protein from four different classes of Chlamydomonas motility mutants that are defective for specific axonemal structures (Fig. 2 A). By doing so, we localized the AKAPs within the axoneme based on whether or not the AKAPs are present in the structural mutants. The analysis revealed that AKAP240 is absent in axonemes from a mutant defective for the central pair apparatus (pf18), and that AKAP97 is absent in axonemes from a mutant lacking the radial spokes (pf14) (Fig. 2 B). Both AKAPs are present in axonemes from a mutant lacking the outer dynein arms and the I1 inner dynein arm (pf28pf30) as well as in axonemes from a mutant defective for the dynein regulatory complex (pf2) (Fig. 2 B). Coomassie blue staining of a corresponding SDS-PAGE gel was performed to verify equivalent protein load. RII binding to the AKAPs was inhibited by addition of the Ht31 peptide (data not shown). The results of the mutant analysis suggest a model in which AKAP240 is associated with the central pair apparatus and AKAP97 is a component of the radial spoke.

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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