Flagellar Radial Spoke Protein 3 Is an a-Kinase Anchoring Protein (Akap)

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.

The radial spokes and central apparatus: Mechano‐chemical transducers that regulate flagellar motility

“…Chemical signaling also includes axonemal enzymes that are not directly sensitive to second messengers including casein kinase 1 (CK1), PP1, and PP2A, all of which appear to be involved in the control of dynein-driven motility [Walczak and Nelson, 1993; Central apparatus-associated polypeptides PF6 238-kDa alanine/proline rich protein; pf6 flagella lack the 1a projection on the C1 microtubule and display only twitching motion [Dutcher et al, 1984;Rupp et al, 2001] PF16 57-kDa armadillo repeat containing protein that localizes to the c1 microtubule; pf16 flagella lack the C1 microtubule and are paralyzed. PF16 may correspond to CP14 [Dutcher et al, 1984;Smith and Lefebvre, 1996] PF20 63-kDa WD-repeat containing protein that localizes to the inter-microtubule bridge connecting C1 and C2; pf20 flagella lack the entire central apparatus and are paralyzed [Adams et al, 1981;Smith and Lefebvre, 1997a] PP1c Localized primarily, but not exclusively, to the C1 microtubule [Yang et al, 2001] KLP1 83-kDa kinesin-like protein that localizes to the C2 microtubule [Bernstein et al, 1994] KRP 110-kDa kinesin related protein associated with the central apparatus, most likely the C1 microtubule [Fox et al, 1994;Johnson et al, 1994;Mitchell and Sale, 1999] AKAP240 240-kDa A-kinase anchoring protein; most likely associated with C2 [Gaillard et al, 2001] Calmodulin Most likely associated with C1, sediments as a complex at 10S [Yang et al, 2001;Dymek et al, 2002] Radial spoke-associated polypeptides RSP2 PF24 gene product, GAF, and calmodulin binding domain; pf24 mutants lack radial spoke heads and have paralyzed flagella Piperno et al, 1981;Yang et al, 2002] RSP3 PF14 gene product, 97-kDa A-kinase anchoring protein most likely localized to the base of the spokes; pf14 mutants lack radial spokes and have paralyzed flagella Piperno et al, 1981;Diener et al, 1993;Gaillard et al, 2001] RSP4 PF1 gene product, 49.8-kDa proline rich polypeptide similar to RSP6, pf1 mutants lack spoke heads and have paralyzed flagella Piperno et al, 1981;Curry et al, 1992] RSP6 PF26 gene product, 48.8-kDa proline rich polypeptide similar to RSP4, pf26 mutants lack spoke heads and have paralyzed flagella Piperno et al, 1981;Curry et al, 1992] LC8 RSP2...…”

“…The radial spokes may be involved in control of cAMPstimulated PKA activity. Of possible relevance, radial spoke protein three has been shown to be an A-kinase anchoring protein, AKAP [Gaillard et al, 2001].…”

“…The reduced microtubule sliding velocity in central pair mutants may be due to mechanism independent of PKA activity. However, a 240-kD central apparatus component is a candidate AKAP [Gaillard et al, 2001]. Therefore, both the radial spokes and central apparatus may serve as scaffolds for the localization of PKA (see Fig.…”

Cofactor fingerprinting with STD NMR to characterize proteins of unknown function: identification of a rare cCMP cofactor preference

“…functional proteomics: RSP2 We next applied the cofactor fingerprinting with STD NMR strategy in a functional proteomics project, devoted to determining functions for the radial spoke proteins in the flagella of Chlamydomonas reinhardtii [12][13][14][15]. One of these proteins is RSP2 (radial spoke protein-2).…”

“…ger functional proteomics project [12][13][14][15]. Dehydrogenases are an excellent gene family for applying functional proteomic methods, since they represent 3-6% of most proteomes, and are easily identified using bioinformatics tools [16,17].…”