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.

Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility. INTRODUCTIONEukaryotic cilia and flagella are highly conserved organelles that are required for diverse and vital motile and sensory functions (Afzelius, 2004;Snell et al., 2004;Pan et al., 2005;Quarmby and Parker, 2005). Motile cilia and flagella are capable of complex, carefully coordinated movements that are important for embryonic development, fertilization, and maintenance of epithelial tracts (Silflow and Lefebvre, 2001;El Zein et al., 2003;McGrath and Brueckner, 2003). Ciliary and flagellar movement is mediated by the axoneme, a highly ordered microtubule-based structure, composed of hundreds of conserved proteins (Avidor-Reiss et al., 2004;Li et al., 2004;Pazour et al., 2005). Within the axoneme, exact spatial and temporal regulation of dynein-driven microtubule sliding is required for the production of precisely formed bends (Satir, 1968;Summers and Gibbons, 1971;Shingyoji et al., 1977;Brokaw, 1991). However, the mechanisms that regulate dynein and modulate the size and shape of the axonemal bend, parameters referred to as waveform, are poorly understood. Experimental studies of Chlamydomonas and other organisms have shown that several key structural components of the axoneme, including the inner dynein arms, central pair apparatus, and radial spokes, are important in the control of axonemal waveform, in a process involving protein phosphorylation (Brokaw et al., 1982;Brokaw and Kamiya, 1987;Porter and Sale, 2000;Kamiya, 2002;Padma et al., 2003;Smith and Yang, 2004;White et al., 2005). Here, we focus on the role of the radial spokes and an associated axonemal cAMP-dependent protein kinase (PKA), which operates to regulate dynein activity and axonemal motility.Previous in vitro studies o...

Radial spokes are critical multisubunit structures required for normal ciliary and eukaryotic flagellar motility. Experimental evidence indicates the radial spokes are mechanochemical transducers that transmit signals from the central pair apparatus to the outer doublet microtubules for local control of dynein activity. Recently, progress has been made in identifying individual components of the radial spoke, yet little is known about how the radial spoke is assembled or how it performs in signal transduction. Here we focus on radial spoke protein 3 (RSP3), a highly conserved AKAP located at the base of the radial spoke stalk and required for radial spoke assembly on the doublet microtubules. Biochemical approaches were taken to further explore the functional role of RSP3 within the radial spoke structure and for control of motility. Chemical crosslinking, native gel electrophoresis, and epitope-tagged RSP3 proteins established that RSP3 forms a dimer. Analysis of truncated RSP3 proteins indicates the dimerization domain coincides with the previously characterized axoneme binding domain in the N-terminus. We propose a model in which each radial spoke structure is built on an RSP3 dimer, and indicating that each radial spoke can potentially localize multiple PKAs or AKAP-binding proteins in position to control dynein activity and flagellar motility.

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9 + 2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation / dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.