Disruption of the A-Kinase Anchoring Domain in Flagellar Radial Spoke Protein 3 Results in Unregulated Axonemal cAMP-dependent Protein Kinase Activity and Abnormal Flagellar Motility

Abstract

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...

Keeping an eye on I1: I1 dynein as a model for flagellar dynein assembly and regulation

“…Consistent with these ideas, the central pair apparatus is asymmetric in structure and composition [Mitchell and Sale, 1999;Mitchell and Yokoyama, 2003;Mitchell, 2003a;Wargo and Smith, 2003;Smith and Yang, 2004;Lechtreck and Witman, 2007] and the position of the central pair correlates with sites of active sliding [Wargo and Smith, 2003;Wargo et al, 2004] and the position of doublets relative to the bend [Mitchell, 2003a;Smith and Yang, 2004]. The results have also revealed a functional role for kinases (PKA, CK1) and phosphatases (PP1, PP2A) that are physically localized in the axoneme Sale, 1996, 1997;King and Dutcher, 1997;Porter and Sale, 2000;Gaillard et al, 2001Gaillard et al, , 2006Gokhale et al, 2006]. Much effort is underway to identify molecular mechanisms for targeting otherwise ubiquitous signaling molecules to precise locations in the axoneme [Gaillard et al, 2001[Gaillard et al, , 2006.…”

“…I1-dynein is the trilobed inner arm just proximal to radial spoke S1 in each repeat (green); it is followed by a series of single headed inner arms (labeled 2-6). PKA is thought to be localized at the base of the radial spokes [Gaillard et al, 2006]; CK1 and PP2A are predicted to be anchored near I1 dynein; a fraction of PP1 located on the outer doublets. The inset shows a diagram of the I1-dynein and its individual subunits [Harrison et al, 1998;Perrone et al, 1998;Yang and Sale, 1998;Porter and Sale, 2000;DiBella et al, 2004a,b;Hendrickson et al, 2004].…”

“…This regulatory process involves changes in IC138 phosphorylation such that in the phosphorylated state, I1 is inactive; when IC138 is dephosphorylated, I1-dynein activity is restored. The critical axonemal kinases and phosphatases involved in this pathway are indicated [Porter and Sale, 2000;Smith and Yang, 2004]. mutants, lacking either the central pair or radial spokes, in which axonemal kinase activity is misregulated, revealing a predicted global change in dynein activity [Gaillard et al, 2001[Gaillard et al, , 2006Gokhale et al, 2006]. The same in vitro measurements of microtubule sliding in wild-type axonemes have not been informative, most likely because the axonemal kinases are tightly and locally controlled.…”

“…As discussed before , the input for the asymmetric central pair-radial spoke signal could be a second messenger, such as calcium impinging upon the central pair [Wargo et al, 2005;Dymek and Smith, 2006], and/or a mechanical interaction with the radial spoke [Warner and Satir, 1974;Smith and Yang, 2004;Yang et al, 2004]. The output signal, presumably mediated by a radial spoke on one doublet microtubule, would be the activation of axonemal kinases located near I1-dynein Gaillard et al, 2006]. Consistent with these ideas, the central pair apparatus is asymmetric in structure and composition [Mitchell and Sale, 1999;Mitchell and Yokoyama, 2003;Mitchell, 2003a;Wargo and Smith, 2003;Smith and Yang, 2004;Lechtreck and Witman, 2007] and the position of the central pair correlates with sites of active sliding [Wargo and Smith, 2003;Wargo et al, 2004] and the position of doublets relative to the bend [Mitchell, 2003a;Smith and Yang, 2004].…”

Effect of Camp Level on Thallus Growth and Gametangia Development in Macroalga Chara Vulgaris (Charales, Charophyta)

“…Work in which endogenous cAMP levels were altered and cAMP level-modifying substances were applied indicates that in unicellular algae this second messenger is involved in regulating the progress of the cell cycle (Aline et al, 1984;Tong et al, 1991;Sakuanrungsirikul et al, 1996;Mohabir and Edmunds, 1999) and performs an essential role in mating processes (Pan and Snell, 2000;2002), gravitaxis (Lebert et al, 1999;Streb et al, 2002), phototaxis (Ntefidou et al, 2003) and flagellum formation and motility (Rubin and Filner, 1973;Jayaswal et al, 1991;Gaillard et al, 2006).…”