We originally identified ASP and ROPN1 in a yeast-two hybrid screen looking for AKAP3 binding partners in testes, and have since determined that both ASP and ROPN1 bind a variety of AKAPs via a conserved R2D2 domain, but, until now, no in vivo
role for these proteins had been identified (Newell et al. 2008
). Because these proteins interact with AKAPs via the PKA binding site, defining their functions separate from PKA signaling is especially complex. Therefore, we reasoned the best way to determine function was production of mice that lacked ASP and/or ROPN1. Our previous studies have shown that ASP is predominantly located in motile cilia while ROPN1 is predominantly located in sperm flagellum (Newell et al. 2008
). In the present study we created mutant mouse lines lacking either ASP or ROPN1; offspring from these mice appeared healthy, and examination of tissues and cells that normally express ASP and ROPN1 [testes/sperm and trachea/ciliated cells] revealed no gross developmental or structural abnormalities. However, we cannot entirely rule out the possibility that the very small amount of ASP still being produced is sufficient to maintain normal development/structure in AspGT
cilia and flagella. The slight upregulation of (hypomorphic) ASP expression in the testes (but not in the trachea) of double mutant (AspGT
) animals may hint that ASP plays such a role in testes, but this cannot be determined with any certainty at present.
mutants that lack R2D2 protein RSP11 have normal flagellar structure, but exhibit impaired motility (Yang and Yang 2006
). This observation, coupled with the localization of ASP and ROPN1 in ciliated/flagellated cells, and the lack of a developmental, structural, or disorientation phenotype in our single mutant animals led us to hypothesize that ASP and/or ROPN1 may function to regulate motility. We thus examined tracheal cilia and indeed determined that lack of ASP results in impaired motility. Asp−/−
mice exhibit reduced basal ciliary motility; statistically significant reductions of this magnitude (approximately 17%) have been shown to be biologically important as even a 2 Hz decrease in cilia beating results in a significant reduction in in vivo
bacterial clearance (Vander Top et al. 2005
). In contrast, lack of ROPN1 had no effect on CBF; this result is perhaps not unexpected due to the exclusion of ROPN1 from the cilia itself and its failure to relocalize in AGT
cilia. In combination with EM data indicating that AspGT
cilia are structurally intact and properly oriented, these data suggest that ASP (but not ROPN1) functions as a critical, non-redundant regulator of ciliary motility in mammalian cells. Further, since CBF is thought to be modulated by the outer dynein arms (ODA) in the axoneme, ASP may thus participate in signaling pathways that affect ODA function.
While lack of ROPN1 has no effect on cilia beat frequency, sperm from Ropn1GT
mice do exhibit altered motility, and these males are subfertile, producing fewer and smaller litters. Strikingly, mice that lack both ASP and ROPN1 (AspGT/Ropn1GT
) are infertile due to sperm immotility. While testes development and testicular spermatogenesis appears normal in these mice, mature sperm have significant structural defects in the principal piece of the flagellum; our current analyses indicate that the axonemes of these sperm are intact, but that the sperm-specific structure of the fibrous sheath (FS) (where ROPN1 is expressed in wild type sperm) is disrupted (Fujita et al. 2000
). Additionally, our data suggest that though ASP does not appear to have an irreplaceable role in sperm motility or fertility (as indicated by the normal fertility parameters exhibited by AspGT
mice presented in ), it may compensate for ROPN1 in Ropn1GT
sperm acting to preserve the structural integrity of the FS, perhaps via AKAP interactions. Further studies into the function of ROPN1 in sperm integrity, motility and fertility are ongoing.
Data presented here indicate that ASP plays a role in ciliary motility, perhaps via regulation of axonemal signaling. We have previously demonstrated that ASP binds a variety of AKAPs, which raises the possibility that AKAP interactions are part of a mechanism by which ASP affects ciliary beat frequency. Two AKAPs have been identified in mammalian ciliary axonemes: AKAP28 and radial spoke protein 3 (RSP3). In Chlamydomonas,
RSP3 forms a homodimer that is located at the base of the spoke stalk domain in the axoneme and is thus in position to mediate the anchoring of PKA or AKAP-binding proteins to control dynein activity and thus ciliary beating (Wirschell et al. 2008
). Studies employing site-directed mutagenesis of the RSP3 gene in the region coding for the PKA binding site suggest that RSP3 is an AKAP required for regulation of axonemal PKA and regulation of flagellar bending by the radial spokes, as these mutants exhibit abnormal flagellar motility (Gaillard et al. 2006b
). The fact that R2D2 proteins share an AKAP binding site with the regulatory subunit of PKA suggests that they may compete with PKA in vivo
for binding to mammalian AKAPs, thus controlling the amount of PKA bound to any particular AKAP at any given time. Controlling PKA/AKAP interaction would regulate the ability of PKA to act on key substrates that may be essential for modifying CBF. We know that both PKA and R2D2 proteins strongly interact with AKAPs in vitro
, but in vivo
associations are less well defined for PKA, and absent for ASP. One mechanism by which AKAP interactions are known to be modulated is via changing binding affinities due to phosphorylation of the AKAP (Fiedler et al. 2008
). Relevant to these studies, it has recently been demonstrated that both ERK1/2 and PKA can phosphorylate RSP3 in a transfected mammalian cell line, and that phosphorylation modulates PKA binding (Jivan et al. 2009
). We have previously determined that both recombinant and endogenously expressed ASP and PKA (from mouse testes) interact with human recombinant RSP3 (Newell et al. 2008
). Further studies will test the hypothesis that phosphorylation of RSP3 differentially modifies its affinity for ASP and PKA.
In addition to RSP3, the only other human ciliary axonemal AKAP that has been identified to date is AKAP28 (Kultgen et al. 2002
). While we speculate herein that AKAPs have changing binding affinities for PKA and R2D2 proteins based on dynamic post-translational modifications such as phosphorylation, an alternate hypothesis is that AKAP28 and RSP3 each interact with a unique subset of binding proteins – perhaps one AKAP with PKA and the other with ASP. We believe that both AKAP28 and RSP3 will prove to be players in ciliary beat regulation along with PKA and ASP, and future studies will seek to identify in vivo
post-translational modifications on AKAPs, and what effect these modifications have on PKA versus ASP binding affinity.
Outside the dimerization/docking domain, there is very little sequence homology between the regulatory subunits of PKA and the R2D2 proteins, thus these proteins are not likely to interact with the catalytic subunits of PKA. We have also demonstrated that none of the R2D2 proteins bind cAMP (Newell et al. 2008
). Sequence analysis indicates that none of these proteins appears to contain intrinsic enzyme activity. Based on these data, we speculate that in addition to participating in PKA regulation via competitive AKAP-binding, R2D2 proteins may also prove to function in a manner similar to the regulatory subunit of PKA — that is, they coordinate signaling events by simultaneously binding not only to AKAPs, but also to one or more signaling molecules. Therefore we predict that, similar to RII, ASP binds to a signaling enzyme and serves as a regulatory subunit (as does RII for PKA), modulating the activity of whatever kinase or phosphatase it interacts with. We speculate that PKC would be a good candidate enzyme for regulation by ASP as lack of ASP results in decreased ciliary motility and it is known that activation of PKC decreases CBF (Salathe 2007
; Salathe et al. 1993
; Slager et al. 2008
). Studies are underway to examine PKC activity in AspGT
Mucociliary clearance is an essential part of the innate defense of the respiratory membranes, airway and lungs from inhaled allergens, pathogens, toxins and pollutants (Mall 2008
; Stannard and O’Callaghan 2006
). In humans, impaired ciliary function can have many negative health consequences; respiratory diseases such as primary ciliary dyskinesia, cystic fibrosis, asthma and chronic obstructive pulmonary disease (COPD) are associated with defects in mucociliary clearance. In many of these diseases, underlying genetic defects in cilia are suspected, though identification of specific mutations and complete understanding of etiology and pathogenesis are largely elusive (Livraghi and Randell 2007
Interestingly, though CBF is decreased in mice that lack ASP, motility can be stimulated (with procaterol, a β agonist), providing further evidence that the cilia in these mice are structurally intact. The vast majority of mouse models that display abnormal ciliary motility have incomplete structures (and missing proteins) within the cilia, often within the axoneme. Because cilia are signaling antennae as well as mechanical removers of pathogens and pollutants, these structural defects may make it difficult to isolate the effects of altered signaling events versus reduced mechanical function. Additionally, genetically engineered mice with primary cilia disorders develop hydrocephalus internus during early brain development. Due to the high and early mortality related to hydrocephalus formation, a detailed analysis of the pulmonary phenotype has been difficult in these mice (Brody et al. 2000
). The phenotype of our AspGT
mice, in which CBF is reduced without an associated structural defect or early death of the animals, provides a rare opportunity to determine the contribution of normal ciliary motility to the mucociliary clearance apparatus, and how its impairment might contribute to the development of ciliopathies and diseases of compromised mucociliary clearance. We show herein that the expression pattern of ASP and ROPN1 in ciliated airway cells of humans and mice is comparable and sequence identity is high, making our mutant mice excellent candidates as models for future studies of the role of R2D2 proteins in human airway function, and the role of dysfunctional ciliary motility in the etiology and pathogenesis of disease.