Several pharmacological studies have identified signaling pathways involved in the regulation of ciliary beat frequency. To better understand human deficiencies in mucociliary clearance, it is necessary to delineate the molecular mechanisms underlying these pathways. Specifically, we are interested in understanding the molecular events linking the production of cAMP, the subsequent activation of PKA, and the associated increase in CBF. Our study provides the first biochemical data showing that a pool of PKA is localized within human ciliary axonemes (Figure ). Under conditions that should inhibit known serine/threonine phosphatases and L-isozymes of alkaline phosphatase, this pool of enzyme is responsible for the specific cAMP-dependent phosphorylation of a 23-kDa substrate (Figure B). Additionally, compartmentalization of type II PKA in the axoneme is likely mediated via AKAP28, a novel axoneme-specific–anchoring protein.
Changes in ciliary beat frequency are likely mediated via a cascade of phosphorylation/dephosphorylation events that alter the activity of axonemal proteins. In our studies of isolated human ciliary axonemes, we have identified one major 23-kDa protein that is phosphorylated in a cAMP-dependent manner (Figure B). Parallels may be drawn to other axonemal systems. For example, Salathe et al. (1993)
studied the in vitro cAMP-dependent phosphorylation of ovine axonemes and found that a protein with an apparent molecular mass of 26 kDa was consistently phosphorylated (Salathe et al., 1993
). Similar results were obtained in studies of Paramecium
(29-kDa), mussel gill (27-kDa), and Tetrahymena
(34-kDa) axonemes (Stommel and Stephens, 1985
; Hamasaki et al., 1989
; Chilcote and Johnson, 1990
Further studies in Paramecium
revealed that p29, the axonemal protein consistently phosphorylated in this species, copurified with the 22S outer arm dynein (Bonini and Nelson, 1990
; Hamasaki et al., 1991
). Additionally, cAMP-dependent phosphorylation of p29 has been linked to both increased swimming velocity of permeabilized paramecia and increased velocity of microtubules gliding across surfaces coated with dynein isolated from phosphorylated axonemes (Barkalow et al., 1994
). In studies of Tetrahymena
, it has been demonstrated that its PKA substrate, p34 also copurifies with 22S dynein (Christensen et al., 2001
). In a number of biochemical tests, Christensen et al. (2001)
elegantly demonstrate that p34 is the functional ortholog of the Paramecium
22S regulatory light chain p29. Functionally it seems the regulation of ciliary motility by PKA involves the phosphorylation of a dynein regulatory light chain. To date, however, the primary sequence of this regulatory light chain has not been determined.
Both type I and type II isoforms of PKA are detectable by Western blot in human ciliary axonemes (Figure C). Although the kinetic and enzymatic specificities of the three catalytic subunits (Cα, β, and γ) of PKA are indistinguishable, different regulatory subunits (RI and RII) display different cAMP-binding affinities and are differentially located within the cell. RI (α and β) is mainly cytoplasmic; however, at times it is compartmentalized. For example, RI holoenzyme is tightly bound to plasma membranes of erythrocytes and is enriched at the neuromuscular junction (Rubin et al., 1972
; Imaizumi-Scherrer et al., 1996
). Additionally, RI is associated with activated B-cell receptors and accumulates at the “cap” site when lymphocytes are stimulated with anti-CD3 antibodies (Skalhegg et al., 1994
; Levy et al., 1996
). In contrast, RII (α and β) often tightly associates with cellular structures and organelles. No studies determining the specific roles of RI and RII within the axoneme have been conducted. Electron microscopic studies of axonemes with RI- and RII-specific antibodies will be necessary to determine where each PKA isotype is localized within this structure. Moreover, we are uncertain whether type I PKA and type II PKA serve overlapping or distinct roles in the axoneme. Pharmacological experiments using isotype-specific agonists will be necessary to delineate the roles of RI and RII in the regulation of CBF.
In this study, we have focused on identifying the protein responsible for anchoring type II PKA in the axoneme. We have determined the molecular identity of AKAP28, an AKAP highly enriched in airway cilia. Due to the insolubility of the axoneme, we are unable to coimmunoprecipate AKAP28 with its binding partners from primary airway cells. Using a heterologous system, however, we have been able to coimmunoprecipate epitope-tagged AKAP28 with both RII and PKA catalytic subunit from HeLa cells (Figure C). We are unable to detect RI in the same immune complexes. This experimental evidence for preferential binding to RII is in agreement with the RI/RII binding predictions made by Miki and Eddy (1999)
in Ala- and Val-scanning mutagenesis studies of AKAP-binding domains. These data show that AKAP28 preferentially binds type II PKA and likely serves as its anchor in the axoneme.
Because type I PKA also copurifies with axonemes (Figure C), RI-specific–anchoring proteins are also likely to be present. Unlike RII-binding proteins, RI-specific AKAPs are not detectable by overlay (Miki and Eddy, 1999
). Identifying RI-binding AKAPs in the axoneme is difficult because the insolubility of this organelle prohibits the coimmunoprecipitation experiments necessary for the detection of RI-binding proteins.
Sequence analysis indicates that AKAP28 is most closely related to a rat testis-specific, developmentally regulated AKAP, TAKAP-80 (Mei et al., 1997
). A 159-amino acid stretch (residues 340–499) at the carboxy terminus of TAKAP-80 shares 50% identity and 67% conserved homology with amino acids 33–192 of AKAP28 (Figure ). In contrast, no similarity is found in the N termini of these proteins. According to Mei et al. (1997)
, TAKAP-80 is exclusively expressed in testis where mRNA expression is up-regulated during a time interval that corresponds to the initiation of spermiogenesis (Mei et al., 1997
). In the study by Mei et al. (1997)
, total RNA from whole lung was probed, however, no message was detected. Either TAKAP-80 is not present in rat lung or the percentage of RNA from ciliated cells was too low to detect TAKAP-80 mRNA expression on the blot. Because tracheal RNA was not tested, we are uncertain whether TAKAP-80 is specifically expressed in rat testis or whether TAKAP-80 is the rat ortholog of human AKAP28. TAKAP-80 protein was detected by Western blot in fibrous sheath preparations from rat sperm, but no staining of rat tissues has been published. In contrast, our staining of airway sections with AKAP28-specific antibodies indicates that AKAP28 is enriched in airway cilia. It is also detectable by Western blot in axoneme preparations. We have not determined the localization of AKAP28 in testis and sperm. Because many components of ciliary and flagellar axonemes are conserved, it is likely that a protein targeted to ciliary axonemes would also localize to the axoneme of sperm flagella. Another possibility, however, is that AKAP28 contains both fibrous sheath- and axoneme-targeting information. In ciliated airway cells, where fibrous sheaths do not exist, the protein would be compartmentalized in the axoneme. In sperm, AKAP28 could preferentially target the fibrous sheath. Further study of AKAP28 in both sperm and testis is needed to resolve these questions.
In addition to AKAP28, we have identified two smaller splice variants expressed from the same gene (National Center for Biotechnology Information accession no. AF514781 and AF514782). The predicted splice variants were initially detected by 5′ and 3′ RACE of human tracheal cDNA and by the examination of expressed sequence tag databases. Their presence was confirmed in both trachea and testis by RT-PCR and the subsequent sequencing of amplified products. All three splice variants of this gene are detected by RT-PCR in both tissues. To date, we have not detected the predicted AKAP28 isoforms by Western blot of axonemes or in whole cell lysates of WD-HBE cells. Either the predicted splice forms are never translated into protein or are expressed at low levels that currently prohibit detection.
Axonemal AKAPs have also been detected in Chlamydomonas
flagella. However, unlike RII overlays of human ciliary axonemes where RII-binding proteins of 50 and 28 kDa are detected (Figure A), AKAPs of 240 and 97 kDa are detected in Chlamydomonas
flagellar axonemes (Gaillard et al., 2001
). Using genetic mutants of Chlamydomonas
, Gaillard et al. (2001)
found that AKAP240 is a component of the central pair apparatus and that AKAP97 is part of the radial spoke stalk. The molecular identity of AKAP240 has not been determined; however, AKAP97 has been defined molecularly as radial spoke protein 3 (RSP3). To date, the human ortholog of RSP3 has not been characterized. Although both human and Chlamydomonas
axoneme systems contain AKAPs, based on size, the AKAPs readily detected in each system do not seem to be the same proteins. One possibility is that human RSP3 may be a lower affinity AKAP than AKAP28 or found at a lower concentration than AKAP28 in human axonemal preparations. Also, this difference could be due to species variations or in differences between flagella and cilia. In Chlamydomonas
, agents that reduce cAMP concentrations or inhibit the activity of PKA increase axoneme motility (Hasegawa et al., 1987
). The inhibitory effect of cAMP on Chlamydomonas
axonemes is opposite of the stimulatory effect seen in other organisms. Moreover, unlike the relatively small phosphoproteins detected in other axoneme-based systems in response to cAMP, two proteins of >270 kDa are phosphorylated in response to cAMP in Chlamydomonas
(Hasegawa et al., 1987
). Although many axonemal proteins are conserved across species, specific signaling pathways and the compartmentalization of particular components vary.
In summary, we have presented the first biochemical data demonstrating the copurification of PKA with human ciliary axonemes. Additionally, we have identified the first human A-kinase anchoring protein targeted to the axoneme. We propose that AKAP28 localizes PKA to a position in the axoneme where it is able to readily interact with its substrate. This compartmentalization likely plays a role in the regulation of outer dynein arm activity and the control of ciliary beat frequency. Future experiments will be designed to examine the physiological role AKAP28 plays in human cilia.