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INTRODUCTION |
Cilia are ubiquitous cellular nanomachines, found in protists and
multicellular eukaryotes, including man, whose repetitive beat depends
on a microtubule-based cytoskeleton, powered by molecular motors, the
outer and inner rows of dynein arms (outer arm dynein, 22 S dynein; and
inner arm dyneins, 14 S dynein; respectively). The arrangement of the
dynein arms along the axonemes is complex (1). Dynein arm
mechanochemistry is thought to regulate beat frequency and beat form by
signal transduction mechanisms that change the parameters of
microtubule sliding within the axoneme, such that the outer arm dyneins
principally regulate beat frequency whereas the inner arm dyneins
control beat form (cf. Refs. 2 and 3).
cAMP specifically increases ciliary beat frequency (4), normally
measured by an increase of swimming speed, in the protozoan ciliate,
Paramecium tetraurelia. The increase occurs in living cells
and in cells that have been permeabilized with Triton X-100 and
reactivated with Mg2+-ATP; it persists in the permeabilized
cells even when cAMP is subsequently removed and it is quenched by
simultaneous addition of Ca2+ to the medium (5-8). We
previously reported on a molecule, p29, whose phosphorylation both
in vivo and in vitro correlated with the
cAMP-dependent Ca2+-sensitive increase in
swimming speed. Further studies revealed that p29 is a component of
outer arm dynein (6, 8, 9), which specifically binds to one heavy chain
isoform of the three-headed 22 S outer arm dynein. Phosphorylation of
p29 increases in vitro microtubule translocation velocity by
outer arm dyneins by 40% (6, 9) via activation of dynein
mechanochemistry (10). Hence, p29 is regarded as a dynein regulatory
light chain of ciliary outer arm dyneins in Paramecium.
Like Paramecium, Tetrahymena thermophila outer
arm dynein consists of three different heavy chain isoforms (11) in a
three-headed bouquet that sediments at 22 S, but is probably compacted
in situ (12), together with intermediate chains and light
chains. Chilcote and Johnson (13) studied phosphorylation of isolated
Tetrahymena 22 S dynein. They were successful in
phosphorylating 22 S dynein using the catalytic subunit of
cAMP-dependent protein kinase from bovine heart with 500 µM ATP and vanadate as a phosphatase inhibitor. They
found that about 0.5 mol of phosphate was added per mol of 22 S dynein,
mainly into proteins at molecular masses 78 and 34 kDa (p78 and
p34, respectively).
Phosphorylated p78 and p34 cosedimented with microtubules in an
ATP-sensitive manner, as expected for dynein constituents. An
endogenous cAMP-dependent protein kinase was present in the preparations. No dephosphorylation was detected, even with the addition
of calcineurin (protein phosphatase type II). Exogenous acid or
alkaline phosphatase was able to partially dephosphorylate the dynein,
which lowered specific activity of dynein ATPase by 30%.
Barkalow et al. (9) showed that p29 from
Paramecium binds to Tetrahymena 22 S dynein,
which suggested that there was a Tetrahymena ortholog of
p29. Accordingly, using a series of fractionation, reconstitution, and
analytical technologies, including
two-dimensional gel electrophoresis and
in vitro motility assays, we have investigated whether p34
as identified by Chilcote and Johnson (13) could be this ortholog.
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EXPERIMENTAL PROCEDURES |
Cell Cultures--
The following strains of T. thermophila were used in this study: a mucus-free mutant, SB 255 (14), and oad-1, a temperature-sensitive outer dynein arm-deficient
mutant (15, 16). SB 255 cells were grown either in 1-liter cultures in
2-liter capacity flat flasks with shaking at 38 °C or in 8-liter
aerated cultures containing Antifoam C (Sigma) at 27 °C.
oad-1 cells were grown in 1-liter cultures either at 28 °C
(permissive temperature) or at 38 °C (non-permissive temperature).
The growth medium was a modified version of a milk medium (17),
supplemented with 0.25% proteose peptone and Fe. Na. EDTA (Sigma).
Isolation and Phosphorylation of Axonemes--
Cilia were
collected according to the dibucaine method by Satir et al.
(18) with minor modifications. Cells were harvested and resuspended in
2% proteose peptone with 3 mM dibucaine and 1 mM phenylmethylsulfonyl fluoride, allowing deciliation to
occur within 5-10 min. Cell bodies were pelleted and discarded by
centrifugation at 1500 × g in axoneme buffer (30 mM HEPES (pH 7.6), 20 mM KCl, 5 mM
MgSO4, 0.5 mM EDTA). Demembranation of cilia
and phosphorylation of axonemes were performed according to Hamasaki
et al. (5) with a few modifications. Demembranation was
accomplished with 0.5% Triton X-100 in axoneme buffer, and axonemes
were collected in axoneme buffer supplemented with 1 mM
dithiothreitol. Axonemes from SB255 cells were in
vitro-thiophosphorylated for 30 min at room temperature in axoneme
buffer containing about 300 nM 5'-
-S thiotriphosphate adenosine (cold labeling) or
5'-
-[35S] thiotriphosphate adenosine (>600 Ci per
mmol; PerkinElmer Life Sciences), Thiophosphorylation prevents
the action of phosphatases during dynein isolation procedures. Axonemes
from oad-1 cells were phosphorylated with [
-32P]ATP.
The reactions were performed in the presence and in the absence of
10-20 µM cAMP and 10
3-10
4
M CaCl2 (pCa 3-4). All steps were performed in
the presence of a mixture of protease inhibitors,
CompleteTM (Roche Molecular Biochemicals).
Isolation of Dynein and p34 from Axonemes--
Dynein
heavy chains 14 S (inner arm dynein) and 22 S (outer arm dynein) were
purified according to the procedure of Hamasaki et al. (6)
with a few modifications. Axonemes were incubated on ice for 30 min in
axoneme buffer with 0.6 M KCl. This separates the dynein
molecules from microtubules, the latter being pelleted by
centrifugation at 27,000 × g for 15 min. Usually, the
dynein-containing supernatant was further incubated on ice for 30 min
in axoneme buffer with 1.2 M KCl, allowing
dynein-regulatory proteins to be separated from the dynein heavy chains
(9). The sample was then loaded onto a Centricon 100 (Millipore,
Milford, MA) and centrifuged at 1000 × g at 4 °C
until the volume of the retentate, containing dynein heavy chains, was
less than 100 µl. The retentate was then carefully layered on top of
a 5-30% linear sucrose gradient and centrifuged at 100,000 × g for 15 h, followed by fractionation of the gradient
into about 20 fractions each having a volume of ~500 µl. The
fractions were assayed for ATPase activity (19) and analyzed by gel
electrophoresis to identify fractions containing 14 S and 22 S dyneins.
Prior to sucrose gradient separation, fractions containing crude 22 S
dynein with thiophosphorylated p34 were incubated with antisera
generated against the 22 S dynein heavy chains and then mixed with
protein-A-Sepharose 3B beads (Zymed Laboratories Inc.,
San Francisco, CA), following procedures modified after Wang and Satir
(20) for immunoprecipitation. Controls included a sample containing
thiophosphorylated p34 but no dynein heavy chains and 22 S dynein mixed
with the-Sepharose beads in the absence of antisera. Purification of
p34 fractions from thiophosphorylated axonemes was performed by the
method used to isolate p29 fractions from Paramecium dynein
(9). Briefly, the Centricon 100 flowthrough from the dynein samples was
concentrated in a Centricon 10, followed by three washings with axoneme
buffer, leading to a final volume of less than 100 µl.
Rebinding of Isolated Thiophosphorylated p34 to Dynein Heavy
Chains--
Reconstitution of p34 to dynein heavy chains from
Tetrahymena and P. tetraurelia was performed
according to Barkalow et al. (9). A 50-µl volume of the
fractions containing non-radiolabeled 14 S or 22 S dynein was
transferred to Eppendorf tubes. To this was added 10 µl of the
fraction containing radiolabeled p34, which had been thiophosphorylated
in the presence of cAMP. The samples were then centrifuged through a
Microcon 100 (Millipore), concentrating the retentate to ~10 µl.
The retentate contains the dynein heavy chains, and the flowthrough
contains free p34. These samples were then analyzed by gel
electrophoresis and autoradiography. If the radiolabeled p34
reconstitutes with the heavy chains it will be located in the
retentate, whereas unbound p34 will localize to the flowthrough. The
flowthrough was concentrated in a Microcon 10 to ~10 µl. In other
experiments we investigated the capability of non-radiolabeled
(cold-labeled) p34 and p29 to compete with the binding of radiolabeled
p34 to 22 S dynein from either Tetrahymena or
Paramecium. These experiments were carried out essentially as described above except that increasing concentrations of
non-radiolabeled p34 and p29 were added to the samples. In all cases
bovine serum albumin (20-50 µg) was added to each reaction tube as a
control for nonspecific protein binding. Reconstitution was carried out at room temperature for 1 h with vertical rotation of the test tubes.
One-dimensional Gel Electrophoresis--
Proteins from SB255
cells were resolved by gel electrophoresis either under denaturation
and reducing conditions
(SDS-PAGE)1 or under native
settings according to NuPAGE minigel procedures for the NOVEX Xcell
(E19001) system (NOVEX, San Diego, CA). Protein samples for SDS-PAGE
were run on 10% NuPAGE bis-tris and on 3-8% NuPAGE Tris acetate gels
with NuPAGE MOPS SDS running buffer (NP0002) and NuPAGE Tris acetate
running SDS buffer (LA0041), respectively. Samples with proteins to be
resolved under native conditions were placed in Tris-glycine native
sample buffer (LC2673). They were then loaded and run on 3-8% NuPAGE
Tris acetate gels with Tris-glycine native running buffer (LC2672).
SDS-PAGE of proteins from oad-1 cells was performed in 10%
Tris-glycine gels. SeeBlueTM prestained standards (NOVEX) were used as
molecular mass markers. The gels were fixed and stained in 45%
methanol, 10% acetic acid, and 0.1% Coomassie Blue R250 for one h,
shrunk in 30% ethanol and 2% glycerol for 30 min, and dried on
Whatman No. 3MM chromatography paper.
Two-dimensional Gel Electrophoresis--
Protein samples were
desalted and concentrated with phenol precipitation (21). The proteins
were then resolved according to the manufacturer's manual for the
Multiphor II horizontal two-dimensional gel electrophoresis system
(Amersham Pharmacia Biotech). The first dimension (isoelectric
focusing) was performed with Immobiline Dry Strip, pH 3-10 linear pH
gradient, and the second dimension (SDS-PAGE) was performed with
ExcelGel SDS XL 12-14% Tris-glycine. The gels were silver-stained
according to Shevchenko et al. (22) with minor
modifications. It was often necessary to go through two steps of
sensitizing and staining to get clear staining of the proteins, as well
as a low background staining.
Detection and Quantification of Radiolabeled Proteins--
Dried
gels were subjected to autoradiography. 32P-labeled
proteins were detected by Kodak x-ray films, and
35S-labeled proteins were detected by Kodak BIOMAX MR film
or BIOMAX MS films with Kodak BIOMAX TranSCreen-LE intensifying screen
(Eastman Kodak Co., Rochester, NY). The intensity of protein
phosphorylation was analyzed by a PhosphorImagerTM using the
ImageQuaNT system (Molecular Dynamics, Sunnyvale, CA).
Microtubule Translocation Assay--
The effect of p34
phosphorylation on microtubule translocation activity was measured
using an in vitro motility assay adapted from Ref. 6. 22 S
dynein was purified from Tetrahymena axonemes that had been
in vitro-thiophosphorylated in the presence and in the
absence of cAMP. The dynein was then perfused into the translocation
chamber. After 5 min of incubation, excess dynein was removed by
washing of the chamber with gliding buffer (50 mM potassium
acetate, 10 mM Tris acetate (pH 7.5), 5 µM
taxol, 3 mM MgSO4, 2 mM
dithiothreitol, 1 mM EGTA). Attached dynein was activated
with gliding buffer supplemented with 1 mM ATP, and this
was followed by perfusion with the same buffer containing microtubules (polymerized from phosphocellulose column-purified bovine
brain tubulin in the presence of 1 mM GTP and stabilized with 5 µM taxol). Movement of the microtubules was
recorded on video. Positions were traced at 4-s intervals, and the
length and average velocity of moving microtubules was measured, based on a minimum of >30 individual determinations for each condition.
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RESULTS |
Effects of cAMP and Ca2+ on in Vitro Protein
Phosphorylation in oad-1 Axonemes--
We initially conducted a series
of phosphorylation experiments with the temperature-sensitive outer
dynein arm-deficient mutant of T. thermophila, oad-1 (Fig.
1). In axonemes isolated from cultures grown at 28 °C (permissive temperature) where outer arm dyneins are
present in this mutant (15, 16) 10 µM cAMP induced the [
-32P]ATP phosphorylation of several proteins in the
p34 range, about 6× over no add controls. In the presence of cAMP,
addition of Ca2+ increased the p34 phosphorylation further
to about 9× control levels, whereas in the absence of cAMP,
Ca2+ did not elicit p34 phosphorylation. Similar changes
were seen in a p78 protein. However, at 38 °C (non-permissive
temperature) where outer arm dyneins are greatly reduced in the mutant,
although similar changes were seen at p78, p34 phosphorylation remained at control levels regardless of whether cAMP and/or Ca2+
were present. These results confirm that p34 is an outer arm dynein-related molecule that is phosphorylated in a
cAMP-dependent manner.

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Fig. 1.
Axonemal outer arm dynein assembly is
required for cAMP-dependent p34 phosphorylation.
Autoradiographic analysis of phosphorylated axonemal proteins after
SDS-PAGE in oad-1 at 28 °C (permissive temperature) or 38 °C
(non-permissive temperature). Protein phosphorylation was performed in
the presence and in the absence of 10 µM cAMP and
1 mM CaCl2. Molecular mass markers
(M; from top): 205, 116, 97, 66, 45, 36, 29, 24, and 16 kDa. The graphs summarize relative levels of p34
phosphorylation based on band densities in the autoradiograms
(average ± S.E.; n = 4).
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p34 Is a 22 S Outer Arm Dynein Light Chain--
Treatment of
axonemes with 0.6 M KCl extracts most of the dynein,
yielding so-called crude dynein. Further incubation of the dynein
fraction in 1.2 M KCl dissociates some of the 22 S dynein into its constituent proteins (9). After high salt treatment, the
dynein-containing fraction, centrifuged through a Centricon 100, produces a retentate containing the dynein heavy chains and a
flowthrough containing axonemal proteins between 10 and 100 kDa,
including dynein light chains.
Fig. 2A shows SDS-PAGE and
autoradiographic analysis of proteins in T. thermophila SB
255 in the salt-extracted thiophosphorylated axonemes, in the retentate
heavy chain containing fraction, and in the 10-100 kDa light
chain-containing fraction. p34 is the major protein of the light chain
fraction that becomes phosphorylated upon stimulation with cAMP.
Phosphorimaging measurements again show a 5× increase on p34 labeling
upon cAMP addition, which is enhanced by Ca2+, whereas in
Ca2+ alone, p34 phosphorylation remains at control levels
(Fig. 2B). Fig. 3 shows ATPase
activity measurements of fractions collected from the crude dynein
within a 5-30% linear sucrose gradient. The ATPase peak in fractions
7-11 corresponds to 14 S inner arm dyneins, and the peak in fractions
14-18 correspond to 22 S outer arm dynein (Fig. 3A), as
confirmed by SDS-PAGE showing the dynein heavy chains (Fig.
3B). The corresponding autoradiogram shows that p34
remaining with the dynein heavy chains comigrates with 22 S dynein and
not with 14 S dynein (Fig. 3B). Some p34 (seen in fractions
1-5) dissociates from the dynein heavy chains during this
procedure.

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Fig. 2.
Preparation of a p34-enriched fraction.
A, Tetrahymena SB 255 axonemes were
thiophosphorylated ± cAMP prior to extraction; after treatment
with 1.2 M KC1 no phosphorylated p34 is retained in the
salt-extracted axonemes. Although some phosphorylated p34 is found in
the heavy chain-containing retentate fraction (HC),
phosphorylated p34 is enriched in a 10-100-kDa light chain-containing
fraction (LC) prepared by passing the dynein-containing
extract through a Centricon 100. Upper panel, Coomassie
Blue-stained gel; lower panel, corresponding autoradiogram.
Molecular mass markers (M; from top): 200, 116, 97, 66, 55, 37, and 31. B, the amount of p34 phosphorylation
in the 10-100-kDa fraction reflects axonemal conditions of
phosphorylation. The graph shows relative levels of p34
phosphorylation based on bands densities from the autoradiograms
(average ± S.E.; n = 3).
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Fig. 3.
Analysis of dyneins fractionated on a 5-30%
linear sucrose gradient. Dyneins were salt-extracted from
Tetrahymena SB255 axonemes thiophosphorylated in the
presence of cAMP. A, ATPase activity measurements used to
identify fractions containing 14 S and 22 S dynein. B,
corresponding SDS-PAGE gel, showing the dynein heavy chain
(HC) region and autoradiograph (autorad)
showing p34.
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To test further whether p34 is a dynein light chain, we
immunoprecipitated 22 S dynein heavy chains using 22 S dynein antisera, protein-A-Sepharose, and p34 coimmunoprecipitated with the dynein heavy
chains (Fig. 4A). The antisera
did not immunoprecipitate thiophosphorylated p34 when fractions
containing thiophosphorylated p34 but no heavy chains (10-100-kDa
light chain-containing fraction) were used for the experiments. As
expected, no immunoprecipitate was produced in the absence of antisera.
We also examined the phosphorylation pattern of 22 S dynein under
native gel electrophoresis conditions. If p34 is a light chain, it
should remain attached to 22 S dynein under native conditions, and upon
denaturation in SDS-PAGE the amount of phosphorylation in p34 plus the
residual radioactivity in the dissociated single-headed dynein heavy
chains and their constituent components should quantitatively match the amount of phosphorylation in the native 22 S complex. After
denaturation, the
and
of dynein heavy chains are seen to be
phosphorylated at a constitutively low level independently of cAMP
addition. A number of additional bands are also phosphorylated.
Although the level of p34 phosphorylation varies, the level of
phosphorylation in the bands other than p34 in fractions 14-17 of the
gel is virtually constant. The phosphorylated bands seen in the
autoradiogram after SDS-PAGE (Fig. 4B) disappear under
native conditions, and fraction by fraction the amount of
phosphorylation under denaturation conditions is switched to the native
22 S complex (Fig. 4C). The variation in p34 phosphorylation
is reproduced in the complex, which supports the conclusion that p34 is
a light chain of native 22 S dynein.

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Fig. 4.
p34 coimmunoprecipitates with 22 S dynein
heavy chains and associates with 22 S dynein under native
conditions. A, autoradiograph showing
coimmunoprecipitation of thiophosphorylated p34 (arrow)
and dynein heavy chains (HC; arrowhead) in
pellet containing protein A-Sepharose beads and crude dynein
incubated with 22 S dynein heavy chain antisera. B,
autoradiograph of thiophosphorylated proteins seen in 22 S dynein
fractions (see Refs. 14-17 and Fig. 3B) under denaturation
and reducing conditions (SDS-PAGE) and without such
treatment (native gel). C, comparative levels of
phosphorylation (counts × 103) from PhosphorImagerTM
measurements in 22 S dynein fractions. In SDS-PAGE, most bands are
constant, illustrated by scan of single-headed and dynein heavy
chains (black bars); however p34 (open bars)
increases in fractions 15 and 16. In the native gel, these changes are
reflected in the phosphorylation of intact 22 S dynein (line with
open circles).
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Reconstitution and Competition Experiments--
To show whether
p29 and p34 are functional orthologs, we performed a series of
experiments on the ability of radiolabeled p34 to reconstitute with
Tetrahymena and Paramecium dyneins. Fig. 5 shows reconstitution of radiolabeled
p34 with Tetrahymena and Paramecium dyneins in
our standard assay. In all cases bovine serum albumin was added to the
reactions both as a control for nonspecific binding of p34 to protein
per se and to prevent dyneins and p34 from binding to the
Centricon membranes. In the absence of any of the dyneins, radiolabeled
p34 identified by autoradiogram was found exclusively in the Centricon
100 flowthrough, as was bovine serum albumin. In the presence of
dyneins, a small amount of bovine serum albumin was found in the
retentate, but this did not affect the retention of p34. 14 S dynein
did not rebind p34 in either ciliate, and labeled p34 was found only in
the flowthrough. On the other hand, p34 reconstituted with both
Paramecium and Tetrahymena 22 S dynein, where
much of the labeled p34 remained with the retentate.

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Fig. 5.
Reconstitution of thiophosphorylated p34 with
Tetrahymena and Paramecium 22 S but
not 22 S dynein. Control (C), no dyneins were present
in the reconstitution reaction. All reactions were carried out in the
presence of 20 µg of bovine serum albumin (BSA).
The abbreviations are as follows: r, retentate from samples
collected with Microcon 100 ( 100-kDa proteins); fl,
flowthrough from Microcon 100, concentrated in Microcon 10 (10-100-kDa
proteins). With 22 S dynein, p34 is found in the retentate, together
with the heavy chains (HC) of the outer arm dynein.
Molecular mass markers (M; from top): 191, 64, 51, 39, 28, and 19 kDa.
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To test whether p34 competes with p29 for binding to 22 S dynein,
we added increasing concentrations of cold-labeled p34 or p29 to a
constant amount of 35S-labeled p34 in our assay (Fig.
6). Cold-labeled p34 competed with the
binding of radiolabeled p34 to Tetrahymena 22 S dynein (Fig.
6B), and cold-labeled p29 competed effectively with the binding of radiolabeled p34 to Paramecium 22 S dynein (Fig.
6A), qualitatively displacing p34 radiolabel from the
retentate to the flowthrough.

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Fig. 6.
Paramecium p29 competes with p34 in 22 S
dynein reconstitution. A, SDS-PAGE (gel) and
autoradiograph (autor) analysis of a competition experiment.
A cold-labeled p29 fraction was added at increasing concentrations to
compete with binding of p34 to Paramecium 22 S dynein. All
reactions mixtures contained 20 µmg of bovine serum albumin. The
abbreviations are as follows: r, retentate from samples
collected with Centricon 100 ( 100-kDa cut-off), containing
radiolabeled p34 bound to dynein heavy chains; fl,
flowthrough from Microcon 100 and concentrated in Microcon 10 (10-100-kDa cut-off), containing free radiolabeled p34. The
graph shows the level of p34 phosphorylation in
retentate (r; black circles) and flowthrough
(fl; open circles) at various concentrations of
p29. B, control showing an autoradiogram of cold-labeled p34
at increasing concentrations competing with binding of radiolabeled p34
to Tetrahymena 22 S dynein.
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p34 Resolved by Two-dimensional Gel Electrophoresis--
Fig.
7 shows a two-dimensional gel analysis of
the 10-100-kDa light chain fraction thiophosphorylated in the absence
and in the presence of cAMP. The silver stain pattern of proteins is reproducible in the two gels, as are virtually all the radiolabeled spots (Fig. 7A). After cAMP treatment, a ladder of at least
five unique phosphorylated spots (Fig. 7B), focusing in the
pH range from 5 to 6, corresponding to faint spots in the
silver-stained gels, is found in the p34 region. These spots increase
slightly in molecular mass, whereas at the same time they decrease in
pI. The p34 spots are the only significant thiophosphorylated molecules in the 10-100-kDa fractions that were consistently radiolabeled in the
presence of cAMP, not seen in the audoradiogram when
thiophosphorylation occurred in the absence of cAMP.

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Fig. 7.
Two-dimensional gel analysis of p34
phosphorylation. A, two-dimensional gel electrophoresis
of radiolabeled proteins in the 10-100-kDa light chain fraction
prepared from axonemes treated ± cAMP. The proteins were resolved
in the first dimension in a pH 3-10 linear gradient (from
left to right) and with a 12-14% Tris-glycine
gel for SDS-PAGE in the second dimension (from top to
bottom). The gels were silver-stained, and radiolabeled
proteins were detected by phosphorimaging. Molecular mass markers
(M; from top): 98, 64, 50, 36, 30, and 16 kDa.
S, cAMP-treated sample resolved in SDS-PAGE as a marker for
phosphorylated p34. B, detail from the phosphorimages of the
p34 region. Positions of fastest (i) and slowest
(ii) migrating thiophosphorylated p34 spots are
marked.
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cAMP Treatment of 22 S Outer Arm Dynein Increases the Microtubule
Translocation Velocity--
The in vitro translocation
velocity of microtubules on 22 S dynein isolated from axonemes that had
been thiophosphorylated in the presence and in the absence of cAMP was
determined. The length of the microtubules that moved on the dynein
varied, although the distribution of the lengths was essentially
similar in all experiments, which allowed us to compare the velocity of
translocation from one experiment to another (6). 22 S dynein from
axonemes thiophosphorylated in the absence of cAMP produced an average in vitro velocity of microtubules of 0.83 ± 0.08 µm s
1 (mean ± S.E.) (n = 37). This contrasted to microtubule velocity produced by 22 S dynein
from axonemes phosphorylated in the presence of cAMP, where the average
velocity increased to 1.42 ± 0.09 µm s
1
(n = 40) (t test, p < 0.0001).
 |
DISCUSSION |
Previous results by Chilcote and Johnson (13) showed that 22 S
outer arm dynein in Tetrahymena is associated with a 34-kDa protein (p34) that is phosphorylated in vitro by
cAMP-dependent protein kinase, resulting in increased
phosphate incorporation into the protein. Accordingly, in our studies,
thiophosphorylation indicated by 35S incorporation probably
represents new phosphate addition, rather than turnover of existing
phosphorylated p34. Two-dimensional gel electrophoresis shows that p34
is largely unphosphorylated prior to cAMP addition. At least five
phosphorylated protein spots appear in a ladder in the p34 region
after cAMP treatment. Sequentially the spots increase slightly in
molecular mass, while at the same time they decrease in pI, which
probably indicates that p34 can be phosphorylated at multiple sites or
alternatively that multiple isoforms of p34 of slightly different mass
and pIs are present in the axoneme. If multiple phosphorylations of p34
occur, this might mean that sequential phosphorylation of a single
dynein regulatory light chain leads to increased outer arm dynein
activity. Attempts are now being made to sequence p34 from these gels.
Our results support the conclusion that p34 is a functional ortholog of
an outer arm dynein regulatory light chain that probably regulates
ciliary beat frequency, found in several cilia, of which p29 in
Paramecium axonemes is the prototype (6, 9). We earlier demonstrated that p29 rebinds to Tetrahymena, as well as to
Paramecium outer arm dynein (9). Here we show that p34 also
rebinds to outer arm dynein, but not inner arm dynein, in both
ciliates, with some differences when binding is to dyneins of the same
versus the contrasting organisms, and that cold-labeled p29
competes with the binding of radiolabeled p34. p34 is a constituent
dynein regulatory light chain, because it coimmunoprecipitates with 22 S dynein heavy chains (20), and it migrates together with heavy chains
of outer arm dynein both in a gradient separation of dyneins and in
native gels. We calculate that ~2% of the molecular weight of outer
arm dynein is p34, assuming that there is one p34 for each
-heavy
chain in the molecule, an assumption consistent with the measurements
of Barkalow et al. (9) on p29. The temperature-sensitive mutant, oad-1, when deficient in outer arms (15, 16) shows no p34
phosphorylation. Further, the in vitro translocation
velocity of microtubules by Tetrahymena 22 S outer arm
dynein increases by about 70% after thiophosphorylation in the
presence of cAMP, an increase that is attributable to
cAMP-dependent phosphorylation of an outer arm dynein
regulatory light chain leading to faster ciliary beat. An increase in
p34 phosphorylation should be directly proportional to a corresponding
increase in ciliary beat frequency (10) over a necessary wide range
of frequencies.
The presence of high Ca2+ (pCa 4) increases cAMP-induced
phosphorylation of p34 in Tetrahymena axonemes by about
60%, although Ca2+ alone has no effect. Similarly,
Chilcote and Johnson (13) found that Ca2+ and calmodulin
alone did not elicit new p34 phosphorylation. Therefore,
Ca2+ in the absence of cAMP must be insufficient to
activate the relevant endogenous kinases, but, speculatively, it could
cause conformational changes to increase the accessibility of p34 to
phosphorylation. In mammalian respiratory cilia (23-25),
Ca2+ also increases ciliary beat frequency synergistically
with cAMP, possibly via a similar action on dynein regulatory light
chain phosphorylation. This is different from Paramecium,
where unusually the effect of Ca2+ on
cAMP-dependent phosphorylation of p29 is inhibitory,
because Ca2+ inhibits the relevant Paramecium
cAMP-dependent protein kinase (5).
Tetrahymena cilia can be readily isolated in larger
quantities than either Paramecium or mammalian cilia, which
may prove significant for the study of certain minor components of the
axoneme, such as p34 and its orthologs. Similar approaches have proven useful in identifying dynein light chains using
Chlamydomonas (26). Gene transformation and knockout
procedures have been developed for Tetrahymena (27, 28). The
biochemical characterization of p34 presented here provides the basis
for a unique opportunity to study function of the dynein regulatory
light chain in further detail through biochemical and molecular genetic
approaches that will yield fundamental information regarding the
molecular mechanisms of beat frequency control in the ciliary axoneme.