1 Department of Oncology, McArdle Lab, University of Wisconsin-Madison, Madison,
Wisconsin 53706, USA
2 Department of Biochemistry, College of Agricultural and Life Science,
University of Wisconsin-Madison, Madison, Wisconsin 53706, USA
* Author for correspondence (e-mail: nelson{at}biochem.wisc.edu )
Accepted 31 January 2002
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Summary |
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We characterized these proteins and Paramecium calmodulin as
substrates for two Ca2+-dependent protein kinases purified from
Paramecium. PCBP-25 and calmodulin were in vitro substrates
for one of the two Ca2+-dependent protein kinases (CaPK-2), but
only PCBP-25
was phosphorylated by CaPK-1. These results raise the
possibility that the biological activities of PCBP-25
and calmodulin
are regulated by phosphorylation.
Key words: Centrin, Phosphorylation, Paramecium, Infraciliary lattice, Cilia, Dynein
![]() |
Introduction |
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The diverse functions of Ca2+-regulated processes are reflected
in the variety of Ca2+-binding proteins (CaBPs), all members of the
EF-hand superfamily (Plattner and Klauke,
2001), found in Paramecium. Calmodulin (CaM) is present
(Walter and Schultz, 1981
;
Momayezi et al., 1986
) and is
known from genetic studies to be involved in the regulation of several ion
channels. Missense mutations in the single CaM gene of
Paramecium lead to striking defects in ion channel function,
regulation of the ciliary beat and swimming behavior
(Preston et al., 1991
). CaM is
the Ca2+ sensor for guanylyl cyclase
(Klumpp et al., 1983a
;
Schultz and Klumpp, 1984
) and
probably for adenylyl cyclase (Gustin and
Nelson, 1987
), and binding studies have identified a number of
other CaM-binding proteins in cilia and cell bodies
(Evans and Nelson, 1989
;
Chan et al., 1999
).
The infraciliary lattice (ICL), a contractile network of cytoskeletal
filaments in the cell cortex (Garreau de
Loubresse et al., 1988), contains and largely comprises six small,
acidic Ca2+-binding proteins
(Klotz et al., 1997
) that
probably account for its ability to contract in response to Ca2+.
The genes for these proteins have been cloned and found to be closely related
to those for centrins of other organisms
(Madeddu et al., 1996
). Klotz
et al. (Klotz et al., 1997
)
showed that mutations in one of these centrin-like genes result in disordered
cortical structure. Allen et al. (Allen et
al., 1998
) used the same anti-centrin antiserum that we used here,
as well as a monoclonal antibody they raised against a Mr
110,000 protein of the striated band, to show definitively that the ICL was
immunologically distinct from the striated band, another filamentous
cytoskeletal system in Paramecium.
It has been proposed that the cytoskeletal rearrangements at cell division
in Paramecium may be associated with a wave of
Ca2+-induced phosphorylation of cytoskeletal proteins
(Sperling et al., 1991;
Beisson and Ruiz, 1992
). Two
Ca2+-dependent protein kinases [CaPK-1, CaPK-2;
(Gundersen and Nelson, 1987
;
Son et al., 1993
;
Kim et al., 1998
)] that are
unlike any found in animal cells are found in Paramecium; they
require micromolar Ca2+, but neither CaM nor phospholipids, for
their activity. Their activation by Ca2+ results from direct
binding of Ca2+ to the kinase
(Gundersen and Nelson, 1987
;
Son et al., 1993
). Cloning and
sequencing of the genes encoding Paramecium CaPKs
(Kim et al., 1998
) revealed
that they, like other CaPKs (Harper et
al., 1991
), contain a CaM-like regulatory domain fused to the
highly conserved protein kinase catalytic domain. The endogenous substrates
for these kinases in Paramecium are not known, and their
physiological roles in mediating Ca2+ signaling are uncertain.
While purifying these kinases from Paramecium
(Gundersen and Nelson, 1987;
Son et al., 1993
), we observed
that several Ca2+-binding proteins of Mr
20,000-25,000 copurified with the kinases and were substrates for
Ca2+-dependent phosphorylation. Here we describe two of these
Ca2+-binding proteins, PCBP-25
(Paramecium
Ca2+-binding protein of 25 kDa) and PCBP-25ß, and compare them
with CaM as substrates for phosphorylation by the purified
Ca2+-dependent protein kinases from Paramecium.
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Materials and Methods |
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Partial purification of PCBP-25
Cells were extracted, and the extract was fractionated on phenyl-Sepharose,
exactly as previously described (Son et
al., 1993). Proteins that bound to phenyl-Sepharose in
Ca2+ and were eluted with EGTA were concentrated, then applied to
an anion-exchange FPLC column (MonoQ HR 5/5, Pharmacia, Piscataway, NJ)
equilibrated in 20 mM bis-Tris propane-Cl, pH 6.7, 0.1 M sucrose, 0.1 mM EDTA
and 1 mM dithiothreitol (DTT). Protein was eluted first with a 24 ml gradient
from 0 to 0.3 M NaCl, then with a 24 ml gradient from 0.3 to 1 M NaCl in the
same buffer.
Preparation of fractions enriched for ICL
ICL-enriched fractions were prepared as described by Garreau de Loubresse
et al. (Garreau de Loubresse et al.,
1988).
Preparation of ciliary fractions
Cells were washed and immobilized in a 1:1 mixture of Dryl's solution [2 mM
Na2HPO4, 2 mM sodium citrate, 1.5 mM CaCl2,
pH 6.8 (Dryl, 1959)] and STEN (0.5 M sucrose, 20 mM Tris-Cl, 2 mM EDTA, 6 mM
NaCl, pH 7.5) using 20 ml of Dryl's/STEN per ml of packed cells. The cilia
were detached by the addition of CaCl2 and KCl to final
concentrations of 10 mM and 30 mM, respectively, plus 0.3 mM PMSF and 1
µg/ml leupeptin. Deciliation was monitored by phase contrast microscopy.
Cell bodies were separated from cilia by centrifugation at 850
g for 2 minutes. The subsequent subciliary fractionations were
performed by published methods (Travis and
Nelson, 1988a).
Phosphorylation assays
Protein kinase activities (CaPK-1 and CaPK-2) were measured using casein as
described (Son et al., 1993).
A unit of protein kinase activity corresponds to 1 pmol of
32Pi incorporated into substrate per minute.
Phosphorylation of PCBP-25
and PCBP-25ß by purified
Paramecium protein kinases was performed under the following
conditions without casein. In brief, the reaction mixture for the CaPK-1 and
CaPK-2 contained 20 mM HEPES-NaOH, pH 7.2, 5 mM magnesium acetate, 1 mM DTT,
0.5 mM EGTA±0.51 mM CaCl2 and 20 µM
[
-32P]ATP (100 Ci/mol) in a final volume of 50 µl. The
reaction was carried out at 30°C for 10 minutes and stopped with 15%
trichloroacetic acid. Acid precipitates were subjected to SDS-PAGE followed by
autoradiography. Assays for synthetic peptides were performed under the same
conditions as for protein substrates except that 50 µM
[
-32P]ATP (50 Ci/mol) was used and the assays were performed
at room temperature. 40 µl of the reaction mixture was spotted onto Whatman
phosphocellulose paper (P81), which was dropped into 75 mM phosphoric acid to
quench the reaction. After four washes in the same solution, the filters were
dried and counted for adsorbed radioactivity.
Immunoblots
Proteins were separated by SDS-PAGE, transferred to nitrocellulose (0.1
µm) and probed with either undiluted monoclonal antibodies or the 1000-fold
diluted polyclonal serum. Transferred proteins were visualized by staining
with Ponceau S (Salinovich and Montelaro,
1986). Blots were incubated in 0.1 µg/ml
alkaline-phosphatase-conjugated goat anti-mouse or goat anti-rabbit IgG
antibody 1000-fold diluted in wash buffer, and the reaction was detected with
substrate solution (0.2 mg/ml 5-bromo-4-chloro-3-indolylphosphate and 0.2
mg/ml nitro blue tetrazolium, 5% methanol in 0.7 M Tris, pH 9.5).
Electron microscopy
Axenically grown Paramecium cells were washed in Dryl's solution
(Dryl, 1959), resuspended in
50 mM HEPES, pH 7.3 and concentrated by low-speed centrifugation (2000
g). They were resuspended in 0.5% glutaraldehyde in 50 mM
HEPES, pH 7.3, at room temperature for 1.5 hours. The cells were washed twice
in buffer before incubation in 0.5% uranyl acetate for 30 minutes. They were
washed once in distilled water, embedded in 1.5% agar at 40°C and then
chilled on ice. The solidified agar pellet containing the cells was cut into
small blocks. The cells were dehydrated in an ethanol series and embedded in
LR White resin (EMS, Fort Washington, PA) according to the manufacturer's
instructions. Silver-gray sections were cut on a Reichert Om U3 ultramicrotome
and placed on Formvar-covered, carbon-coated gold mesh grids. Thin sections
were contrasted with ethanolic uranyl acetate and lead citrate. Lead citrate
staining was omitted in the immunolabeling studies.
For immunolabeling, sections were blocked in 10 mM Tris, 500 mM NaCl, 0.05% Tween-20, 1% bovine serum albumin and 0.02% sodium azide before transfer to buffer containing the primary antibody for 4 hours. Monoclonal antibody was undiluted tissue culture supernatant and the polyclonal antibody was diluted 800-fold in the wash buffer (10 mM Tris, 500 mM NaCl, 0.05% Tween-20, 0.3% bovine serum albumin and 0.02% sodium azide). Grids were washed in wash buffer and then incubated in goat anti-rabbit (or mouse) IgG linked to 10 nm gold particles diluted 1:30 (v/v) in blocking solution for 1 hour. Sections were stained for 10 to 30 minutes in 1% uranyl acetate. Micrographs were taken with a JEOL-100S electron microscope or with a Phillips 300 electron microscope.
Other procedures
45Ca2+ blot overlay assays
(Maruyama et al., 1984) and
silver staining (Poehling and Neuhoff,
1981
) were performed as described by Son et al.
(Son et al., 1993
). The free
[Ca2+] was calculated using the COMICS program
(Perrin and Sayce, 1967
)
translated to BASIC.
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Results |
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|
FPLC anion-exchange chromatography (on MonoQ) resolved the proteins of the
EGTA eluate into four major peaks (Fig.
2). The third major peak contained PCBP-25 and PCBP-10. Subsequent
immunological and phosphorylation studies showed PCBP-25 to consist of two
components, PCBP-25 and PCBP-25ß. PCBP-25
eluted at about
200 mM NaCl and PCBP-25ß between 220 and 250 mM NaCl. Chromatography on
MonoQ completely separated PCBP-25 from the two CaPKs, which were eluted with
a salt gradient as two major earlier peaks. In the MonoQ peak containing
PCBP-25, no protein kinase activity was detected (data not shown).
Chromatography on MonoQ resulted in a considerable purification of
PCBP-25
and PCBP-25ß, as judged by the immunoreactivity of the
whole cell homogenate and MonoQ fractions with antibodies that recognize the
two proteins specifically (described below). From about 3 g of starting
material (120,000 g supernatant), we routinely obtained at least 50
to 100 µg of PCBP-25
and 100 to 200 µg of PCBP-25ß.
|
The amount of Ca2+ bound in overlay blots was similar for
PCBP-25 and PCBP-25ß when equal molar amounts of protein were
compared, and both proteins bound Ca2+ to about the same extent as
an equal amount of CaM (data not shown). The quantification of this binding
was not precise enough to deduce the number of binding sites per molecule.
The mobility shift on SDS-PAGE in the presence and absence of
Ca2+ that is characteristic of many EF-hand-type CaBPs was seen
with PCBP-25ß (which ran faster in the presence of Ca2+).
PCBP-25 showed no comparable shift under identical conditions (data not
shown). In 10% or 12% Laemmli gels with no added Ca2+, the
Mr of both PCBP-25
and PCBP-25ß was 25,000,
and the addition or omission of ß-mercaptoethanol in the sample buffer
did not change the electrophoretic mobility for either protein (data not
shown). In gel filtration on Sephacryl S-300 in 0.1 mM EDTA, PCBP-25ß
behaved like a protein of Mr 36,000 (data not shown),
suggesting that its native aggregation state in the absence of Ca2+
is no larger than a dimer. Since most of the EF-hand CaBPs are highly
asymmetric, the PCBP-25ß may be a monomer like these CaBPs. Gel
filtration clearly separated PCBP-25ß from PCBP-10
[Mr (apparent) 19,000] and CaM [Mr
(apparent) 17,000] (not shown).
PCBP-25 and PCBP-25ß are immunologically distinct
We used a monoclonal antibody (7A9) raised against the EGTA pool from
phenyl-Sepharose (M. Son, PhD thesis, University of Wisconsin-Madison, 1991)
and an antiserum raised against Chlamydomonas centrin
(Baron and Salisbury, 1988) to
distinguish several centrin-like proteins on immunoblots
(Fig. 3). In quick-killed
Paramecium samples (in which a small volume of living cells was
squirted into boiling SDS-PAGE sample buffer and boiled more to minimize
proteolysis), antibody 7A9 recognized a protein of Mr
25,000 and, as expected, the anti-centrin antibody stained a protein of about
Mr 23,000 in whole cell extract of Chlamydomonas
(Fig. 3A). Immunoblots of the
individual MonoQ fractions containing PCBP-25 showed strong reaction of the
early fractions containing PCBP-25
with the anti-centrin antibody. The
later MonoQ fractions (containing PCBP-25ß) reacted strongly with 7A9 but
not with anti-centrin antibody (Fig.
3B). These two antibodies therefore defined two immunologically
distinct proteins that have a similar size and chromatographic properties.
Isoelectric focusing also separated PCBP-25
(pI 4.6) from PCBP-25ß
(pI 4.75-4.85) (data not shown). For comparison, the pI for CaM from
Paramecium is about 4.0 (Plattner
and Klauke, 2001
), and the centrin-like proteins of the ICL focus
at between pH 4.2 and pH 4.7 (Klotz et
al., 1997
).
|
Subcellular localization of PCBP-25 and PCBP-25ß by
blotting
Although PCBP-25 and PCBP-25ß were antigenically distinct,
their subcellular localizations, determined by immunoblotting, were similar.
About two-thirds of each protein remained in the low-speed pellet after cell
disruption and was not extractable with Triton X-100. Both PCBP-25
and
PCBP-25ß were present in the deciliation supernatant and in isolated
cilia (data not shown). The deciliation supernatant contains proteins that are
solubilized when cells are subjected to a Ca2+ shock in the
procedure for releasing cilia (Adoutte et
al., 1980
). This subcellular fraction represents only 1 to 2% of
the total protein of cells, but both PCBP-25
and PCBP-25ß were
found there in significant amounts.
Both PCBP-25 and PCBP-25ß in isolated cilia remained associated
with the axoneme after extraction of soluble proteins and membranes with
Triton X-100 (Fig. 4). About
two-thirds of the axoneme-associated PCBP-25
and PCBP-25ß was
released by high salt, which also solubilized dynein. When dynein was further
separated into 22S and 12S forms by centrifugation through a sucrose gradient
(see Travis and Nelson,
1988a
), PCBP-25ß was detectable in immunoblots of both
fractions, but PCBP-25
was found only in 12S dynein and was much less
prominent there than was PCBP-25ß
(Fig. 5). PCBP-25
in the
12S dynein fraction ran faster on SDS-PAGE than did PCBP-25
isolated on
the MonoQ, probably as a result of proteolysis during isolation. The
anti-centrin antibody also recognized a protein (Mr
90,000) in 22S dynein of unknown identity (data not shown). Baron and
Salisbury (Baron and Salisbury,
1988
) also noted crossreaction of their anit-centrin antibody with
a larger polypeptide.
|
|
The highest specific immunoreactivity of PCBP-25 and PCBP-25ß
was in the crude preparation of ICL, a cytoskeletal structure that lies
beneath the pellicle of Paramecium
(Garreau de Loubresse et al.,
1988
). For a given amount of protein, this fraction was stained at
least ten times more intensely by both antibodies than were extracts of
quick-killed cells (data not shown). Extraction of the crude ICL preparation
with 10 mM EGTA or 1M KCl released nearly all of the PCBP-25
and
PCBP-25ß detectable in immunoblots or 45Ca2+
overlay blots (Fig. 6B,C), but
buffer containing Ca2+, ATP, or urea produced little or no
solubilization (data not shown). This solubility pattern parallels that of the
Ca2+-binding proteins identified as major components of the ICL
(Garreau de Loubresse et al.,
1988
).
|
The anti-centrin antibody also reacted very specifically with a protein of
Mr 115,000 in partially purified ICL preparations, which,
unlike the PCBP-25 and PCBP-25ß, was not solubilized with
EGTA.
Subcellular localization of PCBP-25 and PCBP-25ß by
electron microscopy
To complement the studies of localization by subcellular fractionation, we
determined the localization of PCBP-25 and PCBP-25ß at the
ultrastructural level using colloidal gold-conjugated secondary antibodies.
The anti-centrin antibody labeled two distinct cellular structures the
ICL and the transition zone between the ciliary axoneme and basal body
(Fig. 7A) but did not
label kinetodesmal fibers or other known microtubule-based subciliary
structures. The labeling of the ICL was uniform. A small amount of label was
also observed in mitochondria. Monoclonal antibody 7A9, which is specific for
PCBP-25ß, selectively labeled only the ICL
(Fig. 7B), confirming the
results from subcellular fractionation. Negative controls performed with
normal mouse or rabbit serum gave only very low-level background labeling
(data not shown).
|
Phosphorylation of PCBP-25, PCBP-25ß and CaM by purified
CaPKs
What originally drew our attention to PCBP-25 and PCBP-25ß was
the observation that a protein of Mr 25,000 in the
phenyl-Sepharose eluate was phosphorylated in vitro by one of the CaPKs also
present in that fraction. Although centrin is known to be phosphorylated in
vivo (Salisbury et al., 1984
;
Salisbury, 1989
;
Martindale and Salisbury,
1990
; Lutz et al.,
2001
), the kinase responsible for that phosphorylation has not
been identified. We therefore tested four highly purified protein kinases from
Paramecium [CaPK-1 and CaPK-2
(Fig. 8), cAMP-dependent
protein kinase (PKA) and cGMP-dependent protein kinase (PKG) (data not shown)]
for their ability to phosphorylate PCBP-25
and PCBP-25ß as well as
CaM from Paramecium in vitro.
|
Purified PCBP-25 was phosphorylated by both CaPK-1 and CaPK-2,
whereas PCBP-25ß was not significantly phosphorylated by either. Almost
the same amount of 32P was incorporated into each band of the
doublet representing the purified fraction of PCBP-25
(lane 2), in
which the lower band was probably a proteolytic fragment of PCBP-25
(M.
Son, PhD thesis, University of Wisconsin-Madison, 1991). CaM was
phosphorylated in a Ca2+-dependent manner by CaPK-2 but not by
CaPK-1. The CaM we prepare from axenically cultured cells of
Paramecium always has several (usually three) electrophoretically
distinct forms in a variable ratio, all recognized by a monoclonal antibody
against CaM (B. C. Soltvedt, MS thesis, University of Wisconsin-Madison, 1985;
L. D. DeVito, MS thesis, University of Wisconsin-Madison, 1985). All three
forms bound 45Ca2+ in blot overlay assays and were
phosphorylated by CaPK-2 equally well (i.e., in proportion to their
amounts).
PCBP-25, PCBP-25ß, and CaM were also tested as substrates for
phosphorylation by purified PKA and PKG from Paramecium
(Carlson and Nelson, 1995
). PKG
phosphorylated PCBP-25
but not PCBP-25ß or CaM. None of the three
proteins was a substrate for PKA (data not shown).
Substrate specificity of CaPK-1 versus CaPK-2
CaPK-2 phosphorylated PCBP-25 and CaM, whereas CaPK-1 used only
PCBP-25
as a substrate, so we used these Ca2+-binding
proteins to explore further the substrate specificity of the kinases.
Paramecium CaM overexpressed in E. coli (POK-2) and cloned
centrin from Chlamydomonas were purified and also examined for
phosphorylation by both CaPKs. CaM (POK-2) was phosphorylated by only CaPK-2,
whereas centrin, which is immunologically related to PCBP-25
, was a
substrate for both enzymes (Fig.
9), consistent with the results in
Fig. 8. The extent of
phosphorylation of POK-2 or centrin was almost the same as that for CaM or
PCBP-25
, as determined by quantitative scans of autoradiograms (data
not shown).
|
In preliminary experiments with casein as the substrate, we established that CaPK-1 phosphorylated mainly Thr but also Ser, whereas CaPK-2 preferred Ser over Thr. The requirement for basic residues near the Ser or Thr residue to be phosphorylated is common in second-messenger-dependent serine and threonine protein kinases. Several synthetic peptides of 7 to 17 residues containing the Lys/Arg-X-X-Ser motif were tested as substrates for CaPK-1 and CaPK-2 (Table 1). Peptides 1, 2 and 3 were designed from the sequence of Tetrahymena micronuclear phosphohistone by David Allis and were supplied by him. The remaining peptides were commercially available synthetic substrates for PKC, PKG or PKA, respectively. None of the peptides was stoichiometrically phosphorylated by CaPK-1, although a small amount of 32P was incorporated into the peptide designed as a substrate for PKC (4). This peptide was also the best substrate among those tested for CaPK-2, which must therefore share some substrate specificity with PKC from animal cells. The extent of phosphorylation on the other peptides was very low; the order of phosphorylation activity was: (peptide 4) >> (2), (6), (3), (1) >> (5).
|
Phosphorylation of purified CaM by CaPK-2
The amount of 32P incorporated into CaM (POK-2, produced in
E. coli from the cloned Paramecium CaM gene), increased with
time and peaked at 60 minutes (Fig.
10A,C). The autophosphorylation of CaPK-2 also increased with time
(Fig. 10; band at
Mr 50,000). However, the maximum incorporation was less
than 0.01 mol P per mol CaM. To determine if the CaM concentration affected
the phosphorylation of CaM, increasing amounts of CaM were phosphorylated for
30 minutes (Fig. 10B). In this
experiment, CaM phosphorylation by CaPK-2 was also substoichiometric (1%).
|
Does phosphorylation of CaM affect the function of CaM?
In purified preparations of Paramecium CaM, there were several
electrophoretically separable forms of CaM, each of which could be
phosphorylated by CaPK-2. After phosphorylation by CaPK-2, both natural CaM
and POK-2 (CaM produced in E. coli) showed the
Ca2+-induced increase in mobility (of the labeled species) in
SDS-PAGE that is typical of CaMs (data not shown). The smallest form of CaM
showed the largest Ca2+-dependent mobility shift. This
Ca2+-dependent mobility shift suggested that phosphorylation of CaM
by CaPK-2 did not prevent interaction between CaM and Ca2+.
Several CaM antagonists were tested for inhibition of CaM phosphorylation
by CaPK-2. Neither melittin nor calmidazolium (CaM antagonists) blocked casein
phosphorylation (Gundersen and Nelson,
1987; Son et al.,
1993
) or CaPK-2 autophosphorylation. However, both melittin and
calmidazolium completely inhibited CaM phosphorylation at 5 µM
(Fig. 11). In the presence of
these antagonists, the extent of autophosphorylation was somewhat increased,
as though CaM and CaPK-2 competed as substrates for phosphorylation.
|
Which residue of CaM is phosphorylated by CaPK-2?
Serine is the residue in CaM that is phosphorylated by CaPK-2 (data not
shown), and this is consistent with our results with casein phosphorylation.
Paramecium CaM contains five serines among its 148 amino acids, at
positions 38, 70, 81, 101 and 147. In the mutant cam1, a Phe replaces
Ser101 in Ca2+-binding site III, and Ile136
(in Ca2+-binding site IV) is mutated to Thr in the mutant
cam2. The protein pjk-1 is a truncated CaM, consisting of only the
N-terminal half (residues 1-75), including two Ca2+-binding sites.
Wild-type CaM isolated from Paramecium (PCaM), POK-2, cam1 and cam2
were all equally good substrates for CaPK-2, but pjk-1 was poorly
phosphorylated, eliminating the possibility that Ser101,
Ser38 or Ser70 is the residue phosphorylated. Bovine
CaM, which has a Ser at position 81, was a poor substrate
(Fig. 12), leaving
Ser147 as the probable site of phosphorylation in PCaM.
|
![]() |
Discussion |
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PCBP-25 and PCBP-25ß are different proteins
PCBP-25 and PCBP-25ß are different proteins, not simply two
forms of the same gene product. Antiserum against centrin recognizes only
PCBP-25
, and the monoclonal antibody 7A9 recognizes only PCBP-25ß.
A second, independently selected hybridoma (10D1), produced after immunization
with a mixture of CaBPs from Paramecium, also recognized
PCBP-25ß and not PCBP-25
(M. Son, PhD thesis, University of
Wisconsin-Madison, 1991). It is unlikely that two independently selected
monoclonal antibodies would recognize the same epitope. Furthermore, a
polyclonal serum with broad-enough specificity to recognize centrins from many
species, from the alga Chlamydomonas to human
(Salisbury, 1989
), gave a
strong reaction with PCBP-25
but did not react with PCBP-25ß on
immunoblots.
What are the relationships among PCBP-25, PCBP-25ß and
other EF-hand proteins found in Paramecium and
Tetrahymena?
The proteins of the ICL have been previously described
(Garreau de Loubresse et al.,
1988; Garreau de Loubresse et
al., 1991
; Klotz et al.,
1997
). Klotz et al. (Klotz et
al., 1997
) found 10 small, acidic proteins in crude preparations
of ICL, including six that were stained on immunoblots with an antiserum that
recognizes centrin from many species. Our PCBP-25
is probably related,
or identical, to one of these six proteins. It has about the same
Mr, and, like them, it reacts with anti-centrin
antibodies, binds Ca2+ and is enriched in the ICL. It is most
probably a product of one of the
20 centrin genes of Paramecium
(Madeddu et al., 1996
;
Vayssie et al., 1997
).
Our PCBP-25ß is not one of the six centrins of the ICL, as it does not
cross-react with centrin. Neither is it one of the other four less-acidic
proteins of the ICL, because PCBP-25ß binds to Ca2+ and the
four ICL proteins do not (Klotz et al.,
1997). It is not yet clear whether PCBP-25ß is encoded in one
of the 20 genes in the centrin family described by Vayssie et al.
(Vayssie et al., 1997
).
In another ciliated protozoan, Tetrahymena, three distinct EF-hand
proteins have been described and localized
(Vaudaux, 1976;
Williams et al., 1995
), then
isolated and cloned. These are CaM (Suzuki
et al., 1981
) and two small, acidic proteins called
Tetrahymena Ca2+-binding proteins, TCBP-25
(Hanyu et al., 1995
) and
TCBP-23 (Hanyu et al., 1996
).
Both proteins have four putative EF hands and both bind to Ca2+.
Either could be a homolog of PCBP-25ß: none of the three crossreacts with
centrin but all bind Ca2+. TCBP-23 and TCBP-25 do not crossreact
with our monoclonal antibody against PCBP-25ß (M. Son, PhD thesis,
University of Wisconsin-Madison, 1991), but this is probably not surprising
given the evolutionary distance between the two protozoans. TCBP-25 is present
in cilia, and Watanabe et al. (Watanabe et
al., 1990
) have suggested that it may play a role in the
regulation of the ciliary beat by Ca2+.
Is PCBP-25ß a regulator of the ciliary beat?
One well-characterized Ca2+-dependent function in
Paramecium is the reorientation of the ciliary power stroke, which
causes backward swimming. The Ca2+ receptor protein for this
process remains unknown; it is conceivable that the Ca2+-binding
proteins we describe here play that role.
There is a complex interplay between cyclic nucleotides and Ca2+
in ciliary regulation. Detergent-permeabilized cell models swim forward when
reactivated with Mg2+-ATP but backward when micromolar
Ca2+ is also present (Naitoh
and Kaneko, 1972; Eckert and
Brehm, 1979
). Cyclic nucleotides (cAMP and cGMP) antagonize this
effect of Ca2+ on models, and the fast forward swimming induced by
cyclic nucleotide addition is antagonized by micromolar [Ca2+] (for
reviews, see Bonini et al.,
1991
; Pech, 1995
).
Both adenylyl cyclase and guanylyl cyclase of Paramecium are tightly
regulated by micromolar [Ca2+]
(Klumpp and Schultz, 1982
;
Klumpp et al., 1983a
;
Gustin and Nelson, 1987
).
Dynein ATPase is stimulated two-fold by micromolar [Ca2+], but CaM
does not co-sediment with either 22S or 12S dynein
(Travis and Nelson, 1988b
).
PCBP-25ß is associated with both 22S and 12S dynein, primarily with the
22S species. The outer arm dynein of another unicellular organism,
Chlamydomonas reinhardtii, has a subunit of Mr
18,000 that binds to Ca2+ (King
and Patel-King, 1995
). It is 42% identical in sequence to
Chlamydomonas CaM and 37% identical to centrin, and it may be the
Ca2+ sensor that mediates the transition from asymmetric to
symmetric waveform in the flagellar beat. Centrin is a component of a dynein
regulatory complex of Chlamydomonas flagella, which associates with
the inner arm dyneins (LeDizet and
Piperno, 1995
). It is possible that PCBP-25ß confers
Ca2+ sensitivity on ciliary dynein. It was not seen along the
axoneme by EM immunocytochemistry, but its enrichment in the deciliation
supernatant suggests that it may be loosely associated with some ciliary
structure.
Is the phosphorylation of PCBP-25 and CaM functionally
significant?
In a number of cases, phosphorylation of EF-hand proteins has been observed
in vivo, and this covalent alteration is presumed to be functionally
significant. The centrin of Chlamydomonas undergoes phosphorylation
in vivo in response to rapid changes in the extracellular milieu or in
intracellular [Ca2+] that lead to deflagellation
(Martindale and Salisbury,
1990). The kinase responsible for centrin phosphorylation in
Chlamydomonas has not been identified, but it is of interest that
Chlamydomonas has a CaM-domain kinase like the CaPKs of
Paramecium (Siderius et al.,
1997
). Garreau de Loubresse et al.
(Garreau de Loubresse et al.,
1991
) reported preliminary evidence for the phosphorylation of
polypeptides of Mr 23,000-24,000 of the ICL in vitro by
bovine PKA. Lutz et al. (Lutz et al.,
2001
) recently reported that the phosphorylation of centrin in
cultured vertebrate cells varied strikingly over the cell cycle, peaking at
the G2/M phase. Phosphorylation is on Ser170, the third residue in
from the C terminus, in a sequence typical of those preferred by many Thr/Ser
kinases, with two basic residues preceding the Ser residue: KKTSLY.
Experiments with permeant analogs of cAMP implicate PKA in this
phosphorylation.
PCBP-25 was a substrate for phosphorylation in vitro by CaPK-1,
CaPK-2 and PKG of Paramecium, and Paramecium CaM was
phosphorylated by CaPK-2. CaPK-1 and CaPK-2 from Paramecium also
phosphorylated centrin from Chlamydomonas. The interaction between
CaM and CaPK-2 was sensitive to CaM antagonists, even though the kinase
activity on other proteins is unaffected by these compounds
(Son et al., 1993
). The
phosphorylated C-terminal sequences in Paramecium CaM and two of
three human centrins are similar; of the 10 residues at the C terminus of CaM,
six are identical to the corresponding residues in human centrin, and two
others represent conservative substitutions
(Table 2). The C-terminal
sequences of the three reported centrins from Paramecium are
identical to each other, and of the 13 residues at the C terminus, seven are
identical to those in human centrin and two more are conservative
substitutions. The centrins from Paramecium have a Thr residue near
their C termini (not a Ser as in the other proteins). If this is the residue
phosphorylated in Paramecium centrin, it matches the position of
Ser170 in two of the human centrins. The third human centrin also
lacks a Ser residue near its C terminus
(Table 2) and is presumably not
subject to the same regulation by phosphorylation that occurs with the other
two human centrins.
|
Why is the phosphorylation in vitro of CaM by CaPK-2 substoichiometric?
Paramecium CaM might be already phosphorylated in vivo and therefore
not capable of accepting phosphoryl groups in vitro. This seems unlikely, as
even the Paramecium CaM overexpressed in E. coli could not
be phosphorylated stoichiometrically in vitro. Another possible explanation
for the low stoichiometry is heterogeneity at the C terminus of CaM, produced
by carboxypeptidase action in extracts during CaM purification. Although there
is clearly only one copy of the CaM gene in Paramecium
(Kink et al., 1990), we have
always observed several electrophoretic forms of CaM in our highly purified
preparations (B. C. Soltvedt, MS thesis, University of Wisconsis-Madison,
1985). If the penultimate residue (Ser147) is the one
phosphorylated, and if most molecules lack several residues at their C
termini, low stoichiometry would be expected.
The location of the phosphorylated Ser147 residue in CaM is
similar to that of the Ser170 residue phosphorylated in vertebrate
centrin (Lutz et al., 2001)
just at the C-terminal boundary of the fourth EF hand. In both cases,
the negative charges on the phosphorylated Ser residue would be expected to
force the C-terminal tail away from the negatively charged residues of the EF
hand, or to attract a calcium ion, and might thereby alter Ca2+
binding to that domain. The Mr 18,000 dynein light chain
described by King and Patel-King (King and
Patel-King, 1995
) is a member of the EF-hand superfamily and also
has a C-terminal Ser (Table 2),
but the sequence around this Ser lacks the paired basic residues that often
demarcate a phosphorylation site.
The dramatic reorganization of the cytoskeleton of Paramecium
during cell division and after conjugation
(Iftode et al., 1989) may
involve Ca2+-dependent phosphorylation as an integrating signal to
disassemble, then reassemble the cytoskeleton, including the ICL
(Keryer et al., 1987
;
Sperling et al., 1991
;
Klotz et al., 1997
;
Prajer et al., 1997
). Garreau
de Loubresse et al. (Garreau de Loubresse
et al., 1991
) reported preliminary results showing that the
general protein kinase inhibitor 6-dimethylaminopurine selectively inhibited
disassembly of the ICL. The same kinase inhibitor interfered with the
rearrangement of cytoplasmic microtubule organizing centers and cytokinesis in
Paramecium (Kaczanowska et al.,
1996
). The protein kinase implicated in these events has not been
identified, but there are only three known Ca2+-dependent protein
kinases in Paramecium: the two CaPKs described here and a PKC-like
activity (K. Kim, PhD thesis, University of Wisconsin-Madison, 1994). The
ability of CaPK-2 to phosphorylate centrin, a major component of the ICL, is
consistent with a role for the enzyme in triggering cytoskeletal
rearrangements. The Ca2+ sensitivity of CaPK-2 is also appropriate
for a cytosolic enzyme activated by Ca2+; the enzyme is
half-saturated at 0.2 µM Ca2+
(Son et al., 1993
), and the
cytosolic [Ca2+] is believed to vary between about 0.1 µM to 1
µM during Ca2+-mediated signaling
(Plattner and Klauke, 2001
).
Transformation with appropriate constructs produces gene silencing in
Paramecium (Ruiz et al.,
1998
), and we have cloned the CaPK genes from Paramecium
(Kim et al., 1998
), so it may
be possible to obtain cells without CaPKs and to test the effects of CaPK
action on ciliary reversal and cytoskeletal rearrangement.
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Acknowledgments |
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Footnotes |
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References |
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