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INTRODUCTION |
The centrosome is the major microtubule organizing center of the
cell, and as such it determines the number and polarity of cytoplasmic
microtubules, as well as the general form of the interphase microtubule
array (1). Once in each cell cycle, the centrosome is duplicated to
give rise to two centrosomes (spindle poles) that organize the
microtubule array of the mitotic spindle. Centrosomes consist of a
closely associated pair of centrioles and a pericentriolar matrix that surrounds and connects the individual centrioles to one another and to microtubules. Pericentriolar matrix has the properties of a gel as evidenced by the exclusion of small cytoplasmic particles such as ribosomes and membrane vesicles from the centrosome. At three times during the cell cycle centrioles move apart from one
another. The first time occurs immediately following mitosis when the
centriole pair transiently splits during or just after telophase. At
this time the older centriole remains near the cell center while the
younger centriole wanders extensively throughout the cytoplasm before
returning to reside near its older partner (2). The second separation
of the centriole pair occurs later in G1 phase as cells
pass the restriction point and the pair of centrioles become
disoriented and slightly separated from one another in the first
identifiable event of centrosome duplication (3). The third time
centriole pairs move apart from one another occurs as cells enter
prophase when newly duplicated centrosomes (each containing a pair of
centrioles) separate and migrate to opposite sides of the nucleus where
they function as mitotic spindle poles (4). Changes in centrosome
function at the time of the G2/M transition are regulated
by phosphorylation since centrosomes undergo an increase in protein
phosphorylation at this time (5). It is likely that specific
phosphorylation events also take place at the centrosome at other times
during the cell cycle (9). Numerous kinases known to regulate cell
cycle progression localize at centrosomes and mitotic spindle poles,
including Cdk2 and Cdk4/6, polo-like kinase 1 (PLK1), NIMA-related
kinase (Nek2), pEg2, and cAMP-dependent kinase
(PKA).1 Several of the
centrosome-specific substrates of these kinases have been identified
(for recent reviews, see Refs. 9 and 10). Protein phosphorylation has
been implicated in a variety of centrosome functions including
centrosome duplication, maturation and separation, microtubule
nucleation, and specification of cleavage furrow formation (9).
Cdk2/cyclin E activity coordinates centrosome duplication with the DNA
replication cycle (11).
Centrin is a 20-kDa protein of the EF-hand superfamily of
calcium-binding proteins that is a component of centrioles themselves and the surrounding pericentriolar matrix (16). Gene disruption experiments demonstrate that yeast centrin (Cdc31p) is essential for
cell viability (19). Additionally, two distinct centrin mutations have
been described that result in either failure of centrosome (spindle
pole body) duplication and separation resulting in monopolar spindles
and cell cycle arrest, or precocious and inappropriate centrosome
separation at the time of cell division resulting in cells with the
incorrect number of centrioles/basal bodies (20). Three separate
human centrin genes that encode and express centrin have been
identified (23). A conserved carboxyl-terminal region of centrins
from diverse species includes a consensus motif for protein
phosphorylation that is typical for serine/threonine kinases (26).
Centrin phosphorylation in lower eukaryotes has been shown to correlate
with extension of centrin-containing fibers associated with
centrioles/basal bodies (27, 28). More recently, aberrant centrin
phosphorylation has been demonstrated in human breast tumors that have
amplified centrosomes containing supernumerary centrioles and/or excess
pericentriolar material (29). The objective of this study was to
investigate the cell cycle-dependent phosphorylation of
centrin and to characterize the specific site of phosphorylation on the
protein. Using an antibody that is specific for phosphorylated centrin
and standard biochemical methods, we demonstrate that in cultured
vertebrate cells, centrin is phosphorylated near its carboxyl terminus
at serine residue 170 early in mitosis when the newly duplicated
centrosomes separate to give rise to the mitotic spindle poles. The
spindle pole localization of phosphocentrin remains high until
metaphase and then diminishes to basal levels by telophase. The timing
of centrin phosphorylation suggests that phosphorylation of centrin may
initiate the separation of duplicated centrosomes in preparation for
mitotic spindle formation. Experimental elevation of protein kinase A
(PKA) activity in interphase cells also results in the phosphorylation
of centrin at serine residue 170 and the concomitant movement of
centrioles away from one another in a manner similar to the transient
separation of the pair of centrioles that normally occurs preceding
centrosome duplication which begins at about the time of the
G1/S transition. These observations suggest that centrin
phosphorylation plays a role in mitotic spindle pole and centriole
separation at key stages of the cell cycle.
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EXPERIMENTAL PROCEDURES |
Reagents--
Rat brain protein kinase C was obtained from
Calbiochem, cyclin-dependent protein kinase 1 (Cdc2 kinase)
was obtained from Upstate Biotechnology Inc., and
[
-32P]ATP was from PerkinElmer Life Sciences.
SulfolinkTM coupling gel immobilization kit was obtained
from Pierce Chemical Co. Cellulose-coated (0.1 mm) TLC plates without
fluorescence indicator were from Curtin Matheson Scientific, Inc. The
catalytic subunit of PKA from bovine heart, phosphatidylserine,
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin, calyculin A, dibutyryl-cyclic monophosphate
(Bt2cAMP), citrate lyase, isobutyl-1-methyxanthine (IBMX),
L-phenylalanine hydroxylase, phosphorylase kinase, and fructose-G-phosphate kinase were obtained from Sigma.
Cell Culture--
HeLa and QT6 cells were grown in 100-mm plates
in modified Eagle's medium (Life Technologies, Inc.) supplemented with
10% fetal bovine serum (and 1% chicken serum (Life Technologies,
Inc.) for QT6 cultures), 1 mM sodium pyruvate, and 20 mM HEPES, pH 7.3, at 37 °C and 5% CO2. For
immunofluorescence studies, HeLa cells were plated onto 12-mm diameter
glass coverslips and used before cells reached confluence. Cells were
arrested in S phase of the cell cycle by 2.5 mM thymidine
treatment for 16 h or in G2/M phase by 1 µM nocodazole treatment for 8 h (30). Cell cycle
arrest was analyzed by flow cytometry. For in vivo
phosphorylation studies, cultured cells were grown as described above
except that during the final 4 h of incubation they were washed
and then incubated in phosphate-free Dulbecco's modified
Eagle's medium (Specialty Media) with dialyzed serum containing the
appropriate drug treatments and 0.2 mCi of
32PO4 (Amersham Pharmacia Biotech). Because
high phosphatase activity in HeLa extracts resulted in substantial loss
of centrin phosphorylation, QT6 fibroblasts, which have lower
phosphatase activity in cell lysates, were used in the experiments
reported here involving 32PO4 incorporation
in vivo.
Peptide Synthesis--
A synthetic peptide HCT-12 (EFLRIMKKTSLY)
corresponding to the carboxyl-terminal 12 amino acid residues of human
centrin 2 (CETN2, see Table I) was synthesized by Merrifield solid
phase on 0.5 mmol of 4-hydroxymethyphenoxy (Wang) copolystyrene resin (Advanced Chemtech, Louisville, KY) using methods described previously (31) (Table I). Each peptide was synthesized with
9-fluorenylmethoxycarbonyl-L-amino acid (Fmoc) derivatives
(Advanced Chemtech, Louisville, KY) containing the following side chain
protecting groups:
N
-boc-S-triphenylmethyl (trityl)
for cysteine; O-t-butyl for threonine and tyrosine;
N
-boc for lysine; O-t-butyl ester
for glutamic acid; and
N
-2,2,5,7,8-pentamethylchroman-6-sulfonyl for arginine.
The HCT peptides were assembled with the Fmoc-L-amino acids
on a Rainin PS3 automated peptide synthesizer using
PyBOP/N-methylmorpholine as the coupling reagent, and the hydroxyl
amino acid serine was incorporated into the peptide as its unprotected
side chain derivative Fmoc-Ser-OH. After synthesis, the peptide resins
were weighed, divided into two portions, and one portion of each
peptide resin was used for phosphorylation of the free serine hydroxyl
group by "global" phosphite-triester phosphorylation (32). Both
HCT-12 and HCT-P peptides were also synthesized with a free
amino-terminal cysteine residue for specific coupling to maleimide
activated carriers (keyhole limpet hemocyanin and bovine serum
albumin) and chromatography resins.
Following synthesis and phosphorylation, the peptides were deprotected
and cleaved from the resin by acidolysis with 93% trifluoroacetic acid
containing 1% ethanedithiol, 3% anisole, and 3% ethylmethyl sulfide
for 90 min at room temperature. Each deprotected peptide was filtered
from the resin and precipitated into cold methyl t-butyl
ether. All peptides were purified by reverse-phase high performance
liquid chromatography using a Vydac C8 column (2.2 cm × 25 cm,
Vydac, Hesperia, CA) in 0.1% trifluoroacetic acid/water and a gradient
of 10 to 80% acetonitrile in 0.1% trifluoroacetic acid/water, and
collected as single homogeneous peaks. The composition of each peptide
was verified by mass spectrometry on a Bio-Ion 20 mass analyzer
(PerkinElmer Life Sciences, Forest City, CA).
In Vitro Phosphorylation--
Recombinant human CETN2p expressed
in Escherichia coli was prepared and purified as described
earlier (33). Centrin and HCT-12 (Table I) were phosphorylated in
vitro by PKA at 30 °C in 50 mM Tris-HCl (pH 7.5), 1 mM CaCl2, 10 mM MgCl2,
and 0.4-0.8 mM [
-32P]ATP (4-6 Ci/mmol)
in the presence of 200 units of purified catalytic subunit of PKA for 1 to 2 h. Centrin was phosphorylated by protein kinase C (PKC) at
30 °C in 20 mM Tris-HCl (pH 7.5), 5 mM
MgCl2, 0.5 mM CaCl2, and 0.4-0.8
mM [
-32P]ATP (4-6 Ci/mmol) in the
presence of 10 µg of phosphatidylserine and 0.25 units of purified
PKC from rat brain for 1 to 2 h. Centrin was phosphorylated by
p34cdc2 protein kinase at 30 °C in 50 mM
Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM dithiothreitol, and 0.4-0.8 mmol of
[
-32P]ATP (4-6 Ci/mmol) in the presence of
cyclin-dependent protein kinase 1 Cdc2 from Sea Star
(P. ochraceus) for 1-2 h. Phosphorylation reactions were
started by addition of kinase and were terminated by freezing in liquid
N2 followed by lyophilization.
Phosphopeptide Mapping and Phosphoamino Acid
Analysis--
Lyophilized phosphorylation reaction mixtures were
resolved by 15% SDS-PAGE according to the method of Laemmli (34) or by SDS-Tricine PAGE for centrin peptides according to the method of
Schagger and von Jagow (35), and transferred to Immobulin-P membrane
(Millipore) according to the methods of Hulen and co-workers (36). The
region of the membrane containing phosphocentrin was located by
autoradiography, and subjected to tryptic phosphopeptide and
phosphoamino acid analysis as described by Boyle and co-workers (37).
Antiserum Production and Affinity Purification of
HCT-P
Antibodies--
The synthetic peptide HCT-P (Table I) was conjugated
to keyhole limpet hemocyanin and used to immunize female New Zealand White rabbits. Antisera from the third and fourth bleeds were used for
the experiments described in this work. An IgG fraction was prepared
using a protein-A-SuperoseTM column (Amersham Pharmacia
Biotech) and the methods supplied by the manufacturer. The IgG fraction
was precleared on a SulfolinkTM resin (Pierce Chemical Co.)
that had been coupled with HCT (Table I). Following a 12-h incubation
at room temperature, unbound Ig was washed from the resin,
concentrated, and applied to an affinity column consisting of
SulfolinkTM gel to which had been coupled the
phosphopeptide HCT-P. The bound IgG fraction was eluted (0.05 M glycine, pH 2.5, 0.1% Triton X-100, 0.15 M
NaCl, 0.01 M NaF, 0.01 M Na pyrophosphate, and
0.04% azide), neutralized using 1 M Tris buffer (pH 7.2),
concentrated, and stored in PBS containing 0.04% sodium azide. This
affinity purified IgG fraction is referred to as
HCT-P IgG.
Specificity of affinity purified
HCT-P IgG was characterized in four
separate analyses (Fig. 3). 1) Specificity against the peptides HCT-P
versus HCT was determined by a Western dot blot analysis.
The synthetic peptides HCT and HCT-P (200 ng/well) were blotted onto
polyvinylidene difluoride membrane using carbonate antigen binding
buffer (CB; 0.09 M NaHCO3, 0.01 M
Na2CO3, and 0.02% sodium azide, pH 9.4) and a
Bio-Dot apparatus (Bio-Rad). Membranes were blocked with 400 µl/well
of blocking buffer (0.01 M Tris-HCl, pH 7.4, 0.15 M NaCl, 2% bovine serum albumin, 10% normal goat serum,
0.05% Tween 20, and 0.04% sodium azide) and incubated with Ig
fractions (diluted according to the figure legend) in 10% fetal bovine
serum and 0.04% sodium azide (100 µl/well). Bound Ig was detected by
incubation with alkaline phosphatase-conjugated goat antibodies against
rabbit immunoglobulins (Cappel) using the alkaline phosphate substrate
5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. 2)
Specificity of
HCT-P IgG for phosphorylated versus
nonphosphorylated centrin was determined by autoradiography and Western
blotting of recombinant centrin following in vitro phosphorylation by the catalytic subunit of PKA. 3) Specificity of
HCT-P IgG for phosphorylated centrin versus other
substrates for PKA was determined by autoradiography and Western
blotting following in vitro phosphorylation by the catalytic
subunit of PKA of an equal molar mixture of recombinant centrin,
citrate lyase, L-phenylalanine hydroxylase, and
phosphorylase kinase. 4) Specificity of
HCT-P IgG for centrin
phosphorylated by PKA versus PKC or Cdc2 kinase was
determined by autoradiography and Western blotting following in
vitro phosphorylation of centrin by each of the respective kinases.
Immunoprecipitation and Western Analysis--
Cell lysates were
prepared for immunoprecipitation and Western blot analysis as described
previously (23). For the experiments described here, extracts were
immunoprecipitated using polyclonal serum 26/14-1 that recognizes
centrin regardless of its phosphorylation state or the preimmune serum
from the same rabbit. For Western blot analysis, centrin was resolved
by SDS-PAGE, transferred to Immobulin-P (polyvinylidene difluoride)
membrane (Millipore), and fixed with 0.2% glutaraldehyde followed by
standard procedures according to Towbin and co-workers (59) and
probed using either monoclonal anti-centrin 20H5 that recognizes
centrin regardless of phosphorylation state or
HCT-P IgG.
Immunofluorescence--
HeLa cells grown on glass coverslips
were fixed in cold methanol (
20 °C) for 10 min and processed for
indirect immunofluorescence microscopy as previously described (23)
using
-HCT-P IgG or polyclonal anti-centrin 26/14-1.
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RESULTS |
Analysis of Centrin Phosphorylation Status during the Cell
Cycle--
Immunoprecipitation and Western blot analysis of centrin
(20 kDa) from cultured cells is shown in Fig.
1A. Cell cycle analysis and
the corresponding phosphorylation status of centrin in untreated (control asynchronously cycling) cells or cells subject to cell cycle
arrest by thymidine (S phase) or nocodazole treatment (G2/M phase) are shown in Fig. 1, B and C,
respectively. Incorporation of 32PO4 into
centrin was highest in cultures arrested with 4C DNA (G2/M)
content by nocodazole treatment, while cultures arrested in S phase by
thymidine treatment show the lowest levels of incorporation and control
(cycling) cultures showed an intermediate level of 32PO4 incorporation. These results suggest that
centrin phosphorylation is maximum during mitosis.

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Fig. 1.
Centrin phosphorylation during the cell
cycle. A, centrin (20 kDa) from cultured QT6 cells is
specifically identified by Western blot using monoclonal anti-centrin
20H5 following immunoprecipitation with immune serum anti-centrin
26/14-1 (lane i) but not preimmune serum (lane p)
from the same rabbit. Molecular mass markers (in kDa): 200, 116, 97, 66, 45, 31, 21, 14 (top to bottom). B, flow cytometry of
cultured cells that were either untreated (control asynchronously
cycling), or arrested in the cell cycle in S-phase (thymidine) or
G2/M (nocodazole). Bars indicate 2C and 4C DNA
content. C, incorporation of 32PO4
into centrin in cultured cells treated as above for flow cytometry:
Western, blot of immunoprecipitated centrin;
32P, autoradiograph of the same lanes.
Cont, control cycling cells; thy, thymidine;
noc, nocodazole.
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Phosphorylation of Centrin by PKA in Vitro--
Centrins from a
variety of species share a highly conserved sequence at their carboxyl
termini (Table I) that includes a consensus amino acid motif (KKXS*X) for
phosphorylation by PKA (38). Bacterially expressed centrin and a
synthetic peptide, HCT-12, corresponding to the carboxyl-terminal 12 amino acids of CETN2 (Table I), are readily phosphorylated by PKA
in vitro (Fig. 2). Tryptic
digestion and two-dimensional analysis of the phosphorylated products
reveals a single negatively charged major and a neutral minor
32P-labeled tryptic peptide for both the recombinant
protein and the synthetic peptide (Fig. 2, B and
C, respectively). The two 32P-labeled peptide
fragments comigrate when tryptic digests of centrin and the synthetic
peptide are first mixed and subsequently run together by
two-dimensional peptide analysis (Fig. 2D). Phosphoamino acid analysis demonstrates that serine is the 32P-labeled
residue for both the major and minor peptide fragments (Fig. 2,
E and F). Comparison of migration of tryptic
peptides from centrin with the predicted pattern based on the computer program of Hunter (37) and the fact that there is only one potential serine phosphorylation site in the synthetic HCT peptide, allowed the
unequivocal identification of serine residue 170 in the
carboxyl-terminal sequence KKTSPY as the site for in
vitro phosphorylation of centrin by PKA. The major negatively
charged phosphopeptide (TSPLY) represents the carboxyl
terminus (residues 169-172) of centrin and the minor neutral peptide
fragment (KTSPLY) represents an alternative partial tryptic
product (residues 168-172) resulting from the less efficient trypsin
cleavage activity between adjacent lysine residues (37).

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Fig. 2.
Two-dimensional tryptic peptide maps and
phosphoamino acid determinations for phosphocentrin and synthetic HCT-P
peptide. Centrin and the HCT peptide were phosphorylated in
vitro using PKA and [ -32P]ATP. A,
phosphocentrin was resolved by SDS-PAGE and the HCT-P was resolved by
SDS-Tricine PAGE (not shown). Tryptic phosphopeptide maps for centrin
(B), HCT-P (C), and a mixture of the two
(D) are shown; arrows indicate the minor neutral
and major positively charged peptides, asterisk (*)
indicates the origin, and minus and plus ( and
+) indicate polarity for electrophoresis. E and
F, autoradiographs of chromatograms for phosphoamino acid
determinations of the major and minor peptide spots, respectively. The
positions of ninhydrin-stained phosphoamino acid standards
(phosphoserine, phosphothreonine, and
phosphotryrosine), the origin and free
32PO4 are indicated. Synthetic HCT-P and the
minor neutral and major negative tryptic products are indicated.
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Production and Characterization of Antibodies to Phosphorylated
Centrin--
Because the carboxyl-terminal phosphorylation site is
highly conserved among divergent species (Table I) we raised a
phosphocentrin-specific antibody to study in vivo
phosphorylation of centrin in cultured HeLa cells. We first prepared a
13-amino acid synthetic peptide, HCT-12, which contains the last 12 carboxyl-terminal amino acids of human centrin (residues 160-172, see
Table I) plus an amino-terminal cysteine residue for convenience in
coupling via its sulfhydryl group to either immunogenic haptens or to
affinity matrices. This peptide was prepared either in a
non-phosphorylated form (human centrin
carboxyl-terminus, HCT) or with the serine
residue synthetically phosphorylated (HCT-P). Rabbit antiserum raised
against the phosphorylated peptide HCT-P was found in preliminary
experiments to label mitotic spindle poles. In order to obtain
antibodies that were uniquely specific for phosphorylated centrin and
that showed no cross-reactivity against the nonphosphorylated centrin,
we first precleared the IgG fraction against HCT and then affinity
purified the preparation using resin-conjugated HCT-P as described
under "Experimental Procedures." Analysis using a Western dot blot
assay demonstrated exquisite specificity of the affinity purified
HCT-P IgG fraction for the phosphorylated HCT-P peptide with little
or no reactivity of this IgG fraction against the non-phosphorylated
HCT peptide (Fig. 3A). This
affinity purified IgG fraction was further tested for its specificity
toward the whole protein using recombinant centrin that was first
phosphorylated in vitro by PKA using [32P]ATP
(Fig. 3B). In this experiment, centrin was incubated with PKA under conditions in which only about half of the protein became phosphorylated. Subsequent analysis by SDS electrophoresis in a 15%
polyacrylamide gel resolved centrin into two closely migrating bands of
around 20 kDa that could be detected by Western blot analysis using a
monoclonal anti-centrin ascites (20H5) that recognizes centrin
regardless of its phosphorylation state (Fig. 3B).
Autoradiography (Fig. 3B, 32P-label)
demonstrated that the slower migrating centrin band represented the
phosphorylated protein while the more rapidly migrating band showed no
32P label. Analysis of a companion gel transfer prepared
with the identical sample, but Western blotted with the affinity
purified
HCT-P IgG, demonstrated a remarkable specificity for the
PKA-phosphorylated form of centrin and showed little if any reactivity
with the non-phosphorylated protein (Fig. 3B:
HCT-P).

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Fig. 3.
Specificity of HCT-P
IgG against phosphocentrin. A, dot blot analysis of the
affinity purified HCT-P IgG (dilution series) against the
non-phosphorylated (HCT) and phosphorylated (HCT-P) peptides. The
control in which no primary antibody was used in the
immunoprecipitation step is shown at the far right.
B, Western blot of a descending dilution series (left to
right) of centrin phosphorylated in vitro with PKA and
corresponding 32P autoradiograms of the same gels. Centrin
is resolved into two closely migrating bands in this 15%
polyacrylamide gel. The Western blot stained with monoclonal 20H5
(upper gel) shows two closely migrating centrin bands and
the corresponding autoradiograph demonstrates that only the more slowly
migrating band is phosphorylated. The Western blot stained with
HCT-P IgG (lower gel) shows that only the phosphocentrin
band is recognized by this antibody. C, Western blot
(w) using HCT-P IgG and corresponding autoradiograph
(a) of an equal molar mixture of recombinant centrin,
citrate lyase, L-phenylalanine hydroxylase, and
phosphorylase kinase phosphorylated in vitro using PKA and
[ -32P]ATP. Only centrin at 20 kDa reacts with HCT-P
IgG. D, autoradiograph (32P) and Western blots
(20H5) and ( HCT-P) of centrin phosphorylated in vitro by
PKA, PKC, and Cdc2 kinase. All three kinases phosphorylated centrin,
yet only the PKA product reacts with HCT-P IgG.
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A control experiment was performed in which several known substrates
for PKA were combined at equal molar ratios with centrin, followed by
in vitro incubation under complete reaction conditions with
PKA and [32P]ATP, and subsequent analysis by SDS-PAGE,
autoradiography, and Western blotting (Fig. 3C). In this
experiment the affinity purified
HCT-P IgG specifically and
exclusively recognized phosphocentrin and no other PKA-phosphorylated
proteins (Fig. 3C, Western (w)), even
when present at equivalent levels and having significant 32P incorporation (Fig. 3C,
autoradiograph (a)). A second control experiment
was performed in which centrin was phosphorylated by one of three
distinct protein kinases: PKA, PKC, or p34cdc2 kinase
(Cdc2) and subsequently analyzed by Western blotting with monoclonal
20H5 or
HCT-P IgG (Fig. 3D). Centrin is phosphorylated by
all three kinases in vitro (Fig. 3D,
32P) and analysis by phosphopeptide mapping
demonstrated that each kinase tested phosphorylated distinct sites on
centrin (not shown). Only PKA phosphorylation resulted in gel mobility
retardation of centrin and only the PKA-phosphorylated form of centrin
reacted with the affinity purified
HCT-P IgG (Fig. 3D,
HCT-P). Taken together, these results demonstrate that
affinity purified
HCT-P IgG specifically recognized centrin only
when it was phosphorylated on the extreme carboxyl-terminal amino acid
serine located at residue 170. In addition, this antibody recognized no
other PKA-phosphorylated proteins or centrin itself when it was
exclusively phosphorylated at sites other than serine residue 170.
Phosphorylated Centrin Localizes at Mitotic Spindle Poles in
HeLa--
HeLa cells grown on glass coverslips were processed for
indirect immunofluorescence using affinity purified
HCT-P IgG. When fields of subconfluent cells were observed by indirect
immunofluorescence microscopy a vast majority of the cells showed no
apparent labeling above background when stained with
HCT-P IgG
(Figs. 4C and 5, A
and B). Remarkably, however, dividing cells present
throughout the microscope field showed clear and intense staining of
their mitotic spindle poles (Figs. 4, C-F, and 5,
A and B). The specificity of immunostaining of
mitotic spindle poles by
HCT-P IgG was evaluated following
competition of the antibody preparation with the peptides HCT-P and
HCT. The affinity purified
HCT-P IgG was used at a constant and
known dilution (1.5 µg/ml IgG), preincubated with known amounts of
the phosphorylated peptide HCT-P or the nonphosphorylated peptide HCT,
and subsequently used for indirect immunofluorescence labeling of HeLa
(Fig. 4, A-F). Preincubation of
HCT-P IgG with HCT-P
peptide at a molar ratio of 1:1 (antibody:peptide, calculated based on
antigen-binding equivalents on the IgG) completely eliminated subsequent staining of prophase and metaphase spindle poles (Fig. 4A), while preincubation at a molar ratio of 1:0.1 resulted
in a reduction but not complete elimination of subsequent staining (Fig. 4B), and
HCT-P IgG that was not preincubated with
any peptide resulted in bright staining of spindle poles (Fig.
4C) as described above. However, preincubation of the
HCT-P IgG with the nonphosphorylated peptide HCT showed reduction in
staining of mitotic spindle poles at 1:0.1 (Fig. 4F), 1:1
(Fig. 4E), or even 1:1000 (Fig. 4D) molar ratios
of antibody:peptide (antigen-binding equivalents on the IgG:HCT). These
results demonstrate that spindle pole labeling by
HCT-P IgG is
exquisitely specific for the phosphorylated carboxyl-terminal serine
residue 170 of centrin.

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Fig. 4.
Indirect immunofluorescence micrographs of
HeLa cells stained with HCT-P IgG and
FITC-conjugated secondary antibody following preincubation of the
primary antibody with competing peptides. C, control without
competing peptide reveals HCT-P IgG staining of metaphase spindle
poles but not interphase centrosomes. B, preabsorption of
HCT-P IgG with 0.1:1-fold excess competing HCT-P peptide prior to
staining results in diminution of specific label, while preabsorption
with HCT-P at 1:1 stoichiometry of competing peptide (A)
completely abolishes label. Competition with 0.1:1-, 1:1-, and
1000-fold excess of the nonphosphorylated peptide HCT results in no
diminution of label. Note the bright staining of poles in the metaphase
(m) spindle and lack of staining of centrosomes in
interphase cells (i) and at the poles of the telophase
(t) stage cell in E.
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Careful analysis of a large number of mitotic cells (Fig.
5, A and B) shows
that prophase and metaphase spindle poles stained most intensely with
HCT-P antibodies. Staining of anaphase spindle poles was present,
albeit at diminished levels, and telophase spindle poles (see Fig.
4E) failed to stain at all. That centrin is present in
interphase centrosomes and at the spindle poles during all stages of
mitosis is demonstrated by staining with the polyclonal serum 26/14-1
(Fig. 5C). We interpret these indirect immunofluorescence
studies to demonstrate that HeLa centrin becomes phosphorylated at
serine residue 170 in a cell cycle-specific manner making the
carboxyl-terminal domain recognizable by
HCT-P IgG. Since staining
by
HCT-P IgG is most intense during prophase and metaphase, the
phosphorylation state of centrin is likely to be highest at these
times.

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Fig. 5.
Phosphocentrin is localized at the spindle
poles of HeLa cells. Indirect immunofluorescence micrographs of
HeLa cells stained with HCT-P IgG (A and B)
and 26/14-1 (C) and fluorescein isothiocyanate-conjugated
secondary antibody. A and B, HCT-P IgG only
stains cells in prophase and metaphase intensely with diminishing label
in anaphase cells and only low levels of diffuse label in interphase
cells. Of the 15 cells shown in A, only the two cells in
metaphase near the center of the image show spindle pole labeling.
C, 26/14-1 labels centrin at the centrosome and spindle
poles regardless of the stage of the cell cycle.
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Activation of PKA Stimulates in Vivo Phosphorylation
of Centrin and Centriole Separation in Interphase Cells--
The
observations presented above do not necessarily demonstrate that PKA is
the kinase responsible for phosphorylation of centrin in
vivo, nor do they eliminate the possibility that other kinases may
also phosphorylate distinct sites on centrin either at the time of
mitosis or at other times during the cell cycle. In order to address
the former question, cell cultures were incubated under control
conditions or under treatment conditions that elevate endogenous cAMP
levels to activate PKA. Cultures were allowed to incorporate
32PO4 for 4 h prior to treatment for 2 min
with the membrane permeable cAMP analog (100 µM
Bt2cAMP) that selectively activates PKA, with or without
concomitant treatment with the cAMP phosphodiesterase inhibitor IBMX (1 mM). Cells were lysed, centrin was immunoprecipitated using
26/14, resolved by SDS-PAGE, and subsequently analyzed by Western
blotting and autoradiography for centrin phosphorylation. Control cells
show low but detectable levels of centrin phosphorylation (Fig.
6), while cells treated with
Bt2cAMP alone or Bt2cAMP and IBMX show
substantially increased centrin phosphorylation. The centrin Western
blot appears as a closely spaced double band, with the more slowly
migrating band corresponding to the major phosphorylated product. A
third band also appears following treatment with both
Bt2cAMP and IBMX and represents a minor hyperphosphorylated product migrating higher in the gel. This experiment demonstrates that
treatment of living cells with cAMP analogs to activate PKA results in
an increase in the phosphorylation of centrin. Immunofluorescence staining using
HCT-P IgG of HeLa cells similarly treated to elevate PKA activity (in the presence of calyculin A, an inhibitor of protein
phosphatases 1 and 2A) demonstrates staining of mitotic cells and shows
discrete
HCT-P IgG staining of their spindle poles as seen before.
Remarkably however, in individual interphase cells treated to elevate
PKA activity, centrosomes acquire the phosphocentrin epitope necessary
for labeling with
HCT-P IgG (Fig. 7,
B and C). In addition, the
HCT-P IgG-labeled
interphase centrosomes appear as two spots separated by 2-4 µm,
suggesting interphase centrosomes have separated into two discrete
entities (Fig. 7, B and C). Typically, one of the
spots is brighter than the other. Control cells (not treated with cAMP
analogs) labeled with the polyclonal anticentrin serum 26/14-1 that
recognizes centrin regardless of phosphorylation state show a single
staining spot of a non-separated centrosome (Fig. 7D).
Centrosome separation into two discrete spots in interphase cells was
seen in 44% (22/50) of the Bt2cAMP-treated cells while
only 10% (5/50) of control cells showed separated centrosomes. These
observations demonstrate that elevation of PKA activity in living cells
results in centrin phosphorylation at the carboxyl-terminal serine
residue 170 and concomitant separation of the centrosome into two
discrete units, suggesting that PKA phosphorylation of centrin is
involved in centriole separation.

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Fig. 6.
Stimulation of centrin phosphorylation in
cultured cells following activation of PKA. Western blot using
monoclonal 20H5 antibody and corresponding 32P
autoradiograph of cell lysates from cultures that were untreated
(control) or treated for 2 min to stimulate PKA activity
(Bt2cAMP) and to inhibit phosphodiesterase
activity (Bt2cAMP/IBMX).
Significantly elevated levels of phosphocentrin are seen following
activation of PKA. Double arrows indicate the position of
the two closely migrating centrin bands, arrowhead indicates
minor hyperphosphorylated centrin band.
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Fig. 7.
Stimulation of PKA results in extraordinary
phosphorylation of centrosomes and in centriole separation in
interphase HeLa cells. A, indirect immunofluorescence
of HeLa cells stained with HCT-P IgG following control
(Me2SO) treatment. As before, metaphase spindle poles stain
while interphase cells show no specific label. B and
C, treatment with Bt2cAMP, IBMX, and calyculin A
for 30 min to elevate PKA activity results in HCT-P IgG staining of
interphase centrosomes as well as mitotic spindle poles. Interphase
centrosomes of treated cells show two stained spots (paired
arrows) demonstrating that activation of PKA results in separation
of centrioles from one another. D and E, control
cells stained with 26/14-1 show typical staining of interphase
centrosomes. Bar in A for A, B, and
D; and in E for C and E, 20 µm.
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DISCUSSION |
The primary objective of this investigation was to study the
in vivo phosphorylation of the centrosomal protein centrin
during the cell cycle. Observations presented here demonstrate that
centrin serves as a substrate for in vitro phosphorylation
by PKA at its carboxyl-terminal serine residue 170, and that a
phosphocentrin specific antibody (
HCT-P IgG) recognized centrin only
when it was phosphorylated at this site. Analysis of cultured cells for 32PO4 incorporation into centrin demonstrated a
significant increase in the level of centrin phosphorylation in
G2/M phase. Indirect immunofluorescence staining of HeLa
cells using the phosphocentrin-specific antibody resulted in intense
labeling of mitotic spindle poles during prophase and metaphase of the
cell division cycle, while the staining was diminished in anaphase and
was absent in telophase and interphase centrosomes. These observations
suggest that centrin phosphorylation is largely restricted to the
period of the cell cycle at G2/M when centrosome separation
and mitotic spindle assembly occur. Furthermore, cultured cells showed
a dramatic and rapid increase in centrin phosphorylation following
experimental treatment (Bt2cAMP and IBMX) that elevates PKA
activity, suggesting that this kinase can phosphorylate centrin
in vivo. Artificial elevation of in vivo cAMP
levels resulted in phosphocentrin-specific antibody staining of
centrosomes in interphase HeLa cells in addition to the mitotic spindle
pole label seen without drug treatment. Surprisingly, elevated cAMP
also resulted in precocious separation of centrosomes into two distinct
spots that we interpret to represent the displacement of individual
centrioles a short distance from one another.
Taken together these results indicate human centrin is phosphorylated
near its the carboxyl terminus at serine residue 170 early in mitosis
by PKA or another protein kinase with a substrate specificity similar
to PKA. Observations which favor the in vivo phosphorylation
of centrin by PKA include the following: 1) a conserved PKA consensus
phosphorylation motif in the carboxyl terminus of centrin; 2) the
in vitro phosphorylation of centrin by PKA at this site; and
3) the stimulation of centrin phosphorylation in vivo by
cell permeable cAMP analogs and treatments that reduce phosphodiesterase and phosphatase activity (IBMX and calyculin A,
respectively). Moreover, PKA is localized at the centrosome in
vertebrate cells, including HeLa (39, 40) through interaction of its
regulatory subunit (RII) and the protein kinase A anchoring coiled-coil
domains of AKAP450 and the centrosomal structural protein pericentrin
(41). Tethering of PKA to protein kinase A anchoring proteins is
thought to target the enzyme to the proximity of relevant substrates,
thereby conveying spatial specificity to cAMP/PKA signaling.
Numerous studies demonstrate that cellular levels of cAMP in HeLa and
other cultured cells are maximal in G1 of the cell cycle and minimal in G2/M (44). High levels of cAMP seen
during G1 phase correspond to the time of transient
centriole separation following mitosis. However, the period when
centrin phosphorylation levels appear highest (G2/M)
corresponds to the time when cellular cAMP levels are at their lowest.
Furthermore, cAMP has been shown to delay G2 progression,
inhibit cell proliferation, and negatively regulate mitotic Cdc2 kinase
activity (47). These observations confound a possible role for
PKA-mediated phosphorylation of centrin at the onset of mitosis. It is
possible that PKA acting at the centrosome is regulated through local
changes in the cAMP pool that are not reflected in the overall cellular
levels of cAMP. This could be accomplished through the activation of
adenylate cyclase at times when the cAMP pool is low as is seen at
G2/M (44), and/or through competition between PKA and
protein phosphatases (50, 51) that are tethered at the centrosome near
their substrates. Alternatively, a kinase(s) other than or in addition
to PKA may phosphorylate centrin at G2/M.
As indicated earlier, ~20 kinases have been localized at the
centrosome, some only transiently and others throughout the cell cycle
(9). As far as kinases are concerned the centrosome is a very crowded
place. The cyclin-dependent kinases Cdk2 and Cdk4/6 are of
particular interest because of their role in regulation of cell cycle
progression and because centrosome duplication has been demonstrated to
be dependent on Cdk2/cyclin E or cyclin A activity in
Xenopus cell-free extracts and somatic cells, respectively (3, 12, 15, 52). Our studies show that centrin can indeed be
phosphorylated by Cdk in vitro, however, at a site that is
distinct from serine 170 recognized by the phosphocentrin-specific
antibody described in this study.
The functional consequences of centrin phosphorylation are not known.
Genetic studies in lower eukaryotes suggest centrin plays an essential
role in centrosome duplication and/or separation (20). Yeast
centrin, Cdc31p, is a component of the spindle pole body half-bridge
that extends between the nascent and the pre-existing spindle pole body
during its duplication, and might play a role in the templated process
involved in new spindle pole body formation (53). In higher eukaryotes
centrin is a component of centrioles themselves and of fibrous material
connecting centrioles/basal bodies to one another (28, 54, 55).
Similarly disposed fibers in vertebrate epithelial cells lengthen and
shorten during the centriole duplication cycle (56). Studies in budding
yeast have implicated centrin (Cdc31p) in activation of a protein
kinase (Kic1p) that is required for cell integrity and bud morphology, in addition to its role in spindle pole body duplication (57). A
unifying functional theme has recently emerged (53) wherein the
centrins may act as small regulatory molecules that modulate the
activity of target proteins in centrosome-associated contractile fiber
systems, in a manner similar to that of another member of the
calmodulin superfamily, troponin C, which regulates the
Ca2+-dependent contraction of skeletal muscle.
In this regard, calcium binding by centrin stimulates
centriole-associated fiber contraction while centrin phosphorylation
and Ca2+ release are associated with fiber extension (27, 28, 58). Thus, the observations reported in the present work suggest
that centrin phosphorylation may play a role in solation or extension
of the gel-like pericentriolar matrix at during G1 phase
when centriole pairs undergo disorientation initiating centrosome
duplication and at G2/M when newly duplicated centrosomes
separate from one another to form the bipolar mitotic spindle.