From the a University of Edinburgh, Division of Biomedical and Clinical Laboratory Sciences, Hugh Robson Building, George Square, Edinburgh EH8 9XD, the c Division of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, the d Royal Free and University College Medical School, Royal Free Campus, London NW3 2PF, the e Department of Pharmacology and the g Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom, the i University of Alabama at Birmingham, Neurobiology Research Center and the Department of Cell Biology, Birmingham, Alabama 35294-0021, the j Polypeptide Laboratory, Department of Experimental Medecine, McGill University, Montreal, Quebec H3A 2B2, Canada, and the k Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122
Received for publication, November 2, 2000, and in revised form, February 20, 2001
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ABSTRACT |
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Mammalian casein kinases I (CKI) belong to a
family of serine/threonine protein kinases involved in diverse cellular
processes including cell cycle progression, membrane trafficking,
circadian rhythms, and Wnt signaling. Here we show that CKI The casein kinase I
(CKI)1 family of
serine/threonine kinases is ubiquitously expressed in a range of
eukaryotes including yeast and humans as well as in plants (reviewed in
Ref. 1). Seven isoforms from distinct genes are expressed in mammals
(CKI Yeast CKI isoforms are involved in DNA repair (2-4). Recently, many
reports (5-12) indicate that they also play a role in cytokinesis and
in vesicle trafficking especially in endocytosis. The functions of the
mammalian isoforms are less well understood, but based on high homology
with their yeast counterparts, they may have similar biological
functions. CKI Several substrates, including nuclear and cytosolic proteins and
membrane receptors, have been reported to be phosphorylated at least
in vitro by a CKI activity (reviewed in Ref. 1). CKI isoforms are thought to be constitutively active and second
messenger-independent. However, it has been shown that CKI Although the yeast CKI isoforms have been well
characterized, the functions of the mammalian CKI isoforms are much
less known. Therefore, the identification of mammalian CKI substrates
and CKI-binding proteins should help to clarify their cellular
function(s). CKI In the present study, we have shown that CKI cDNA Cloning--
The cDNA corresponding to rabbit
muscle CKI
For the experiment performed in Fig. 7B, pET3c CKI Identification of Centaurin- Recombinant Protein Purification--
Escherichia
coli carrying GST-centaurin- In Vitro binding between Purified CKI In Vitro Kinase Assays--
One µg of purified GST,
GST-centaurin- In Vitro Transcription and Translation--
CKI Cell Culture and Transfection--
COS-7 cells were obtained
from the European Collection of Cell Cultures. They were maintained in
Dulbecco's modified Eagle's medium (Sigma) supplemented with 10%
fetal calf serum (Life Technologies, Inc.) and 1%
penicillin/streptomycin (Life Technologies, Inc.) at 37 °C in a
humidified chamber with 5% CO2. Cells were transfected using Fugene (Roche Molecular Biochemicals) for 24-36 h in 60-mm diameter Petri dishes with HA-tagged CKI Co-immunoprecipitation--
Cells were lysed with 1 ml of lysis
buffer (25 mM Tris, pH 7.5, 137 mM NaCl, 1%
Nonidet P-40, 10% glycerol, 1 mM DTT) containing a mixture
of protease inhibitors (Roche Molecular Biochemicals), 1 mM
sodium fluoride, 1 mM sodium orthovanadate, and 10 mM sodium pyrophosphate. Lysates were pre-cleared with
Pansorbin cells (Roche Molecular Biochemicals) and centrifuged for 20 min at 15,000 × g at 4 °C. Mouse anti-FLAG M2
antibodies (Sigma) were added to the lysates for 2 h. Protein-A/G
coupled to Sepharose (Amersham Pharmacia Biotech) was then added for an
additional 1 h of incubation. The beads were washed 4 times with 1 ml of lysis buffer, and the proteins associated with the beads were
resolved on 10% SDS-PAGE. Proteins were transferred onto
nitrocellulose (Bio-Rad), and the presence of HA-CKI Affinity Chromatography with the CKI Peptide--
Two rat brains
were homogenized in 20 ml of lysis buffer (50 mM Tris, pH
7.5, 100 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 2 mM DTT) containing a mixture of
protease inhibitors (Roche Molecular Biochemicals). Nonidet P-40 was
then added to a final concentration of 0.1%, and the mixture was
incubated at 4 °C for 2 h with constant agitation and
subsequently clarified by centrifugation at 15,000 × g
for 30 min, followed by ultracentrifugation at 100,000 × g for 1 h. The resulting high speed supernatant was
loaded onto a 1-ml Sulfo-Link (Pierce) column to which 1 mg of a
peptide corresponding to residues 214-233
(C-214FNRTSLPWQGLKAATKKQKY233) of CKI A Protein of 45-kDa Co-purifies with CKI Identification of the 45-kDa Protein as Centaurin- CKI
In order to confirm this interaction,
[35S]methionine-labeled CKI
Therefore, two distinct experiments showed that CKI
In order to verify the specificity of this interaction,
[35S]methionine-labeled CKI CKI Centaurins Are Not Phosphorylated by CKI Centaurin- Centaurin-
In conclusion, our results mapped biochemically the
centaurin- Centaurin- In this report we have identified centaurin- CKI isoforms are characterized by a conserved core kinase domain and by
variable amino- and carboxyl-terminal tails. We report here that
centaurin- Centaurin- Centaurin- Members of the ARF-GAP family contain several domains for
protein-protein interactions, and they have been shown to associate with a number of different proteins. This suggests that ARF-GAP proteins may act as scaffold proteins in addition to their function as
GAP proteins. Whether other ARF-GAP proteins interact with CKI is not
known. The ARF-GAP proteins Git1 and Git2 have been reported to
regulate the internalization of some G-protein-coupled receptors
(56-58). CKI Gcs1, the budding yeast homologue of centaurin- Our data suggest that CKI In conclusion, we have shown an interaction between CKI
co-purifies with centaurin-
1 in brain and that
they interact in vitro and form a complex in cells. In
addition, we show that the association is direct and occurs through the
kinase domain of CKI within a loop comprising residues 217-233. These
residues are well conserved in all members of the CKI family, and we
show that centaurin-
1 associates in vitro
with all mammalian CKI isoforms. To date, CKI
represents the first
protein partner identified for centaurin-
1. However, our
data suggest that centaurin-
1 is not a substrate for
CKI
and has no effect on CKI
activity. Centaurin-
1
has been identified as a phosphatidylinositol
3,4,5-trisphosphate-binding protein. Centaurin-
1
contains a cysteine-rich domain that is shared by members of a newly
identified family of ADP-ribosylation factor guanosine
trisphosphatase-activating proteins. These proteins are involved in
membrane trafficking and actin cytoskeleton rearrangement, thus
supporting a role for CKI
in these biological events.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
,
1,
2,
3,
, and
), four in
Saccharomyces cerevisiae (Hrr25, Yck1, Yck2, and Yck3), and
five in Schizosaccharomyces pombe (Cki1, Cki2, Cki3, Hhp1,
and Hhp2). The CKI family is characterized by a conserved core kinase
domain and variable amino- and carboxyl-terminal tails.
and CKI
play a role in the regulation of p53 (13,
14). CKI
has also been implicated in circadian rhythms in
Drosophila (15, 16) and in development by transducing the
Wnt pathway (17, 18). CKI
might play a role in cytokinesis and/or in
membrane trafficking (19). CKI
has been shown to play a role in cell
cycle progression (20) and in membrane trafficking (21, 22). Recently,
CKIs have been shown to be implicated in regulating the
nucleocytoplasmic localization of some substrates (23, 24).
and
CKI
are regulated by autophosphorylation (25-28). CKI
is also
autophosphorylated, but whether this has an effect on its activity is
not well defined. CKI
is negatively regulated by
PtdIns(4,5)P2 (21). Moreover, CKI isoforms have been
reported to phosphorylate some of their substrates only if they were
previously phosphorylated by another kinase two or three residues
carboxyl-terminal to the CKI phosphorylation site. In this way, the
effect of CKI is dependent on other kinases. CKI
is present in cells
in different spliced forms (1, 29) exhibiting different substrate
specificities and differences in their protein-protein interactions.
interacts with NF-AT4 (23), the paired helical
filaments (30), G-protein-coupled receptors (31), and the AP-3 complex (22). CKI
also forms a complex with certain splicing factors but
these interactions may be indirect (32).
interacts with
centaurin-
1. Centaurin-
1 is a
PtdIns(3,4,5)P3-binding protein containing two PH domains
(33-35) and a zinc finger motif similar to the one found in a newly
identified family of ADP-ribosylation factor (ARF) guanosine
trisphosphatase-activating proteins (GAP) (reviewed in Refs. 36-39).
The yeast protein that shows the highest homology to
centaurin-
1, Gcs1, also contains a zinc finger motif that confers its ARF-GAP activity (40). Members of this family are
involved in membrane trafficking and in actin cytoskeleton rearrangement. Our results suggest that CKI
plays a role in membrane trafficking and/or actin cytoskeleton rearrangement, thus confirming previous reports (21, 22).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was originally cloned in the pET 3 vector (Novagen) (41).
CKI
cDNA from this clone was amplified by the polymerase chain
reaction (PCR) using two oligonucleotides
(5'-gggcatatggcgagcagcag-cggctccaagg-3') and
(5'-gggggatccttagaaacctgtgggggtttgggc-3') in order to
create a 5' NdeI site and a 3' BamHI site (both
are underlined in sequences). Amplified cDNA was inserted in a
pET-16b vector (Novagen) at NdeI/BamHI restriction sites to express CKI
as a histidine-tagged protein. The
CKI
cDNA was inserted in a pcDNA3 vector (Invitrogen) from the original clone after being amplified by PCR using two
oligonucleotides (5'-gggggatccgccaccatggcctacccctacgacgtgcccgactacgcccccgcgagcagcagcggctcc-3') and (5'-gcggcggccgcttagaaacctgtgggggtttgggc-3') in order to
create a 5' BamHI site and a 3' NotI site (both
are underlined in sequences). The Kozak sequence is shown in italics
and the HA- tagged sequence in bold. CKI
mutants were generated by
PCR and cloned in the pSP72 vector (Promega) at the EcoRI
and BamHI sites downstream of the T7 promoter. The
oligonucleotides (the EcoRI and BamHI sites are
underlined) used are
5'-ggggaattcgccaccatgtacaaactggtacggaagatcgg-3';
5'-ggggaattcgccaccatgtacagagacaacagaacaaggc-3'; 5'-ggggaattcgccaccatgagcatcaatgcacatcttgg-3';
5'-ggggaattcgccaccatgaccagcctgccgtggcaagg3'; 5'-ggggaattcgccaccatgtacgagaagattagcgaaaagaagatgtcc-3';
5'-gggggatccttaggtcctgaaaaggatgcgg-3'; 5'-gggggatccttagaaacctgtgggggtttgggc-3'. The
construction of GST-centaurin-
1 has been described
previously (42). Centaurin-
1 cDNA from that vector
was recovered after digestion with EcoRI and XbaI
and subcloned in a FLAG-cmv2 vector. HA-tagged CKI
(D136N) in
pcDNA3 is a gift from F. McKeon (23). FLAG-centaurin-
is from
Trevor R. Jackson.
(41),
pET8c CKI
(25), pET8c CKI
317 (25), pET8c CKI
1 (19), pSV2Zeo CKI
2 (from Louise Larose), pET8c CKI
3 (19), and pSP72 CKI
were used. pSP72 CKI
construct was subcloned using human CKI
plasmid (pV405) provided by Dr. David Virshup (27). CKI
cDNA was amplified by PCR using two oligonucleotides
(5'-ggaagatctatggagctacgtgtggggaacaag-3') and
(5'-ggaaaagctttcacttcccgagatggtcaaatgg-3') in order to create a 5'
BglII site and a 3' HindIII site and inserted
into pSP72 vector.
1 by Mass
Spectrometry--
After the final chromatography step during the
purification of CKI
as a 14-3-3 kinase from brain (Sephacryl S-100
gel exclusion), fractions containing this kinase activity were pooled
and loaded on 12.5% SDS-PAGE (43). Gels were stained for 5-10 min and
then destained for the minimum time. The 45-kDa band was excised and subjected to trypsin (L-1-tosylamido-2-phenylethyl
chloromethyl ketone-treated, Worthington) digestion. The extracts were
then dried in a Speedvac vacuum centrifuge and made up to the injection volume with water for on-line liquid chromatography mass spectrometry. Electrospray mass spectrometry of in-gel digested protein was carried
out as described (43), and the peptide map data were used in the
PeptideSearch program.
1, GST-centaurin-
,
or histidine-tagged CKI
plasmid were grown overnight at 37 °C in
Liquid Broth medium containing 50 µg/ml ampicillin and were diluted
the following day (1/10) in the same medium. Culture was then continued
until the absorbance (600 nm) of the bacterial growth reached
0.6. Expression of the tagged proteins was induced with 0.5 mM isopropyl
-D-thiogalactopyranoside for
3-5 h at 25 °C. The fusion proteins were purified by affinity
chromatography on glutathione-Sepharose beads or nickel columns
(Amersham Pharmacia Biotech). Proteins were further purified on a
MonoS column using the AKTA purifier (Amersham Pharmacia Biotech) and
stored in 20 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 100 mM NaCl, and 50% glycerol at
20 °C. Recombinant 14-3-3
was purified as described previously (43).
and Members of the
Centaurin-
Family--
GST, GST-centaurin-
1, or
GST-centaurin-
(0.2 µM final concentration) were
incubated with histidine-tagged CKI
in binding buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 10% glycerol, 1 mM DTT, 0.1% Nonidet P-40) for 2 h at 4 °C.
Glutathione-Sepharose beads were added and incubated for a further
1 h. Bead precipitates were then washed 4 times with binding
buffer and once with kinase buffer (50 mM Hepes, pH 7.0, 10 mM MgCl2, 100 mM NaCl, 0.1%
Nonidet P-40, 1 mM DTT, and 100 µM cold ATP).
The washed beads were incubated with kinase buffer (without NaCl and
0.1% Nonidet P-40) containing 40 µM of a CKI-specific
phosphopeptide substrate (New England Biolabs) and
[
-32P]ATP (2 µCi/point) (Amersham Pharmacia Biotech)
in a final volume of 30 µl. Reactions were performed at room
temperature for 30 min. After centrifugation, 20 µl was spotted on
P81 paper squares (Whatman) and washed four times with 1% phosphoric
acid. Radioactivity retained on the papers was quantified by liquid
scintillation counting.
, GST-centaurin-
1, and GST-14-3-3
were subjected to an in vitro kinase assay using purified
histidine-tagged CKI
as described previously (43). Proteins were
analyzed on 10% SDS-PAGE. Gels were stained with Coomassie Blue,
dried, and autoradiographed. To study the effect of
centaurin-
1 on CKI
activity, different amounts of
centaurin-
1 (0.1, 1, or 10 µg) were preincubated with
histidine-tagged CKI
for 15 min at room temperature. The reaction
was initiated by the addition of ATP (50 µM final, 1 µCi/point) and 14-3-3
or GST-14-3-3
or phosvitin (0.3 µg/point) as CKI substrates. After 30 min at room temperature, the
reactions were stopped and analyzed on 10% SDS-PAGE. Gels were stained
with Coomassie Blue, dried, and autoradiographed.
and 14-3-3
subcloned in pcDNA3 were expressed in vitro using a
T7 TNT-coupled transcription/translation reticulocyte lysate assay
(Promega Corp., Madison, WI). The reactions (50 µl) were performed
following the manufacturer's instructions using [35S]methionine (Amersham Pharmacia Biotech) for 90 min
at 30 °C. The reactions were then diluted 4-fold with binding buffer
(containing 0.1 or 1% Nonidet P-40 as indicated in figure legends) and
split in two for incubation (as indicated in the figure legends) with 1 or 10 µg of GST, GST-centaurin-
, or GST-centaurin-
1
at 30 °C for 15 min. Glutathione-Sepharose beads and binding buffer (300 µl) were added to the reactions and incubated at room
temperature for an additional 1 h. The beads were washed 5 times
with 1 ml of binding buffer and electrophoresed on SDS-PAGE. After
staining/destaining, the gels were incubated with AmplifyTM
(Amersham Pharmacia Biotech), dried, and exposed to film.
(D136N) and/or FLAG-tagged centaurin-
/centaurin-
1 and/or empty vectors (4 µg
of total DNA).
was detected by
Western blotting with a rat anti-HA (Roche Molecular Biochemicals)
antibody and ECL detection (Amersham Pharmacia Biotech).
was coupled (according to the manufacturer's instructions). A cysteine
was added at the amino terminus to allow efficient coupling to
Sulfo-Link. Brain extract was also loaded onto a control column. The
columns were washed with 50 ml of lysis buffer containing 0.1% Nonidet
P-40 and with 50 ml of phosphate-buffered saline. Bound proteins were
eluted with 10 ml of 50 mM Tris, pH 7.5, 1 M
NaCl, 10% glycerol, 1 mM EDTA, and 2 mM DTT.
Eluted fractions and the last 5-ml washes were concentrated on
Centricon-10 (Amicon) and analyzed by immunoblotting. The presence of
14-3-3 proteins was detected using a rabbit polyclonal 14-3-3 Pan
antibody (KK 1106) from our laboratory. Centaurin-
1 was
detected using a rabbit polyclonal antibody (J49) that was raised
against an amino-terminal peptide of centaurin-
1
(33).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in Brain--
We have
previously reported that 14-3-3
was phosphorylated on a novel site
(44) and identified the kinase as CKI
after four conventional
chromatography steps (43). A total of seven proteins with molecular
masses ranging from 25 to 80 kDa were shown to co-purify with
CKI
(Fig. 1), and we postulated that they may form protein complex(es) in brain. A protein migrating at 45 kDa represented the most abundant co-purifying protein as judged by
Coomassie Blue staining (Fig. 1). The putative association between
CKI
and the 45-kDa protein would appear to be of high affinity
because the complex was observed after elution with 0.5 M
NaCl during the two cationic exchange chromatography steps and after
elution with 0.6-0.8 M NaCl from the Affi-Gel blue column (the chromatography steps are described in Ref. 43). Experiments were
performed to identify the 45-kDa protein and to elucidate whether it
associates with CKI
.
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Fig. 1.
Co-purification of a 45-kDa protein with
CKI . CKI
was purified from pig brain
after four chromatography steps (43). The fractions containing CKI
activity were electrophoresed on 10% SDS-PAGE (lane 2), and
the gel was stained with Coomassie Blue. CKI
was previously
identified by mass spectrometry (43). A total of seven other proteins
co-purified with CKI
(indicated by arrows), suggesting
that they may form protein complex(es). The major co-purifying band
migrates at 45 kDa. Molecular mass standards (lane 1) are
shown in kDa.
1,
a PtdIns(3,4,5)P3-binding Protein--
The
45-kDa protein band was subjected to in-gel trypsin digestion, and the
mass of each peptide was measured by electrospray mass spectrometry.
Analysis of the peptide mass map using the "PeptideSearch" program
identified the 45-kDa protein as a PtdIns(3,4,5)P3 binding
protein. We identified nine peptides that matched with centaurin-
1 and eight peptides that matched with
centaurin-
(Fig. 2A).
Centaurin-
1 shares high homology to centaurin-
.
Centaurin-
contains an extended carboxyl-terminal tail and only one
PH domain compared with centaurin-
1 (Fig.
2B). Therefore, peptide mass analysis differentiated these
two proteins and identified the 45-kDa protein unequivocally as
centaurin-
1 (42). Centaurin-
1 has been
given different names in the literature including p42IP4
(34) and PIP3BP (35). However, for clarity in the rest of this paper,
we refer to it as centaurin-
1.
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Fig. 2.
Identification of the 45-kDa protein as
centaurin- 1, a
PtdIns(3,4,5)P3-binding protein. A,
the band corresponding to the 45-kDa protein (Fig. 1) was excised and
subjected to trypsin digestion and electrospray mass spectrometry. Nine
peptides were recovered (shown in boxes). By using the
"PeptideSearch" program, they all matched with
centaurin-
1 and eight of them with centaurin-
. Both
aligned sequences are from rat (33, 45). Note that the first peptide is
not identical in sequence to the one that we found (from pig brain),
but for easier interpretation of the figure, we aligned the sequence of
centaurin-
and -
1 from the same species (rat). The
45-kDa protein was purified from pig brain, and the actual sequence of
the peptide is VPPFYYRPSASDCQLLR (the different amino acids
are underlined). This peptide is identical in the pig
centaurin-
1 (34). B, a schematic
representation of centaurin-
and centaurin-
1. Both
these proteins contain a zinc finger motif (Zinc) related to
the one found in ARF-GAP proteins. Centaurin-
differs from
centaurin-
1 by the presence of only one PH domain
(PH) rather than two and by a 43-amino acid extension at the
carboxyl-terminal end (the differences are shown by dashed
boxes). The mass spectrometric analysis was able to differentiate
between these two proteins. Indeed, one peptide (KFVLTER) is only
present in centaurin-
1 (in its first PH domain) and not
in centaurin-
.
Associates Physically with
Centaurin-
1--
We tested the potential interaction
between CKI
and centaurin-
1 using purified
recombinant proteins. We show that CKI
associates with
GST-centaurin-
1 and not with GST (Fig.
3A). In addition, CKI
also
binds directly to centaurin-
(Fig. 3A).
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Fig. 3.
CKI associates
physically with centaurin-
and
centaurin-
1. A,
GST, GST-centaurin-
(GST-
), and
GST-centaurin-
1
(GST-
1) were incubated for 2 h at 4 °C with purified histidine-tagged CKI
.
Glutathione-Sepharose beads were then added. Beads were washed and
subjected to an in vitro kinase assay using a CKI-specific
phosphopeptide substrate. The presence of 32P incorporated
into the peptide was quantified by liquid scintillation counting and
represents CKI
activity associated with the beads. B,
CKI
was expressed and 35S-labeled using a T7 TNT-coupled
transcription/translation reticulocyte cell-free system and incubated
with 1 µg of GST, GST-centaurin-
1
(GST-
1) or GST-centaurin-
(GST-
) for 15 min at 30 °C in the presence of 0.1%
Nonidet P-40. Glutathione-Sepharose beads were added and incubated at
room temperature for an additional 1 h. Beads were washed, and
samples were analyzed by 10% SDS-PAGE followed by autoradiography
(panel Pull down assay). An aliquot of the lysate from the
in vitro transcription/translation assay was loaded on the
gel (panel Input). C, CKI
and 14-3-3
were
synthesized as described above (panel Input). They were
incubated with 10 µg of GST or GST-centaurin-
1
(GST-
1) for 15 min at 30 °C in
the presence of 1% Nonidet P-40. Beads were washed, and samples were
analyzed by 10% SDS-PAGE followed by autoradiography (panel Pull
down assay).
was synthesized using a
cell-free coupled transcription/translation system and incubated with
GST, GST-centaurin-
, or GST-centaurin-
1 (Fig.
3B). The results show that CKI
associates with
centaurin-
and with centaurin-
1.
interacts
physically with both centaurins. The levels of CKI
that associated with centaurin-
1 remained the same in the presence of
0.1-1% Nonidet P-40 (data not shown).
and 14-3-3
were
incubated with either GST or GST-centaurin-
1 (Fig.
3C). By using higher stringency (1% detergent),
centaurin-
1 indeed associates with CKI
but not with
14-3-3
even though the latter is expressed at much higher levels
than CKI
(Fig. 3C, panel Input), thus showing
the specificity of the interaction between centaurin-
1
and CKI
. In conclusion, centaurin-
and centaurin-
1
associate specifically and directly with CKI
. As rat centaurin-
(except its unique carboxyl-terminal tail) shares 94% identity with
rat centaurin-
1 (Fig. 2, A and B),
one could imagine that they bind to CKI
via a common domain, thus
eliminating the importance of the first PH domain of
centaurin-
1 and the carboxyl-terminal domain of
centaurin-
.
Associates in Cells with Centaurin-
and
Centaurin-
1--
We tested whether CKI
associates in
cells with centaurin-
1. For that purpose, we transiently
transfected different cell lines in order to check the expression of
ectopically expressed CKI
. However, the expression of CKI
was not
detectable in cell lysates of PC12 and NIH3T3 cells, was low in HEK 293 cells, and only really detectable in COS-7 cells (data not shown).
Therefore, we have used COS-7 cells for the transient transfection
experiments as the other cell lines were not suitable for
co-immunoprecipitation experiments due to the low expression level of
CKI
. COS-7 cells were co-transfected with HA-tagged CKI
and with
FLAG-tagged centaurin-
or centaurin-
1. FLAG-tagged
proteins were immunoprecipitated, and CKI
in the immunoprecipitates
was revealed by Western blot using HA antibodies. This experiment shows
that CKI
is pulled down in centaurin-
(Fig.
4, 3rd lane) and
centaurin-
1 (Fig. 4, 4th lane)
immunoprecipitates and not in the controls (Fig. 4, 1st, 2nd
and 5th lanes). Therefore, the experiment demonstrates that
CKI
associates with both centaurins in COS-7 cells.
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Fig. 4.
CKI associates
in vivo with centaurin-
and
centaurin-
1. COS-7 cells were
co-transfected with plasmids expressing the proteins indicated at the
top of the figure or empty vectors. After 36 h, cells
were harvested in lysis buffer containing 1% Nonidet P-40. Centaurins
were immunoprecipitated using mouse monoclonal antibody
(mAb) M2 anti-FLAG antibodies (Ab), and the
presence of CKI
was detected by Western blotting with a rat anti-HA
monoclonal antibody (clone 3F10).
--
We tested whether
CKI
phosphorylates centaurin-
and
centaurin-
1 in vitro using recombinant
proteins. Fig. 5A shows that GST-centaurin-
and GST-centaurin-
1 were not
substrates for CKI
in vitro. As a positive control,
14-3-3
, a CKI substrate (43), was shown to be phosphorylated (Fig.
5A, lane 14-3-3
). In addition, recombinant
centaurin-
1 is not phosphorylated by CKI
indicating that the GST tag itself does not confer conformational restraint and
has an effect on the result (data not shown). However, CKI isoforms
have been shown to phosphorylate some substrates only if they have been
previously phosphorylated by another kinase two or three residues
carboxyl-terminal to the CKI site. Therefore, our in vitro
results do not completely eliminate the possibility that centaurins are
not substrates for CKI
. To investigate this possibility, FLAG-tagged
centaurin-
and centaurin-
1 immunoprecipitated from
COS-7 cells were subjected to an in vitro kinase assay using purified histidine-tagged CKI
. However, immunoprecipitated proteins were not phosphorylated by purified CKI
(data not shown). Therefore, we conclude from our experiments that centaurin-
and
centaurin-
1 either purified or immunoprecipitated from
COS-7 cells are not substrates for CKI
.
View larger version (43K):
[in a new window]
Fig. 5.
Centaurins are not phosphorylated by
CKI and have no effect on
CKI
kinase activity. A, 1 µg
of purified GST, GST-centaurin-
(GST-
), and
GST-centaurin-
1
(GST-
1) were assessed for in
vitro phosphorylation by purified histidine-tagged CKI
as
described under "Materials and Methods." Reactions were stopped and
electrophoresed on 10% SDS-PAGE. The gel was dried and
autoradiographed. 14-3-3
was phosphorylated by CKI
as a positive
control (lane 14-3-3). B, 0.1, 1, or 10 µg of
recombinant centaurin-
1 or buffer (b) was
preincubated for 15 min with CKI
. The reaction was initiated with
the addition ("+") of the CKI substrate 14-3-3
. No substrate
was added in ("
") as a control in case some degraded forms of
centaurin-
1 would be phosphorylated by CKI
.
C, 0, 0.1, 1, or 10 µg of recombinant
centaurin-
1 or the corresponding volume of buffer
(b) was incubated with CKI
as described above. The
reaction was initiated with the addition of GST-14-3-3
or
phosvitin.
1 Does Not Affect CKI
Activity--
As
centaurin-
1 does not represent a substrate for CKI
,
we have tested whether centaurin-
1 affects the kinase
activity of CKI
. We have shown that different amounts of recombinant
centaurin-
1 (0.1, 1, or 10 µg) have no effect on CKI
activity using 14-3-3
(Fig. 5B), GST-14-3-3
, or
phosvitin (Fig. 5C) as CKI substrates.
1 Interacts with Residues 217-233 of
CKI
--
Mammalian CKI
belongs to a family of seven isotypes
that show a high degree of homology in their kinase domains and have variable amino- and carboxyl-terminal tails. Therefore, it would be
interesting to identify the centaurin-
1-binding site in
CKI
in order to elucidate whether centaurin-
1
associates in a region specific to CKI
or one that is also present
in other CKI isotypes. To address this question, we constructed a set
of CKI
deletion mutants (Fig.
6A). These mutants were
synthesized and labeled with [35S]methionine in a
cell-free coupled transcription/translation system (Fig. 6B)
and incubated with GST or GST-centaurin-
1 (Fig. 6C). The different mutants were expressed in similar amounts
apart from mutants 217-325 and 233-325 that were less well
synthesized (Fig. 6B). The results show that the amino- and
carboxyl-terminal domains (mutants 17-325 and 17-287) of CKI
are
not required for the association. In addition,
centaurin-
1 binds with high efficiency to all mutants
apart from mutant 233-325. Although the levels of expression of
mutants 217-325 and 233-325 in cell lysates were the same, mutant
217-325 bound to centaurin-
1 whereas mutant 233-325
did not (Fig. 6, C and D), demonstrating that the
binding occurs between residues 217 and 233 within the kinase domain of CKI
. These residues belong to a loop between helices E and F of
CKI
that has been suggested to be the target region for
protein-protein interactions (46). In order to confirm that residues
217-233 represent the site of interaction with
centaurin-
1, a brain extract was loaded onto a column
containing this peptide or a control column. Centaurin-
1
was found to associate to the peptide column (Fig. 6E). This
association is highly specific as we did not detect the presence of
14-3-3 proteins that represent 1% of total brain proteins.
View larger version (50K):
[in a new window]
Fig. 6.
Centaurin- 1 associates with
the residues 217-233 of CKI
.
A, schematic representation of a series of deletion mutants
of CKI
(WT, wild type). Residues 17-287 constitute the
kinase domain. Residues 164-189 represent a loop containing a kinesin
homology domain (1). Residues 217-233 are found in a loop that has
been proposed to be responsible for the interaction of CKI
with
other proteins (46). The calculated molecular masses of wild type and
deletion mutants of CKI
are shown in kDa. B, wild type
and mutant CKI
were synthesized using an in vitro
transcription/translation assay, and an aliquot of the reaction (0.5%
input) was analyzed on 15% SDS-PAGE. C, the rest of the
reaction was incubated with 10 µg of GST or
GST-centaurin-
1 using the same protocol as described in
Fig. 3B. The binding of CKI
was determined by analysis on
15% SDS-PAGE and autoradiography. D, a longer exposure of
the experiment shown above is presented for the mutants 217-325 and
233-325 because of their lower expression compared with the other
mutants, thus providing means of a better interpretation of the
results. E, brain extracts were loaded onto a 1-ml peptide
affinity column to which a peptide corresponding to residues 214-233
(C-214FNRTSLPWQGLKAATKKQKY233) of CKI
was
coupled (CKI peptide) or a control column
(control). The columns were washed, and bound proteins were
eluted with buffer containing 1 M NaCl. Eluted fractions
(elut) and the last washes (wash) were subjected
to SDS-PAGE and analyzed by immunoblotting using
centaurin-
1 or 14-3-3 antibodies. A brain extract was
also analyzed as a positive control for the antibodies (brain
lysate).
1-interacting site on a single loop of CKI
that had been proposed previously from the three-dimensional x-ray
structure to be the site of interaction for CKI partners.
1 Associates with all Members of the CKI
Family--
Residues 217-233 of CKI
are well conserved in all CKI
isoforms as indicated in Fig.
7A. Therefore, it was
interesting to determine whether other CKI isoforms were able to
associate with centaurin-
1. To that purpose, all
mammalian CKI isoforms were synthesized and labeled with
[35S]methionine in a cell-free coupled
transcription/translation system and incubated with GST or
GST-centaurin-
1 (Fig. 7B). All mammalian CKI
isoforms were capable of associating with
GST-centaurin-
1 and not with GST alone (Fig.
7B). Interestingly, a mutant of CKI
deleted of its
carboxyl-terminal domain (CKI
317) binds as well as wild type
CKI
to centaurin-
1 (Fig. 7B). This result
is in agreement with data from Fig. 6C that show that the kinase domain of CKI
is sufficient for its association to
centaurin-
1. These data have led us to strongly believe
that centaurin-
1 interacts with residues 217-233 of
CKI
that are well conserved in all CKI isoforms.
View larger version (49K):
[in a new window]
Fig. 7.
Centaurin- 1 associates with
all members of the CKI family. A, sequence alignment of
residues 217-233 of CKI
with those corresponding to the other CKI
isoforms (
-
). B, CKI isoforms (
,
,
317,
,
1,
2, and
3)
were synthesized as described in Fig. 3B. They were
incubated with 10 µg of GST or GST-centaurin-
1
(GST-
1) as described in Fig.
3C in the presence of 1% Nonidet P-40 and 0.1% bovine
serum albumin. Beads were washed, and samples were analyzed by
12.5% SDS-PAGE followed by autoradiography (pull
down assay). Aliquots representing 2% of total lysate were
analyzed as a control (lysate 2%).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 as a
novel CKI
partner based on the following evidence: (a)
they co-purified from brain after elution from four chromatography
steps; (b) centaurin-
1 associates in
vitro with CKI
indicating that the binding is direct; (c) the binding is specific as centaurin-
1
does not interact in vitro with 14-3-3
under the
same conditions; (d) they form a protein complex
in COS-7 cells as shown in immunoprecipitation experiments;
(e) centaurin-
1 interacts with residues
217-233 of CKI
using deletion mutants of CKI
; and (f)
centaurin-
1 elutes from a peptide affinity
chromatography column containing residues 214-233 of CKI
.
1 interacts with the kinase domain and not with the unique tails of CKI
. Moreover, a mutant of CKI
deleted of its carboxyl-terminal domain binds to centaurin-
1 as
well as does CKI
, suggesting that the kinase domain of CKI
represents the centaurin-binding site. In addition, the site of
interaction within the kinase domain (residues 217-233) is present in
a loop between two helices which has been proposed to represent an
interaction domain for CKI targets (46). The residues within that loop
are well conserved among the CKI family. Indeed, we have shown that all
mammalian CKI isoforms are able to associate with
centaurin-
1 in vitro. This suggests that the
same loop, present in all CKI isoforms, is responsible for the
interaction with centaurin-
1.
1 and centaurin-
have been identified
as PtdIns(3,4,5)P3-binding proteins (33-35).
Phosphatidylinositol 3-kinase is mainly responsible for the synthesis
of PtdIns(3,4,5)P3 by phosphorylating
PtdIns(4,5)P2 at the 3-OH position (47).
Phosphatidylinositol 3-kinase is involved in regulating various
biological processes including membrane ruffling, membrane trafficking,
and actin cytoskeleton regulation (48-50). It is interesting to note
that CKI
has recently been shown to interact with the clathrin
adaptor AP-3 (22), another PtdIns(3,4,5)P3-binding protein
(51). PtdIns(4,5)P2 has been shown to inhibit CKI
activity in vitro (21). However, the physiological relevance
of the inhibition of CKI
by these two phospholipids remains to be
demonstrated. Another link between CKI and the phosphoinositide pathway
has been reported in S. pombe. The authors showed that a
yeast CKI homologue, Cki1, phosphorylates and inhibits PtdIns(4)P
5-kinase (52).
1 belongs to a newly identified family of
ARF-GAP proteins (reviewed in Refs. 36-39). Members of this family
share a cysteine-rich GAP domain and contain several other domains such as PH domains, SH3 domains, and ankyrin repeats. These proteins are
involved in vesicle trafficking and in actin cytoskeleton rearrangement. Therefore, our data support a role for CKI
in these
biological events, in agreement with previous reports (21, 22). Indeed,
CKI
interacts with and phosphorylates the clathrin adaptor AP-3
(22), which is involved in endocytosis. It is interesting to note that
a genetic interaction between yeast CKI and AP-3 was identified
previously (8). Moreover, CKI
has been found to co-localize in
neurones with synaptic vesicle markers and phosphorylates some vesicle
synaptic associated proteins (21). Interestingly, centaurin-
1 has been shown to associate with presynaptic
vesicular structures (53). An actin-associated protein kinase shown to be a member of the CKI family phosphorylates actin in vitro
(54). The molecular mass of the kinase (37 kDa) suggests that it could be CKI
, and we have shown that recombinant CKI
indeed
phosphorylates actin.2 In
addition, the protein DAH (Discontinuous Actin Hexagon) that interacts
with the actin cytoskeleton has been shown to be phosphorylated by CKI
in vitro (55).
has been shown to interact with and phosphorylate these G-protein-coupled receptors (31, 59). In addition, most of the
identified ARF-GAP proteins are involved in the Pak signaling pathway
(reviewed in Ref. 36). Intriguingly, CKI
2 has been found to interact
with the adaptor molecule Nck (60) in a complex with Pak1 (61), thus
raising the possibility that CKI may associate with other ARF-GAP
proteins. Therefore, it would be important to investigate whether
ARF-GAP proteins interact with CKI isoforms.
1, also
contains a cysteine-rich domain that is necessary for its ARF-GAP activity (40). As yet, no ARF-GAP activity has been reported for
centaurin-
1, but it is able of rescuing a
Gcs1 strain
mutant indicating that centaurin-
1 and Gcs1 may have
similar function(s) (62). Gcs1 has been shown to be necessary for the
resumption of cell proliferation from stationary phase (63) and is
involved in endocytosis (7). Gcs1 also plays a role in actin
cytoskeleton regulation in vivo and binds to actin in
vitro (64). As vesicle trafficking is closely associated to actin
organization in yeast, Gcs1 may link vesicle trafficking and the actin
cytoskeleton (64). Yeast CKIs (Yck1 and Yck2) were shown to suppress
the Gcs1 blockage effects on cell proliferation and endocytosis (7).
The membrane association of Yck2 was necessary for this effect (7).
Yck1/2 is involved in cytokinesis, in bud development (5, 6), and regulation of the actin cytoskeleton as yckts mutants fail
to depolarize the actin cytoskeleton during mitosis (5). Another link
between Gcs1 and CKI is the ankyrin repeat protein Akr1p. Gcs1 has been
shown to interact with Akr1p in yeast two-hybrid experiments (65).
Akr1p and Yck1/2 regulate yeast endocytosis, and Akr1p regulates the
plasma membrane localization of Yck1/2 (11). These authors proposed
that the Yck1/2 membrane localization may involve other proteins such
as Gcs1 (65).
does not phosphorylate centaurin-
and
centaurin-
1. In addition centaurin-
1 has
no effect on CKI activity. Therefore, what is the functional relevance
of the interaction between CKI
and these
PtdIns(3,4,5)P3-binding proteins? As CKI
does not
contain a lipid binding domain, it may associate with membranes through
interaction with other proteins. Centaurin-
1 may
represent one of these proteins, as has been proposed for its yeast
counterpart (see above and Ref. 65). CKI
may also represent a
downstream target for centaurin-
1 as suggested by the
results in budding yeast showing that CKIs suppress Gcs1 mutant phenotypes.
and
centaurin-
1, a member of the ARF-GAP protein family that
is involved in membrane trafficking and actin cytoskeleton regulation. Our present results are in agreement with data reported previously suggesting a role for CKI in membrane trafficking and/or regulation of
the actin cytoskeleton. Our findings are further supported by evidence
of a genetic link between CKI and Gcs1 in budding yeast.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Frank McKeon (Department of Cell
Biology, Harvard Medical School, Boston) and David Virshup (Department
of Oncological Sciences, University of Utah, Salt Lake City, UT) for
the CKI(D136N) and CKI
(pKF182) plasmids, respectively. We are
also grateful to Alex Peden for critical reading of this manuscript. We
also thank C. Hyde for CKI peptide synthesis.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Medical Research Council (to A. A., E. Z, P. K., S. H., and T. D.) and a Program grant award (to A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b To whom correspondence should be addressed. Tel.: 44 131 650 3877; Fax: 44 131 650 3711; E-mail: tdubois@srv4.med.ed.ac.uk.
f BBSRC David Phillips Research Fellow.
h Lister Institute Research Fellow.
l Supported by National Institutes of Health Grant DK27221.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M010005200
2 T. Dubois, S. K. Maciver, and A. Aitken, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CKI(s), casein kinase(s) I; PtdIns (3, 4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; ARF, ADP-ribosylation factor; GAP, guanosine trisphosphatase-activating protein; PH, pleckstrin homology; HA, hemagglutinin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol.
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