Characterization of a Novel Giant Scaffolding Protein,
CG-NAP, That Anchors Multiple Signaling Enzymes to Centrosome and
the Golgi Apparatus*
Mikiko
Takahashi
,
Hideki
Shibata§,
Masaki
Shimakawa§,
Masaaki
Miyamoto
,
Hideyuki
Mukai§, and
Yoshitaka
Ono
§¶
From the
Department of Biology, Faculty of Science,
and the § Graduate School of Science and Technology,
Kobe University, Kobe 657-8501, Japan
 |
ABSTRACT |
A novel 450-kDa coiled-coil protein, CG-NAP
(centrosome and Golgi localized
PKN-associated protein), was
identified as a protein that interacted with the regulatory region of
the protein kinase PKN, having a catalytic domain homologous to that of
protein kinase C. CG-NAP contains two sets of putative RII (regulatory
subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly
bound to RII
in HeLa cells. Furthermore, CG-NAP was
coimmunoprecipitated with the catalytic subunit of protein phosphatase
2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously
expressed in COS7 cells. CG-NAP also interacted with the catalytic
subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence
analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RII
were found to be accumulated. These data indicate that CG-NAP serves as
a novel scaffolding protein that assembles several protein kinases and
phosphatases on centrosome and the Golgi apparatus, where physiological
events, such as cell cycle progression and intracellular membrane
traffic, may be regulated by phosphorylation state of specific protein substrates.
 |
INTRODUCTION |
Stimulation of various signaling cascades results in activation of
protein kinases and phosphatases, which alter phosphorylation states of
their respective substrates, leading to diverse physiological responses. Many serine/threonine protein kinases and phosphatases have
relatively broad and overlapping substrate specificity. One of the
mechanisms to organize such enzymes into individual signaling pathways
is targeting them to discrete subcellular locations by anchoring
proteins. For instance, type II cyclic AMP (cAMP)-dependent protein kinase (PKA)1 is
targeted to intracellular compartments through association of its
regulatory subunit RII with protein kinase A anchoring proteins (AKAPs)
(1, 2). Three types of targeting proteins for protein kinase C (PKC)
have been described (3-5). Three classes of phosphatase-targeting
subunits have been identified that are specific for protein phosphatase
1 (PP1), PP2A, and PP2B (6).
Recently, a new class of multivalent adapter proteins that coordinate
the location of multienzyme signaling complexes has been identified.
For example, the pheromone mating response in yeast proceeds
efficiently by clustering the successive members in the
mitogen-activated protein kinase cascade on the scaffold protein STE5
(7). AKAP79 anchors not only PKA but also PKC and PP2B at the
postsynaptic densities of mammalian synapses (8). AKAP250 (gravin)
targets both PKA and PKC to the membrane cytoskeleton and filopodia of
cells (9).
PKN is a serine/threonine protein kinase, having a catalytic domain
homologous to the PKC family in the C-terminal region and a unique
regulatory region in the N-terminal region (10). PKN is activated by a
small GTPase Rho (11-13), unsaturated fatty acids such as arachidonic
acid (14, 15), and by truncation of the N-terminal regulatory region
(14, 16). Since PKN represents broad substrate specificity in
vitro (10), PKN function may be regulated by intracellular
targeting as well as by specific interaction with its substrates. We
previously demonstrated that PKN associates with and phosphorylates
intermediate filament proteins in vitro (17, 18), which may
be physiological substrates for PKN. PKN interacts with the actin
cross-linking protein
-actinin, but does not efficiently
phosphorylate it in vitro (19), suggesting that
-actinin
serves as a scaffolding protein that targets PKN to specific
cytoskeletal substrates.
In the present study, a cDNA encoding a novel coiled-coil protein
with predicted molecular mass of 450 kDa was identified as a
PKN-interacting protein by a yeast two-hybrid screen using the
N-terminal regulatory region of PKN as bait. This protein was localized
to centrosome throughout the cell cycle and the Golgi apparatus at
interphase. Therefore, it was designated CG-NAP (centrosome
and Golgi localized PKN-associated
protein). CG-NAP interacted with various signaling enzymes
including protein kinases (PKN and PKA) and phosphatases (PP1 and
PP2A), and thus, may function as a novel multivalent adapter
protein at these organelles.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screening--
The N-terminal region (amino
acids (aa) 1-540) of PKN fused to the Gal4 DNA binding domain (Gal4bd)
was used as bait to screen a million clones of a human brain cDNA
library fused to the Gal4 transcription activation domain (Gal4ad) as
described (17). Screening to isolate PR130 was performed using a human
fetal kidney cDNA library fused to the Gal4ad
(CLONTECH) with a fragment of PKN (aa 136-306)
fused to the LexA DNA binding domain (LexAbd) as bait. The yeast
expression plasmids for proteins fused to the LexAbd and those to the
VP16 transcription activation domain (VP16ad) were constructed by
subcloning the corresponding cDNA fragments into pBTM116 and pVP16, respectively.
Isolation of cDNA Clones--
The cDNA clones encoding
CG-NAP were isolated by screening human neuroblastoma and HeLa cDNA
libraries with a 32P-labeled probe prepared from the insert
of clone 2-43. To obtain the entire cDNA sequence, 5'-RACE and
3'-RACE methods were employed using a Marathon-Ready cDNA library
(human hippocampus) according to the manufacturer's instruction
(CLONTECH). Mammalian expression plasmid for
full-length CG-NAP was constructed by assembling these clones into
pTB701-HA (20).
Full-length cDNAs of human RII
and human PR130 were obtained by
PCR cloning using cDNA libraries of a human lung cancer cell line
and human fetal kidney, respectively.
Preparation of Recombinant Proteins in Escherichia
coli--
Expression plasmids for proteins fused to glutathione
S-transferase (GST) were constructed by subcloning the
corresponding fragments into pGEX4T (Amersham Pharmacia Biotech). An
expression plasmid for the deletion mutant HH tagged with
(His)6-epitope was constructed by subcloning the
corresponding fragment into pRSET A (Invitrogen). GST-fused and
(His)6-tagged recombinant proteins were expressed in
E. coli and purified by using glutathione-Sepharose 4B
(Amersham Pharmacia Biotech) and nickel-NTA-agarose (Qiagen), respectively, according to the manufacturer's instruction.
In Vitro Binding
Assay--
[35S]Methionine-labeled PKNN2 (aa 1-474 of
PKN)(17) was incubated with GST-P#2-43 in a buffer containing 20 mM Tris-HCl at pH 7.5, 0.5 mM dithiothreitol,
150 mM NaCl, 0.05% Triton X-100, 1 mM EDTA,
and 1 µg/ml leupeptin at 4 °C for 1 h. After addition of
glutathione-Sepharose 4B, the reaction was continued for an additional
30 min. The resin was extensively washed with the same buffer, then
bound proteins were eluted, resolved by SDS-PAGE, and the radioactive
bands were visualized using a Fuji BAS1000 imaging analyzer.
For in vitro homodimer formation, the purified deletion
mutants (His)6-tagged HH and GST-fused HH were incubated in
a buffer containing 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1% Triton X-100, 3 mM
MgCl2, 1 mM EDTA, and 1 mM
dithiothreitol at 4 °C for 1 h. Then proteins bound to
glutathione-Sepharose 4B were analyzed by immunoblotting with anti-His antibody.
Cell Culture, Transfection, and Drug Treatments--
COS7 and
HeLa cells were grown in Dulbecco's modified Eagle's medium
containing 10% heat-inactivated fetal bovine serum, 50 units/ml
penicillin, and 50 µg/ml streptomycin at 37 °C in a humidified 5%
CO2 atmosphere. Mammalian expression plasmids for HA- and
FLAG-tagged proteins were constructed by inserting the corresponding
cDNA fragments into pTB701-HA and pTB701-FLAG (20), respectively. Plasmids pMhPKN3 (10) and pRc/CMV/PKN-FL (16) were used to express PKN
and FLAG-tagged PKN, respectively. For transient expression studies,
COS7 cells were transfected with expression plasmid(s) by
electroporation using GenePulser II (Bio-Rad). To disrupt the Golgi
structure, HeLa cells were treated with 20 µg/ml nocodazole (Sigma)
for 90 min or 10 µg/ml brefeldin A (BFA) (Wako, Japan) for 15 min.
Antibodies--
Polyclonal antisera against CG-NAP designated as
EE and
BH were prepared by immunizing rabbits with bacterially
synthesized GST-fused fragments of aa 423-542 and 2875-2979,
respectively (see Fig. 3). For immunofluorescence analysis,
EE was
affinity-purified using antigen-coupled Sepharose beads according to
the manufacturer's instruction (Amersham Pharmacia Biotech).
Polyclonal antisera against PKN,
C6 and
N2, were previously
described (21). The following antibodies were purchased:
anti-
-tubulin GTU88, anti-Golgi 58K protein, and anti-
-tubulin
DM1A (SIGMA); anti-PKA-RII
, anti-PP2A-C, and anti-PP1 (Transduction
Laboratories); mouse anti-HA 12CA5, and rat anti-HA 3F10 (Roche
Molecular Biochemicals); anti-FLAG M2 (Eastman Kodak); anti-His
RGSHis antibody (Qiagen); rhodamine-conjugated anti-rabbit
IgG, and DTAF-conjugated anti-mouse IgG (Chemicon International);
peroxidase-conjugated secondary antibodies (Santa Cruz).
Immunoprecipitation and Immunoblotting--
Cells were lysed
with a buffer containing 20 mM Tris-HCl at pH 7.5, 1%
Nonidet P-40, 0.15 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl
fluoride, 20 µg/ml aprotinin, and 10 µg/ml leupeptin. Cleared
lysates were incubated with the appropriate antibody at 4 °C for
2 h, then Protein G-Sepharose (Amersham Pharmacia Biotech) was
added and the reaction was continued for another 1 h. After
extensively washing the resin with the same buffer, the bound proteins
were resolved by SDS-PAGE and then subjected to immunoblotting as
described (10). Blots were visualized by enhanced chemiluminescence method.
Northern Blotting--
Polyadenylated RNA was prepared from HeLa
cells using QuickPrep mRNA Purification Kit (Amersham Pharmacia
Biotech). The blot of HeLa mRNA (3 µg) was incubated with the
32P-labeled probe prepared from the cDNA insert of
clone 2-43, followed by extensive washing. The radioactive band was
then visualized using a Fuji BAS1000.
Immunofluorescence Microscopy--
Cells grown on cover glasses
were extracted with 0.1% Triton X-100 in 80 mM Pipes, pH
6.9, 5 mM EDTA, and 1 mM MgCl2 at
room temperature for 2 min, then fixed with cold MeOH for 3 min, or cells were directly fixed with 3.7% formaldehyde in 0.2 M
Na-PO4, pH 7.2, and permeabilized using 0.1-0.3% Triton
X-100. Cells were blocked with 5% normal donkey serum in
phosphate-buffered saline with Tween 20 (20 mM
Na-PO4, pH 7.5, 150 mM NaCl, and 0.03% Triton X-100), then incubated with the relevant antibody for 1 h at room temperature. Cells were washed with phosphate-buffered saline with
Tween 20, and primary antibody is visualized by subsequent incubation
with the appropriate secondary antibody conjugated with either
rhodamine or DTAF. DNA was visualized by adding
4',6-diamidino-2-phenylindole dihydrochloride at concentration of 0.1 µg/ml. The fluorescence of rhodamine and DTAF was observed under a
confocal laser scanning fluorescent microscope (Zeiss), the former at
543 nm argon excitation using a 590-nm long pass barrier filter and the
latter at 488-nm argon excitation using a 510-525-nm band pass barrier filter.
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RESULTS |
Yeast Two-hybrid Screen for PKN Interacting Proteins--
The
N-terminal region (aa 1-540) of PKN was used as bait to screen a human
brain cDNA library by yeast two-hybrid system as described (17).
Clone 2-43 contained a 1.3-kilobase pair cDNA insert encoding a
novel and partial amino acid sequence, which was named P#2-43. Other
combinations of two-hybrid constructs further confirmed the specific
interaction between the N-terminal region of PKN and P#2-43 (Fig.
1A).

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Fig. 1.
Interaction between PKN and P#2-43.
A, analysis by yeast two-hybrid system. L40 cells were
cotransfected with expression plasmids encoding various proteins fused
to LexAbd (left) and those fused to VP16ad
(right) as indicated in left panel. Right panel
shows developments of blue color 1 h after initiating filter
assays. Murine tumor suppressor p53 and SV40 large T antigen were used
as controls. B, analysis by in vitro binding
assay. 35S-Labeled in vitro translated PKNN2 (aa
1-474 of PKN) was incubated with bacterially synthesized GST or
GST-fused P#2-43. After removing aliquots (Input), proteins
bound to glutathione-Sepharose 4B were collected (Output).
Proteins in "Input" and "Output" preparations were separated on
SDS-PAGE followed by visualization of radioactive bands using a Fuji
BAS1000. C, analysis by immunoprecipitation. HA-tagged
P#2-43 and full-length PKN were coexpressed in COS7 cells and
immunoprecipitated (IP) with anti-PKN ( N2),
normal rabbit serum (NRS), anti-HA 12CA5 ( HA),
or normal mouse immunoglobulin (NMIg). P#2-43 and PKN in
immunoprecipitates and in extracts ( ) were visualized by
immunoblotting with anti-HA 3F10 ( HA) and N2,
respectively.
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Binding of PKN to P#2-43--
To investigate whether PKN directly
interacts with P#2-43 in vitro, GST pull-down assay was
performed. In vitro translated PKNN2 (aa 1-474 of PKN)
specifically bound to GST-fused P#2-43 (Fig. 1B, lane
4). To confirm the interaction between PKN and P#2-43 within the
cellular context, immunoprecipitation was performed using COS7 cells
coexpressing HA-tagged P#2-43 and full-length PKN (Fig. 1C).
Anti-PKN antibody (
N2) coimmunoprecipitated P#2-43 (lane
3) with PKN, and conversely, anti-HA 12CA5 coimmunoprecipitated PKN (lane 5) with P#2-43. These results indicate that PKN
directly interacts with P#2-43 through the N-terminal region.
Primary Structure of CG-NAP--
We obtained the presumptive
full-length coding sequence from human cDNA libraries of
neuroblastoma, hippocampus, and HeLa cells by conventional
hybridization screening in combination with 5'- and 3'-RACE methods.
The combined cDNA sequence contained an open reading frame of
11,700 bp encoding a polypeptide of 3,899 amino acids with a predicted
molecular mass of 451,803 daltons (Fig.
2A). We designated this giant
protein as CG-NAP (centrosome- and
Golgi-localized PKN-associated
protein). CG-NAP was a novel protein; however, BLAST search
yielded two proteins that are highly homologous to partial regions of
CG-NAP: human yotiao (22) and rabbit AKAP120 (23), corresponding to aa
1-1626 and 2049-3060, respectively (Fig.
3). CG-NAP also represents limited and
relatively weak homology with pericentrin (Fig. 3), a centrosomal
protein (24). CG-NAP contains four leucine zipper-like motifs (Fig. 2A) and many stretches of coiled-coil structure (Fig.
2B). These structural features are thought to be involved in
association with other proteins and/or
homodimerization/homo-oligomerization (25). Homodimerization of P#2-43
was suggested by the yeast two-hybrid assay using a combination of
P#2-43-LexAbd and P#2-43-VP16ad (data not shown). Therefore,
homodimerization of the N-terminal region of CG-NAP was examined
in vitro using a deletion mutant HH (aa 17-859).
(His)6-tagged HH was copurified with GST-fused HH by
glutathione-Sepharose (Fig. 2C), suggesting the
homodimerization (or homo-oligomerization) of this protein.
Furthermore, we independently confirmed the homodimerization by
coimmunoprecipitation experiment using different epitope-tagged
constructs of CG-NAP deletion mutant (aa 1-1280) coexpressed in COS7
cells (data not shown).

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Fig. 2.
Primary structure and expression of
CG-NAP. A, primary structure of CG-NAP. Predicted amino
acid sequence of full-length CG-NAP is shown with dark
boxes indicating leucine residues in leucine zipper-like
motifs. The coding region of the original clone 2-43 is
shaded. B, coiled-coil analysis of CG-NAP by
COILS program (43). C, homodimerization of the N-terminal
region of CG-NAP. GST or GST-fused HH (aa 17-859 of CG-NAP) was
incubated with (His)6-tagged HH. After removing aliquots
(Input), proteins bound to glutathione-Sepharose 4B were
collected (Output). Proteins in "Input" and "Output"
preparations were analyzed by immunoblotting with anti-His antibody.
D, Northern blots of CG-NAP. Polyadenylated RNA from HeLa
cells was probed with cDNA insert from clone 2-43. Position of
mRNA is indicated by arrowhead. E,
immunoblots of recombinant and endogenous CG-NAP. HA-tagged CG-NAP
expressed in COS7 cells was immunoprecipitated with 12CA5
( HA) or with normal mouse immunoglobulin
(NMIg). Immunoprecipitates and extracts of HeLa cells
(H) and U937 cells (U) were separated on 4.5%
SDS-PAGE, followed by immunoblotting with EE, BH, or control
rabbit serum (NRS) as indicated.
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Fig. 3.
Schematic representation of various
constructs of CG-NAP and the corresponding positions of cDNA
sequences yielded by BLAST search. Schematic representation of the
structure of CG-NAP is shown on the top. LZ,
leucine zipper-like motif; PP1, putative PP1 binding motif;
RII, putative RII binding motif. Aligned below are locations
of polypeptide P#2-43 encoded by the original clone 2-43, the EE and BH
fragments bacterially expressed as GST-fused proteins to generate
rabbit polyclonal antisera EE and BH, respectively, and various
deletion constructs of CG-NAP. cDNAs representing high sequence
homology obtained by BLAST search, yotiao (22), AKAP120 (23), and
pericentrin (24) are also shown at corresponding regions of CG-NAP with
percentage of amino acid sequence homology in parentheses.
Start and end positions of each fragment are indicated.
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Expression of CG-NAP mRNA and Protein--
Northern blots
using polyadenylated RNA of HeLa cells revealed a single band longer
than 12 kilobase pairs (Fig. 2D). CG-NAP mRNA of the
identical size was ubiquitously expressed at low abundance in human
tissues (data not shown).
To examine whether the cDNA sequence obtained here encoded the
complete CG-NAP, we performed immunoblotting using antiserum
EE
raised against a CG-NAP fragment EE (Fig. 3). Recombinant CG-NAP
expressed in COS7 cells (Fig. 2E, lane 2) co-migrated with the endogenous CG-NAP in HeLa (lane 3) and U937 (lane
4) cells. The size of the band appeared to agree with the
calculated molecular mass of 450 kDa. Another antiserum,
BH, raised
against a different part of CG-NAP (Fig. 3) also detected a band of the
same size (Fig. 2E, lane 6). These results indicate that the
cDNA sequence obtained here encodes full-length CG-NAP.
Localization of CG-NAP to Centrosome, the Midbody, and the Golgi
Apparatus--
Subcellular localization of CG-NAP was examined by
immunofluorescence analysis using
EE in HeLa cells at various phases
of the cell cycle (Fig. 4A).
Cells were extracted with nonionic detergent before fixation to
visualize proteins of low abundance associated with intracellular
structures. In interphase cells, CG-NAP was localized to one spot at
the perinuclear region and to a dispersed network near the spot (Fig.
4A, a), presumably corresponding to centrosome and the Golgi
apparatus, respectively. In mitotic cells, CG-NAP was localized to the
spindle poles (Fig. 4A, d, g, and j), and to extremities of the midbody in the cells at
telophase/cytokinesis (Fig. 4A, j). Antiserum
BH also gave the identical staining (Fig. 4B,
c), whereas normal rabbit serum did not (data not shown). Subcellular distribution of CG-NAP was similar in other cell lines, such as SaOS2, TIG1, HEK293, and NIH3T3 cells (data not shown). Localization of CG-NAP to centrosome and the midbody was confirmed by
double-staining, using
EE or
BH with an antibody against a
centrosomal protein
-tubulin (26) (Fig. 4B). CG-NAP was
also co-stained with Golgi 58K protein at perinuclear area (Fig.
4C, a and b). We further examined the
relationship between CG-NAP and the Golgi apparatus using the
microtubule-destabilizing agent nocodazole (27) and the fungal
metabolite BFA (28), which are known to disrupt the Golgi apparatus by
distinct mechanisms. Nocodazole treatment of HeLa cells dispersed the
perinuclear CG-NAP staining into scattered pattern throughout the
cytoplasm (Fig. 4C, d), which is characteristic
for the Golgi staining of nocodazole-treated cells. BFA treatment
disrupted the perinuclear CG-NAP staining (Fig. 4C,
e). BFA-induced Golgi disruption is reversible (28), and the
CG-NAP staining was also recovered by incubation in the absence of the
drug (Fig. 4C, f). Either treatment did not
change the centrosomal staining of CG-NAP. These results indicate that CG-NAP is localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase.

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Fig. 4.
Immunocytochemical localization of
CG-NAP. A, subcellular localization of CG-NAP during
the cell cycle. HeLa cells grown on cover glasses were lightly
extracted with nonionic detergent, then fixed with cold MeOH. Cells
were triple-stained with affinity-purified EE (a, d, g,
and j) for CG-NAP, anti- -tubulin (b, e, h, and
k) for microtubules, and DAPI (c, f, i, and
l) for chromosomal DNA. Cells are at the following stages of
the cell cycle: interphase (a-c), prophase
(d-f), metaphase (g-i), and
telophase/cytokinesis (j-l). B, localization of
CG-NAP to centrosome and the midbody. HeLa cells were double-stained
with EE (a) for CG-NAP, and anti- -tubulin
(b) for centrosome. Cells were also double-stained with
another antiserum for CG-NAP, BH (c), and
anti- -tubulin (d). C, localization of CG-NAP
to the Golgi apparatus. a and b, HeLa cells were
double-stained with EE (a) for CG-NAP, and anti-Golgi
58K protein (b) for the Golgi apparatus. Insets
show the cell in mitotic phase. c-f, HeLa cells were
untreated (c) or treated with nocodazole for 90 min
(d) or BFA for 15 min (e) or BFA for 15 min
followed by recovery in the absence of BFA for 90 min (f).
Then the cells were stained with EE for CG-NAP. D,
subcellular localization of recombinant CG-NAP. HA-tagged full-length
CG-NAP was transiently expressed in COS7 cells. Cells were fixed with
(b) or without (a) prior detergent extraction,
then stained with 12CA5.
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Immunostaining of recombinant CG-NAP expressed in COS7 cells detected
an intense spot at the perinuclear area (Fig. 4D,
a), which was obvious when cells were fixed after detergent
extraction (Fig. 4D, b). This spot was
colocalized with
-tubulin (data not shown), suggesting that
recombinant CG-NAP is predominantly localized to centrosome.
Interaction of PKN with Full-length CG-NAP--
We next examined
the interaction between PKN and full-length CG-NAP. PKN was
coimmunoprecipitated with full-length CG-NAP when both proteins were
exogenously expressed in COS7 cells (Fig. 5A). Immunofluorescence
analysis revealed that a certain population of PKN was localized to
centrosome in the cells fixed after detergent extraction (Fig.
5B), although this protein is predominantly located in the
soluble fraction of the cells (21). These results suggest that PKN is
associated with CG-NAP under physiological condition.

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Fig. 5.
Interaction of PKN with CG-NAP.
A, coimmunoprecipitation of PKN with CG-NAP. HA-tagged
full-length CG-NAP was coexpressed with full-length PKN in COS7 cells,
and immunoprecipitated (IP) with BH or normal rabbit
serum (NRS) as a control. PKN in immunoprecipitates and
extracts ( ) was visualized by immunoblotting with C6. Position of
PKN is indicated by arrowhead. B, localization of
PKN to centrosome. HeLa cells fixed as described in Fig. 4A
were double-stained with C6 (a) for PKN and
anti- -tubulin (b) for centrosome.
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CG-NAP as AKAP--
AKAP120 (23) shows high sequence homology with
a part of CG-NAP (Fig. 6A),
and a putative RII-binding motif forming an amphipathic helix (29) was
conserved in CG-NAP at aa 2540-2558 (Motif 2 in Fig.
6A). Another RII-binding motif was also found at aa
1438-1455 (Motif 1 in Fig. 6A). We therefore
examined whether these regions could bind to RII
. Deletion mutants
ES (aa 1229-1917, Fig. 3) and MB (aa 2380-2876, Fig. 3) were
coimmunoprecipitated with RII
(Fig. 6B). Furthermore,
interaction between RII
and full-length CG-NAP was observed using
exogenously expressed proteins in COS7 cells (Fig. 6C, lane
3), and using endogenous proteins in HeLa cells (Fig. 6C,
lane 5). These data indicate that CG-NAP binds to RII
at high
affinity and constitutively, and thus, is a novel AKAP.
Immunofluorescence analysis detected RII
in cytosol and in other
organelles, when cells were fixed without detergent extraction (Fig.
6D, b). On the other hand, RII
was found to be
colocalized with CG-NAP in the cells fixed after detergent extraction
(Fig. 6D, c and d).

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Fig. 6.
Interaction of RII subunit of PKA with
CG-NAP. A, homology between CG-NAP and AKAP120, and
alignment of putative RII-binding motifs in CG-NAP and other AKAPs.
Amino acid sequence homology between CG-NAP and AKAP120 is shown as a
matrix (MacVector). Putative RII-binding motifs in CG-NAP (Motif
1 and Motif 2) are shown in alignment with those of
AKAP120 (23) and Ht31 (29). Positions of the sequences are indicated in
parentheses. B, coimmunoprecipitation of RII
with deletion fragments of CG-NAP containing putative RII binding
motifs. HA-tagged RII and FLAG-tagged ES (containing motif 1) or
FLAG-tagged MB (containing motif 2) were coexpressed in COS7 cells and
extracts were immunoprecipitated with anti-FLAG M2 ( FL),
12CA5 ( HA), or normal mouse immunoglobulin
(NMIg). Extracts ( ) and immunoprecipitates were analyzed
by immunoblotting with M2 ( FL) or 3F10
( HA). Open and closed
arrowheads indicate positions of FLAG-tagged deletion
fragments and HA-RII , respectively. C,
coimmunoprecipitation of RII with full-length CG-NAP. HA-tagged
RII was coexpressed with full-length CG-NAP in COS7 cells. Extracts
of the COS7 cells (left panel) or nontransfected HeLa cells
(right panel) were immunoprecipitated with anti-CG-NAP
( BH) or normal rabbit serum (NRS) as a
control, then analyzed by immunoblotting with anti-RII . Position of
RII is indicated by arrowhead. D, colocalization of
CG-NAP and RII in HeLa cells. HeLa cells grown on cover glasses were
fixed with (+) or without ( ) prior detergent extraction. Cells were
then double-stained with EE and anti-RII
( -RII).
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Association of CG-NAP with PP2A--
Several AKAPs associate
not only with PKA but also with other signaling enzymes including PP2B
(8). We have isolated another cDNA clone encoding aa 203-1150 of
PR130 as PKN-interacting protein by yeast two-hybrid screen (Fig.
7A). PR130 is one of the B
subunits of PP2A, and its mRNA is expressed ubiquitously at low
levels (30); however, a heterotrimeric PP2A holoenzyme containing PR130 has not been described. We found that a complex consisting of PR130,
the A subunit (PP2A-A), and the catalytic subunit (PP2A-C) was formed
in insect cells in an equal molar ratio and exhibited phosphatase
activity.2 These data
indicate that PR130 is a functional B subunit of the PP2A
holoenzyme.

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Fig. 7.
Interaction of PP2A or PP1 with CG-NAP.
A, interaction of PKN and PR130, a regulatory subunit of
PP2A, in yeast two-hybrid assay. Yeast L40 cells were cotransfected
with combinations of expression vectors as indicated: PKN (aa 136-396
of PKN) or p53 fused to LexAbd (left) and PR130 (aa
203-1150 of PR130) or SV40 large T antigen fused to VP16ad
(right). Blue color developed 1 h after initiation of
the filter assay are shown. p53 and SV40 large T antigen were used as
controls. B, coimmunoprecipitation of PR130 with PKN.
HA-tagged full-length PR130 (HA-PR130) and FLAG-tagged
full-length PKN (FL-PKN) were coexpressed in COS7 cells and
immunoprecipitated with M2 ( FL), 12CA5
( HA), or normal mouse immunoglobulin (NMIg).
Extracts ( ) and immunoprecipitates were analyzed by immunoblotting
with M2 ( FL) or 3F10 ( HA). C,
coimmunoprecipitation of PR130 with CG-NAP. FLAG-tagged PR130
(FL-PR130) was expressed in COS7 cells. Endogenous CG-NAP in
cell extracts was immunoprecipitated with BH or normal rabbit serum
(NRS) as a control. FL-PR130 in extracts ( ) and
immunoprecipitates was visualized by immunoblotting with M2.
D, localization of PR130 binding site in deletion fragment
BS of CG-NAP. FL-PR130 was coexpressed with HA-tagged deletion mutant
of CG-NAP, BB or BS (see Fig. 3) in COS7 cells and immunoprecipitated
with M2 ( FL) or normal mouse immunoglobulin
(NMIg) as a control. Extracts ( ) and immunoprecipitates
were analyzed by immunoblotting with 3F10 ( HA).
E, coimmunoprecipitation of PP2A-C with CG-NAP through
binding with PR130. FL-PR130 and HA-tagged BS were coexpressed in COS7
cells and immunoprecipitated with M2 ( FL), 12CA5
( HA), or normal mouse immunoglobulin (NMIg).
The extracts ( ) and the immunoprecipitates were examined for the
presence of endogenous PP2A-C by immunoblotting. PP2A-C in lane
6 implies that PP2A-C interacted with HA-BS through binding with
PR130. F, interaction of the catalytic subunit of PP1 with
CG-NAP. FLAG-tagged P#2-43 expressed in COS7 cells was
immunoprecipitated with M2 (lane 3). HeLa cell extracts were
immunoprecipitated with BH (lane 2) or normal rabbit
immunoglobulin (NRS, lane 1) as a control. Imunoprecipitates
and extracts ( ) of the COS7 cells (C, lanes 5 and 6) and HeLa cells (H, lane 4) were analyzed
by immunoblotting with anti-PP1-C ( PP1) for endogenous
PP1-C, or with M2 ( FL) for P#2-43.
|
|
Next, we examined the interaction between PKN and PR130 by
coimmunoprecipitation using full-length proteins. PR130 and PKN associated weakly but significantly (Fig. 7B). Since the
efficiency of coimmunoprecipitation was relatively low and direct
interaction between PKN and PR130 in vitro was hardly
detected by GST-pull down assay (data not shown), we speculated that
this interaction is indirect and mediated by some adapter protein such
as CG-NAP. Thus, we examined whether PR130 interacted with CG-NAP using
COS7 cells expressing PR130.
BH coimmunoprecipitated PR130 with
endogenous CG-NAP (Fig. 7C, lane 3), suggesting
that PR130 associates with PKN through binding with CG-NAP. Deletion
analysis of CG-NAP located the binding site for PR130 in the deletion
BS (Fig. 7D, lane 5, and Fig. 3).
These results raised the possibility that PP2A holoenzyme could also
associate with CG-NAP. We performed immunoprecipitation using COS7
cells coexpressing FLAG-tagged PR130 and HA-tagged BS. Endogenous
PP2A-C was coimmunoprecipitated with BS by anti-HA (Fig. 7E,
lane 6). Since binding of endogenous PP2A-C with PR130 was
confirmed in the same lysate (Fig. 7E, lane 3),
it is implicated that PP2A-C and probably PP2A-A were coprecipitated
with CG-NAP (in this case, its deletion BS) through binding with PR130.
Association of CG-NAP with the Catalytic Subunit of PP1--
By
searching for binding motifs for other signaling enzymes, we found a
possible PP1 binding motif R/KVXF (31) at aa 1053-1056 of
CG-NAP. Immunoprecipitation revealed that the endogenous catalytic subunit of PP1 (PP1-C) interacted with P#2-43 (containing the putative
PP1 binding motif) expressed in COS7 cells (Fig. 7F, lane 3), and furthermore, with endogenous CG-NAP in HeLa
cells (Fig. 7F, lane 2). These results indicated
that PP1-C associates with CG-NAP under physiological condition.
 |
DISCUSSION |
The present study discovered a novel 450-kDa protein CG-NAP
ubiquitously expressed in human tissues. CG-NAP is localized to centrosome throughout the cell cycle, the midbody at telophase, and the
Golgi apparatus at interphase in cultured cell lines. We have
demonstrated that CG-NAP interacts with PKN, RII
subunit of PKA,
PP2A through its regulatory B subunit PR130, and the catalytic subunit
of PP1. Therefore, CG-NAP may function as a scaffolding protein for the
subcellular targeting of these enzymes, and thus may be a novel
multivalent adapter protein for signaling enzymes as well as a new
AKAP.
AKAPs localized to centrosome (AKAP350) (32) and the Golgi apparatus
(AKAP85) (33) have been identified by RII overlay of human lymphoblast
lysates. The relationship between these AKAPs and CG-NAP
remains unclear, since amino acid sequences of these AKAPs are
currently unknown. In the course of this study, two novel proteins,
rabbit AKAP120 (23) and human yotiao (22), were discovered, both of
which represent high sequence homology with partial regions of CG-NAP
(Fig. 3). In addition, contiguous BAC clones of human genome, RG293F11
(from chromosome 7q21-22) and GS541B18 (from chromosome 7q21), were
found to cover aa 20-1639 and 1793-3768, respectively, of CG-NAP,
suggesting that CG-NAP is encoded by a single gene located at
chromosome 7q21-22. AKAP120 may be coded by a rabbit homolog of
CG-NAP. Yotiao might be a partial clone or the product of the
alternative splicing or post-translational proteolytic processing
of CG-NAP.
Phosphorylation of centrosomal proteins is suggested to be
involved in the regulation of centrosomal function (34). Various protein kinases and phosphatases are localized to centrosome (6), and
some of the kinases are implicated in the regulation of centrosome separation (35, 36) and microtubule nucleation (37). Protein phosphorylation is also implicated in mitotic Golgi fragmentation (38)
and membrane traffic (39, 40). However, it remains largely unknown how
protein kinases and phosphatases are localized to these organelles and
how these enzymes are coordinated to fulfill the physiological
processes. We have demonstrated that a certain population of PKN and
RII
are localized to centrosome (Fig. 5B) and
centrosome/the Golgi apparatus (Fig. 6D, c and
d), respectively. In addition, immunocytochemical study of
human brain tissues showed that PKN is also enriched in the Golgi
bodies (41). Therefore, CG-NAP may represent the candidate that
coordinates the location and activity of these enzymes at centrosome
and the Golgi apparatus.
Coimmunoprecipitation studies using various deletion mutants of CG-NAP
indicated that the four enzymes bind to CG-NAP at distinct sites: PKN
on P#2-43, PKA on ES and MB, PP1 on P#2-43, and PP2A on BS (see Fig.
3). Furthermore, PP2A and PKN appears to bind simultaneously to CG-NAP
(Fig. 7B). Since we found that dephosphorylation of PKN by
PP2A decreased its kinase activity in
vitro,3 it is possible
that dephosphorylation of PKN by PP2A closely located on CG-NAP affects
the activity and/or localization of PKN under physiological condition.
In other words, CG-NAP may provide a scaffold to facilitate
interactions among the bound enzymes. On the contrary, another possible
function of CG-NAP is that it retains the bound enzymes as inactive
pools until they are activated by appropriate signals. Such inhibitory
effect is demonstrated in other multiadaptor proteins, AKAP79 on PKC
(8) and PP2B (42), and gravin on PKC (9). CG-NAP was phosphorylated by
PKN and PKA at distinct regions in vitro (data not shown). Changes in the phosphorylation state of CG-NAP may lead to its dynamic
structural alterations, which may result in the changes in its binding
affinity with the target compartment(s) or with other enzyme(s). This
might explain why the association of PKN, PP2A, or PP1 with full-length
CG-NAP is relatively weak compared with that with the deletion fragment
of CG-NAP. Studies to elucidate the role of CG-NAP and the bound
signaling enzymes in the centrosome and Golgi functions will provide
further understanding of the regulatory mechanisms of signal
transduction occurring at these organelles.
 |
ACKNOWLEDGEMENT |
We thank Y. Nishizuka for encouragement.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Ministry
of Education, Science, Sports and Culture, Japan, the "Research for
the Future" program, the Japan Society for the Promotion of Science,
the Japan Foundation for Applied Enzymology, and Kirin Brewery Co. Ltd.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.
The nucleotide sequence reported in this paper has been submitted
to the DDBJ/GenBankTM/EMBL Data Bank with accession number
AB019691.
¶
To whom correspondence should be addressed: Dept. of Biology,
Faculty of Science, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe
657-8501, Japan. Tel.: 81-78-803-5792; Fax: 81-78-803-5782; E-mail:
yonodayo{at}kobe-u.ac.jp.
2
H. Shibata, M. Takahashi, H. Mukai, and Y. Ono,
manuscript in preparation.
3
C. Yoshinaga, H. Mukai, M. Toshimori, M. Miyamoto, and Y. Ono, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
PKA, protein kinase
A;
AKAP, protein kinase A anchoring protein;
PKC, protein kinase C;
PP1, protein phosphatase 1;
PP2A, protein phosphatase 2A;
PP2B, protein
phosphatase 2B;
CG-NAP, centrosome- and Golgi-localized PKN-associated
protein;
GST, glutathione S-transferase;
aa, amino acid(s);
BFA, brefeldin A;
HA, hemagglutinin;
RACE, rapid amplification of
cDNA ends;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis;
bp, base pair(s);
Pipes, 1,4-piperazinediethanesulfonic acid;
ad, activation domain;
bd, binding
domain;
DTAF, dichlorotriazinyl amino fluorescein.
 |
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