Characterization of a Novel Giant Scaffolding Protein, CG-NAP, That Anchors Multiple Signaling Enzymes to Centrosome and the Golgi Apparatus*

Mikiko TakahashiDagger , Hideki Shibata§, Masaki Shimakawa§, Masaaki MiyamotoDagger , Hideyuki Mukai§, and Yoshitaka OnoDagger §

From the Dagger  Department of Biology, Faculty of Science, and the § Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 RIIalpha 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 RIIalpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -actinin, but does not efficiently phosphorylate it in vitro (19), suggesting that alpha -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 RIIalpha 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 alpha EE and alpha 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, alpha EE was affinity-purified using antigen-coupled Sepharose beads according to the manufacturer's instruction (Amersham Pharmacia Biotech). Polyclonal antisera against PKN, alpha C6 and alpha N2, were previously described (21). The following antibodies were purchased: anti-gamma -tubulin GTU88, anti-Golgi 58K protein, and anti-alpha -tubulin DM1A (SIGMA); anti-PKA-RIIalpha , 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (alpha N2), normal rabbit serum (NRS), anti-HA 12CA5 (alpha HA), or normal mouse immunoglobulin (NMIg). P#2-43 and PKN in immunoprecipitates and in extracts (-) were visualized by immunoblotting with anti-HA 3F10 (alpha HA) and alpha N2, respectively.

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 (alpha 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 (alpha 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 alpha EE, alpha 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 alpha EE and alpha 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.

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 alpha 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, alpha 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 alpha 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 alpha 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 alpha EE or alpha BH with an antibody against a centrosomal protein gamma -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 alpha EE (a, d, g, and j) for CG-NAP, anti-alpha -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 alpha EE (a) for CG-NAP, and anti-gamma -tubulin (b) for centrosome. Cells were also double-stained with another antiserum for CG-NAP, alpha BH (c), and anti-gamma -tubulin (d). C, localization of CG-NAP to the Golgi apparatus. a and b, HeLa cells were double-stained with alpha 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 alpha 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.

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 gamma -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 alpha BH or normal rabbit serum (NRS) as a control. PKN in immunoprecipitates and extracts (-) was visualized by immunoblotting with alpha 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 alpha C6 (a) for PKN and anti-gamma -tubulin (b) for centrosome.

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 RIIalpha . Deletion mutants ES (aa 1229-1917, Fig. 3) and MB (aa 2380-2876, Fig. 3) were coimmunoprecipitated with RIIalpha (Fig. 6B). Furthermore, interaction between RIIalpha 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 RIIalpha at high affinity and constitutively, and thus, is a novel AKAP. Immunofluorescence analysis detected RIIalpha in cytosol and in other organelles, when cells were fixed without detergent extraction (Fig. 6D, b). On the other hand, RIIalpha 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 RIIalpha with deletion fragments of CG-NAP containing putative RII binding motifs. HA-tagged RIIalpha 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 (alpha FL), 12CA5 (alpha HA), or normal mouse immunoglobulin (NMIg). Extracts (-) and immunoprecipitates were analyzed by immunoblotting with M2 (alpha FL) or 3F10 (alpha HA). Open and closed arrowheads indicate positions of FLAG-tagged deletion fragments and HA-RIIalpha , respectively. C, coimmunoprecipitation of RIIalpha with full-length CG-NAP. HA-tagged RIIalpha 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 (alpha BH) or normal rabbit serum (NRS) as a control, then analyzed by immunoblotting with anti-RIIalpha . Position of RIIalpha is indicated by arrowhead. D, colocalization of CG-NAP and RIIalpha in HeLa cells. HeLa cells grown on cover glasses were fixed with (+) or without (-) prior detergent extraction. Cells were then double-stained with alpha EE and anti-RIIalpha (alpha -RII).

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 (alpha FL), 12CA5 (alpha HA), or normal mouse immunoglobulin (NMIg). Extracts (-) and immunoprecipitates were analyzed by immunoblotting with M2 (alpha FL) or 3F10 (alpha 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 alpha 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 (alpha FL) or normal mouse immunoglobulin (NMIg) as a control. Extracts (-) and immunoprecipitates were analyzed by immunoblotting with 3F10 (alpha 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 (alpha FL), 12CA5 (alpha 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 alpha 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 (alpha PP1) for endogenous PP1-C, or with M2 (alpha 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. alpha 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, RIIalpha 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 RIIalpha 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.

    REFERENCES
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ABSTRACT
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DISCUSSION
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