A-kinase-anchoring Protein AKAP95 Is Targeted to the Nuclear Matrix and Associates with p68 RNA Helicase*

Lakshmi Akileswaran, Justin W. Taraska, Jonathan A. Sayer, Jessica M. Gettemy, and Vincent M. CoghlanDagger

From the Neurological Sciences Institute, Oregon Health Sciences University, Beaverton, Oregon 97006

Received for publication, February 6, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cell nucleus is structurally and functionally organized by the nuclear matrix. We have examined whether the nuclear cAMP-dependent protein kinase-anchoring protein AKAP95 contains specific signals for targeting to the subnuclear compartment and for interaction with other proteins. AKAP95 was expressed in mammalian cells and found to localize exclusively to the nuclear matrix. Mutational analysis was used to identify determinants for nuclear localization and nuclear matrix targeting of AKAP95. These sites were found to be distinct from previously identified DNA and protein kinase A binding domains. The nuclear matrix-targeting site is unique but conserved among members of the AKAP95 family. Direct binding of AKAP95 to isolated nuclear matrix was demonstrated in situ and found to be dependent on the nuclear matrix-targeting site. Moreover, Far Western blot analysis identified at least three AKAP95-binding proteins in nuclear matrix isolated from rat brain. Yeast two-hybrid cloning identified one binding partner as p68 RNA helicase. The helicase and AKAP95 co-localized in the nuclear matrix of mammalian cells, associated in vitro, and were precipitated as a complex from solubilized cell extracts. The results define novel protein-protein interactions among nuclear matrix proteins and suggest a potential role of AKAP95 as a scaffold for coordinating assembly of hormonally responsive transcription complexes.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nuclear matrix was first described in 1974 by Berezney and Coffey (1, 2) as the insoluble, proteinaceous structural component of the nucleus that remains after digestion and extraction of chromatin and associated nuclear factors. Since then it has become increasingly apparent that the matrix plays an important functional role, coordinating many nuclear activities including DNA replication, RNA processing, and transcription (3-4). Integration of these events is thought to occur within a high level of chromatin structure where the nuclear matrix organizes DNA into functional loops by binding specific sequences or "matrix attachment regions." Not surprisingly, these regions are often found flanking actively transcribed genes and may serve as localization sites for assembly of factors regulating gene expression (5-7).

Structurally, the intact nuclear matrix includes nuclear lamina, residual nucleoli, and ribonucleoprotein particles bound via a fibrous network of proteins, RNA, and polysaccharides. Although lamins and heterogeneous nuclear RNA proteins are the predominant protein constituents, the core structure contains more than 100 distinct nuclear matrix proteins, many of which bind DNA (8-10). To date, relatively few nuclear matrix proteins have been characterized in detail, and little is known about how they are incorporated into the matrix. Complicating the analysis is the fact that the nuclear matrix is a dynamic structure, and its protein composition is affected by environmental stimuli and growth conditions. Indeed, several nuclear matrix proteins have been identified as useful markers for specific types of cancer (11, 12). Detailed ultrastructural analysis of purified matrices has confirmed that despite this variability, all nuclei contain a similar structural network that constitutes a core nuclear matrix (13-16).

Although the structure of the nuclear matrix has been carefully observed, the mechanisms for its assembly and stabilization remain largely unknown. The fact that the core matrix remains after removal of nucleic acids indicates that protein-protein interactions play an important part in maintaining the structure. This principle has led us to propose that nuclear matrix proteins contain unique domains for association with binding partners within the nuclear matrix. These domains may provide sufficient specificity to direct proper assembly of the nuclear matrix, an event that occurs after each mitotic cycle. This hypothesis is consistent with the nuclear matrix serving as a scaffold upon which chromatin and other nuclear components are subsequently incorporated and ultimately organized.

We have examined the binding domains within AKAP95,1 a zinc finger protein found in the nuclear matrix of a wide variety of mammalian cells (17, 18). In addition to binding DNA, AKAP95 contains a C-terminal amphipathic helix that serves as an anchoring domain for the cAMP-dependent protein kinase (PKA). Thus, AKAP95 may play a key role in linking the nuclear matrix to specific cell-signaling events. In this report, we define domains within AKAP95 that are critical for nuclear import and for interactions within the nuclear matrix. We identify several AKAP95 binding partners in purified nuclear matrix and characterize the specific interaction with p68 RNA helicase, a important component of transcriptional initiation complexes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutagenesis-- Deletion mutants were constructed by polymerase chain reaction amplification of rat AKAP95 cDNA (GenBankTM accession number UO1914) using native Pfu thermostable DNA polymerase (Stratagene) and synthetic oligonucleotide primers (Genosys). Primers were designed to amplify selected fragments of AKAP95-coding sequence with incorporation of suitable restriction sites for subcloning. The polymerase chain reaction products were digested with restriction enzymes, gel-purified (Geneclean II; Bio 101), and cloned directly into either pcDNA3 (Invitrogen) or pEGFP-N2 (CLONTECH), a mammalian expression vector incorporating a C-terminal green fluorescent protein (GFP) fusion. Deletion mutant vectors were designated based on the amino acids of AKAP95 that they encode. DNA modification and restriction enzymes were obtained from New England Biolabs or Promega. For site-directed mutagenesis, a polymerase chain reaction-based method (QuikChange, Stratagene) was used to introduce codon changes in AKAP95 cDNA that resulted in substitutions of acidic or neutral residues for selected basic amino acids. Basic regions of AKAP95 were designated alphabetically and correspond to A (amino acids 283-289), B (301), C (366), D (373), E (438), and F (444). Predicted amino acid sequences of the substitutions were A (RDWPRRR to QDWRSSR), B (RKRK to REQK), and CD (KKRREKQRRRDR to KESSEKQRSRDR). Proper subcloning and sequence integrity of all constructs was confirmed by dideoxy sequencing (Thermosequenase, Amersham Pharmacia Biotech). Mutated vectors were transiently transfected into cultured cells 24 h before fixation.

Cell Culture-- Tissue culture cells were grown in a humidified incubator at 37 °C at 7% CO2. Cell lines used were: COS7 (SV40-transformed African green monkey kidney; ATCC CRL-1651), CHO-K1 (Chinese hamster ovary; ATCC, CCL-61), HEK293 (human embryonic kidney; ATCC CRL-1573), GH3 (rat pituitary tumor, ATCC; CCL-82.1), and Swiss 3T3 (Swiss albino mouse fibroblasts; ATCC, CCL-92). COS7, 293, and 3T3 cells were grown in Dulbecco's modified Eagle's medium (Cellgro) containing 10% fetal bovine serum (JRH Biosciences), 100 units/ml penicillin-streptomycin (Sigma). CHO-K1 cells were grown in F-12K medium (Cellgro) supplemented with 10% fetal bovine serum and penicillin-streptomycin. GH3 cells were grown in Dulbecco's modified Eagle's medium with 15% HS (JRH Biosciences) and 2.5% fetal bovine serum.

For transfections, cells were split onto sterile, poly-L-lysine (Sigma)-coated coverslips in 6-well tissue culture dishes. When 60-80% confluent, the cells were incubated with lipid-DNA complexes in serum-free medium (Opti-MEM, Life Technologies) for 4-5 h, then grown in serum-containing growth medium for 18-24 h. COS7, CHO-K1, and 293 cell lines were transfected using Pfx-6 lipid and GH3 cells with Pfx-4 lipid as described by the manufacturer (Invitrogen). 3T3 cells were transfected using standard calcium phosphate reagents (Life Technologies).

Nuclear Matrix Preparation-- Isolation of the nuclear matrix in situ was carried out using a modification of the procedure described by Penman and co-workers (13). Briefly, transfected cells grown on glass coverslips were washed with Tris-buffered saline (TBS) and extracted with cytoskeletal buffer (CS; 300 mM sucrose, 3 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 10 mM MOPS, pH 7.0) containing 0.5% Triton X-100 and protease inhibitors (2 mg/ml leupeptin, 1 mM benzamidine, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride). After a 10-min incubation at room temperature, coverslips were rinsed three times with CS buffer and incubated in digestion buffer (300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 50 mM NaCl, 10 mM MOPS, pH 7.0) containing 200 units/ml DNase I and 1 unit/ml RNase A (Roche Molecular Biochemicals). After 1 h, the digestion buffer was brought to 0.25 M (NH4)2SO4 by dropwise addition from a 1 M stock, and the coverslips were incubated for an additional 30 min at 4 °C. After two final CS buffer rinses, the extracted nuclear matrix was fixed in formalin for 10-15 min and placed in phosphate-buffered saline (PBS) before immunochemical analysis.

Nuclear matrix was prepared from rat brain as described (13). Nuclear matrix was prepared similarly from cultured cells, except CS buffer containing 1% Triton X-100, 0.5% SDS, 5 mM EDTA, and 5 mM EGTA was used to extract nuclei because this buffer was found to partially solubilize AKAP95. Briefly, nuclei from ~1 × 107 cells were lysed in 1 ml of the supplemented CS buffer at 4 °C by passage through a 22-gauge needle (3×). After 5 min on ice, the lysate was passed again through the needle then centrifuged at 600 × g for 5 min. The supernatant (solubilized fraction) was removed, and the pellet was resuspended in digestion buffer. After incubation with DNase I (200 units/ml) and RNase A (1 unit/ml) for 30 min at 20 °C, the mixture was brought to 0.25 M (NH4)2SO4, incubated for 5 min on ice, and centrifuged at 600 × g for 5 min. The supernatant (chromatin fraction) was removed, and the pellet was resuspended in CS buffer containing 2 M NaCl. After 5 min on ice, the mixture was centrifuged at 1000 × g for 5 min. The supernatant (high salt extract) was removed, and the pellet (insoluble matrix) was resuspended in 10 mM Tris-HCl, 0.2 mM MgCl2, pH 7.5.

For immunoprecipitations, solubilized extracts were obtained from COS7 cells 16 h after transfection with expression vectors for AKAP95 (AKAP95-pCDNA) or p68 RNA helicase (RH-pCMV/GST; ~1 µg each) as described above. The extract was pre-incubated with protein A-agarose for 30 min at 4 °C and clarified by centrifugation (14,000 × g for 5 min at 4 °C). AKAP95 was immunoprecipitated from the clarified extracts using affinity-purified rabbit antibodies and excess protein A-agarose as previously described (18). Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis (12%) and the presence of RH-GST fusion was determined by Western blot analysis using anti-GST (Amersham Pharmacia Biotech) and PAb204 anti-RH antibodies.

Fluorescence Microscopy-- For fluorescence detection of GFP, coverslips bearing transfected cells were rinsed with PBS containing 1 mM CaCl2 and 1 mM MgSO4 and fixed in a dark chamber for 15 min in formalin (4% formaldehyde, 1.5% methanol, 2% sodium acetate). For indirect immunofluorescence, cells were rinsed and fixed as above, then membrane-extracted with ice-cold 100% acetone (high performance liquid chromatography grade, Sigma) and blocked for 1 h in PBS containing 0.5% bovine serum albumin (Fraction V, Fisher). Coverslips were incubated in primary antibodies (rabbit anti-green fluorescent protein (Molecular Probes), mouse anti-tubulin monoclonal antibody (Zymed Laboratories Inc.), rabbit anti-AKAP95 antibodies (17), or mouse anti-p68 RNA helicase monoclonal antibody (PAb204, generously provided by Dr. H. Stahl, Universitat Konstanz, Federal Republic of Germany), each diluted 1:200 in PBS containing 0.1% bovine serum albumin) for 2-4 h at 37 °C. After rinsing 3× with PBS, coverslips were incubated with secondary antibody (Texas Red- or fluorescein isothiocyanate-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch) diluted 1:300 in PBS) for 1 h at 37 °C. The coverslips were rinsed with PBS, incubated for 1 min in 1 µg/ml Hoechst 33258 (Sigma) to stain chromatin, rinsed briefly in distilled H2O, and wet-mounted onto slides using Fluoromount-G (Molecular Probes). Slides were viewed on a Nikon E600 epifluorescence microscope using 40× or 60× PlanFluor objectives, and images were acquired using a digital camera (Diagnostic Instruments).

Protein Overlays-- For in situ binding studies, recombinant AKAP95-(1-687), AKAP95-(1-386), AKAP95-(378-687), and purified bovine serum albumin (New England Biolabs) were diluted to 0.5 mg/ml in 0.05 M bicarbonate buffer, pH 8.5, and labeled with N-hydroxysuccinimide-fluorescein for 2 h on ice as described by the manufacturer (Pierce). After quenching the reactions with 0.25 M Tris-HCl, pH 8.5, labeled proteins were purified in the dark using a 2-ml desalting column (Excellulose; Pierce) equilibrated with TBS. All preparations had an F/P ratio (mol of fluorochrome/mol of protein) of 0.5-0.8. The labeled proteins (0.5 µg/ml in TBS) were layered over bovine serum albumin-blocked coverslips containing either formalin-fixed, acetone-extracted Swiss 3T3 cells or isolated nuclear matrix. After incubation for 2 h at room temperature, the coverslips were washed extensively with TBS and processed for fluorescence observation as described above.

Far Western blot assays were performed as described previously (19). Recombinant proteins were produced in bacteria with N-terminal hexahistidine tags using the pET system (Novagen). For labeling, AKAP95 protein fragments were produced with a consensus PKA phosphorylation site and radiolabeled using [gamma -32P]ATP and the catalytic subunit of PKA as described previously (19, 20). The labeled proteins were used to probe polyvinylidene difluoride membranes (Millipore) containing proteins transferred from 10% SDS-polyacrylamide gels. After incubation at room temperature for 3 h, the radiolabeled AKAP95 probe was removed, and the membranes were washed three times with TBS before exposing to film for 16 h. Alternatively, a nonradioactive overlay was employed where recombinant proteins were labeled using activated digoxigenin and detected using peroxidase-labeled anti-digoxigenin antibodies with enhanced chemiluminescence as described previously (38). Cell extracts for analysis were prepared in TBS containing protease inhibitors (2 mg/ml leupeptin, 1 mM benzamidine, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride). Direct in vitro binding assays using recombinant proteins immobilized on slot-blots were similarly performed and have been described previously (20). All radiolabeled recombinant AKAP95 protein fragments were of similar specific activity (1-2 × 105 cpm/pmol), and incubations were performed using 105 cpm/ml probe in TBS containing 5% nonfat dried milk.

Yeast Two-hybrid Cloning-- A fragment of AKAP95 encoding amino acids 109-201 was amplified using polymerase chain reaction and cloned as a fusion with the Gal4 DNA binding domain in the yeast expression vector pAS-2. This construct was used as bait to screen a library (~1.3 × 106 independent clones) of mouse embryo cDNAs, each fused to the Gal4 activation domain in the vector pACT2 (Matchmaker, CLONTECH). The yeast reporter strain AH109 (CLONTECH) was used as the host, and cells positive for growth on selective medium (-Ade/-His/-Leu/-Trp) were examined for beta -galactosidase activity using a colony filter-lift assay (21). Positive interactions were confirmed by re-introducing cDNA (and sub-cloned derivatives) of retrieved clones into yeast Y190a and performing control crosses with yeast harboring the AKAP95-pAS2 construct or control vectors pAS, AKAP79-pAS, and RII-pAS as described previously (22).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A heterologous expression system was used to investigate the subcellular localization of AKAP95. We initially examined the expression of rat AKAP95 cDNA in human embryonic kidney cells (HEK293) because this cell line has well defined morphology, and our antibodies do not cross-react with the endogenous AKAP95. As shown in Fig. 1, immunocytochemical staining indicated that the expressed AKAP95 protein (amino acids 1-687) localized exclusively to the nucleus (Fig. 1A), where it co-localized with DNA (Fig. 1B) but was excluded from nucleoli. The staining pattern is identical to that previously observed for the native protein in a variety of cell types (17-18), suggesting that the recombinant protein is imported into the nucleus and incorporated within the nuclear matrix. Similar results were obtained using cultured COS7 and CHO-K1 cells (not shown). Based on these results, we hypothesized that the AKAP95 polypeptide contains specific information (e.g. signal sequences) for both intracellular and intranuclear targeting.


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Fig. 1.   Expression of rat AKAP95 in mammalian cells. After 18 h, human embryonic kidney (HEK293) cells transiently transfected with AKAP95 cDNA-pCDNA3 expression vector were formalin-fixed, acetone-extracted, and incubated with rabbit anti-AKAP95 antibodies (1:200) and mouse anti-tubulin (1:200) for 2 h at room temperature. A, fluorescent staining for AKAP95 (green) using fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:300) secondary antibodies. B, staining for tubulin (red) using Texas Red-conjugated anti-mouse secondary antibodies (1:300) and nucleic acid (blue) using Hoechst stain (1 µg/ml).

Initially, we used deletion mutagenesis to map regions of AKAP95 involved in intracellular targeting. Analysis of the AKAP95 sequence revealed six highly basic regions (sequentially designated A-F) that exhibit similarity to known nuclear import signal sequences (23). Various fragments of AKAP95 cDNA containing selected basic regions were subcloned into mammalian vectors and expressed in COS7 cells as fusions with GFP. In transfected cells, an N-terminal fragment of AKAP95 (amino acids 1-386) containing the first four basic regions (A-D) was sufficient to direct the GFP reporter into the nucleus (Fig. 2a), where it co-localized with DNA (Fig. 2b) and was absent from other cellular compartments (Fig. 2c). Thus, the basic regions E and F, the zinc fingers, and the PKA binding domain of AKAP95 are not essential for nuclear import.


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Fig. 2.   Identification of a nuclear import signal for AKAP95. A fragment containing the first 386 amino acids of AKAP95 was transiently expressed as a fusion with GFP in COS-7 cells. a, cellular distribution of AKAP95-GFP fusion. b, nucleic acids stained with Hoechst stain (1 µg/ml). c, same cell observed under phase contrast. Site-specific mutations were introduced into basic regions (designated A-F) within AKAP95. Transiently transfected cells were observed under fluorescence for GFP fusions with mutations in basic region A (d), region B (e), regions C and D (f) and compared with cells expressing GFP alone (g). ZF, zinc finger.

To further define the nuclear import signals of AKAP95, site-specific mutations were introduced into the N-terminal fragment that resulted in substitutions of neutral or acidic amino acids for basic residues within basic regions A-D. Mutations introduced into the A, C, and D regions of AKAP95 had no effect on nuclear import of the AKAP-GFP fusion protein (Fig. 2, d and f). In contrast, mutations within the basic region B drastically impaired nuclear import of the fusion protein (Fig. 2e) and resulted in a subcellular distribution similar to GFP (Fig. 2g), where the fluorescent reporter was detected throughout the cell. The results indicate that basic region B (amino acids 301-305) is an important determinant for nuclear import of AKAP95.

Deletion mutants were also used to define signals for intranuclear targeting of AKAP95. Because expressed AKAP95-(1-386) and full-length proteins exhibited identical localization patterns, we proposed that the nuclear matrix targeting site (NMTS) of AKAP95 was contained within the N terminus. Immunolocalization studies of purified nuclear matrix confirmed this hypothesis. Like the full-length protein (Fig. 3a), the AKAP95-(1-386)-GFP fusion protein (Fig. 3b) remained associated with the nuclear matrix isolated in situ from transfected COS7 cells. In control experiments, GFP expressed by itself was not associated with the nuclear matrix (Fig. 3c). Antibodies to GFP were used to specifically detect GFP and GFP fusion proteins because the harsh conditions used to extract the nuclear matrix were found to bleach GFP fluorescence. The results not only indicate that the AKAP95 NMTS is located within the N terminus, they also demonstrate that nuclear-matrix association is independent from DNA and PKA binding since the AKAP95-(1-386) fragment is missing the zinc finger domains and the PKA binding amphipathic helix (17).


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Fig. 3.   Identification of an NMTS for AKAP95. The nuclear matrix was prepared as described under "Experimental Procedures" from COS7 cells expressing full-length AKAP95 (a), AKAP95-(1-386)-GFP (b), GFP (c), or a series of AKAP95 N-terminal deletion mutants fused to GFP including amino acids 50-386 (d), 110-386 (e), 140-386 (f), and 170-386 (g). Immunolocalization was performed using anti-GFP primary antibodies, except in a, where anti-AKAP95 antibodies were used. Fluorescence detection was achieved using fluorescein isothiocyanate-conjugated secondary antibodies. ZF, zinc finger. NLS, nuclear localization signal.

To further define the NMTS of AKAP95, a series of N-terminal deletions were constructed and fused to the GFP reporter. When transiently expressed in cultured cells, deletion mutants missing 50 or 110 N-terminal amino acids of AKAP95 localized and bound to the nuclear matrix (Fig. 3, d and e). In contrast, deletion mutants missing 140 or 170 amino acids from the N terminus failed to associate with the nuclear matrix (Fig. 3, f and g). These results indicate that amino acids in the region between positions 110 and 140 contain critical determinants for association of AKAP95 with the nuclear matrix. This region is highly conserved in rat, mouse, and human AKAP95 (Fig. 4). Comparative analysis revealed similar sequences in the known nuclear matrix protein ZAN75 (24) and in the genetic neighbor of AKAP95, NAKAP95 (25). Sequence similarities, including a distinct pattern of aromatic residues, were found with AKAP95 from amino acids 127 to 152, and we propose that this region constitutes the NMTS of this protein family.


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Fig. 4.   Comparison of NMTS sequences within the AKAP95 family. Sequences from human (hum), mouse (mou), and rat AKAP95 NMTS (amino acids 127-152) are compared with the nuclear matrix protein ZAN75 (amino acids 70-95; Ref. 20) and genetic neighbor NAKAP95 (amino acids 136-161; Ref. 21). Strong sequence similarities are highlighted.

The most likely role for the NMTS is to facilitate protein-protein interactions between AKAP95 and specific nuclear matrix components. Direct binding of AKAP95 N-terminal fragment to the nuclear matrix was confirmed using an in situ overlay where fibroblasts grown on glass coverslips were sequentially extracted and incubated with fluorescein-labeled recombinant AKAP95-(1-386) protein. In whole cells (Fig. 5a) with intact, DNA-containing nuclei (Fig. 5b), the labeled AKAP95 fragment bound to sites within the nucleus, with some staining of extranuclear structures (Fig. 5c). More significantly, the labeled fragment bound the nuclear matrix after chromatin and other cellular structures were fully extracted from these cells (Fig. 5, d-f). Identical results were observed using the full-length protein (not shown). In contrast, a fluorescein-labeled AKAP95-(378-687) fragment missing the NMTS (Fig. 5, g-i) and a similarly labeled albumin control (not shown) did not bind isolated matrix. These results are consistent with NMTS-directed association of AKAP95 with specific nuclear matrix proteins and support our previous result that nuclear matrix incorporation is independent from nucleic acid binding.


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Fig. 5.   Binding of AKAP95 to the nuclear matrix in situ. Swiss 3T3 fibroblasts grown on glass coverslips were either formalin-fixed and permeabilized with acetone (a-c) or sequentially extracted to isolate the nuclear matrix as described under "Experimental Procedures"(d-i). Coverslips were incubated with either fluorescein (FITC)-labeled AKAP95-(1-386) N-terminal fragment (a-f) or a control fluorescein-labeled AKAP95-(378-687) C-terminal fragment that is missing the NMTS (g-i). Nucleic acids were stained with Hoechst stain, and slides were observed under phase contrast or epifluorescence as indicated.

To identify potential binding partners within the nuclear matrix, we probed various tissue extracts with 32P-labeled recombinant AKAP95-(1-386) protein using a Far Western blot assay (see "Experimental Procedures"). No significant AKAP95 binding activity was observed in a survey of crude protein extracts from several rat tissues known to express AKAP95 including forebrain, cerebellum, and kidney (Fig. 6A). However at least three protein bands corresponding to AKAP95-binding proteins (AKBPs) of 45, 52, and 69 kDa were detected in purified rat brain nuclear matrix using the radiolabeled AKAP95 probe (Fig. 6A). Identical results were obtained using recombinant AKAP95 labeled with digoxigenin (not shown), suggesting that the interactions are independent of AKAP95 phosphorylation. It is likely that the identified AKBPs represent distinct proteins since they were consistently observed in several preparations of nuclear matrix isolated in the presence of protease inhibitors. No binding proteins were detected using a truncated recombinant AKAP95-(170-386) probe (Fig. 6b), supporting the hypothesis that AKBP association occurs via the identified NMTS. Traditional Western blot analysis of similar blots confirmed that AKAP95 was greatly enriched in the nuclear matrix preparation (Fig. 6c), suggesting that AKBP-45, AKBP-52, and AKBP-69 are potential physiological binding partners for AKAP95.


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Fig. 6.   Far Western analysis of AKAP95. Protein extracts (50 µg) from rat forebrain (br), cerebellum (cb), kidney (kn), and purified brain nuclear matrix (nm) were separated by SDS-polyacrylamide gel electrophoresis (10%) and transferred to polyvinylidene difluoride membranes. The blots were probed with either 32P-labeled AKAP95-(1-386) protein to detect AKBPs (A), 32P-AKAP95-(170-386) protein missing the NMTS as a control (B), or affinity-purified antibodies to detect AKAP95 (C). Autoradiographs (A and B) were exposed for 16 h. Peroxidase-conjugated secondary antibodies and chemiluminescent detection was used for immunoblotting (C).

We employed the yeast two-hybrid method of interaction cloning to isolate cDNAs encoding AKBPs. Using an expressed Gal4 fusion of AKAP95 fragment (amino acids 109-201) containing the NMTS as bait, we recovered three positive clones from a mouse embryo library (1.3 × 106 independent clones total). Sequence analysis of the recovered cDNAs revealed that, although two clones encoded unique proteins, one clone encoded the C-terminal portion of p68 RH (amino acids 426-614). Control experiments confirmed that this region of RH specifically interacted with AKAP95; dihybrid crosses of the RH clone in yeasts co-expressing the Gal4 DNA binding domain alone fused to a nonrelevant AKAP (AKAP79) or fused with an AKAP95 fragment missing the NMTS (AKAP95-(378-687)) were negative, whereas crosses with yeast expressing AKAP95 fragments containing the NMTS (AKAP95-(1-386), (109), and (1)) were positive (Table I). Hence, the results indicate that the C-terminal fragment of RH specifically associates with AKAP95 in yeast, and the NMTS region of the AKAP is essential for the interaction.

                              
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Table I
Yeast two-hybrid assays

Direct binding of RH and AKAP95 was investigated in vitro using recombinant proteins produced in bacteria. On Far Western blots, radiolabeled full-length AKAP95 (amino acids 1-687) bound immobilized RH C-terminal fragment (amino acids 426-614) and the Type II regulatory subunit of PKA (RII) (Fig. 7A). In contrast, a C-terminal fragment of AKAP95 (amino acids 378-687) missing the NMTS bound RII but not RH. These results confirm that RH and RII binding sites within AKAP95 are distinct and provide further evidence that the RH interaction occurs via the AKAP95 NMTS region. Given these results and comparisons of Far Western blots of nuclear matrix extracts (e.g. Fig. 6) with Western blots using anti-p68 monoclonal antibody Pab204 (not shown), where the bands co-migrate, we propose that AKBP-69 is p68 RH.


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Fig. 7.   Association of RH with AKAP95. A, in vitro binding of radiolabeled AKAP95 fragments to recombinant p68 RNA helicase (amino acids 426-614) (RH) and Type II PKA regulatory subunit (RII). Recombinant AKAP95 fragments were radiolabeled to comparable specific activity with the catalytic subunit of PKA as described under "Experimental Procedures" and used to probe the indicated amounts of RH and RII immobilized on nitrocellulose slot blots. Washed blots were exposed to film for 12 h. Amino acids contained in each fragment are indicated in parentheses. B, co-localization of AKAP95 and RH. Swiss 3T3 fibroblasts transiently expressing RH were grown on glass coverslips, fixed with formalin, and permeabilized (top row) or extracted to isolate the nuclear matrix (bottom row) before staining with rabbit anti-AKAP95 (left) or mouse monoclonal anti-RNA helicase (center) antibodies. Texas Red anti-rabbit and fluorescein isothiocyanate anti-mouse secondary antibodies were used for fluorescent detection. Hoescht stain was used to stain DNA (right). C, isolation of AKAP95-RH complexes. Solubilized (Sol.), chromatin (Chr.), high salt (H.S.) and insoluble matrix (I.M.) fractions were prepared from transfected COS7 cells as described under "Experimental Procedures." Proteins (10 µg) from each fraction were separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot using anti-AKAP95 antibodies (alpha 95, upper panel). Precipitates were prepared from the solubilized fraction using either pre-immune sera (Pre.) or anti-AKAP95 sera (Imm.) and were analyzed for the presence of RH by Western blot (alpha RH, lower panel). Arrows indicate the expected positions of expressed AKAP95 (upper panel) and RH (lower panel).

To determine whether interaction occurs in mammalian cells, we examined the subcellular distribution of AKAP95 and RH using immunocytochemistry. RH (426) transiently expressed in mouse fibroblasts as a GST fusion localized exclusively to the nucleus, where it co-localized with DNA and endogenous AKAP95 (Fig. 7B, top row). Staining for the expressed RH (using anti-GST antibody) was specific, because no staining or signal cross-over was observed in untransfected cells (e.g. lower cells, top row). More significantly, RH and AKAP95 co-purified with the nuclear matrix extracted from transfected cells and exhibited staining patterns that were indistinguishable (Fig. 7B, bottom row). Similar results were obtained by staining for the helicase using Pab204 anti-RH antibody, whereas no nuclear staining was observed in cells expressing GST alone (not shown). Collectively, the results demonstrate that RH and AKAP95 occupy the same subnuclear compartment.

To further investigate cellular interaction, we isolated AKAP95-RH complexes from extracts of COS7 cells transfected with AKAP95-(1-687) and RH (426)-GST. Using buffer containing detergents and chelating agents (see "Experimental Procedures") we were able to partially extract AKAP95 from the nucleus (Fig. 7C, top panel). Analysis of Western blots confirmed that the expressed RH was also present in the solubilized fraction (Fig. 7C, bottom panel). Moreover, affinity-purified anti-AKAP95 antibodies co-precipitated RH from the preparation, whereas RH was not precipitated by pre-immune sera (Fig. 7C, bottom panel). These results suggest formation of a RH-AKAP95 complex in mammalian cells. Because the complex was isolated from the solubilized preparation, the results also suggest the interaction is stable in detergent and does not require divalent cations.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously identified both DNA binding and PKA binding domains within AKAP95 (17). Herein we identified distinct domains for nuclear import and interaction with nuclear matrix components. The single cluster of basic amino acids (RKRK) found to be important for nuclear import of AKAP95 conforms to the SV40-like class of nuclear localization signal found in many nuclear proteins (23), suggesting that the AKAP utilizes standard pathways for nuclear import. This result was not obvious given that nuclear matrix proteins form the structural foundation of the nucleus and might be expected to localize before functional assembly of the nuclear import machinery. However, the finding is consistent with our previous immunocytochemical studies of mitotic cells, where AKAP95 was found to enter newly formed daughter nuclei late in the mitotic cycle, after the nuclear envelope is formed but before chromatin is decondensed (18). Hence, AKAP95 is likely to be incorporated into the nuclear matrix after some initial structural framework is established.

Analysis of deletion mutants demonstrated that association of AKAP95 with the nuclear matrix was independent from DNA binding. A similar result was reported for the transcription factors AML/CBF (26) and YY1 (27), where nuclear matrix targeting and DNA binding domains were found to be distinct. In contrast, the RNA-binding protein ZNF74 was shown to require its zinc finger domain for nuclear matrix targeting (28). However, this domain may have dual functions since it was found to be important for both nucleic acid binding and interactions of ZNF74 with RNA polymerase II (28). Our findings with AKAP95 support the general concept that assembly of the nuclear matrix is highly dependent on specific protein-protein interactions and may occur in the absence of nucleic acids.

The identified NMTS of AKAP95 was found to be highly conserved and present in the genetic neighbor NAKAP95. A search of sequence data bases did not reveal other proteins containing this motif. Furthermore, the AKAP95 sequence is not significantly similar to nuclear matrix targeting signals found in the matrix-associated transcription factors AML/CBF and YY1 (26, 27). These findings are consistent with each nuclear matrix protein having a distinct set of binding partners within the nuclear matrix. Because the NMTS was found to mediate interactions with other proteins, we would predict that NAKAP95 associates with the same AKBPs as AKAP95. Interestingly, Westberg et al. (29) recently retrieved NAKAP95 (called HAP95 in their study) in a two-hybrid screen for proteins that bind RNA helicase A. Although the p68 RH identified as an AKBP in the present study shares little sequence similarity with RH helicase A, both proteins exhibit identical activities and belong to the same family of DEAD/H box helicases. Hence, several distinct AKAP95/RNA helicase complexes may be present in mammalian cells.

AKAPs are often found to associate with multiple binding partners, a characteristic that has led to their description as "scaffold" proteins (30). Because so few nuclear matrix interactions are known, it will be very informative to characterize other AKBPs and determine their specific binding properties. An intriguing aspect of these interactions is that they are likely to be regulated, since the nuclear matrix is reorganized at mitosis. This temporal regulation, the relatively small amounts of enzymes present in the nuclear matrix, and the harsh extractions needed to solubilize AKAP95 may hinder isolation of ternary complexes containing both AKBPs and PKA. Indeed, preliminary attempts to purify RH-AKAP95-PKA complexes have been unsuccessful.2 Nevertheless, the results described herein do not exclude formation of a complex containing AKAP95, PKA, DNA, and an AKBP, since AKAP95 contains distinct binding sites for each interaction. Such complexes would be exclusive to AKAP95, as other members of the family (ZAN75 and NAKAP95) do not contain a PKA-binding site.

The functional role of AKAP95 scaffolds may ultimately depend on the specific components bound. Recently, Tasken and co-workers (31) show that AKAP95 functions near condensing chromatin to recruit Eg7, a 150-kDa component of the 13 S-condensing complex. Association of AKAP95 with Eg7 was found to occur exclusively at mitosis and was not mediated by DNA. Although our studies failed to identify a 150-kDa AKAP95-binding protein, it is possible that one of the AKBPs may function as a "bridging" protein between AKAP95 and Eg7 at this stage of the cell cycle.

Our results, suggesting that p68 RH associates with AKAP95 within the nuclear matrix of interphase cells, implies a novel physiological role for this AKAP scaffold. Although the helicase was widely reported to be a nuclear protein, it was not previously known to be a component of the nuclear matrix. Localization of RH to this subnuclear compartment is consistent with models of the nuclear matrix as the major site of mRNA synthesis and processing (32, 33). Moreover, RNA helicases are known components of specific transcriptional complexes. Most notably, RH helicase A has been shown to mediate association of cAMP-response element-binding protein (CREB)-binding protein (CBP) with RNA polymerase II (34). Likewise, p68 RH has been found to associate with CBP (35). Our finding that AKAP95 may participate in such complexes through its interaction with RH provides a plausible mechanism for cAMP-regulated gene expression since the AKAP also provides a docking site for PKA, optimally targeting the kinase for its established role in regulating CBP/CREB transcriptional initiation complexes (36, 37). Further study of these complexes may help establish a relationship between hormonally regulated modifications within the nuclear matrix and induced changes in chromatin structure that result in altered patterns of gene expression.

    ACKNOWLEDGEMENT

We thank Dr. H. Stahl of the Universitat Konstanz for providing the PAb204 antibody.

    FOOTNOTES

* This work was supported by United States Public Health Service Grant DK52491 from the National Institutes of Health (to V. M. C.).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.

Dagger To whom correspondence should be addressed: Neurological Sciences Institute, Oregon Health Sciences University, 505 N. W. 185th Ave., Beaverton, OR 97006. Tel.: 503-418-2585; Fax: 503-418-2501; E-mail: coghlanv@ohsu.edu.

Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M101171200

2 J. A. Sayer and V. M. Coghlan, unpublished result.

    ABBREVIATIONS

The abbreviations used are: AKAP, A-kinase-anchoring protein; PKA, protein kinase A; GFP, green fluorescent protein; CHO, Chinese hamster ovary; TBS, Tris-buffered saline; CS buffer, cytoskeletal buffer; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; RH, RNA helicase; GST, glutathione S-transferase; NMTS, nuclear matrix targeting site; AKBP, AKAP95-binding protein; CBP, CREB-binding protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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