From the Neurological Sciences Institute, Oregon Health Sciences University, Beaverton, Oregon 97006
Received for publication, February 6, 2001
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ABSTRACT |
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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.
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.
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 [ 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
( 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
Ade/
His/
Leu/
Trp) were examined for
-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
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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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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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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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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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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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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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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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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENT |
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We thank Dr. H. Stahl of the Universitat Konstanz for providing the PAb204 antibody.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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.
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