From the Institute of Molecular Medicine and Genetics, Departments
of § Medicine, Surgery, and
** Cellular Biology and Anatomy, Medical College of Georgia
and the Augusta Veterans Affairs Medical Center, Augusta, Georgia
30912, the
Department of Cell and Molecular Physiology,
University of North Carolina, Chapel Hill, North Carolina 27599, and the ¶ Oncology Gene Therapy Program, The Toronto Hospital and
Department of Medical Biophysics, University of Toronto,
Toronto, Ontario M5G 2M1, Canada
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ABSTRACT |
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Protein kinase A-anchoring proteins (AKAPs)
localize the second messenger response to particular subcellular
domains by sequestration of the type II protein kinase A. Previously,
AKAP120 was identified from a rabbit gastric parietal cell cDNA
library; however, a monoclonal antibody raised against AKAP120 labeled
a 350-kDa band in Western blots of parietal cell cytosol. Recloning has
now revealed that AKAP120 is a segment of a larger protein, AKAP350. We
have now obtained a complete sequence of human gastric AKAP350 as well as partial cDNA sequences from human lung and rabbit parietal cells. The genomic region containing AKAP350 is found on chromosome 7q21 and is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, a protein associated with the
N-methyl-D-aspartate receptor. Rabbit parietal
cell AKAP350 is missing a sequence corresponding to a single exon in
the middle of the molecule located just after the yotiao homology
region. Two carboxyl-terminal splice variants were also identified.
Both of the major splice variants showed tissue- and cell-specific expression patterns. Immunofluorescence microscopy demonstrated that
AKAP350 was associated with centrosomes in many cell types. In
polarized Madin-Darby canine kidney cells, AKAP350 localized asymmetrically to one pole of the centrosome, and nocodazole did not
alter its localization. During the cell cycle, AKAP350 was associated
with the centrosomes as well as with the cleavage furrow during
anaphase and telophase. Several epithelial cell types also demonstrated
noncentrosomal pools of AKAP350, especially parietal cells, which
contained multiple cytosolic immunoreactive foci throughout the cells.
The localization of AKAP350 suggests that it may regulate centrosomal
and noncentrosomal cytoskeletal systems in many different cell types.
Transduction of signals from extracellular stimuli is most
commonly accomplished via ligand-receptor binding and generation of a
second messenger response. While increases in intracellular second
messengers have traditionally been viewed as global cellular events,
second messenger effects are often limited to particular regions or
organelles within cells. Investigations over the past decade have led
to a greater understanding of the mechanisms responsible for the
compartmentalization of second messenger effects. These studies have
identified a diverse group of scaffolding proteins that sequester both
protein kinases and protein phosphatases within specific cellular
domains (1, 2). In the case of cAMP-dependent protein
kinases, protein kinase A-anchoring proteins
(AKAPs)1 tether the protein
kinase A holoenzyme through binding to the regulatory subunit dimer. A
growing group of AKAPs that bind the regulatory subunit of type II
protein kinase A (RII) have been reported over the past
several years. The first RII-binding protein was identified
over 15 years ago when microtubule-associated protein 2 (MAP-2) was
described (3, 4). Since that time, several AKAPs have been identified,
localizing the type II protein kinase A to thyroid cytoskeleton (5),
mitochondria (6), the Golgi apparatus (7, 8), centrosomes (8, 9), and
microtubules (7, 10), among other places. More recently, an AKAP with binding capacity for the regulatory subunits of type I protein kinase A
has also been reported (11).
A number of AKAPs are associated with the cell cytoskeleton and appear
to regulate plasma membrane events possibly through anchoring at the
sites of interaction between cytoskeletal elements and membrane
receptors and channels. Ezrin, a member of the ERM family of
F-actin-associated proteins that is phosphorylated in response to
mediators that elevate cAMP responses in gastric parietal cells (12),
contains an amphipathic We have previously reported the cloning and initial characterization of
a protein kinase A-anchoring protein, AKAP120, from a rabbit gastric
parietal cell cDNA library (18). It is now apparent that the
initial AKAP120 sequence is a fragment of a larger 350-kDa AKAP
(AKAP350). We have completed the sequence of human AKAP350, which
represents a multiply spliced family of proteins coded for by a single
gene sequence on human chromosome 7q21. Immunolocalization demonstrates
that AKAP350 is associated with centrosomes in many cells and also
shows noncentrosomal organization in certain epithelial cells,
including gastric parietal cells.
Materials--
Recombinant RII was purified from
bacteria expressing the protein based on a pET11D-RII
expression vector (a gift of Dr. John Scott, Vollum Institute). A human
tracheal epithelia-enriched LambdaZap cDNA library was a gift
from Drs. S. Gabriel and D. Fenstermacher (University of North
Carolina, Chapel Hill). Cultured CalU3 and HBE16Eo cells were a gift of
Dr. J. Stutts (University of North Carolina, Chapel Hill). Polyclonal
anti- Monoclonal Antibody Production--
Recombinant
polyhistidine-tagged AKAP120-(183-1022) was prepared by the methods of
Dransfield et al. (18). Recombinant protein was used to
immunize mice, and serum titers were monitored by enzyme-linked
immunosorbent assay using the recombinant protein (19). Splenocytes
were harvested and fused with myeloma cells. Culture supernatants from
the resulting hybridomas were screened by enzyme-linked immunosorbent
assay, and productive cell wells were cloned to monoclonal lines by
serial dilutional screening. Four monoclonal antibodies were prepared
that also detected protein in Western blots. The 14G2 monoclonal
antibody, an IgG1, was used for the present studies because
of its detection sensitivity. Monoclonal antibodies were concentrated
by preparation of purified IgG from culture supernatants
(University of Georgia Monoclonal Facility, Athens, GA).
EGFP-AKAP120--
Full-length AKAP120 nucleotide sequence
previously cloned into pBluescript (18) was excised with an
EcoRI restriction digest and ligated in-frame into the
pEGFP-C2 vector (CLONTECH). 100 ng of plasmid DNA
was transiently transfected into MDCK cells (35-mm dishes) with the
Effectene reagent (Qiagen) according to the manufacturer's
instructions (20). Cells were grown to confluency and lysed with 1%
SDS stop solution.
Western Blot Analysis--
Sample extracts from cells and
tissues were resolved by SDS-PAGE (3-10% gradient gels) and
transferred for 2 h at 750 mA to nitrocellulose (0.22 µm) for
subsequent Western blotting. Nitrocellulose blots were blocked with 5%
nonfat dry milk in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl for 16-24 h at 4 °C. Blots were then probed in
0.5% nonfat dry milk in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl for 1 h at room temperature with a monoclonal
antibody against AKAP350 (14G2; 1:500). After the primary incubation,
the blots were washed three times for 15 min each with 25 mM Tris-HCl, pH 7.5, 150 mM NaCl and then
incubated with horseradish peroxidase-conjugated anti-mouse IgG
(1:2500) for 1 h at room temperature. The blots were finally
washed three times for 15 min each with 25 mM Tris/HCl, pH 7.5, 150 mM NaCl, and immunoreactivity was detected
with chemiluminescence (Renaissance; NEN Life Science Products) and autoradiography.
Cloning and Sequencing of AKAP350--
Both 3'- and 5'-rapid
amplification of cDNA ends (RACE) were used to clone the human
gastric AKAP350 beginning with sequence information from the human
genomic BAC AA004013, which showed homologies with the original AKAP120
sequence. A single 3'-RACE and multiple rounds of 5'-RACE were
performed with the Marathon system (CLONTECH) to
complete the recloning of human AKAP350 from an end-adapted human
gastric cDNA (Marathon cDNA, CLONTECH).
Similarly, a rabbit parietal cell cDNA with Marathon adapters was
constructed from poly(A) mRNA from a greater than 95% pure rabbit
parietal cell preparation. All RACE products were cloned into
pBluescript-T (21). Further human sequence information was obtained
from the total sequencing of an expressed sequence-tagged cDNA from
the human promyeloblast cell line KG1a, PMY2245 (22). Finally,
biotinylated recombinant RII was used to screen a human
tracheal cDNA library in LambdaZap using a standard protein overlay
technique. Phage plaques were identified by incubation in
streptavidin-alkaline phosphatase followed by colorimetric detection
with a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate
substrate. Clones were rescued into pBluescript and sequenced. One
clone showed homology with rabbit AKAP120 and was sequenced completely.
Northern Blot Analysis--
Total rabbit RNAs were prepared from
>95% pure parietal cells, gastric fundic mucosa, and lung (23).
mRNA was resolved on 1% agarose/formaldehyde gels and transferred
to Magnagraph (MSI, Westboro, MA). A human multitissue Northern blot
was purchased from CLONTECH. Rabbit blots were
probed with a 1650-nt probe corresponding to the 3'-end of rabbit
AKAP350 labeled by random priming in the presence of
[ Splice Variant Analysis--
Total RNAs were prepared from
tissues and cells using RNASTAT-60 (23). The Advantage RT for PCR kit
(CLONTECH) was used to construct cDNA from 2 µg of total RNA using random hexamer primers. Rabbit cDNAs were
constructed for parietal cell, spleen, liver, kidney, lung, heart,
forebrain, hind brain, and cerebellum and for mucosa from fundus,
antrum, duodenum, jejuneum, ileum, and colon. Human cDNAs were also
prepared from total RNA obtained from stomach, lung, and kidney, as
well as the CalU3 and HBE16Eo cell lines. Gene-specific primers were
constructed immediately upstream and downstream from the splice 1 exon
(sense, GAACAGTTGGAAGATATGAGACAGGAAC; antisense,
GATGCCTCTGCTTCTGACCTCCA), and products were amplified using Advantage
Polymerase (CLONTECH) with 40 cycles: 95 °C for 15 s, 60 °C for 10 s, and 68 °C for 60 s.
Amplified products were separated on 2% Nuseive 3:1 agarose gels. To
amplify the alternative 3'-end coding sequences (splice 2) in human
samples, a common sense primer (GGTCGGCCGTCAGAGTATCC) paired with
antisense primers specific for either the gastric sequence
(AGGATTATCTTCTCATGCCAGCA) or the sequence derived from human lung
(ATCCTAATGAGTGTGAAAGAATT) were employed using the same amplification
protocol described above. The identities of amplified products were
confirmed by gel isolation and DNA sequencing.
Immuncytochemistry in Rabbit Tissues--
For investigation of
tissue distribution, under anesthesia New Zealand White rabbits were
canulated through the distal aorta and perfused sequentially with
phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in
phosphate buffer, pH 7.4. Tissues were excised and post-fixed for
2 h in 4% paraformaldehyde and then infiltrated with sucrose and
embedded in OCT medium. Five-µm frozen sections were cut and mounted
on gelatin-coated slides. For staining of gastric glands, isolated
gastric glands were prepared from New Zealand White rabbits as
described previously (24). Glands were fixed in 4% paraformaldehyde
for 30 min at 4 °C, washed in cold PBS, and then permeabilized with
0.3% Triton X-100 in PBS. Glands were then adhered to
polylysine-coated slides.
For staining glands and frozen tissue sections, slides were blocked
with 17% donkey serum for 30 min and then incubated with 14G2
monoclonal antibody (1:50) or a class-matched nonspecific monoclonal
IgG (Sigma) for 2 h at room temperature or overnight at 4 °C.
Following washing, antibody localization was detected with incubation
with Cy3-donkey anti-mouse IgG antibodies for 30 min at room
temperature. Slides were mounted with Prolong Antifade solution
(Molecular Probes). Tissue sections were examined under a Zeiss Axiphot
microscope equipped with a Sensys digital camera (Photometrics).
Gastric glands were examined with confocal fluorescence microscopy
(Molecular Dynamics, IMMAG Imaging Core Facility) using maximum
intensity projections assembled from 40 0.29-µm optical sections.
Immunocytochemistry in Cultured Cells--
HCT116 colon
adenocarcinoma cells were plated sparsely on number 1 glass coverslips
for 24 h. Cells were washed in PBS and briefly permeabilized in
1% Triton X-100, 45 mM Pipes, 45 mM Hepes, pH
6.9, 10 mM EGTA, 5 mM MgCl2, 0.5 mM AEBSF for 60 s at 4 °C followed by fixation in
For immunolocalization in MDCK cells, MDCK (type II) cells were grown
on 23-mm Transwell clear filters for 3 days to establish highly
polarized monolayers. HBE cells were cultured on Transwell filters for
3 days. Cells were then fixed as above for HCT116 cells. Cells were
permeabilized and blocked in 10% donkey serum and then incubated with
the following primary antibody: 14G2 monoclonal antibodies (1:30);
polyclonal rabbit anti-RII (1:1000); rat monoclonal anti-ZO-1 (1:100, a tight junction marker); rabbit anti- Parietal Cell Preparations--
Isolated gastric glands and
isolated parietal cells were prepared from New Zealand White rabbits as
described previously (25). Subfractions of gastric parietal cells were
prepared by sequential centrifugation of homogenates at 1000, 4000, 15,000, and 100,000 × g to prepare four microsomal
pellets and a final high speed supernatant fraction (26). Parietal
cells were maintained in primary culture on Matrigel-coated coverslips,
as described previously (27). For immunocytochemistry, cultured cells
were fixed as for HCT116 cells. Cells were dual-stained as described
above with 14G2 and polyclonal anti- Identification of Endogenous AKAP120
Immunoreactivity--
Recombinant polyhistidine-tagged
AKAP120-(183-1022) was used to make a monoclonal antibodies against
the AKAP. Western blots of rabbit parietal cell cytosolic proteins
probed with the monoclonal antibody for AKAP120 (14G2) revealed a major
band at 350 kDa and a variably detected 250-kDa band (Fig.
1). A 200-kDa band was also
intermittently noted. In parietal cells, the variable observation of
the 200- and 250-kDa immunoreactive bands in multiple preparations has
suggested that they are proteolytic breakdown products. In preparations
of protein from rabbit parietal cell subfractions, more than 85% of
the total AKAP350 was found in the high speed supernatant (data not
shown). A similar banding pattern was seen in a survey of other rabbit
tissues by Western blots of protein from duodenum, jejunum, ileum,
colon, and liver (Fig. 1). While the most prominent immunoreactive
species in all rabbit tissues was a 350-kDa band, 250- and 200-kDa
species were also observed. Western blots of cultured epithelial cell
lysates also were examined, including nonpolarized HeLa cells and
polarized MDCK cells (Fig. 1). Both cultured cell lines demonstrated
the 350- and 250-kDa immunoreactive bands. In the MDCK cells, the
250-kDa species was consistently more prominent, although the ratio
between the 250- and 350-kDa bands varied considerably.
Although AKAPs tend to migrate in acrylamide gel electrophoresis to
higher than predicted molecular weights (8), in light of the Western
blots, it was necessary to investigate the possibility that the
full-length sequence of the AKAP had not been cloned. A single
experiment provided the necessary evidence that the sequence was
incomplete. The known AKAP120 sequence was cloned into the pEGFP-C2
vector and transiently transfected into MDCK cells. Duplicate Western
blots of cell lysates were then probed with anti-AKAP120 (14G2) and
anti-EGFP antibodies. The 14G2 antibody recognized the endogenous 250- and 350-kDa bands as well as a 150-kDa band; however, only the 150-kDa
band was seen when probing with anti-EGFP (data not shown). This result
indicated that AKAP120 cDNA sequence could not account for
endogenous immunoreactive proteins, and therefore the full-length
sequence had not been cloned.
Cloning of AKAP350--
We sought to complete the cloning from
rabbit parietal cells and human gastric cDNAs. Both 3'- and 5'-RACE
were used to identify the remainder of the nucleotide sequence. Because
a human genomic DNA sequence from chromosome 7q21 corresponding to
AKAP120 became available at the time, we chose to focus on the human
clone. This has led to the cloning of the full-length human sequence
composed of 11,490 base pairs with an open reading frame of 10,593 nucleotides (Fig. 2). The 5'-end of the
sequence contains a strong consensus initiation sequence, which is
preceded by two upstream in-frame stop codons. From the first in-frame
ATG, this sequence codes for 3531 amino acids with a predicted
molecular mass of 409 kDa and a pI of 4.8. Because of its migration in
SDS-PAGE, we have named this new full-length sequence AKAP350. The
sequence contains 58 phosphorylation sites for protein kinase C, 56 for
casein kinase II, and three for tyrosine kinase. protein kinase A
phosphorylation sites are predicted at residues 1860, 2285, 2727, and
3076. An RII binding region previously identified in the
AKAP120 sequence is found from amino acid 2174 to 2187 in the human
AKAP350 (Fig. 2). These sites are identical except for a single
isoleucine to valine substitution, which should not affect the
integrity of the amphipathic helix. Four leucine zipper motifs are
present between amino acids 313 and 334, 391 and 412, 2651 and 2672, and 3211 and 3239 (Fig. 2).
A closer examination of the peptide sequence for AKAP350 reveals a
structure involving multiple coiled-coil domains throughout the
protein, especially from amino acid 739 to 2270. The AKAP350 protein is
15.3% glutamate, 9.3% glutamine, and 12.4% leucine by total
amino acid composition. In 62 individual locations, two consecutive
glutamate residues are found, and in 21 locations, two consecutive
glutamine residues are found.
By conducting searches of on-line data bases including Fasta, PROSITE,
and BLAST, other proteins possessing significant homology with the
human AKAP350 were identified. The original rabbit parietal cell
AKAP120 protein sequence is 78% identical to the human gastric AKAP350
(HGAKAP350) sequence. Yotiao, a recently described ligand of the NR-1
subunit of the NMDA receptor (29) shares the same genomic DNA region as
AKAP350. This protein is 99% identical to amino acids 1-1249 (YHR,
Fig. 3) of HGAKAP350. The human AKAP350 cDNA maps onto chromosome 7q21 in the genomic DNA of BACs 4013, 0066, 3086, and 0120 while splicing in and out of yotiao exons. Approximately 50% of the coding sequence for yotiao is found in exons
for AKAP350. Another protein with significant homology is pericentrin,
a 220-kDa coiled-coil protein found in centrosomes and pericentriolar
material. Pericentrin shares a 21% identity and 47% similarity with
AKAP350. Other centrosomal associated proteins sharing similarity with
AKAP350 include CENP-F (20.7%), and CEP250 (20.2%). Other
cytoskeletal proteins with 20-24% identity include two intermediate
filament binding proteins, plectin and tricohyalin, as well as a number
of other proteins with extensive coiled-coil domains including myosin
heavy chain, giantin, and the Golgi antigen GCP372.
Additional sequence for AKAP350 was acquired by examination of other
cDNAs obtained from human lung, rabbit parietal cell, and the
longest expressed sequence tag with homology to the known AKAP
sequence, PMY2245 (Fig. 3). Sequencing of the PMY2245 clone from
a human promyeloblast cell line revealed 100% identity to the human
gastric AKAP350 (HGAKAP350) over approximately 4 kb of sequence at the
3'-end (Fig. 3). Human lung cDNA yielded a clone (HLAKAP350)
with 99% protein identity and 95% DNA identity over a 4-kb segment
also at the 3'-end of the AKAP. From rabbit parietal cell cDNA, a
partial sequence was obtained from 5'- and 3'-RACE products, accounting
for all but the 1000 nt at the 5'-end, 91% of the total human gastric
AKAP350, with 87% protein identity and 87% DNA identity to HGAKAP350.
AKAP350 Genomic Structure--
A comparison of the resolved
cDNA sequences from human stomach, human lung, rabbit parietal
cells, and the expressed sequence tag cDNA (PMY2245) with the
genomic sequences have allowed us to map the exons of this gene. The
AKAP350 exons are scattered over 200 kb of genomic DNA (Fig.
4a). This genomic region
apparently codes for a multiply spliced gene that includes DNA for the
yotiao protein and several forms of AKAP350. Upstream from the first yotiao exon, there is a GC-rich region that probably represents a
TATA-less promoter. The first exon of AKAP350 lies between exons 9 and
10 of yotiao. Upstream from the AKAP350 first exon is a promoter region
that contains two putative TATA box elements.
At least three major sites of alternate splicing exist in the AKAP350
sequence. The first location is a highly spliced region where three
separate splice choices are made including a read-through to complete
the final yotiao exon or a choice in AKAP350 of two different splice
acceptors for a common splice donor (Fig. 4b). The latter
splice variant (Fig. 4b) was observed in the rabbit parietal
cell AKAP350 cDNA sequence, representing the loss of an entire exon
immediately following the yotiao homology region. To examine the
incidence of this splice variant, this region was amplified from
multiple rabbit tissue cDNAs with specific primers (Fig.
5a). Most mucosal tissues from
rabbit gastrointestinal tract predominantly express the transcript with
the S1 region deleted. Overloading of samples reveals that parietal
cells and other gastrointestinal mucosae do express a small amount of
the larger transcript. In contrast with the mucosal samples, in smooth
muscle from the gastric wall the larger splice variant predominates.
Other rabbit tissues such as heart and brain reveal the presence of the
larger variant exclusively. The third (highest) band observed in
forebrain, hind brain, and cerebellum was sequenced and found to be an
artifactual amplification of phosphofructokinase.
Alternative splicing also was found at the C terminus of the protein
(Fig. 4c). While the human gastric, rabbit parietal cell, and promyeloblast cDNAs all showed the same 3' sequences, the cDNA obtained from the tracheal cDNA library (HLAKAP350)
demonstrated a different 3' sequence resulting from a read-through
extending past the splice donor site (similar to that seen for the
final yotiao exon). This "missed splice" allows the cDNA
sequence to continue for another 435 base pairs before terminating. The
incidence of these variants was again surveyed using locus-specific
primers and amplification from cDNA of several human tissues and
the Calu-3 and HBE cultured cell lines (Fig. 5b). Both
splice variants were observed in pancreas, gastric and lung cDNAs
(Fig. 5b). However, the 497-base pair product, which is
characteristic of the cDNA derived from the human tracheal
cDNA, was not observed in either Calu-3 or HBE cells. Only the
397-base pair product was observed in these cell lines.
Finally, comparison of the PMY2245 cDNA from promyeloblasts with
the sequences from human lung and human stomach showed a loss of 8 amino acids (24 nt). Examination of the genomic sequence showed that a
variation in the choice of splice donor sites accounted for this small
sequence deletion. There are also several instances in the rabbit
AKAP350 sequence where 1-3 amino acids have been lost or gained.
Because the rabbit genomic sequence is not available, we cannot
presently evaluate whether these differences are due to splice
variation or interspecies variations.
Northern Blot Analysis of AKAP350 Expression--
Northern
blotting with the 3' end of HGAKAP350 as a probe, revealed an 11-kb
message found in rabbit parietal cells and rabbit fundus, as well as in
rabbit lung (Fig. 6). Human multitissue Northern blots probed with either a 1.6-kb fragment from the 5'-end of
HLAKAP350 (Fig. 6) or a 1.0-kb fragment of the 3'-end of rabbit parietal cell AKAP350 (data not shown) demonstrated two bands, 9.5 and
11 kb, most prominent in kidney and skeletal muscle and to a lesser
extent in lung. Only the 9.5-kb message was noted in liver, and only
the 11-kb message was observed in heart and brain.
AKAP350 Localization in Rabbit Tissues--
To investigate the
subcellular localization of AKAP350, the 14G2 monoclonal antibody was
used to stain rabbit tissue sections and isolated gastric glands. In
the gastric fundus, parietal cells demonstrated multiple foci of
immunostaining throughout the cytosol (Fig.
7a). Confocal fluorescence
microscopic examination of isolated gastric glands also demonstrated
multiple foci of staining in parietal cells (Fig. 7b). This
immunostaining in parietal cells did not coincide with staining for
either the H/K-ATPase-containing tubulovesicles or F-actin-staining
secretory canalicular membranes (data not shown). In the esophagus,
basal cells appeared to show a single point of bright fluorescence
(Fig. 7c). In the jejunum, the most prominent staining was
observed in submucosal lymphoblasts, which showed a single focus of
perinuclear staining (Fig. 7d). In the ileum and colon,
epithelial cells of the deep crypts were strongly stained with multiple
bright foci just beneath the apical membranes (Fig. 7, e and
f). In pancreatic islets, strong AKAP350 immunostaining was
present in all of the endocrine cells of the islet (Fig.
7g). In many cells, multiple foci of staining were apparent
in the perinuclear region. In contrast, adrenal medullary cells
demonstrated only a single focus of AKAP350 immunostaining in all cells
(Fig. 7h). In the kidney collecting duct, AKAP350 staining
was observed as a single focus of staining deep to the apical membrane
(Fig. 7i). Single foci of immunostaining also were observed
in primary and secondary germ cells in the testes (Fig. 7j).
Finally, in the epithelial cells lining the ductus efferens and the
bronchial epithelium, mutiple foci of AKAP350 immunostaining were
observed deep to the apical membranes (Fig. 7, k and
l).
AKAP350 Is Associated with Centrosomes--
The perinuclear
localization of AKAP350 in many cells suggested its association with
centrosomes. We therefore studied the localization of AKAP350 in
HCT116, a moderately differentiated colonic adenocarcinoma cell line.
In interphase cells, AKAP350 immunostaining colocalized with
Since HCT116 cells are not polarized, we also sought to investigate
AKAP350 distribution in polarized cells. MDCK cells, a well established
polarized kidney cell line, were grown on permeable filters. While
multiple foci of immunostaining for RII were present throughout the cytosol, AKAP350 staining was only present in one focus
per cell colabeling with major points of RII staining (Fig. 9b). Colabeling of MDCK cells
for AKAP350 and
Finally, since several rabbit cells including gastric parietal cells
and bronchial epithelium showed multiple foci of AKAP350 staining, we
also examined in greater detail AKAP350 staining in primary cultures of
rabbit parietal cells and human bronchial epithelial cells. In parietal
cells, multiple foci of AKAP350 staining were observed throughout the
cell (Fig. 10). One perinuclear focus
coincided with staining by antibodies against Protein kinase A-anchoring proteins represent a diverse
superfamily of scaffolding proteins. This report clarifies the
characteristics of a novel multiply spliced AKAP family. Monoclonal
antibodies raised against AKAP120 identified proteins of 350 and 250 kDa in most tissues and cells. AKAP120 was originally cloned from a
rabbit parietal cell cDNA expression library using
32P-labeled RII as a probe. The single clone
isolated yielded an open reading frame of 3500-nt cDNA coding for a
recombinant expressed protein migrating with an apparent molecular mass
of 120 kDa (18). As described above, eukaryotic expression of this AKAP
sequence as a fusion with the EGFP indicated that the AKAP120 clone
could not account for the endogenous 350-kDa immunoreactive species. The results of our cloning experiments reported here show that the
original clone was not full-length and included an erroneous stop
codon. The full-length sequence of AKAP350 contains at least two major
areas for alternative splicing. In addition, genomic sequence also
contains a major spliced product that codes for the previously
described yotiao protein (29). The present sequence length matches the
major mRNA length observed in rabbit and human tissues of over 11 kb. Our results demonstrate that splice variants may be differentially
distributed among tissue and cell types. Indeed, since smaller 9.5-kb
species of mRNA have been identified in Northern blots, it is
possible that further splice variants may be present.
Previously, a multiply spliced murine AKAP gene has been described,
yielding six isoforms, collectively known as AKAP-KL (30). Parallel
cloning of the AKAP350 in both rabbit and human has revealed multiple
splice variants. Additionally, the existence of the yotiao protein
coding region as a 5'-end splice variant indicates that the AKAP350
genomic region produces a complex family of protein products from
internal alternative exon splicing as well as alternative splicing at
both the 5'- and 3'-ends. The function of specific spliced exons
remains to be determined. It is tempting to suggest that specific
spliced exons may account for proper targeting of the AKAP or
interaction with specific proteins. This possibility appears especially
possible for the highly spliced region within BAC AA0066, which
contains the alternate splice point for the terminal exon of yotiao as
well as the major internal alternatively spliced exon in AKAP350,
immediately following the yotiao homology region. The AKAP350 sequence
lacking this exon was the major sequence in rabbit parietal cells as
well as in most gastrointestinal mucosae. In contrast, the sequence
including this 140-nt sequence was predominant in gastric wall smooth
muscle and brain tissue. Interestingly, this exon includes a pair of
adjacent cysteines, perhaps indicative of a possible covalent
cross-linking point either for homodimerization or interaction with
other proteins.
A second major splice variation occurs at the C terminus of AKAP350 as
described above. The AKAP350 sequence isolated from the tracheal
cDNA library results in an alternative 16 carboxyl-terminal amino
acids not found in the human gastric or the promyeloblast sequence due
to a splice variant in the next to last exon. Since the end of this
exon coincides with multiple consecutive adenosines, it is unlikely
that this is the true 3'-end in this mRNA. It is more probable that
oligo(dT) priming occurred internally at the polyadenine in the
mRNA 3'-untranslated region. Analysis of this splice variant has
demonstrated its presence in several tissues. However, while both 3'
splice variant sequences were detected in whole lung cDNA, when we
examined cDNA from CalU3 cells (a lung serous cell line) and a
human bronchial epithelial cell line, we were unable to detect the 3'
splice variant originally isolated from the bronchial epithelial cell
library. Since the library was constructed from only an enriched
population of tracheal epithelial cells, it seems likely that the
message must emanate from a nonepithelial lung cell. These data further
suggest that there is a cell-specific distribution of these AKAP350
isoforms. In addition to the two major splice variants, the PMY2245
expressed sequenced tag cDNA also demonstrated a minor splice
variant yielding a deletion of eight amino acids (24 nt) because of an
alternate splice donor choice. Given the theoretical function of AKAPs,
it appears reasonable that these splice variants may define either
specific intracellular targeting for particular AKAP350 isoforms in
different cell types or their association with specific scaffolding components.
Alternative splicing of this nature has been well characterized in
several complex spliced protein families including odorant receptors
(31), neurexins (32), and myofibrillar proteins (33). Contractile
protein genes provide a model system for study of this process. Either
a particular gene among members of a multigene family is selected for
expression or a spectrum of different proteins may be generated from a
single gene. A prime example of both is found in troponin T, a member
of the thin filament of vertebrate sarcomere with specific subtypes in
cardiac as well as fast and slow skeletal muscle (Tc, Tf, Ts). Each
subtype is coded by a gene with a unique promoter region and terminates
at a single polyadenylation site. Within each subtype, however, a
series of adjacent exons are intermittently spliced out, forming a
large number of isoforms (32 for Tf) coded by the same gene. Gene
transcripts are assembled from variations in both donor and acceptor
splice site selection (33).
The results of our investigations have shown that AKAP350 and yotiao
are products of the same genomic region on chromosome 7q21. Yotiao was
identified as a protein in brain that interacts with the NMDA receptor
subunit NR1 (29). Interestingly, the regions that mediate this
interaction also are present in the amino-terminal region of AKAP350.
Examination of the genomic sequence region upstream of the yotiao
coding region suggests that there is a GC-rich putative promoter
region. The genomic region upstream of the 5'-end of the AKAP350
sequence contains a putative TATA box and other putative regulatory
elements. Thus, the two proteins probably have separate transcriptional
regulators. The 14G2 antibody that we have used in our
immunolocalization studies recognizes a region of AKAP350 that is not
contained in yotiao. Thus, our localization studies are specific for
AKAP350. In contrast, the antibodies used in studies of yotiao are
raised against regions that are predicted to cross-react with AKAP350
(29). Indeed, it is interesting to note that the size of the major
species observed in whole brain was considerably larger than the size
of yotiao protein exogenously expressed in fibroblasts (29). Some of
the assignment of yotiao staining and distribution may need to be reevaluated with more specific antisera against the far amino or
carboxyl termini of yotiao. In addition, it will be of interest to
identify whether AKAP350 in brain also interacts with an NMDA receptor.
An extensive amount of study has been dedicated to elucidating the
function of the centrosome and its components as they relate to the
general physiology of the cell. The centrosome is the principal microtubule organizing center in most cells, composed of a pair of
centrioles and a surrounding matrix, also referred to as the pericentriolar material. Each centriole has bilateral appendages at one
pole known as transitional fibers (34). AKAPs are well characterized as scaffolding proteins, sequestering the
RII subunit and consequently the catalytic subunit of the
protein kinase A in a location where phosphorylation of a particular
substrate will be needed for some key regulatory process (39, 40).
Centrosomes, as the site of microtubule nucleation and mitotic spindle
formation as well as a binding site for several centrosome-associated
proteins, provide a host of possible substrates for regulation by
protein kinase A phosphorylation. A role for AKAPs in microtubule
nucleation has previously been established in the case of MAP-2 (3). In
immunocytochemical analysis, Confocal immunofluorescence microscopy demonstrated AKAP350 staining
associated with large globular complexes scattered throughout the
cytosol of parietal cells, lending support to the idea that this
protein is part of a complex of regulatory molecules. The presence of
multiple AKAP350-containing structures in parietal cells may reflect
its involvement in the massive cytoskeletal reorganization that takes
place when parietal cells are stimulated to secrete acid (27). Other
less differentiated cell types, especially those that have undergone
transformation into a phase of unregulated growth, may require AKAP350
simply for microtubule nucleation at the centrosome; hence, the
distribution is strictly limited to this organelle. Examination of the
mitotic spindle apparatus in the stages of mitosis in HeLa and HCT116
colon adenocarcinoma cells demonstrated the presence of AKAP350 not
only at the centrosome throughout mitosis but also at the cleavage
furrow in anaphase and telophase for both cell types. This finding
might suggest a possible role for AKAP350 in establishment or
activation of contraction to form the contractile ring in telophase
cells. Interestingly, no RII was observed in association
with AKAP350 in the cleavage furrow. This may suggest that an alternate
splice variant, different from centrosomal AKAP350, is associated with
a different cytoskeletal system. Of note, the extensive coiled-coil
structure in AKAP350 shows homology not only to centrosome-associated
proteins (e.g. pericentrin) but also to intermediate
filament-associated proteins and myosins. In addition to alternate
cytoskeletal association, it is also likely that AKAP350 is scaffolding
other regulators beyond type II protein kinase A. Along these lines,
AKAP79 is associated with both protein phosphatase and protein kinase C (28). Further investigations will be required to elucidate components of the AKAP350 scaffolding complex and the basis of its cytoskeletal targeting.
In summary, we have cloned and characterized a new family of 350-kDa
AKAPs that are the products of a multiply spliced gene region that also
produces the previously described yotiao protein. In most tissues,
AKAP350 localizes to the centrosome. However, in several epithelial
cell types, multiple extracentrosomal foci are present. These results
suggest that AKAP350 may be a multifunctional scaffolding protein.
INTRODUCTION
Top
Abstract
Introduction
References
-helix that binds RII (13). AKAP100, which is enriched in cardiac and skeletal muscle (14), localizes the type II protein kinase A to the sarcoplasmic reticulum and is hypothesized to play a role in the regulation of membrane channel activity. Similarly, protein kinase A anchoring to the postsynaptic densities (15) by AKAP79 is required for modulation of
AMPA-kainate currents in hippocampal neurons (16). AKAP15 targets type
II protein kinase A to L-type calcium channels in transverse tubules of
skeletal muscle and influences rapid voltage-dependent potentiation (17). MAP-2 binds type II protein kinase A and sequesters
it in association with dendritic microtubules (4). Phosphorylation of
MAP-2 by protein kinase A inhibits microtubule nucleation in
vitro (3). The distribution of all of these AKAPs suggests that
specific scaffolding functions can account for subcellular signaling specificity.
EXPERIMENTAL PROCEDURES
-tubulin antibody was purchased from Babco (Berkeley, CA).
Monoclonal anti-
-tubulin was purchased from Sigma. Cy2-, Cy3-, and
Cy5-conjugated secondary antibodies were purchased from Jackson
ImmunoResearch Labs (West Grove, PA). Effectene transfection reagent
was obtained from Qiagen (Valencia, CA). Prolong Antifade,
4',6-diamidino-2-phenylindole, and Alexa 488-conjugated secondary
antibodies were from Molecular Probes, Inc. (Eugene, OR). E-GFP-C2
vector, Advantage Taq, and Marathon cloning kits were
purchased from CLONTECH. All DNA sequencing was
performed using dye terminator chemistry automated sequencing in the
Molecular Biology Core Facility at the Medical College of Georgia or
the DNA Sequencing Facility, University of North Carolina.
Oligonucleotides were also synthesized by the Molecular Biology Core
Facility. [
-32P]dCTP was purchased from NEN Life
Science Products. Random priming kits were purchased from Amersham
Pharmacia Biotech. HCT116 cells were obtained from ATCC.
-32P]dCTP. Human blots were probed with both a
1650-nt probe corresponding to the 3'-end of human gastric AKAP350 or a
1600-nt probe corresponding to the 5'-end of human lung AKAP350. Blots
were probed overnight at 42 °C and then washed to high stringency
(0.1× SSC, 65 °C). Dried blots were exposed to either x-ray film at
70 °C or PhosphorImager screens for 72 h.
20 °C methanol for 6 min. Cells were blocked with 17% donkey
serum, 0.1% Tween 20 in PBS and then incubated simultaneously with
14G2 (1:30) and rabbit anti-
-tubulin (1:400) for 2 h at room
temperature. The cells were then simultaneously incubated with
ALEXA-488-conjugated anti-mouse IgG and Cy5-conjugated anti-rabbit IgG
for 60 min. Following washing in PBS, the cells were incubated in
4',6-diamidino-2-phenylindole (1 mM) for 5 min and washed
in PBS. Coverslips were inverted and mounted with Prolong Antifade.
Cells were examined for triple labeling on an Axiophot microscope as
described above.
-tubulin (1:400); or murine anti-
-tubulin (1:250). Antibodies were visualized with secondary antibodies conjugated with Cy2, Cy3, and Cy5.
Immunostained cells were examined by confocal microscopy using maximum
intensity projections of 40 0.29-µm optical sections.
-tubulin.
RESULTS
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Fig. 1.
Immunoreactivity with 14G2 monoclonal
antibody. Western blots demonstrated a 350-kDa immunoreactive band
(upper arrow), as well as the intermittently
detected 250- (lower arrow) and 200-kDa bands in
protein extracts (50 µg/lane). Left, immunoreactivity was
compared between isolated rabbit parietal cells (PC) and the
following rabbit gastrointestinal tissues: duodenum (D),
jejunum (J), ileum (I), colon (C), and
liver (L). Right, comparison of rabbit parietal
cells with lysates from HeLa cells (H) and MDCK
(M) cells.
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Fig. 2.
Alignment of full nucleotide and amino acid
sequence for human gastric AKAP350. The positions of the
RII binding site (underlined) and the four
leucine zippers (highlighted) are noted.
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Fig. 3.
Comparison of cDNAs coding for
AKAP350. The HGAKAP350 is compared with the structures of the
PMY2245 promyeloblast expressed sequence clone, the HLAKAP350 cDNA,
rabbit parietal cell AKAP350 (RAKAP350), and the original
rabbit AKAP120 sequence (RAKAP120). The percentages of
protein and DNA sequence identities versus HGAKAP350 are
shown at the right. The HGAKAP350 sequence contained an
amino-terminal yotiao homology region (YHR) followed
immediately by a site of major splice variants (S1). The
RAKAP350 sequence is missing a single exon at the S1 splice site (see
Fig. 5b). Note also the region recognized by the 14G2
monoclonal antibody to AKAP350 (amino acids 1478-1547 in RAKAP350 and
1854-1958 in HGAKAP350) and the position of the RII
binding site.
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Fig. 4.
Multiple splicing in AKAP350.
a, exons composing the DNA sequence for both yotiao and
AKAP350 are labeled according to the BAC in which they are found.
S1 and S2 denote the sites of alternative
splicing to generate isoforms. Note that the figure shows
BAC AA000066 in the reverse orientation to its numbering in its
GenBankTM entry. b, map of the S1 splice region
and corresponding amino acid sequence. The shaded
region codes for the terminal 3' end of yotiao. A splice
before this sequence to the next exon occurs in human gastric AKAP350,
whereas in rabbit AKAP350 this exon is spliced out. c, map
of S2 splice region and corresponding amino acid sequence. The 3' end
of both human and rabbit AKAP350 splice into the last exon at a point
read through by the AKAP350 sequence in human lung. The HLAKAP350
therefore contains 18 additional amino aicds, resulting in a larger
3'-end in this isoform.
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Fig. 5.
Tissue survey for splice variants by the
polymerase chain reaction method. a, the S1 region in
Fig. 4 was amplified in an array of rabbit tissues including parietal
cells (PC), fundus (F), gastric wall smooth
muscle (G), antrum (A), jejunum (J),
ileum (I), colon (C), liver (V),
spleen (S), kidney (K), lung (L),
heart (H), forebrain (FB), hind brain
(HB), cerebellum (CE). b, The S2
region in Fig. 4 was amplified from cDNA from human pancreas
(P), stomach (G), lung (L), Calu-3
cells (C), and human bronchial epithelium (B).
For each cDNA, two reactions were performed: one with an antisense
primer specific for the longer 3' end splice variant (1) and
a second time with an antisense primer specific for the shorter splice
(2).
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Fig. 6.
Northern blots of AKAP350 mRNA
expression. Rabbit (right) tissues were probed with the
3'-end of HGAKAP350, revealing an 11-kb message for rabbit parietal
cell (PC), fundus (F), and lung (L).
Human tissue, including kidney (K), skeletal muscle
(S), liver (V), lung (L), placenta
(P), brain (B), and heart (H), was
probed with a 1-kb fragment of HLAKAP350.
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Fig. 7.
Distribution of AKAP350 in rabbit
tissues. The distribution of AKAP350 was examined using the 14G2
monoclonal antibody in tissue sections and isolated gastric glands
(b). AKAP350 staining was localized in frozen sections of
gastric fundic mucosa (a), esophagus (c), jejunum
(d), ileum (e), colon (f), pancreatic
islet (g), adrenal medulla (h), renal collecting
duct (i), testes (j), ductus efferens
(k), and tracheal epithelium (l). Nonspecific
staining of large submucosal macrophages in ductus efferens sections
was also observed in controls incubated with no primary antibody.
Multiple points of AKAP350 immunofluorescence were observed in parietal
cells of gastric glands (b) visualized through confocal
fluorescence microscopy with maximum intensity reconstruction of 40 0.29-µm optical sections. Bar, 12 µm (a,
g, i, and l), 3 µm (b),
24 µm (c, d, e, f,
h, j, k).
-tubulin staining (Fig. 8). During metaphase, AKAP350 immunostaining was concentrated in both centrosomes colabeling with
-tubulin. However, in both anaphase and telophase cells, while AKAP350 immunostaining colocalized with
-tubulin-staining centrosomes, several discrete foci of staining
also were present in the cleavage furrow of dividing cells. An
identical distribution during the cell cycle was also observed in HeLa
cells (data not shown). These results indicate that AKAP350 is
associated with centrosomes in rapidly dividing cells.
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Fig. 8.
Distribution of AKAP350 during mitosis.
HCT116 cells were plated at low density for 24 h and then fixed.
Cells were triple labeled with polyclonal anti- -tubulin, monoclonal
anti-AKAP350 (14G2) and 4',6-diamidino-2-phenylindole. A triple label
overlay of the staining patterns is shown at the right.
Based on the 4',6-diamidino-2-phenylindole staining, cells were
identified according to their mitotic phase as interphase, metaphase,
anaphase, or telophase. At interphase, a single nidus of AKAP350
staining coincided with the
-tubulin staining adjacent to the
nucleus. In metaphase, AKAP350 stained both of the centrosomal poles
and, in addition, a diffuse cytosolic staining was also observed.
During anaphase and telophase, AKAP350 immunostaining was associated
with the centrosomes, but discrete foci of staining were also present
in the cleavage furrow. Bar, 3 µm.
-tubulin showed clear colocalization at the
centrosomes; however, there was obvious asymmetry between the poles.
Higher resolution projections demonstrate that AKAP350 staining
predominates in one centriole as a projection radiating toward the
nucleus. Examination of centrosomal staining with RII
antibodies (Fig. 9, m-o) showed a similar RII
immunostaining projection from one pole of the
-tubulin-staining
centrosome. Treatment of MDCK cells with nocodazole (33 µM) to disrupt microtubules did not alter the association
of either AKAP350 or RII with the centrosomes (data not
shown).
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Fig. 9.
Association of AKAP350 with centrosomes in
polarized MDCK cells. MDCK cells were grown on permeable filters
for 3 days and then fixed. a-c, cells were triple stained
with polyclonal anti-RII (a), murine monoclonal
anti-AKAP350 (14G2) (b), and rat monoclonal anti-ZO-1 (tight
junctions) (c). Cells were imaged as maximum intensity
projections of 40 0.29-µm optical sections. The arrowheads
show regions of colocalization of AKAP350 staining with staining for
RII. d-l, cells were triple labeled with murine
monoclonal anti-AKAP350 (d, g, j),
polyclonal anti- -tubulin (e, h, k)
and rat monoclonal anti-ZO-1 (f). AKAP350 labeled the poles
of the centrosomes assymetrically (arrowheads). Cells were
imaged as maximum intensity projections of 40 0.29-µm optical
sections. X-Y (g-i) and X-Z
(j-l) projections of a single pair of centrioles shows that
AKAP350 immunoreactivity was concentrated as a projection from one of
the centrioles (dual label overlaps in i and l).
The projection always was oriented away from the apical membrane. A
similar distribution was observed for RII immunostaining
(m) in association with
-tubulin staining centrioles
(n). The dual label image (o) of the
X-Z reconstruction of a pair of centrioles shows a similar
pattern of a projection from one pole of the centriole. Bar,
5 µm (a-c), 3 µm (d-f), 1 µm
(g-o).
-tubulin, but the vast
majority of AKAP350 foci did not correspond with the centrosomes.
AKAP350 foci also did not coincide with F-actin staining of the
intracellular canaliculus (data not shown). In human airway epithelial
cells, the major staining was associated with centrosomes; however,
prominent staining of foci associated with the lateral aspects of the
subapical regions also were present. A similar pattern of
noncentrosomal staining was observed in T84 and HCA-7 colon
adenocarcinoma cells (data not shown).
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Fig. 10.
Noncentrosomal AKAP350 in cultured
cells. Primary cultures of gastric parietal cells (a,
b) and cultured human bronchial epithelial cells
(c, d) were dual stained with antibodies against
AKAP350 (a, c) and -tubulin (b-d).
Single 0.29-µm optical sections were used to evaluate the
distribution of the immunostaining. In parietal cells, while AKAP350
staining could be seen in association with centrosomes
(arrowheads), the majority of the staining was not
associated with
-tubulin staining. In airway epithelial cells, the
majority of AKAP350 staining was associated with centrosomes, but
discrete foci of AKAP350 staining were also present in the apical
region of the cell, especially near the lateral borders
(arrows). Bar, 3 µm.
DISCUSSION
-Tubulin is a major
component of the centriole cores in a variety of cells (35), and
several studies suggest that this protein may be essential for
microtubule nucleation from centrosomes (36). As one might expect for
an organelle central to cytoskeletal organization, a number of
regulatory molecules also are associated with the centrosome. The
importance of protein phosphorylation in microtubule nucleation is
underscored by the presence of the p34cdc2 kinase and type II
protein kinase A at the centrosome (7, 8, 37). Keryer et al.
(9) subsequently studied the distribution of RII isoforms
and
in a human lymphoblast cell line, finding the
RII-
localized to the Golgi-centrosomal area. The
characteristics of the 350-kDa AKAP that we have cloned appear similar
to those of the centrosomal AKAP350 identified by Keryer et
al. (9) in an RII blot overlay of isolated centrosome
proteins from the KE37 human lymphoblast cell line. These investigators
immunoprecipitated the protein using the serum 0013 polyclonal
antibody, previously reported as a specific marker for human
centrosomes (20, 22, 38). No further characterization of this protein
in the literature has been submitted, however, since the initial
report. Nevertheless, several lines of evidence suggest that this
protein may indeed be identical to the protein cloned here. First,
Western blots with our 14G2 monoclonal antibody show a similar pattern
of 250- and 350-kDa proteins as seen by Keryer et al. (9)
with the 0013 polyclonal serum and RII overlays. Second,
immunocytochemistry with the 14G2 monoclonal antibody also shows
prominent localization of the protein with the centrosomes of both
polarized and nonpolarized cells. Third, as noted for the AKAP350
protein previously described, the distribution of 14G2 immunoreactive
protein in MDCK cells was not altered by treatment of cells with nocodozole.
-tubulin has become the marker of
choice for the centrosomes; however, it is now clear that many cell
types also have noncentrosomal pools of
-tubulin. This does not seem
to be the case in the MDCK cell line, where
-tubulin staining and
AKAP350 were limited to the discrete pair of centrioles forming the
centrosome. Parietal cells, at the other end of the spectrum, display
multiple foci of AKAP350 labeling throughout the cell. These other
sites do not correlate with
-tubulin staining in parietal cells.
While most tissues and cells demonstrate AKAP350 distribution in
association with centrosomes, several epithelial cells also showed
noncentrosomal staining patterns. In the human airway epithelial cell
line, for example, the majority of AKAP350 staining appears at the
centrosome; however, it is also apparent in a lateral subapical
pattern. Similar permutations of AKAP350 distribution in these and
other epithelial cells (e.g. colonic epithelial cells) allow
us to speculate that the protein kinase A is probably targeted to
cytoskeletal elements other than centrosomes in many cell types.
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ACKNOWLEDGEMENTS |
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We thank Carolyn Leithner and the Molecular Biology Core Facility for assistance with DNA sequencing, Drs. John Parente and Catherine Chew for cultured gastric parietal cells, and Julie Woodrum and Jennifer Navarre for outstanding technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health (NIH) Grants DK48370 and DK43405 (to J. R. G.), a Department of Veterans Affairs Merit Award (to J. R. G.), NIH Grant DK50744 (to S. L. M.), Cystic Fibrosis Foundation Grant MILGR9710 (to S. L. M.), a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to R. G. H.), a Career Development Fellowship Award from the Canadian Red Cross Society (to J. O. C.), and the Edward Christie Stevens Fellowship from the Faculty of Medicine, University of Toronto (to J. O. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U26360 (rabbit AKAP350), AF083037 (human gastric AKAP350), AF091711 (human lung AKAP350), and AA471131 (PMY2245).
To whom correspondence should be addressed: Institute of
Molecular Medicine and Genetics, CB2803 Medical College of Georgia, 1120 15th St., Augusta, GA 30912-3175. Tel.: 706-721-0693; Fax: 706-721-7915; E-mail: jgolden{at}mail.mcg.edu.
The abbreviations used are: AKAP, protein kinase A-anchoring protein; HGAKAP30, human gastric AKAP350; HLAKAP350, human lung AKAP350; BAC, bacterial artificial chromosome; MDCK, Madin-Darby canine kidney; RACE, rapid amplification of cDNA ends; nt, nucleotide(s); PBS, phosphate-buffered saline; Pipes, 1,4-piperazinediethanesulfonic acid; kb, kilobase pair(s); EGFP, enhanced green fluorescent protein; NMDA, N-methyl-D-aspartate.
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REFERENCES |
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