From the Department of Molecular Pharmacology, Atran
Laboratories and the § Department of Medicine, Albert
Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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We have cloned cDNA that encodes six novel A kinase anchor proteins (collectively named AKAP-KL). AKAP-KL diversity is generated by alternative mRNA splicing and utilization of two translation initiation codons. AKAP-KL polypeptides are evident in lung, kidney, and cerebellum, but are absent from many tissues. Different isoforms predominate in different tissues. Thus, AKAP-KL expression is differentially regulated in vivo. All AKAP-KL isoforms contain a 20-residue domain that avidly binds (Kd ~ 10 nM) regulatory subunits (RII) of protein kinase AII and is highly homologous with the RII tethering site in neuronal AKAP75. The distribution of AKAP-KL is strikingly asymmetric (polarized) in situ. Anchor protein accumulates near the inner, apical surface of highly polarized epithelium in tubules of nephrons. Both RII and AKAP-KL are enriched at an intracellular site that lies just below the plasma membrane of alveolar epithelial cells in lung. AKAP-KL interacts with and modulates the structure of the actin cytoskeleton in transfected cells. We also demonstrate that the tethering domain of AKAP-KL avidly ligates RII subunits in intact cells. AKAP-KL may be involved in (a) establishing polarity in signaling systems and (b) physically and functionally integrating PKAII isoforms with downstream effectors to capture, amplify, and precisely focus diffuse, trans-cellular signals carried by cAMP.
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INTRODUCTION |
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Protein kinase A (PKA)1
mediates actions of hormones and neurotransmitters that activate
adenylate cyclase (1-4). Signals carried by cAMP are often directed to
effectors that accumulate at discrete intracellular sites (5-7).
Targeting of signals to these sites can be achieved by generating a
non-uniform distribution of PKA molecules. This occurs when PKAII
and II
isoforms are attached to cytoskeleton or organelles by A
kinase anchor proteins (AKAPs) (5, 6). Prototypic anchor proteins
(AKAPs 75, 79, and 150) have a binding site for regulatory (RII)
subunits of PKAII isoforms and distinct domains that mediate
non-covalent coupling of AKAP·PKAII complexes to the
microtubule-based dendritic cytoskeleton of neurons and the
cortical actin cytoskeleton of non-neuronal cells (5-13). Both
cytoskeletal locations are closely apposed to the plasma membrane.
Thus, anchored PKAII is positioned near a signal generator (adenylate
cyclase) and multiple PKA substrate/effector proteins (e.g.
myosin light chain kinase, microtubule-associated protein-2, ion
channels, serpentine receptors that couple with Gs) (5, 6,
14). Effector proteins in cAMP signaling pathways can also be
associated with organelles, specialized regions of plasma membrane, or
sites in cytoskeleton that are separated from adenylate cyclase by
substantial distances. Novel RII-binding proteins apparently
participate in the assembly of "distal signaling modules" that are
associated with mitochondria, Golgi membranes, peroxisomes, and
centrioles (5, 6, 15, 16).
Anchored PKAII isoforms may be essential for dissemination of cAMP signals in highly polarized epithelium. Epithelial cells of lung and kidney provide examples of maximally polarized signal transduction, in which hormone-activated adenylate cyclase and downstream target/effectors can be located at opposite ends of the cell. Tight junctions between adjacent cells block communication by diffusible molecules and create two functionally distinct regions of plasma membrane. In epithelial layers of nephrons the basolateral surface is accessible to hormones in plasma and contain receptors and adenylate cyclase; the apical surface is enriched in channel and transporter proteins that mediate absorption/elimination of ions, nutrients, metabolites, and water (17, 18). Activities of many channels and transporters that traverse the apical membrane are regulated by hormones that stimulate adenylate cyclase, thereby promoting PKA activation (17-21). Several considerations suggest that C subunits derived from cytoplasmic PKAs may not mediate trans-epithelial signaling. Signals (cAMP) generated by modest, physiological levels of hormone at the basolateral surface can become weak, diffuse, and insufficient to activate target PKA molecules dispersed (at a relatively low concentration) in cytoplasm. This is due to the transient and intermittent nature of both hormone-release and hormone-mediated stimulation of adenylate cyclase in situ, rapid desensitization of receptor-G protein interactions, dilution of cAMP into the large volume of the cytoplasm, and degradation mediated by cAMP phosphodiesterases. Moreover, studies on knock-out mice and cultured neurons indicate that the ability of PKA isoforms to respond to small changes in cAMP content is often crucial for regulation in vivo (14, 22, 23). Another constraint is that channels/transporters cluster into patches that constitute only a small portion of the apical membrane surface (24, 25). A mechanism that confers increased sensitivity in a specific microenvironment involves concentrating and colocalizing PKAII isoforms with substrate effectors via AKAPs. AKAP·PKAII complexes could serve as sensor/transducers that capture highly diffuse and relatively small trans-cellular signals (cAMP generated at the basolateral surface) with enhanced sensitivity (because of the elevation in local PKAII concentration). Signals can then be rapidly amplified and focused directly on the co-localized effector channel (also at high local concentration) to achieve targeted physiological regulation.
The observation that PKAII is associated with fragments of membrane/cytoskeleton derived from the apical portion of kidney epithelium (21) suggests that the preceding model may be operative in polarized tissue. To gain insights into precise physiological roles for anchored PKAII in establishing and mediating polarized signal transduction, it is essential to identify and characterize AKAPs that are (a) expressed in highly polarized epithelial cells, (b) targeted to the vicinity of the apical surface, and (c) able to avidly bind RII (PKAII) in the context of intact cells. We now describe the discovery and characterization of a novel AKAP, which is expressed in lung and kidney (AKAP-KL) and fulfills the listed criteria.
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EXPERIMENTAL PROCEDURES |
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Isolation of cDNAs Encoding AKAP-KL--
A full-length
cDNA clone encoding murine RII was obtained as described
previously for RII
(26). The cDNA was subcloned into the yeast
bait plasmid pAS1 (CLONTECH) to generate a
fusion gene. The gene encodes a hybrid protein in which the yeast GAL4 DNA-binding domain is appended at the N terminus of RII
. A rat brain
cDNA library in the yeast expression plasmid (library vector) pGAD10 was obtained from CLONTECH and screened via
the two-hybrid interaction assay as described by Fields (27) and Durfee
et al. (28). Growth of auxotrophic yeast on medium lacking
Trp, His, and Leu and
-galactosidase assays were used to isolate and characterize cDNAs encoding candidate AKAPs. A fragment of cDNA (1.4 kilobase pairs) encoding a portion of a novel RII-binding protein
was excised from recombinant pGAD10 (by digestion with EcoRI) and was used as a template to generate a
random-primed, 32P-labeled probe. This probe was used to
screen a 5'-stretched mouse brain cDNA library in bacteriophage
gt11 (CLONTECH) as indicated in previous papers
(29, 30). Six positive recombinant phage clones were plaque purified
and the cDNAs (0.9-3.8 kilobase pairs) were subcloned in plasmids
pGEM7Z (Promega) and pBluescript (Stratagene) and sequenced.
DNA Sequence Analysis-- cDNA inserts were sequenced by a dideoxynucleotide chain termination procedure (31) using T3, T7, and custom synthetic oligonucleotide primers as described previously (30).
Computer Analysis-- Analysis of sequence data, sequence comparisons, and data base searches were performed using PCGENE-Intelligenetics software (Intelligenetics, Mountainview, CA) and BLAST programs (32, 33) provided by the NCBI server at the National Institutes of Health.
Electrophoresis of Proteins-- Proteins were denatured in gel loading buffer and subjected to electrophoresis in 9 or 10% polyacrylamide gels containing 0.1% SDS as described previously (8). Myosin (Mr = 210,000) phosphorylase b (97,000), transferrin (77,000), albumin (68,000), ovalbumin (45,000), and carbonic anhydrase (29,000) were used as standards for the estimation of Mr values.
Western Immunoblot Assays-- Size-fractionated proteins were transferred from denaturing polyacrylamide gels to an Immobilon P membrane (Millipore Corp.) (34). Blots were blocked, incubated with antiserum directed against AKAP-KL (1:2000), and washed as described previously (34, 35). Antigen-IgG complexes were visualized by an indirect chemiluminescence procedure (34, 35). Signals were recorded on Kodak XAR-5 x-ray film.
Overlay Assay for RII Binding--
Overlay binding assays have
been described previously (8, 9). In brief, a Western blot is probed
with 32P-labeled RII (using a subunit concentration of
0.3 nM and 1 × 105 cpm of 32P
radioactivity/ml). Complexes of 32P-RII
and AKAPs are
visualized by autoradiography. Results were quantified by scanning
densitometry (Pharmacia-LKB Ultroscan XL laser densitometer) or
PhosphorImager analysis (Molecular Dynamics) (35).
Equilibrium Binding Assay--
Aliquots (80 ng) of highly
purified partial AKAP-KL protein (residues 354-741, Fig.
2A) were used for binding assays. Residues 586-605
constitute the high-affinity RII-binding site in AKAP-KL (see
"Results"). RII
was expressed, purified, and labeled with 32P as described previously (8, 26). Assays were performed in 250 µl of buffer A (10 mM Tris-HCl, 50 mM
sodium phosphate, pH 8.0, 0.1 M NaCl).
32P-Labeled RII
was varied from 0.1 to 75 nM. Incubations were carried out at 0 °C for 90 min.
Subsequently, 40 µl of a 50% suspension of Ni2+-chelate
Sepharose 4B beads (Talon resin, CLONTECH),
which was pre-equilibrated with Buffer A, was added and the incubation
continued for 30 min. Next, samples were diluted to 1 ml with buffer A
and the beads were pelleted by centrifugation at 2,000 × g for 5 min at 4 °C. The beads were washed an 5 additional times by resuspension in 1 ml of buffer A and centrifugation
at 2,000 × g at 4 °C. Bound 32P-labeled
RII
was eluted from the beads in 0.2 ml of 1% SDS and radioactivity
was determined in a scintillation counter. 32P
radioactivity in an aliquot of the first supernatant solution was
determined to measure the amount of free RII
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Growth and Transfection of HEK-293 and AV-12 Cells--
Human
embryonic kidney fibroblasts (HEK293 cells) and a cell line derived
from a hamster subcutaneous tumor (AV-12) were obtained from the
American Type Culture Collection. Cells were grown in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum. Full-length
AKAP-KL2 and AKAP-KL3 cDNA inserts were excised from recombinant
pBluescript plasmids by digestion with SpeI and
ClaI and ligated into the pEBG mammalian expression vector
(36), which was cleaved with the same enzymes. This placed the
cDNAs downstream from a powerful EF1 promoter and a
GST gene and upstream from a poly(A) addition signal. Thus, the vector
promotes high level expression of GST-AKAP-KL in mammalian cells.
Transfections were performed via calcium phosphate precipitation as
described previously (13, 35). Stable transformants were obtained by selection with 1 mg/ml G418 for 14 days (34).
Expression and Purification of a Fusion Protein That Contains the
RII-binding Domain of AKAP-KL--
AKAP-KL cDNA (nucleotides 1573 to 2736, Fig. 2A) that encodes amino acids 354-741 in the
anchor protein was cloned into expression plasmid pET14b as described
in previous papers (26, 37). This enabled abundant synthesis of a
His-tagged partial AKAP-KL fusion protein in Escherichia
coli BL21 (DE3) that was transformed with recombinant plasmid and
induced with isopropyl-1-thio--D-galactopyranoside (26,
37). Induced bacteria were disrupted in a French press and the soluble
His-tagged AKAP-KL protein was purified to near homogeneity by affinity
chromatography on Ni2+-chelate-Sepharose 4B beads
(Pharmacia) as described previously (26). Two mg of protein were
purified from a 600-ml culture.
Production of IgGs Directed against AKAP-KL-- Samples of AKAP-KL fusion protein were injected into rabbits (0.4-mg initial injection; 0.2 mg for each of four booster injections) at Covance Laboratories (Vienna, VA) at 3-week intervals. Serum was collected at 3-week intervals after the first injection.
Deletion and Site-directed Mutagenesis of AKAP-KL-- Deletion mutagenesis was performed via polymerase chain reaction as described for AKAP75 and S-AKAP84 (11, 15). Amino acid substitutions were introduced into the RII-binding domain of AKAP-KL via site-directed mutagenesis, as described previously (11).
Determination of AKAP-KL Localization by Immunofluorescence Analysis and Immunoperoxidase Histochemistry-- HEK293 cells that were stably transfected with an AKAP-KL transgene were fixed and incubated with anti-AKAP-KL antibodies using procedures described by Li et al. (13). AKAP-KL·IgG complexes were visualized by incubation with fluorescein isothiocyanate-coupled secondary antibodies and the utilization of a laser scanning confocal microscope system as described previously (13). F-Actin was visualized by its interaction with rhodamine-tagged phalloidin, as previously reported (13). Mice were sacrificed by in situ fixation (12). Sections of kidney and lung were prepared, probed with antibodies directed against AKAP-KL, and stained via an indirect peroxidase procedure as previously reported (12). A black precipitate is formed at sites containing AKAP-KL·IgG complexes.
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RESULTS AND DISCUSSION |
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Discovery of a Novel Anchor Protein, AKAP-KL--
A cDNA
encoding full-length RII was cloned into the yeast plasmid
pAS1-CYH2. This recombinant "bait" vector was used to screen a rat
brain cDNA library inserted into the yeast "library" vector pGAD10. Interaction of the GAL4 DNA-binding domain-RII
fusion protein encoded by the bait vector with RII-binding proteins
fused to the activation domain of GAL4 (encoded by library
vector) reconstitutes a transcription factor that stimulates
GAL4-dependent promoters to transcribe
HIS3 and lacZ-reporter genes. The combination of chimeric genes in plasmids and interaction-dependent
HIS3 gene expression enable selection of candidate AKAPs by
growth of auxotrophic yeast colonies on
Trp
Leu
His
medium. Yeast
carrying four different cDNAs were obtained from 106
transformants. Each yeast clone produced positive results in
-galactosidase assays. Library plasmids were recovered from yeast and amplified in E. coli. Reconstitution assays showed that
plasmids encoding candidate AKAPs did not activate HIS3 or
lacZ alone, or in combination with irrelevant bait plasmids.
In contrast, co-transformation of yeast with selected library plasmids
and the original bait plasmid enabled growth on "triple minus"
medium and
-galactosidase expression. RII-binding assays were
performed on samples of total protein from yeast carrying positive,
recombinant plasmids. One fusion protein bound ~10-fold more
32P-labeled RII
than the others (Fig.
1, lane 8). When the
corresponding cDNA was sequenced it yielded a derived protein
comprising 415 residues. A translation termination codon was evident,
but the 5' end of the cDNA corresponded to an open reading frame.
Therefore the cDNA insert was used to screen a 5'-stretched mouse
brain cDNA library in bacteriophage
gt11 via standard DNA
hybridization procedures. Six overlapping cDNA clones (including
one near the full-length clone) were obtained and sequenced (Fig.
2A). A translation initiation
codon was detected and the amino acid sequence of the murine
RII-binding protein (named AKAP-KL) was elucidated. The name
AKAP-KL was selected because of the expression and highly
asymmetric targeting of the anchor protein in epithelial cells of
kidney and lung (see below).
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Structure-function Relationships in AKAP-KL--
AKAP-KL is
composed of 885 amino acids and has a calculated
Mr of 98,000 (Fig. 2A). The sequence
of the acidic polypeptide (pI ~ 5.0) is not homologous with
sequences of previously characterized proteins. However, several
domains that could potentially contribute to functional roles for
AKAP-KL are evident. The sequence between residues 586 and 605 (Fig.
2A) of AKAP-KL aligns with the RII-binding site of AKAP75
(11) to yield 45% overall identity (Fig.
3A). A central core of 7 amino
acids (Leu593 to Gln599 in AKAP-KL) is nearly
invariant (86% identity) in the two proteins. Furthermore, five amino
acids with large aliphatic side chains, which coordinately regulate RII
binding affinity in AKAP75 (11), are conserved in AKAP-KL
(Leu586, Leu593, Val594,
Ile598, and Ile602). A partial AKAP-KL protein
(residues 354-741) that includes the putative tethering site binds
both RII (Fig. 3B, lane 1) and RII
(not shown). Two
approaches verified the functionality of the potential RII-binding site
in AKAP-KL. First, a hydrophobic residue (Ile598) predicted
to be essential for binding RII (by analogy with AKAP75) was mutated to
Ala. This substitution diminished RII binding activity of partial
AKAP-KL by >95% (Fig. 3B, lane 3). A similar effect was
observed when the corresponding Ile in AKAP75 was replaced with Ala
(11). Mutation of an amino acid predicted to be non-essential (Ala590 to Ser) had little effect on the tethering of RII
(Fig. 3B, lane 2). Partial AKAP-KL proteins that lack
residues 586-605 have no RII binding activity.2
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Six Isoforms of AKAP-KL Are Produced in Mammalian Cells-- Sequencing of 3' ends of AKAP-KL cDNAs revealed two alternative modes of AKAP-KL mRNA splicing. In the first instance, a splice donor sequence at nucleotide 2878 (Fig. 2A) is joined to an acceptor site at position 3097, thereby deleting 218 nucleotides in cDNA. This deletion shifts the reading frame after codon 789 so that the succeeding 21 nucleotides encode a novel, C-terminal hexapeptide (Fig. 2B) and a translation termination signal. As a result, the C terminus of the anchor protein is truncated by 90 residues. AKAP-KL proteins that lack 90 C-terminal residues are designated AKAP-KL3; AKAP-KL proteins that retain this region are designated AKAP-KL1. A second mode of RNA splicing joins a donor sequence at nucleotide 3058 (Fig. 2A) with the acceptor site at nucleotide 3097. This splicing reaction excises 13 codons (for amino acids 849-861) and does not alter the reading frame (Fig. 2B). Anchor proteins that lack residues 849-861 are named AKAP-KL2.
Further diversity in AKAP-KL structure is introduced by utilization of either of two translation start codons. Like codon 1 in Fig. 2A, Met codon 125 is included within the context of a consensus translation initiation sequence (ANNATGG, nucleotides 883-889). Both start sites are used in cultured cells and in vivo (see Fig. 4, below). Thus, AKAP-KL proteins can differ by retention (form A) or deletion (form B) of a 124-residue N-terminal domain. Together, alternative splicing of AKAP-KL mRNA and utilization of two translation start codons generate 6 discrete AKAP-KL isoforms. Sizes and nomenclature for AKAP-KL isoforms are presented in Table I.
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Preparation and Specificity of Antibodies Directed against AKAP-KL-- AKAP-KL cDNAs were cloned from a brain cDNA library. Since a high proportion of the complete constellation of mammalian genes is expressed in the central nervous system, the efficiency of screening is maximized. However, isolation of reverse transcripts of AKAP-KL mRNAs from this library does not imply that brain is the principal site of accumulation of the anchor protein. To determine the tissue and cellular distributions, as well as functions of AKAP-KL it is essential to generate highly specific anti-AKAP-KL IgGs.
A fragment (1164 base pairs) of AKAP-KL cDNA encoding amino acids 354-741 (Fig. 2A) was amplified by the polymerase chain reaction using 5' and 3' primers that introduced NdeI and BamHI restriction sites, respectively. This enabled cloning of the cDNA fragment into the pET14b expression plasmid. The inserted cDNA lies downstream from a bacteriophage T7 promoter and DNA encoding a fusion peptide composed of 20 amino acids. Included in the fusion peptide are six consecutive His residues, which constitute a Ni2+-binding site. E. coli BL21 (DE3) was transformed with recombinant pET14b plasmid and induced to synthesize fusion protein with 1 mM isopropyl-1-thio-Expression of AKAP-KL Is Tissue-specific and Isoform-selective-- Analysis of P1 fractions from various tissues disclosed that AKAP-KL is abundantly expressed in lung (Fig. 5A, lane 1). In contrast, the anchor protein is either absent or produced at very low levels in multiple tissues, including liver, heart, and cerebral cortex (Fig. 5A, lanes 3-5). Moderate levels of AKAP-KL were detected in thymus and cerebellum. AKAP-KL isoforms observed in cerebellum (Fig. 5A, lane 6) do not co-migrate with anchor proteins that accumulate in lung. Thus, expression of the AKAP-KL gene is tightly regulated. Accumulation of AKAP-KL polypeptides is restricted to certain cell/tissue types and anchor protein isoforms that predominate in a given tissue are evidently determined by a combination of regulated AKAP-KL mRNA splicing and utilization of two translation initiation codons.
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Properties of AKAP-KL in Vitro and in Cells-- Extraction with buffers containing 0.5% Triton X-100 failed to solubilize the anchor protein, indicating that is not embedded in a lipid bilayer (Fig. 6A). Buffers containing 0.5% sodium deoxycholate (which disrupts association of proteins in cytoskeleton) efficiently solubilize AKAP-KL. Confocal immunofluorescence microscopy of HEK293 cells that were stably transfected with cDNA encoding full-length AKAPs KL2A and 2B revealed the intracellular distribution of the anchor protein. AKAP-KL accumulates in regions of cortical cytoskeleton that appear as projections or large clusters of antigen (Fig. 6B). These structures are not present in control HEK293 cells or cells transfected with unrelated transgenes (13). In contrast, AKAP75 is dispersed throughout the cortical cytoskeleton in the same cells (13). Thus, AKAP-KL appears to be targeted to specific microenvironments in cytoskeleton. It is possible that AKAP-KL actively promotes assembly/organization of specialized structures via interactions with other proteins in cytoskeleton. The differential clustering of AKAP-KL in subdomains of cytoskeleton in HEK293 cells may also reflect properties involved in targeting of the anchor protein to the vicinity of the apical surface of polarized epithelial cells (see below).
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AKAP-KL Is Targeted to the Apical Surface of Polarized Epithelial
Cells--
Anti-AKAP-KL IgGs were used in an indirect immunoperoxidase
staining procedure to determine the location of AKAP-KL in sections of
rat kidney. The anchor protein is expressed in epithelial cells and
accumulates exclusively at the apical surface, which abuts the lumen
(e.g. see epithelial cells in proximal tubules, Figs. 7, A and B). RII
is also concentrated at the apical surface of epithelial cells in renal
proximal tubules (21). In lung, AKAP-KL accumulates in long, linear
structures just below the lumenal surface of alveolar epithelial cells
(Fig. 7C). In parallel, a substantial amount of RII is also
concentrated in the elongated structures near the apical surface of
alveolar epithelial cells (Fig. 7D). The preceding results
suggest that AKAP-KL and RII are co-localized and co-enriched in two
types of polarized epithelium. Confirmation of this working hypothesis
will ultimately require high-resolution, immunoelectron microscopy and
manipulation of the system by molecular genetics (e.g.
disrupting AKAP-KL·RII complexes by expressing AKAP-KL mutants that
bind RII, but are mis-targeted, or by ablating AKAP-KL gene expression)
in future studies. However, the present observations support the idea
that AKAP-KL incorporates PKAII into distal signaling modules at sites near junctions of cytoskeleton and apical plasma membrane. Such modules
might include PKA substrate/effector proteins (e.g.
transporters) which span the apical membrane. This mode of organization
can greatly facilitate cAMP-mediated trans-epithelial signal
transduction.
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AKAP-KL Binds RII with High Affinity in Intact Cells--
The pEBG
expression vector (36) was used to assess the ability of the tethering
domain AKAP-KL to sequester RII in intact cells. pEBG contains a
powerful EF1 promoter that lies upstream from the
glutathione S-transferase (GST) gene and a 3' multiple cloning region. AKAP-KL cDNA was inserted, in-frame with the GST gene, and the vector was co-transfected with RC/CMV (an
expression plasmid which contains the neoR gene under
control of an SV40 promoter) into hamster AV-12 cells. Stable cell
lines expressing GST-AKAP-KL were isolated by selection with G418.
Typically, ~80% of GST-AKAP-KL appeared to be anchored, whereas 20%
was present in cytosol. Cytosolic GST·AKAP-KL complexes were purified
to near homogeneity by affinity chromatography on GSH-Sepharose 4B.
Western blot analysis revealed that a large proportion of available
cytosolic RII was complexed with the anchor protein (Fig.
8). Two conditions were varied to exclude
the possibility that AKAP-KL·RII complexes assembled after cell
homogenization. Since AKAP-KL·RII complexes dissociate very slowly at
4 °C,2 cells were disrupted: (a) in a volume
of lysis buffer that was increased 20-fold to reduce concentrations of
free RII (PKAII) and anchor protein or (b) in a standard
volume of buffer that contained a vast excess (15 µg/ml) of
His-tagged AKAP-KL partial protein (see Fig. 4A). The
partial anchor protein avidly binds RII (Fig. 3C), but is
not sequestered by GSH-Sepharose 4B resin. Neither dilution nor
post-lysis competition with an RII-binding protein altered the results
obtained in Fig. 8 (data not shown). Thus, stable AKAP-KL·RII
complexes are efficiently produced in the environment of the internal
milieu of intact cells.
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Conclusions and Implications-- AKAP-KL isoforms are differentially expressed and selectively targeted to apical regions of polarized lung and kidney epithelial cells. This family of novel anchor proteins efficiently sequesters RII (PKAII) in vitro and in situ. AKAP-KL proteins may be involved in (a) establishing polarity in trans-epithelial signaling systems and (b) creating/organizing distal PKA·effector complexes that receive, amplify, and focus trans-cellular signals carried by cAMP. Generation of six AKAP-KL isoforms by post-transcriptional processes creates substantial anchor protein diversity and suggests possible mechanisms by which intracellular localization of PKAII isoforms may be modulated in different cell types and in response to environmental stimuli.
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ACKNOWLEDGEMENTS |
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We thank Dr. Joseph Avruch, Diabetes Unit, Massachusetts General Hospital and Harvard Medical School, for providing the pEBG vector. Ann Marie Alba provided expert secretarial assistance. Drs. L. Fowler and S. Jaken (Alton Jones Cell Science Center, Lake Placid, NY) provided invaluable assistance in the initial phase of characterization of the distribution of AKAP-KL in kidney.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM 22792 (to C. S. R.) and the Lucille P. Markey Charitable Trust (to C. S. R.).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) AF033274, AF033275, and AF033276.
¶ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, F-229, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2505; Fax: 718-430-8922; E-mail: rubin{at}aecom.yu.edu.
1 The abbreviations used are: PKA, protein kinase A; RII, regulatory subunits of type II protein kinase A isoforms; AKAP, A kinase anchor protein; GST, glutathione S-transferase.
2 F. Dong and C. S. Rubin, unpublished observations.
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REFERENCES |
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