From the Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Classical A kinase anchor proteins (AKAPs)
preferentially tether type II protein kinase A (PKAII) isoforms to
sites in the cytoskeleton and organelles. It is not known if distinct
proteins selectively sequester regulatory (R) subunits of type I PKAs, thereby diversifying functions of these critical enzymes. In
Caenorhabditis elegans, a single type I PKA mediates all
aspects of cAMP signaling. We have discovered a cDNA that encodes a
binding protein (AKAPCE) for the regulatory subunit
(RCE) of C. elegans PKAICE.
AKAPCE is a novel, highly acidic RING finger protein
composed of 1,280 amino acids. It binds RI-like RCE with
high affinity and neither RII nor RII
competitively inhibits
formation of AKAPCE·RCE complexes. The
RCE-binding site was mapped to a segment of 20 amino acids in an N-terminal region of AKAPCE. Several hydrophobic
residues in the binding site align with essential Leu and Ile residues in the RII-selective tethering domain of prototypic mammalian AKAPs.
However, the RCE-binding region in AKAPCE
diverges sharply from consensus RII-binding sites by inclusion of three
aromatic amino acids, exclusion of a highly conserved Leu or Ile at
position 8 and replacement of C-terminal hydrophobic amino acids with
basic residues. AKAPCE·RCE complexes
accumulate in intact cells.
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INTRODUCTION |
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A kinase anchor proteins
(AKAPs)1 avidly bind
regulatory subunits (RII and RII
) of the type II isoforms of
cAMP-dependent protein kinase (PKAII
and PKAII
)
(1-3). The various AKAPs are functional and not structural homologs;
they possess partly homologous RII-binding domains, but the remaining
portions of their sequences are divergent. Moreover, different AKAPs
target PKAII isoforms to distinct intracellular loci including
mitochondria, peroxisomes, Golgi, and plasma membranes and sites in
cytoskeleton (1-6). This accomplishes incorporation of AKAP·PKAII
complexes into signaling modules that are either juxtaposed with or
spatially segregated from hormone-activated adenylate cyclase.
Substrate-effector proteins are thought to be clustered in proximity
with anchored PKAII.
RI is expressed in most tissues, where it mediates (as PKAI
)
actions of many hormones. It is the only R isoform that is essential
for the progression of embryogenesis (7). Persistent synthesis of low
levels of free RI
serves as a critical homeostatic mechanism to
maintain the total R:PKA catalytic subunit (C) ratio at 1 (7). If
excess C accumulates it is bound by RI
, and the t1/2 of RI
increases 5-fold, thereby inhibiting
otherwise unregulated and potentially toxic C activity. It is possible
that RI
(PKAI
) functions are further diversified by interactions
with anchor proteins. This topic received little attention because
fractionation of cell and tissue homogenates and immunocytochemical
localization analysis indicate that the bulk of PKAI is dispersed in
cytoplasm. However, RI is bound to the plasma membrane of human
erythrocytes, and PKAI is recruited to a multi-protein complex at the
"cap" site of activated T lymphocytes (8, 9). Proteins and
mechanisms involved in sequestration of PKAI in blood cells have not
been characterized. However, high affinity RII tethering sites in
several AKAPs also sequester RI
subunits with low avidity (10-12).
Huang et al. (10) reported in vitro binding of
RII
or RI
with a single site on an isoform of S-AKAP84 (4) known
as D-AKAP1 (10) or AKAP121 (6). S-AKAP84 targets and tethers PKAII to the outer membrane of mitochondria and contains the same RII/RI-binding site as D-AKAP1/AKAP121 (4, 6). Moreover, high concentrations of RI
competitively inhibit the binding of RII subunits with D-AKAP1 and Ht31
(10, 11). Although RII
is bound with 25-500-fold higher affinity
than RI
in the cited studies (10, 11), the results suggest that
(a) RI dimers have a site available for interactions with
potential anchor proteins and (b) some PKAI molecules may be
immobilized within cells.
The preceding observations raise additional pertinent questions. Are
there high affinity, RI-specific anchor proteins? If so, what is the
biochemical basis for the binding and anchoring of RI (PKAI)? To what
degree are structures of RI anchor proteins homologous with or
divergent from RII-selective AKAPs? The nematode Caenorhabditis
elegans provides an advantageous system for addressing these
questions. The pathways, protein components, and mechanisms of cell
signaling systems are conserved between C. elegans and mammals (13, 14). However, the C. elegans genome contains only one C gene and one R gene (15, 16). C. elegans C is
82% identical with mammalian C. C. elegans R
(RCE) is closely related to mammalian RI
(~60%
overall identity) but not the RII isoforms (~30% identity) (16).
Like RI
(17), RCE has a pseudosubstrate site and two
N-terminal Cys residues that covalently cross-link RCE
subunits in
vitro.2 Moreover,
RCE has (a) an N-terminal region that is
homologous with the RI dimerization domain and (b)
cAMP-binding sites that are 100% (site 1) and 87% (site 2) identical
with corresponding regions in RI
(16). These properties strongly
indicate that RCE is a homolog of RI.
A basic conundrum is as follows: how can one PKA holoenzyme mediate all aspects of cAMP-regulated signaling? Fractionation of C. elegans homogenates revealed that only 40% of PKA is isolated in cytosol, whereas 60% partitioned with the particulate fraction (16). Therefore, RCE anchor proteins might play a key role in diversifying and adapting C. elegans PKA for multiple functions. We now report the discovery and characterization of an RCE-binding protein, AKAPCE.
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EXPERIMENTAL PROCEDURES |
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Isolation of cDNAs Encoding
AKAPCE--
Approximately 500,000 plaques from a C. elegans cDNA library in the expression vector ZAPII
(CLONTECH) were screened by the procedure of
Bregman et al. (18, 19). Full-length,
32P-labeled RCE (1.5 × 105
cpm/ml) (see below) was used as a probe. Complexes of radiolabeled RCE with
-galactosidase-AKAP fusion proteins were
detected by autoradiography (18). Positive plaques were purified to
homogeneity. Recombinant pBluescript SK phagemids that contained anchor
protein cDNAs were excised from the phage in Escherichia
coli (20), amplified, and purified. Two related cDNA clones
were isolated. The larger cDNA (4.3 kbp) contained the complete
coding and 3'-untranslated sequences for AKAPCE mRNA,
as well as a segment of the 5'-untranslated region. The entire sequence
of the smaller cDNA (3.3 kbp) is included within the larger
insert.
DNA Sequence Analysis-- cDNA inserts were sequenced by a dideoxynucleotide chain termination procedure (21) as described previously (6).
Computer Analysis-- Analysis of sequence data, sequence comparisons, and data base searches were performed using PCGENE-Intelligenetics software (Intelligenetics, Mountainview, CA) and BLAST programs (22, 23) provided by the NCBI server at the National Institutes of Health.
Expression, Purification, Phosphorylation, and Mutagenesis of C. elegans RCE--
Complementary DNA encoding C. elegans RCE was cloned and sequenced as described in a
previous paper (16). A DNA insert that encodes amino acids 1-73 in
RCE was amplified by the Pfu DNA polymerase (Stratagene) chain reaction (PCR). The 5' primer contained a
NotI restriction sequence upstream from an ATG codon in the
context of an NdeI cleavage site. This was followed by 18 nucleotides corresponding to codons 2-7 of RCE (16). The
3' primer corresponded to the complement of codons 67-73 in
RCE. A unique BspE1 site is embedded in codon
71. Amplified DNA was digested with NotI and
BspE1 and cloned into a recombinant pBluescript plasmid
(which contains full-length RCE cDNA) that was cleaved
with NotI (a 5' vector site) and BspE1. Fidelity
of the PCR amplification was verified by DNA sequencing. Next,
full-length RCE cDNA was released from pBluescript by
digestion with NdeI and KpnI (a 3' vector site)
and cloned into the plasmid pGEM3Z (Promega) that was digested with the
same restriction enzymes. Finally, RCE cDNA was excised from the pGEM3Z plasmid by cleavage with NdeI and
BamHI (a 3' vector site) and cloned into the expression
plasmid pET14b (Novagen), which was cut with the same restriction
endonucleases. In pET14b RCE cDNA (375 codons) is
preceded by DNA that encodes a 20-residue N-terminal fusion peptide
that includes 6 consecutive His residues. Transcription of the chimeric
gene is governed by a promoter sequence for bacteriophage T7 RNA
polymerase, and synthesis of the polymerase is induced with
isopropyl-1-thio--D-galactopyranoside (IPTG). E. coli BL21 (DE3) was transformed with the expression plasmid, and
synthesis of His-tagged RCE was induced with 1 mM IPTG for 2 h at 37 °C. Bacteria were harvested,
disrupted, and separated into soluble and particulate fractions as
described previously (24). The RCE fusion protein was
purified to near homogeneity by Ni2+-chelate chromatography
as described by Li and Rubin (25).
Deletion Mutagenesis of AKAPCE--
Deletion
mutagenesis was performed via PCR as described for AKAP75 and S-AKAP84
(4, 6, 26). Mutants were verified by DNA sequencing. Amplified
cDNAs were cloned into the expression plasmid pGEX-KG (27) after
digestion of insert and vector with EcoRI and
HindIII. This placed cDNAs encoding partial
AKAPCE polypeptides downstream from and in-frame with the
3' terminus of a glutathione S-transferase (GST) gene in the
vector. Transcription of the GST fusion gene is driven by an inducible
tac promoter. High levels of chimeric GST partial
AKAPCE polypeptides were produced when E. coli
DH5 was transformed with recombinant pGEX-KG plasmids and then
induced with 0.5 mM IPTG as described previously (28). Amino acid substitutions were introduced into the
RCE-binding domain of AKAPCE via site-directed
mutagenesis, as described previously (26).
Growth of C. elegans and AV-12 Cells-- C. elegans was grown at 20 °C as described previously (29). A cell line (AV-12) derived from a hamster subcutaneous tumor was grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
Transfection of AV-12 Cells-- Full-length RCE and AKAPCE cDNA inserts were excised from recombinant, pBluescript plasmids by digestion with NotI and ApaI and ligated into the pCIS2 mammalian expression vector (30), which was cleaved with the same enzymes. This placed the cDNAs downstream from a powerful cytomegalovirus promoter and upstream from a poly(A) addition signal. Transient transfections of AV-12 cells were performed by calcium phosphate precipitation (31). Cells were harvested 60 h after transfection.
Expression and Purification of a GST Fusion Protein That Contains
the RCE-binding Domain--
A fragment of
AKAPCE cDNA (nucleotides 526-1227, Fig. 2A)
that encodes amino acids 176-409 in the anchor protein was cloned into
the expression plasmid pGEX-KG. The methodology is described above for
"Deletion Mutagenesis of AKAPCE." This enabled
synthesis of a GST-AKAPCE fusion protein in E. coli DH5 that was transformed with recombinant plasmid and
induced with IPTG (24, 27). After induction, bacteria were disrupted in
a French press, and the soluble GST-AKAPCE (176-409)
protein was purified to near homogeneity by affinity chromatography on
GSH-Sepharose 4B beads (Amersham Pharmacia Biotech) as previously
reported (24).
Production and Purification of IgGs Directed against
AKAPCE and RCE--
Samples of
AKAPCE (176-409) 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. Purified GST
partial AKAPCE antigen was coupled to CNBr-Sepharose 4B as
described by Bregman et al. (19) to yield a final
concentration of 1.7 mg of protein bound per ml of resin. Antibodies
that bind GST were eliminated by passing serum (2 ml) over a column of
GST-Sepharose 4B (24). Adsorbed serum was applied to a column (2 ml) of
partial AKAPCE (176-409)-Sepharose 4B. The resin was
washed extensively, and anti-AKAPCE IgGs were then isolated
by successive elutions at pH 2.5 and pH 11.8, as described previously
(24). Sufficient 1 M Na2HPO4 or 0.5 M Hepes was added to column fractions to adjust the pH to
~7.5. The IgG concentration was estimated from the absorbance at 280 nm. IgGs were dialyzed against 10 mM sodium phosphate, pH
7.5, containing 0.15 M NaCl and 50% (v/v) glycerol and
were stored at 20 °C.
Electrophoresis of Proteins-- Proteins were denatured in gel loading buffer and subjected to electrophoresis in 7.5 or 10% polyacrylamide gels containing 0.1% SDS as described previously (18, 19). Myosin (Mr = 210,000), phosphorylase b (Mr = 97,000), transferrin (Mr = 77,000), albumin (Mr = 68,000), ovalbumin (Mr = 45,000), and carbonic anhydrase (Mr = 29,000) were used as standards for the estimation of molecular weight values.
Western Immunoblot Assays-- Size-fractionated proteins were transferred from denaturing polyacrylamide gels to an Immobilon P membrane (Millipore Corp.) as described previously (18). Blots were blocked, incubated with affinity purified IgGs directed against AKAPCE (1:4000, relative to serum), and washed as described previously (26, 32). Antigen-IgG complexes were visualized by an indirect chemiluminescence procedure (32, 33). Signals were recorded on Kodak XAR-5 x-ray film.
Overlay Assay for RCE Binding Activity-- Overlay binding assays have been described in several papers (18, 19). In brief, a Western blot is probed with 32P-labeled RCE (using a subunit concentration of 0.3-0.7 nM and 2 × 105 cpm 32P radioactivity/ml). Complexes of 32P-RCE and RCE-binding proteins are visualized by autoradiography. Results were quantified by scanning laser densitometry (Amersham Pharmacia Biotech XL laser densitometer) or PhosphorImager analysis (Molecular Dynamics) as described previously (26, 33).
Equilibrium Binding Assays-- Aliquots (200 ng) of highly purified, GST partial AKAPCE protein (residues 176-409, Fig. 2A) were used for binding assays. Residues 236-255 constitute the high affinity RCE-binding site in AKAPCE (see "Results"). Full-length C. elegans RCE was expressed, purified, and labeled with 32P as described above. Binding assays were performed in 250 µl of buffer A (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.5 mM EDTA, 1 mM dithiothreitol) supplemented with 5% (w/v) fat-free powdered milk proteins. The amount of 32P-labeled RCE was varied from 0.1 to 25 nM. Incubations were carried out at 0 °C for 90 min. Subsequently, 40 µl of a 50% suspension of GSH-Sepharose 4B beads, which was pre-equilibrated with buffer A plus 5% milk proteins, was added and the incubation was continued for 30 min. Next, the samples were diluted to 1 ml with buffer A plus 5% milk, and the beads were pelleted by centrifugation at 2,000 × g for 5 min at 4 °C. The beads were then washed twice by resuspension in 1 ml of buffer A, 5% milk and centrifugation at 2,000 × g at 4 °C. Finally, partial AKAPCE·RCE complexes on the beads were washed three times by resuspension in 1 ml of buffer A containing 0.1% Triton X-100 and centrifugation at 2,000 × g. Bound 32P-labeled RCE was eluted from the beads in 0.2 ml of 1% SDS, and radioactivity was determined by a scintillation counter. 32P radioactivity in an aliquot of the first supernatant solution was also determined to measure the amount of free RCE. Similar results were obtained when the beads were collected and rapidly washed on glass fiber filters.
Immunoprecipitation of AKAPCE·RCE Complexes-- Cytosol was prepared from transfected AV-12 cells as described previously (31). Affinity purified IgGs directed against RCE (3 µl, 1:1.5, relative to serum) or preimmune serum (2 µl) were mixed with ~100 µl of cytosol containing 150 µg of protein, and the samples were incubated for 1 h at 4 °C. Next, 30 µl of a 50% (v/v) suspension of protein A-Sepharose 4B beads (Amersham Pharmacia Biotech) was added, and the samples were rotated at 4 °C for 16 h. Subsequently, samples were diluted with 0.9 ml of buffer A containing 0.1% Tween 20 and centrifuged at 4,000 × g for 5 min at 4 °C. The beads were collected and washed five times (1 ml per wash) with buffer A, 0.1% Triton X-100 by resuspension and centrifugation at 4,000 × g. Proteins in immune complexes bound to protein A-Sepharose 4B were dissolved and denatured in gel loading buffer, fractionated according to size by denaturing electrophoresis, and transferred to an Immobilon P membrane as described above. The Western blots were probed with anti-AKAPCE IgGs and AKAPCE·IgG complexes were visualized as described above.
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RESULTS |
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Isolation and Characterization of cDNA Encoding a Novel AKAP
from C. elegans--
The overlay binding assay described by Bregman
et al. (18, 19) detects -galactosidase-AKAP fusion
proteins in
phage plaques by their ability to bind cognate,
32P-labeled R subunits. To execute functional screening of
a C. elegans cDNA expression library, it was essential
to produce and purify the nematode RI-like, cAMP-binding subunit,
RCE. A cDNA that encodes full-length RCE
(375 amino acids) (16) was cloned into the expression plasmid pET14b.
This enabled high level synthesis (in E. coli) of a soluble
fusion protein in which the RCE polypeptide is preceded by
a 20-residue N-terminal peptide encoded by plasmid DNA. The fusion
peptide contains a block of six consecutive His residues that
constitute a divalent metal-binding site.
His6-RCE fusion protein was purified to near
homogeneity by affinity chromatography on a Ni2+-chelate
resin (Fig. 1). Approximately 1.5 mg of
purified fusion protein was isolated from a 250-ml culture of E. coli.
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Structural Properties of AKAPCE-- The derived amino acid sequence of AKAPCE is presented in Fig. 2A. AKAPCE is a novel, highly acidic (pI ~4.4) protein composed of 1,280 amino acids (Mr = 144,000). There are no homologs of AKAPCE in current data bases. However, several regions of potential functional significance are evident in the AKAPCE sequence. Residues 544-596 constitute a RING finger motif (37) (CX2CX11CX2CX4CX2CX23CX2C, where C indicates Cys and X indicates any amino acid; the lengths of sequences between C2 and C3, and C6 and C7 are long and variable, typically 9-39 residues and 4-48 residues, respectively). RING finger domains fold into a configuration in which zinc is tightly bound, via tetrahedral coordination with 4 Cys residues (37). Thus, each RING domain sequesters two zinc atoms. The RING finger region of AKAPCE is flanked by two candidate PKA phosphorylation sites (residues 522-526 and 613-617, respectively) (Fig. 2A). A third PKA target site is located between residues 711 and 716. A partial protein containing residues 445-745 of AKAPCE is a substrate for PKA in vitro.2 Finally, a binding site for RCE was identified and characterized as described below.
Organization of the AKAPCE (kap-1) Gene--
During
the course of our investigations, the C. elegans genome
project (34) deposited DNA sequences for large segments of chromosome
II in the GenBankTM data base. A data base search with 5'
and 3' AKAPCE cDNA sequences revealed that cosmid D1022
contained the complete AKAPCE (kap-1) gene. (No
previous studies have addressed experimentally any aspects of
kap-1 gene expression or properties and functions of the
AKAPCE protein.) Alignment of the complete sequence of
AKAPCE cDNA with the DNA sequence of cosmid D1022
disclosed the intron/exon organization of the cognate gene (Table
I). The remarkably compact C. elegans AKAPCE structural gene contains 17 exons but
spans only 5.3 kbp of DNA because 15 of the 16 introns are 58 bp in
length (Table I). The AKAPCE locus lies slightly to the
right of center on chromosome II, where it is flanked by genes named
col-6 and sro-1 (34).
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Residues 236-255 in C. elegans AKAPCE Constitute an
RCE-binding Site--
In order to verify the
RCE binding activity of AKAPCE and initiate
determination of structural features that constitute the tethering
site, several fragments of AKAPCE (segments composed of
residues 176-475, 445-745, 711-1011, and 976-1280, Figs.
2A and 3A) were expressed as GST fusion proteins.
Partial AKAPCE (176-475) avidly bound
32P-RCE, whereas the other anchor protein
fragments had no tethering activity (Fig.
3B). Subsequently, N- and
C-terminal deletion mutagenesis was employed to establish boundaries
for the RCE-binding domain according to the strategy
outlined in Fig. 3A. All GST fusion proteins that bind
RCE contain amino acids 236-255 (Fig. 3,
B-D). This region has an overall organization
that is somewhat similar to the RII-binding site in AKAP75, although
little sequence identity is evident. Four aliphatic hydrophobic amino
acids, including three that are essential for RII binding in AKAP75
(26), align with hydrophobic residues in the AKAPCE-binding
domain (Fig. 4A). The putative
tethering domain of AKAPCE is also predicted to be an
amphipathic -helix with a hydrophobic face (Fig. 4B). The AKAPCE tethering domain includes a Tyr and 2 Phe residues,
which may be involved in recognition and binding of RI-like proteins. Aromatic amino acids are absent from RII-binding sites of most previously characterized AKAPs. Moreover, the rare Phe or Tyr residues
(no more than one per binding site) observed in RII tethering regions
have not been implicated in ligand binding activity.
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AKAPCE Binds RCE with High
Affinity--
If AKAPCE subserves targeting and tethering
of C. elegans PKA in vivo, then the
AKAPCE·RCE complex should have a low
dissociation constant, indicative of highly stable protein-protein
interactions. This parameter was measured in solution by incubating GST
partial AKAPCE (residues 176-409) to equilibrium with
various concentrations of 32P-labeled RCE.
Subsequently, GSH-Sepharose 4B beads were added and
GST-AKAPCE·32P-RCE complexes were
isolated and washed. 32P radioactivity on the beads (bound
RCE) and in the supernatant solution (free RCE)
was determined by scintillation counting, and data were analyzed by the
method of Scatchard (38). Representative results shown in Fig.
6A yield a
KD value of 7 nM. Thus, RCE
is tightly bound by AKAPCE. In typical eukaryotic cells
total R subunit concentration is ~300 nM (39). If the
concentration of AKAPCE is assumed to be 10-100
nM (e.g. in a specific cell type), then >85%
of the binding sites would be occupied with RCE (C. elegans PKA). (See Ref. 40 for discussion of such calculations.) Mammalian RII and RII
do not competitively inhibit binding of RCE with AKAPCE (Fig. 6B).
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Production and Purification of Antibodies Directed against
AKAPCE--
cDNA encoding residues 176-409 of
AKAPCE was cloned downstream from the GST gene in the
expression plasmid pGEX-KG. After transformation of E. coli
DH5 and induction with 0.5 mM IPTG, the soluble
GST-AKAPCE-(176-409) fusion protein was purified to homogeneity via affinity chromatography on GSH-Sepharose 4B (Fig. 7A). Approximately 4 mg of
fusion protein was purified from a 1-liter culture of E. coli. Antibodies directed against the partial AKAPCE
protein were produced in rabbits. IgGs in the antiserum yield a robust
signal with 25 ng of AKAPCE partial protein (cleaved from
the fusion protein by thrombin to eliminate GST epitopes) on a Western
blot (Fig. 7B). An excess of structurally unrelated protein
(RCE) is not bound by the antibodies. Excess antigen
eliminates the positive signals, and preimmune serum did not
complex any polypeptides on the blot.
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AKAPCE·RCE Complexes Are Isolated from Transfected Cells-- Extracts were prepared from AV-12 cells that were co-transfected with both AKAPCE and RCE transgenes. Samples were incubated with affinity purified IgGs directed against RCE, and immune complexes were isolated on protein A-Sepharose 4B beads. Proteins in immunoprecipitates were fractionated according to size by denaturing electrophoresis and transferred to a polyvinylidene difluoride membrane. The Western blot was probed with anti-AKAPCE IgGs. Substantial amounts of AKAPCE were co-immunoprecipitated with RCE and anti-RCE IgGs (Fig. 8, lanes 1 and 2). Lane 3 (Fig. 8) shows that AKAPCE directly precipitated by anti-AKAPCE IgGs is indistinguishable from the anchor protein isolated in complexes with RCE (Fig. 8, lanes 1 and 2). Results were confirmed by 32P-labeled RCE overlay binding assays (data not shown). No signals were observed when immunoprecipitates from control (non-transfected) AV-12 cells were assayed by the same procedures (Fig. 8, lane 4). AKAPCE was not precipitated by preimmune serum (Fig. 8, lane 5) or anti-RCE immune IgGs preincubated with excess antigen (Fig. 8, lane 6). Thus, stable AKAPCE·RCE complexes are generated in the physiological context of intact cells.
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DISCUSSION |
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We have discovered and characterized cDNAs that encode a
C. elegans A kinase anchor protein, AKAPCE.
AKAPCE avidly binds (KD = 7 nM) the RI-like regulatory subunit (RCE) of
the sole PKA that is expressed in the nematode (16). Since neither
mammalian RII
and RII
nor Drosophila RII2
efficiently compete for the RCE-binding site in the
C. elegans anchor protein, it appears that
AKAPCE is the first example of an RI-specific
targeting/tethering protein.
AKAPCE is a novel protein composed of 1280 amino residues
(Mr = 144,000). However, the nematode anchor
protein shares two overall properties that are characteristic features
of mammalian AKAPs; the polypeptide is highly acidic (pI ~4.4) and
exhibits an erroneously high apparent molecular weight (200,000) in
denaturing polyacrylamide gels. Moreover, several potentially important
motifs are evident within a segment of AKAPCE that spans
amino acids 200-725 (Fig. 2A). Residues 544-596 are
predicted to fold into a classical RING finger structure (see
"Results"). Initially, it was thought that RING finger proteins
were sequence-specific DNA-binding proteins. A substantial body of
evidence indicates that this is not the case (37). RING finger domains
are present in many cytoplasmic, cytoskeleton-associated and integral
membrane proteins. Emerging concepts are that (a) proteins
containing RING fingers are usually components of multi-protein
complexes and (b) the RING domain plays a key role in the
organization and assembly of such complexes (37). A pertinent example
of a RING finger protein is STE5, the scaffolding protein of yeast that
sequesters and co-localizes subunits of a G protein, MEKK, MEK,
and mitogen-activated protein kinase. This multi-protein complex
couples a pheromone-generated signal at the cell surface to gene
transcription in the nucleus (37). The RING finger domain in
AKAPCE is not essential for binding RCE (Fig. 3
and text of "Results"). Observations and concepts summarized above
suggest the speculation that this region of the anchor protein mediates
immobilization of RCE·AKAPCE complexes at
specific intracellular sites and/or clustering of the complexes with
other signaling proteins.
Three consensus PKA phosphorylation sites (residues 522-526, 613-617, 711-716, Fig. 2A) are included in a fragment of AKAPCE (residues 445-745) that is phosphorylated by the catalytic subunit of PKA in vitro.2 Once the phosphorylation sites are precisely mapped and mutated, it should be possible to determine if PKA-mediated phosphorylation reversibly regulates the ability of AKAPCE to bind with "docking" proteins (or RCE), thereby modulating scaffolding or targeting functions.
The high affinity RCE-binding site was mapped to a short
segment of AKAPCE that encompasses amino acids 236-256.
Comparison of the RCE-binding region with the prototypic
RII binding module of bovine AKAP75 disclosed several similarities.
Binding domains contain ~20 amino acids, and both are predicted to
fold as -helices that contain one large, predominantly hydrophobic
face. Studies on mammalian AKAP75 and AKAP79 indicate that a large
hydrophobic surface at the tethering site and a complementary apolar
surface on RII
and RII
dimers are essential for formation of
stable AKAP·PKAII complexes (25, 26, 41). Binding of
AKAPCE with RCE may also be driven by
hydrophobic interactions. Although sequence identity between the
aligned binding domains is minimal, three aliphatic hydrophobic amino
acids (Leu236, Ile248, and Leu252)
of AKAPCE match large aliphatic hydrophobic residues that
are essential for the RII binding activity of AKAP75 (26). Mutation of
Ile248 to Ala eliminated the tethering activity of
AKAPCE; the corresponding mutation in AKAP75 yields the
same result. Thus Ile248 and its highly conserved
counterparts in AKAP75 and other AKAPs (1-5) apparently play a central
role in maintaining the structural integrity R subunit-binding
sites.
Other features of the RCE-binding domain diverge sharply from properties conserved in RII-selective mammalian AKAPs. Substitution of Leu252 with Ala had little effect on the RCE tethering capacity of AKAPCE, whereas the comparable replacement in AKAP75 compromises binding activity. Positions occupied by Phe253 and Ser254 in AKAPCE are almost invariably filled by Leu, Ile, or Val in mammalian AKAPs. Moreover, mutagenesis of Phe253 (to Ala) revealed that this aromatic hydrophobic residue is essential for maintenance of high affinity for RCE. Two additional aromatic amino acids (Tyr237 and Phe239) are included in the RCE binding module, whereas aromatic residues are excluded from most RII-binding AKAPs. Finally, basic amino acids (Arg255 and Lys256) at the C terminus of the binding region in AKAPCE align with hydrophobic amino acids in AKAP75 (Fig. 4). Mutagenesis and binding analyses are needed to establish or exclude roles for Tyr237, Phe239, Arg255, Lys256, and Ser254 in controlling the affinity and selectivity of the RCE-binding domain and to define further unique features of an RI-selective binding module. Similarities and differences between the R-binding sites in AKAPCE and S-AKAP84/D-AKAP1 (4, 6, 10) are essentially the same as those described above for AKAPCE and AKAP75.
The discovery of the RCE-binding domain in a C. elegans protein, the recent identification and characterization of an RII-selective binding site in a Drosophila AKAP (28), and conservation of structural features shared with mammalian RII tethering sites (see above) indicate the R binding modules and R subunits co-evolved in a broad range of eukaryotes. It is possible that current R binding modules are derived from a common ancestor domain that was incorporated into a variety of polypeptides through the process of exon shuffling. Conservation of R binding modules during evolution also provides further support for the idea that anchoring/targeting of PKA isoforms plays a pivotal role in diversifying and adapting A kinases for a variety of physiological functions. This concept is especially pertinent for C. elegans. This nematode expresses only one type of PKA molecule (RCE2 CCE2), yet the kinase is nearly equally distributed between cytosolic and particulate (i.e. membrane/cytoskeleton) fractions of tissues (16). Moreover, C. elegans PKA must mediate a myriad of conserved regulatory functions that are operative in all eukaryotes. The demonstration that AKAPCE avidly binds RCE in intact cells (Fig. 8) suggests that interactions of PKA with anchoring/targeting proteins(s) provide a mechanism for diversification and specialization in PKA signaling pathways in the nematode.
The cDNA expression library (3 × 106 independent clones) has not been exhaustively screened for AKAPs (which are typically encoded by low abundance mRNAs) because (a) only one cDNA in three has the appropriate reading frame for translation, and (b) partial cDNA clones may lack the 20-residue RCE-binding site. Thus, it is possible that additional AKAPs mediate targeting and tethering of C. elegans PKAI to organelles and cytoskeleton. An alternative targeting mechanism involves the possibility that distinct domains in the large (144 kDa) AKAPCE polypeptide bind with protein ligands that accumulate in certain organelles or portions of cytoskeleton. Such interactions would enable the distribution of PKAI·AKAPCE complexes to multiple intracellular sites. These possibilities are currently under investigation.
Immunoprecipitation experiments revealed that some AKAPCE is recovered in cytosol. This does not preclude a tight association of the anchor protein with cytoskeleton in situ. Many components of highly organized cytoskeletal structures are isolated in cytosol (e.g. microtubule associated protein-2, paxillin, actin-binding proteins, 15-30% of AKAP75) when cells/tissues are disrupted in standard, low ionic strength buffer (as used in our studies) in the absence of certain stabilizing agents. The development of stabilizing conditions for C. elegans cytoskeleton and the application of confocal immunofluorescence microscopy should facilitate assessment of the distribution of AKAPCE among various subcellular fractions in future experimentation. It is also possible that AKAPCE is a "cytoplasmic anchor" or "scaffold" that either (a) directs cAMP signaling to substrate/effector proteins in cytoplasm, (b) inhibits PKA-mediated signaling at certain organelles or sites in cytoskeleton, or (c) co-assembles tethered PKA with other substrate and effector proteins that are directly bound to other domains of the anchor protein. The cloning of AKAPCE cDNA and the availability of the kap-1 gene and its flanking (regulatory) regions will now permit enhancement, inhibition, mislocalization, and ablation of AKAPCE in situ in C. elegans via recently developed molecular genetics technology. Such approaches may ultimately reveal the exact physiological roles for anchored PKA.
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ACKNOWLEDGEMENT |
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We thank Ann Marie Alba for expert secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM22792 (to C. S. R.) and a National Institutes of Health Pharmacological Sciences Training Grant GM07260 stipend (to R. A.).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) AF045926.
To whom correspondence should be addressed: Dept. of Molecular
Pharmacology, F-229, Albert Einstein College of Medicine, 1300 Morris
Park Ave., Bronx, New York 10461. Tel.: 718-430-2505; Fax: 718-430-8922; E-mail: rubin{at}aecom.yu.edu.
1
The abbreviations used are: AKAP, A kinase
anchor protein; R, regulatory subunits; C, catalytic subunit; PKA,
protein kinase A; RII, regulatory subunits of type II PKA isoforms;
RI, regulatory subunit of type I
PKA; RCE, regulatory
subunit of C. elegans PKAI; GST, glutathione
S-transferase; PCR, polymerase chain reaction; IPTG,
isopropyl-1-thio-
-D-galactopyranoside; kbp, kilobase
pair; bp, base pair.
2 R. Angelo and C. S. Rubin, unpublished results.
3 In accordance with standard C. elegans nomenclature, genes are named with three lowercase letters and a number. kap is derived from kinase anchor protein.
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REFERENCES |
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