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INTRODUCTION |
Cyclic AMP-dependent protein kinases
(PKAs)1 mediate actions of
hormones that stimulate adenylate cyclase (1-4). Signals carried by
cAMP often regulate activities of proteins that accumulate at discrete
intracellular locations (e.g. ion channels in plasma membrane) (5-8). A kinase anchor proteins (AKAPs) guide transmission and routing of cAMP signals to such microenvironments. AKAPs have an
avid binding site for regulatory subunits (RII) of PKAII
isoforms2 and distinct
domains that target the tethered PKA holoenzyme to docking sites in
organelles or cytoskeleton (5, 6, 9, 10). Accumulation of a high
concentration of anchored PKAII in proximity with clustered
substrate-effector protein promotes efficient reception, amplification,
and precisely focused transmission of signals borne by cAMP (5-8).
Several fundamental questions about AKAP·RII complexes remain
unresolved. Current models suggest that AKAPs statically associate with
docking sites in organelles. However, it is possible that AKAP·RII
complexes shuttle dynamically between intracellular loci. This
proposition has neither been excluded nor systematically investigated.
Many physiological processes are coordinately regulated by groups of
hormones. Homeostatic control is often achieved through synergistic or
antagonistic interactions among signaling pathways that are activated
by different second messenger molecules. A key question is how signals
transmitted via AKAP·PKAII complexes are integrated with inputs from
pathways controlled by other second messengers in order to regulate
common effector proteins. PKA anchoring may be regulated by
pretranslational mechanisms. In thyroid-derived cells, activation of
adenylate cyclase elicits AKAP121 gene transcription and accumulation
of AKAP121 mRNA and protein (11). However, hormonal activation of
transcription of other AKAP genes has not been demonstrated. Could the
concentration of anchored PKAII be governed by a post-transcriptional mechanism?
Drosophila melanogaster provides an attractive model system
for addressing questions posed above. Signaling pathways, molecules, and mechanisms used in Drosophila are conserved in mammals
(12). Classical and molecular genetics indicate that accumulation and activation of PKA at discrete intracellular locations are essential for
normal development and reproduction in Drosophila (13-15). Consequences of localized activation of PKA are proper
anterior-posterior patterning in developing tissues, maintenance of
intercellular bridges between nurse cells and oocytes, and remodeling
of the microtubule-based cytoskeleton that changes microtubule polarity in embryos and facilitates differential segregation of mRNAs
(14-18). Drosophila express PKAI-, PKAII-, and high
affinity RII-binding proteins (13, 19, 20). RII binding assays reveal
that Drosophila has about five polypeptides with
characteristic features of
AKAPs.3 Thus, systematic
characterization of the entire constellation of Drosophila
AKAPs is feasible. Such studies could ultimately yield comprehensive
knowledge of regulatory properties, integrative functions and precise
physiological roles of anchored PKA in the context of individual cells
of an intact organism.
Recently, we characterized DAKAP550, a large Drosophila
anchor protein that is asymmetrically positioned in neurons, gut cells, and trachea (13). To further the aim of elucidating the complete spectrum of AKAP structure and function in Drosophila, we
now report on the characterization of a novel, multifunctional anchor protein named DAKAP200. Differential splicing of DAKAP200 gene transcripts produces a second polypeptide (
DAKAP200) that lacks the
ability to bind and target PKAII but appears to subserve functions analogous to those of the mammalian myristoylated alanine-rich C kinase
substrate (MARCKS) protein.
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EXPERIMENTAL PROCEDURES |
Screening of cDNA Libraries--
Expression libraries of
Drosophila melanogaster (Canton S strain) cDNAs were
searched for inserts encoding A kinase anchor proteins by functional
(RII binding) assays and DNA hybridization. cDNA libraries were
generated by reverse transcription of mRNAs isolated from embryos
0-24 h after fertilization. Initially, a cDNA library in
bacteriophage 
gt11 (CLONTECH) was screened by the procedure of Bregman et al. (21). AKAP-
-galactosidase
fusion proteins in phage plaques are detected by their ability to bind 32P-labeled RII. A 5' EcoRI-XcmI
fragment (386 bp) from the largest cDNA (1.7 kbp) was employed as a
template to synthesize a random-primed, 32P-labeled probe
for further screening. Two additional libraries were used: a 5'
stretched Drosophila embryo cDNA library in
bacteriophage gt11 (CLONTECH) and a gridded
library in the plasmid pNB40 (22, 23). Screening via DNA hybridization
(24) yielded 19 independent cDNA clones. Eight of these cDNAs
were characterized as described under "Results."
DNA Sequence Analysis--
cDNA inserts were subcloned into
plasmids pGEM7Z, pGEM5Z (Promega), or pBluescript SKII (Stratagene) and
sequenced by a dideoxynucleotide chain termination procedure (25, 26)
using T7, T3, SP6, and custom oligonucleotide primers. Taq
Dye Deoxy Terminator Cycle Sequencing Kits (Applied Biosystems) were
used according to the manufacturer's instructions. DNA products were
analyzed in a model 377 automated DNA Sequencer (Applied Biosystems) in
the DNA Analysis Facility of Albert Einstein College of Medicine.
Computer Analysis--
Analysis of sequence data and data base
searches were performed using PCGENE-IntelliGenetics software
(IntelliGenetics, Mountainview, CA), BLAST programs (27, 28) provided
by the NCBI server (National Institutes of Health), and programs
provided by the Berkeley Drosophila Genome Project server
(29).
Assay for RII Binding Activity--
Overlay binding assays have
been described in several papers (21, 30). In brief, a Western blot is
probed with 32P-labeled RII (using a subunit
concentration of 0.3 nM and 2 × 105 cpm
of 32P radioactivity/ml), and RII-binding proteins are
visualized by autoradiography. Results were quantified by scanning
laser densitometry (Amersham Pharmacia Biotech Ultroscan XL laser
densitometer) or PhosphorImager analysis (Molecular Dynamics, Inc.,
Sunnyvale, CA) as described previously (31).
Expression and Purification of a DAKAP200 Fusion
Protein--
cDNA encoding amino acid residues 475-753 (see Fig.
1) was synthesized via the polymerase chain reaction as described
previously (26). The 5' primer contained an NdeI restriction
site followed by nucleotides 1577-1598 of DAKAP200 cDNA; the 3'
primer consisted of the inverse complement of nucleotides 2396-2417 in
DAKAP200 cDNA preceded by a BamHI restriction site.
Product DNA was digested with NdeI and BamHI and
cloned into the bacterial expression plasmid pET-14b (Novagen) that was
cleaved with NdeI and BamHI. The cDNA insert
is preceded by vector DNA that encodes a 20-residue N-terminal peptide.
This peptide includes six consecutive His residues, which constitute a
Ni2+-binding site. Fusion gene transcription is driven by
the bacteriophage T7 RNA polymerase. Escherichia coli BL21
(DE3), which contains a genomic copy of T7 RNA polymerase under control
of the lac operon, was transformed with recombinant pET14b.
Transformed bacteria were grown and induced with
isopropyl-1-thio--D-galactopyranoside as described
previously (32). Bacteria were harvested, disrupted, and separated into
soluble and particulate fractions as described in previous studies
(32). The partial DAKAP200 fusion protein (designated p-DAKAP70) was
recovered in the soluble fraction and was purified to near homogeneity
by nickel-chelate chromatography (32). Approximately 3 mg of
His6 fusion protein was obtained from a 300-ml culture of
E. coli.
Expression and Purification of His-tagged Drosophila RII
(RIIDR)--
NdeI and BamHI
restriction sites were appended to the 5'- and 3'-ends of cDNA
encoding full-length Drosophila RII (RIIDR). After cloning the cDNA into pET14b, His-tagged RIIDR
was expressed in E. coli and purified to near homogeneity by
using the strategy described above for the partial DAKAP200 protein.
Production of Antibodies Directed against DAKAP200--
Purified
p-DAKAP70 protein (see above) was injected into rabbits (0.35-mg
initial injection; 0.2 mg for each of three booster injections) at
Covance Laboratories (Vienna, VA) to generate antisera. Serum was
collected at 3-week intervals.
Affinity Purification of Anti-RIIDR
Immunoglobulins--
Purified His-tagged RIIDR fusion
protein was coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia
Biotech) (21) to yield a final concentration of 1.5 mg of protein bound
per ml of resin. Antiserum (2 ml) generated against murine RII
was
incubated with 2 ml of RIIDR fusion protein-Sepharose 4B
for 2 h at 20 °C. Next, the resin was packed into a column and
washed with 20 mM Tris-HCl, pH 7.6, 0.5 M NaCl
until the flow-through reached an A280 of 0. The
column was successively eluted with 5 ml of 0.5% acetic acid, pH 2.5, 0.15 M NaCl, and 5 ml of 0.1 M diethylamine, pH
11.8, to release IgGs. Sufficient 1 M Na2
HPO4 or 0.5 M Hepes was added to column
fractions to adjust the pH to ~7.5. IgGs were dialyzed against PBS
containing 50% glycerol, quantified by absorbance at 280 nm, and
stored at 
20 °C.
Preparation of Cytosolic and Particulate Proteins from
Drosophila--
Drosophila embryos, larvae, pupae, or
separated fly heads and bodies were suspended in 4 volumes of buffer A
(20 mM sodium phosphate, pH 7.4, 20 mM NaCl,
0.2 mM dithiothreitol, 1 mM EDTA, 0.2 mM EGTA, 10 µg/ml soybean trypsin inhibitor, 40 µg/ml
aprotinin, 10 µg/ml pepstatin A, and 40 µg/ml leupeptin) and were
disrupted in a Polytron homogenizer (two 30-s cycles of homogenization
at the maximum setting). All operations were performed at 0-4 °C. The homogenate was centrifuged at 12,000 × g for 20 min, and the supernatant solution (cytosol) was collected. The pellet
was resuspended in the original volume of buffer A, homogenized, and
centrifuged as described above. The pelleted, particulate fraction of
Drosophila homogenates was dispersed in the starting volume
of buffer A by a final round of homogenization.
Preparation of Cytosol and Particulate Fractions from Drosophila
S2 Cells and Hamster AV12 Cells--
Cytosolic and particulate
proteins were prepared as indicated above, with several modifications.
Buffer A was replaced with buffer B (20 mM Hepes-NaOH, pH
7.4, 2 mM DTT, 5 mM EDTA, 1 mM EGTA, 20 mM NaCl, 10 µg/ml pepstatin A, 20 mM
benzamidine·HCl, 100 µg/ml pefablock). Cells were disrupted in a
Dounce homogenizer (tight) with 50 strokes of the pestle.
Centrifugation was performed at 150,000 × g for 30-60 min.
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 (21).
Myosin (Mr = 200,000) 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.
Immunoprecipitations--
Anti-DAKAP200 serum (2 µl) was added
to samples (300-500 µg of protein) of cytosolic proteins or Triton
X-100 solubilized, membrane proteins from S2 or AV12 cells. After
incubation at 4 °C for 16 h, 40 µl of a 50% suspension of
protein A-Sepharose 4B beads was added to the samples. The beads were
washed three times with phosphate-buffered saline, incubated 1 h
with 5% milk proteins in phosphate-buffered saline, and washed three
more times with PBS prior to addition to the sample containing
antigen-IgG complexes. After 2 h on ice, beads were recovered by
centrifugation at 2000 × g for 1 min. The resin was
then washed (4 °C) three times with 1 ml of phosphate-buffered
saline lacking detergent. Finally, the beads were suspended in gel
loading buffer and heated for 5 min at 95 °C in preparation for
denaturing electrophoresis. The same procedure was used to
immunoprecipitate RIIDR·DAKAP200 complexes with 4 µg of
affinity-purified anti-RIIDR IgGs.
Cell Culture and Transfections--
Hamster AV12 cells (derived
from a subcutaneous tumor) were obtained from the American Type Culture
Collection. Cells were grown at 37 °C in Dulbecco's modified
Eagle's medium containing 10% fetal calf serum. Drosophila
embryo-derived Schneider S2 cells were grown at 27 °C in
Schneider's medium containing 10% fetal calf serum.
Full-length cDNA encoding DAKAP200 was excised from plasmid pGEM7Z
by digestion with BamHI and XbaI. The
BamHI overhang was made blunt with Klenow DNA polymerase
prior to digestion with XbaI. The cDNA fragment was then
cloned into the mammalian expression vector pCIS2 (33), which had been
cleaved with NotI, blunted, and then cleaved with
XbaI This placed DAKAP200 cDNA downstream from a
constitutively active cytomegalovirus promoter and upstream from a
polyadenylation signal. AV12 cells were transfected with recombinant
pCis2 plasmid via calcium phosphate precipitation (34).
Full-length RIIDR cDNA was excised from pGEM5Z by
digestion with SalI and SpeI. The cDNA
fragment was cloned into the Drosophila expression plasmid
pMK33 HS (generously provided by Dr. Nick Baker, Department of
Molecular Genetics, Albert Einstein College of Medicine) that was
cleaved with XhoI and SpeI. This placed the
RIIDR cDNA downstream from a strong,
copper(II)-inducible metallothionein promoter/enhancer and upstream
from a polyadenylation signal. pMK33 HS also contains a hygromycin
resistance gene that is controlled by a constitutively active
Copia promoter. S2 cells were transfected by the calcium
phosphate·DNA co-precipitation method, and stable transfectants were
obtained by growth in 0.3 mg/ml hygromycin for 14 days.
Protein Determination--
Protein concentrations were
determined with the Coomassie Plus Protein Assay Reagent (Pierce) using
bovine albumin as a standard.
Western Immunoblot Analysis--
Western blots of cytosolic and
particulate proteins from AV12 and S2 cells were blocked, incubated
with antiserum, and washed as described previously (35). DAKAP200,
DAKAP200, and RIIDR polypeptides were visualized by
indirect chemiluminescence using an enhanced chemiluminescence (ECL)
kit from Amersham Pharmacia Biotech. Membranes were incubated with
horseradish peroxidase coupled with goat immunoglobulins directed
against rabbit IgG heavy and light chains (Amersham Pharmacia Biotech)
in 20 mM Tris-HCl, pH 7.4, containing 0.15 M
NaCl and 0.1% (w/v) Tween 20 (10 ml of solution/gel lane) for 90 min
at 22 °C. Filters were then washed, incubated with Luminol, and
exposed to x-ray film for 2-30 s to record chemiluminescence signals.
Relative amounts of proteins were determined by scanning densitometry
(31, 35). Standard curves were prepared with 0.5-100 ng of recombinant
protein standards. Amounts of protein in experimental samples were
obtained from the linear portion of the standard curve.
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RESULTS |
Discovery of cDNAs Encoding a Novel Drosophila
AKAP--
Candidate cDNA clones for Drosophila AKAPs
were isolated from an embryo cDNA expression library in
bacteriophage gt11. -Galactosidase-AKAP fusion proteins in phage
plaques were detected by their ability to bind 32P-labeled
RII subunits (21). Twenty-one cDNA clones were isolated from 4 × 105 phage. DNA hybridization analysis revealed that
eight cDNAs encoded portions of the previously characterized
DAKAP550 protein (13). The cDNA inserts from five distinct,
cross-hybridizing clones were ligated into the plasmid pGEM7Z and
sequenced. The largest cDNA (1736 bp) contained an open reading
frame for 476 codons, a translation stop signal, and 3'-untranslated
sequence. Sequences of the smaller cDNAs were included within the
1.7-kbp cDNA. A 5'-stretched Drosophila embryo cDNA
library in bacteriophage gt11 and a gridded, high density library in
the plasmid pNB40 (22, 23) were screened by DNA hybridization to obtain
sequences of upstream codons and the 5'-untranslated region. Nineteen
cDNA clones were retrieved from the libraries. Eight nearly
full-length or overlapping partial cDNAs were subcloned into pGEM7Z
or pBluescript plasmids and sequenced to determine a derived amino acid
sequence for an anchor protein named DAKAP200.
The sequence of the largest DAKAP200 cDNA (3053 bp) is deposited in
GenBankTM (accession number AF132884). A predicted
initiator Met codon (nucleotides 155-157) is incorporated within an
optimal translation start motif (ANNATGG, nucleotides
152-158) (36). The putative 154-bp 5'-untranslated portion of the
cDNA lacks alternative ATG sequences but includes translation stop
codons in all reading frames. In addition, amino acid residues 1-7
constitute an acceptor site for myristoylation (see Fig.
1), a modification that occurs at the N
terminus of proteins. An open reading frame of 752 codons follows the
initiator ATG and precedes a translation stop signal at nucleotides
2414-2416. The 3'-untranslated region comprises 614 bp and is followed
by a polyadenylate tail. Processing of the 3'-end of DAKAP200 mRNA
appears to be governed by either of two overlapping, atypical poly(A)
addition signals (GATAAA or AATATA, nucleotides 3004-3014) that
precede the polyadenylate tail by 22 or 17 nucleotides.
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Fig. 1.
Identification of structural motifs in the
DAKAP200 protein that predict specific domain functions. The
complete amino acid sequence of DAKAP200 is presented. Residues that
constitute a target site for N-myristoyl transferase are
shown in boldface type and dashed underline at
the N terminus. Three conserved amino acids that ensure a maximal level
of myristoylation are marked with asterisks. A highly basic,
positively charged segment of DAKAP200 (residues 119-148) is depicted
in boldface type. Two Pro-rich regions are indicated with
boldface italic type. An RII binding site (residues
511-530) is enclosed in a rectangle. The sequence encoded
by a large, alternatively spliced exon (exon 5) is marked by a
solid underline and includes amino acids 345-725. The amino
acid composition is shown below the DAKAP200 sequence. Amino acids that
are unusually abundant in DAKAP200 are indicated with boldface
type; residues that are unusually rare are shown in boldface
italic type. The amino acid sequence of DAKAP200 is
obtained by linking residues 1-344 with amino acids 726-753.
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Several Structural Motifs Are Evident in the Derived Amino Acid
Sequence of DAKAP200--
DAKAP200 is composed of 753 amino acids and
has a calculated Mr of 79,075 (Fig. 1). The
anchor protein is exceptionally acidic (pI ~3.8) and has an atypical
amino acid composition. Glu, Asp, Ser, Thr, Ala, and Pro account for
61% of the residues in DAKAP200. In contrast, Cys, Met, Trp, and Tyr
are included at only five positions (0.7% of total amino acids) in the
polypeptide chain. DAKAP200 has an apparent Mr
of 200,000 upon electrophoresis under denaturing conditions (see
below). High net negative charge and aberrantly reduced electrophoretic
mobility are properties shared between DAKAP200 and mammalian AKAPs (5,
9, 37). Most AKAPs have been assigned names that correspond to their
apparent Mr values (5, 6). Thus, the name
Drosophila A
kinase anchor protein of
200 kDa, or DAKAP200, is formulated in accord with standard
nomenclature for anchor proteins.
The DAKAP200 sequence is not highly homologous with sequences of
previously studied polypeptides. However, several segments of the
sequence shown in Fig. 1 provide clues about potential targeting,
tethering (RII binding), and regulatory domains. The N terminus of
DAKAP200 includes a Gly residue adjacent to the initiator Met and a
Ser-Lys dipeptide at positions 6 and 7. This sequence
(MGXXXSK) constitutes an optimal target site for the ubiquitous enzyme N-myristoyl-CoA transferase (38). A
segment of DAKAP200 that encompasses residues 119-148 (Fig. 1)
includes a large cluster of Lys residues that create a highly basic (pI ~11.5) and positively charged (+12) domain in the midst of a protein with a predicted overall charge of 116 at pH 7. The compositions of
the basic region in DAKAP200 and a central portion of MARCKS and
MARCKS-related proteins (MacMARCKS, F52, MRP) (39, 40) are similar,
although the degree of amino acid identity is modest (
30%) when the
indicated sequences are aligned. The basic region of MARCKS proteins
(named the phosphorylation site domain (PSD)) is a major target for
protein kinase C-catalyzed phosphorylation in many mammalian cells (39,
40). The Drosophila anchor protein also possesses Pro-rich
regions (amino acids 328-332 and 468-479; Fig. 1) that may serve as
docking sites for proteins with Src homology 3 domains (41).
Organization and Location of the DAKAP200 Gene--
Fragments of
genomic DNA that are cloned in P1 vectors are being sequenced and
ordered by the Berkeley Drosophila Genome Project (29).
Screening of the Berkeley Drosophila Genome Project Data base with the full-length DAKAP200 cDNA sequence revealed that P1
clone DS02110 (GenBankTM accession number AC004423)
contains the DAKAP200 gene as well as complete 5'- and 3'-flanking
regions. (No previous studies have analyzed this locus to
(a) elucidate the nature and organization of component genes
or (b) investigate the expression, properties, and functions
of the encoded mRNAs and proteins). Alignment of DAKAP200 cDNA
with portions of the DS02110 sequence revealed the organization of the
cognate gene. The DAKAP200 structural gene spans 15,220 bp and is
composed of six exons and five introns (Table
I). The first exon serves as a template
for transcription of a short segment of 5'-untranslated mRNA. Exon
1 precedes a large intron (~11 kbp) that accounts for >70% of the
nucleotides at this locus. Exons 2-6, which encode the DAKAP200 open
reading frame and 3'-untranslated nucleotides in mRNA, contain a
total of 2773 bp. Small and medium size introns (introns 2-5, Table I)
contribute only 1200 bp of intervening DNA sequence in this region.
Exon 5 is atypically large and includes a block of 381 codons that
direct the incorporation of ~50% of the constituent amino acid
residues into the DAKAP200 polypeptide. A Pro-rich region and the RII
tethering domain are encoded by exon 5 (Fig. 1). In situ
hybridization assays performed by the Berkeley Drosophila Genome Project group disclosed that the anchor protein gene is located
on the left arm of chromosome 2 at the cytological position designated
29C3. The DAKAP200 gene lies between genes named fuzzy and
gurken at positions 2-32 on the physical (genome) map.
Elucidation of the intron/exon organization of the DAKAP200 gene, as
well as complete 5'- and 3'-flanking sequences, provides the first comprehensive picture of an AKAP locus; comparable information is not
available for any of the mammalian AKAP genes.
Identification of an RII Binding Site in DAKAP200--
An
approximate location for the PKAII tethering site in DAKAP200 was
established by sequencing five cDNAs that directed synthesis of
RII-binding, fusion proteins in plaques of a bacteriophage expression
library (see above). The sequence of the smallest cDNA (147 bp)
encodes a region of the anchor protein bounded by Asp490
and Glu538 (Fig. 1). The same sequence was present in each
of the larger (1.1-1.7 kbp) cDNAs. Tethering sites in mammalian
AKAPs are composed of ~20 amino acids and contain a precisely spaced
group of residues with large, aliphatic side chains (Leu, Val, Ile, and
Thr; Fig. 2A) that
cooperatively governs sequestration of RII and RII subunits (35).
Residues 511-530 in DAKAP200 can be aligned with tethering regions of
mammalian AKAPs (9, 35, 42), so that Ile511,
Ile518, Val519, Thr523, and
Val530 are in register with essential hydrophobic residues
in previously characterized RII binding sites (Fig. 2A). A
final conserved position is occupied by a smaller hydrophobic residue
(Ala527) in the Drosophila anchor protein. RII
binding domains of mammalian AKAPs are predicted to fold as an
amphipathic -helix that contains one markedly hydrophobic surface
(43). This feature is also evident in the segment of DAKAP200 that
encompasses residues 511-528 (Fig. 2B). Folding algorithms
predict that this partial polypeptide is organized into an -helix in
which 9 of 10 side chains on one surface have hydrophobic character
(Fig. 2B). Thus, amino acids 511-530 constitute a candidate
RII binding site in DAKAP200.
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Fig. 2.
Identification of a predicted RII
binding site in DAKAP200. A, six hydrophobic
amino acids in a segment of DAKAP200 (residues 511-530) align in
register with key hydrophobic residues in the RII tethering domains of
AKAP75 (residues 392-413; Ref. 35), S-AKAP84 (residues 306-325; Ref.
9), and AKAP-KL (residues 586-605; Ref. 42). Critical hydrophobic
residues are shown in boldface type. B, a
helical wheel diagram of the
orientation of hydrophobic and hydrophilic amino acids in the RII
binding site of DAKAP200. Amino acids that contribute to an extended
hydrophobic surface are underlined.
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To further characterize the tethering domain, a fragment of DAKAP200
cDNA (nucleotides 1577-2314) that encodes amino acids 475-753
(Fig. 1) was amplified by polymerase chain reaction. The cDNA
fragment was cloned into the bacterial expression plasmid pET14b,
thereby creating an in-frame fusion with plasmid DNA that encodes a
20-residue N-terminal peptide (see "Experimental Procedures"). The
fusion peptide includes six consecutive His residues (His tag) that
constitute a high affinity binding site for divalent metals. A
substantial amount of soluble, His-tagged, partial DAKAP200 was
synthesized in transformed E. coli during a 2-h incubation with 1 mM
isopropyl-1-thio--D-galactopyranoside (Fig.
3A, lane 2). The acidic, recombinant protein exhibited an abnormally
large apparent Mr (70,000) during denaturing
electrophoresis; the calculated Mr value of the
partial DAKAP200 protein is 34,000. The soluble fusion protein (named
p-DAKAP70) was purified to near homogeneity by affinity chromatography
on Ni2+-chelate Sepharose 4B resin (Fig. 3A,
lane 3).
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Fig. 3.
Expression, purification, and RII binding
activity of a DAKAP200 partial protein (p-DAKAP70). A,
a His-tagged fusion protein that contains residues 475-753 (Fig. 1) of
DAKAP200 was expressed in E. coli and purified by affinity
chromatography as described under "Experimental Procedures" and
"Results." Samples of total soluble protein from transformed and
induced E. coli (25 µg, lane 2),
proteins in the column flow-through (25 µg, lane 1), and protein eluted with 1 M imidazole (1 µg, lane 3) were size-fractionated by
denaturing electrophoresis. A 10% polyacrylamide gel that was stained
with Coomassie Blue is shown. B, Samples of p-DAKAP70 were
subjected to electrophoresis in a 0.1% SDS, 10% polyacrylamide gel.
Proteins were then transferred to an Immobilon P membrane and incubated
with 32P-labeled RII (0.3 nM) (overlay assay,
see "Experimental Procedures") to determine the binding activity of
the fusion protein. Lanes 1-6 received 20, 10, 5, 2.5, 1, and 0 ng, respectively, of the partial anchor protein. An autoradiogram
is presented. Only the relevant portion of the gel is shown; no other
radioactive bands were observed. C, a Western blot was
prepared using samples of purified p-DAKAP70 and bovine brain AKAP75.
The blot was probed with 32P-labeled RIIDR.
32P-RIIDR·AKAP complexes were
visualized by autoradiography. Lanes 2-4
contained 100, 200, and 150 ng, respectively, of p-DAKAP70;
lane 5 received 150 ng of AKAP75. Lane 1 was loaded with 1 µg of transferrin
(Mr = 77,000) and 1 µg albumin
(Mr = 67,000) and served as a negative
control.
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Small amounts of immobilized p-DAKAP70 avidly bound
32P-RII (human) in an overlay assay performed with a low
concentration (0.3 nM) of labeled ligand (Fig.
3B). Similar results were obtained with radiolabeled murine
RII, thereby indicating that the tethering domain of DAKAP200
complexes both RII isoforms with high affinity. Experiments that
definitively map N and C-terminal boundaries of the RII binding site
and reveal individual amino acids that are essential for high affinity
ligation of RII by DAKAP200 are presented in an accompanying paper
(44).
A caveat is that both binding studies and functional screening of
cDNA expression libraries were designed and executed on the basis
of a logical but unproved assumption, that mammalian RII isoforms are
interchangeable substitutes for authentic Drosophila RII
(RIIDR) subunits. To verify this assumption and demonstrate more directly the physiological relevance of the novel fly anchor protein, it was essential to examine the ability of DAKAP200 to bind
RIIDR. Recently, the Kalderon
laboratory4 cloned and
sequenced cDNA that encodes the 376-residue RIIDR polypeptide. Structural features that govern subunit dimerization and
create a docking surface for AKAPs are located near the amino terminus
(residues 1-50) of mammalian RII isoforms (32, 45-49). Alignment of
the N termini of RIIDR and RII reveals that the two
sequences are quite divergent (only 44% identity). However, groups of
aromatic residues (Phe and Tyr) and amino acids with large aliphatic
side chains (Leu, Val, and Ile) contribute the essential functional
properties of dimerization and AKAP binding regions in RII and
RII (32, 45-49). Amino acids with these characteristics are
conserved at all corresponding positions within residues 1-50 of
RIIDR.
His-tagged RIIDR was expressed in E. coli by
using the strategy outlined above for p-DAKAP70. Approximately 1 mg of
nearly homogeneous RIIDR (apparent
Mr = 54,000) was obtained from a 350-ml culture
of E. coli. Like mammalian RII subunits, RIIDR
contains the PKA phosphorylation site sequence
(RRXSX) in a linker region between the
dimerization-AKAP binding domain and the cAMP binding sites. Thus,
RIIDR was labeled by incubation with
Mg-[
-32P]ATP and the catalytic (C) subunit of PKA.
32P-Labeled RIIDR binds with low levels of
p-DAKAP70 (Fig. 3C, lanes 2-4) and
also forms a stable complex with bovine AKAP75 (Fig. 3C,
lane 5). Thus, structural features that mediate
interactions between RII subunits and AKAPs have co-evolved and are
conserved from flies to humans. Binding interactions between RII,
RII, or RIIDR and various AKAPs are sufficiently similar
to enable their interchangeable use. Since recombinant mammalian RII
isoforms are available in plentiful supply and these proteins are more thoroughly characterized than RIIDR, they were employed for
most of the studies presented herein. Repetition of experiments with RIIDR yielded similar results in all instances.
Alternative Splicing of DAKAP200 mRNA Produces a Predicted
Protein (DAKAP200) That Lacks an RII Tethering Domain--
A novel,
1.9-kbp insert was discovered by sequencing candidate DAKAP200
cDNAs obtained from phage and bacterial libraries (see above). The
sequence of 5'-untranslated nucleotides and a large contiguous DNA
segment comprising 344 codons was identical with nucleotides 94-1186
in DAKAP200 cDNA (GenBankTM accession number AF132884).
However, nucleotide 1186 was directly linked to a 3' sequence that
matches perfectly with nucleotides at positions 2330-2964 in DAKAP200
cDNA. Alignment of the 1.9-kbp cDNA (GenBankTM
accession number AF132885) and DAKAP200 gene sequences revealed that
the smaller cDNA was produced by the precise excision of exon 5 (Table I) from the DAKAP200 transcript. The shorter cDNA and its
cognate polypeptide were named "deleted DAKAP200," or DAKAP200, to indicate their derivation from DAKAP200 via exon elimination. Excision of exon 5 results in the loss of a block of 381 residues that includes the RII binding region and a Pro-rich sequence
that is a candidate Src homology 3 binding site. Other structural
features of DAKAP200 are retained in the 372-residue DAKAP200
polypeptide (Fig. 1). DAKAP200 is a highly acidic protein (pI
~4.2) in which Glu, Asp, Ala, Pro, Ser, and Thr contribute 62% of
total amino acids. The protein lacks Tyr, whereas Met, Cys, and Trp
appear only once in the conceptual translation of the protein (Fig. 1).
DAKAP200 has a calculated Mr of 38,000, but
its atypical migration in denaturing electrophoresis yields an apparent
Mr of 95,000 (see below). Moreover, the
N-terminal myristoylation site (residues 1-7), the highly basic
PSD-like region (residues 118-148), and a Pro-rich (and Thr-rich)
segment (residues 316-335) are present in both DAKAP200 and the
larger DAKAP200 isoform.
DAKAP200 and DAKAP200 Are Expressed throughout Drosophila
Development and in an Embryo-derived Cell Line--
The purified
279-residue protein fragment corresponding to the central and
C-terminal portion of DAKAP200 (amino acids 475-753, p-DAKAP70; Fig.
1) was injected into rabbits to produce antibodies. A high dilution
(1:3000) of antiserum yielded robust signals on lanes of a Western blot
that contained low levels of partial DAKAP200 protein (Fig.
4A). Thus, anti-DAKAP200 IgGs
bind with high affinity and provide a sensitive assay for monitoring
expression of the Drosophila anchor protein.
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Fig. 4.
Anti-DAKAP200 IgGs avidly bind recombinant
anchor protein synthesized in bacteria, mammalian cells, and
Drosophila S2 cells. A, a Western blot
was prepared from a denaturing 10% polyacrylamide gel as described
under "Experimental Procedures." The lanes were loaded with the
indicated amounts of partial DAKAP200 (p-DAKAP70). The blot was probed
sequentially with anti-DAKAP200 serum (1:3000) and peroxidase-coupled
secondary antibodies directed against rabbit IgGs. p-DAKAP70·IgG
complexes were visualized by an enhanced chemiluminescence procedure
(see "Experimental Procedures"). B, hamster
AV12 cells were stably transfected with a full-length DAKAP200
transgene as described under "Experimental Procedures." Samples (30 µg) of cytosolic (lanes 1, 5, and
7) and total particulate proteins (lanes 2, 6, and 8) were isolated from AV12
cells (see "Experimental Procedures") transfected with the DAKAP200
transgene. Proteins were then size-fractionated by SDS-PAGE (7.5% gel)
and transferred to an Immobilon P membrane. Samples (30 µg) of
cytosolic (lane 3) and particulate
(lane 4) proteins from nontransfected AV12 cells
were processed in the same manner. Lanes 1-4
were probed with anti-DAKAP200 serum (1:3000); lanes 7 and 8 were incubated with anti-DAKAP200 serum
(1:3000) in the presence of 3 µg of p-DAKAP70 antigen; and
lanes 5 and 6 were incubated with
preimmune serum (1:1500). After incubation with secondary antibodies
coupled to peroxidase, DAKAP200·IgG complexes were detected by an
enhanced chemiluminescence procedure. Signals recorded on x-ray film
are shown. Only the relevant portion of the blot is shown. No other
immunoreactive proteins were observed. C, cytosolic and
total particulate fractions were prepared from embryo-derived S2 cells
as described under "Experimental Procedures." Samples (20 µg) of
cytosolic (lanes 1, 3, and
5) and particulate (lanes 2,
4, and 6) proteins were fractionated in an
SDS-7.5% polyacrylamide gel and transferred to an Immobilon P
membrane. Lanes 1 and 2 were probed
with anti-DAKAP200 serum (1:3000); lanes 3 and
4 were incubated with anti-DAKAP200 serum (1:3000) in the
presence of excess antigen (3 µg p-DAKAP70); lanes 5 and 6 were exposed to preimmune serum (1:3000).
DAKAP200-IgG and DAKAP200-IgG complexes were detected and recorded
as described for B.
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The apparent Mr of the polypeptide encoded by
full-length DAKAP200 cDNA was determined by a
transfection/expression experiment. A cDNA insert containing the
complete anchor protein open reading frame was ligated into the
multiple cloning site of mammalian expression vector pCIS2 (see
"Experimental Procedures"). Recombinant vector was introduced into
a hamster tumor cell line (AV12) as a calcium phosphate-DNA
co-precipitate (37). A protein with an apparent
Mr of 200,000 was detected by anti-DAKAP200 IgGs
in both the cytosolic and particulate fractions of AV12 cells that contained the transgene (Fig. 4B, lanes
1 and 2). A slightly smaller immunoreactive band
(Mr ~195,000) was also evident in the pellet fraction. These proteins were not detected in cytosol and particulate fractions isolated from nontransfected AV12 cells (Fig. 4B,
lanes 3 and 4). No signals were
obtained when lanes 1 and 2 were
duplicated and probed with preimmune serum (Fig. 4B,
lanes 5 and 6) or immune serum in the
presence of excess (3 µg) antigen (Fig. 4B,
lanes 7 and 8). Thus, the antibodies
are highly specific and indicate that the 3-kbp DAKAP200 cDNA
directs synthesis of a 753-residue protein (Fig. 1) with an
unexpectedly large apparent Mr (~200,000). The
appearance of a DAKAP200 doublet (Fig. 4B, lane
2) suggests that the anchor protein may be a target for
post-translational modification and/or proteolytic cleavage.
An embryo-derived Drosophila cell line named Schneider S2
cells (hereafter designated "S2 cells") expresses endogenous
DAKAP200 that is visualized as a cluster of three or four polypeptide
bands with apparent Mr values in the range of
190,000-205,000 (Fig. 4C, lanes 1 and
2). The intensity of the smallest of these bands varies
widely among samples and may arise from proteolysis or dephosphorylation. In contrast, the 200/205-kDa doublet reproducibly accounts for the bulk of the anchor protein. Anti-DAKAP200 IgGs also
bind with a protein that exhibits an apparent Mr
of 95,000 (Fig. 4C, lanes 1 and
2). Together, highly specific IgG binding (Fig.
4C, compare lanes 1 and 2 with lanes 3-6) and the size of the protein
(i.e. ~50% of apparent Mr of
DAKAP200) suggest that the 95-kDa band corresponds to the 372-residue
DAKAP200 polypeptide (Fig. 1). The 95-kDa protein is detected by the
IgGs because 28 amino acids at the C terminus of the p-DAKAP70 antigen
are shared by DAKAP200 and DAKAP200 (Fig. 1). This segment of the
anchor protein provides a common epitope(s) that is recognized by
anti-DAKAP200 IgGs in the absence of the remainder of the
polypeptide.5 As expected,
expression of the 1.9-kbp DAKAP200 cDNA in AV12 cells elicits
accumulation of a recombinant protein with an apparent Mr of 95,000 (data not shown).
DAKAP200 and DAKAP200 (95 kDa) proteins are distributed nearly
equally between cytosolic and total particulate fractions derived from
S2 cell homogenates (Fig. 5A,
lanes 2 and 3). Cytosolic and
particulate RII binding activity was associated exclusively with a
tight cluster of proteins that exhibit an apparent
Mr of ~200,000 (Fig. 5B,
lanes 2 and 3). To determine the
relative contribution of DAKAP200 to this tethering activity, cytosol
was incubated with antibodies directed against the anchor protein for
16 h. Immune complexes were harvested with protein A-Sepharose 4B
beads and assayed via Western blot and overlay binding procedures.
Essentially all immunoreactive proteins (200-kDa cluster and 95-kDa
band) were depleted from cytosol by immunoprecipitation (Fig.
5A, lane 4). DAKAP200 and DAKAP200
proteins were recovered in high yield in the immunoprecipitate (Fig.
5A, lane 5). Some of the larger polypeptides appeared to be partly degraded during the 16-h incubation used for immunoprecipitation, whereas the 95-kDa DAKAP200 protein was evidently less susceptible to proteolysis. Virtually 100% of RII
binding activity was co-isolated with DAKAP200 proteins in the
immunoprecipitate (Fig. 5B, lanes 4 and 5). No RII tethering activity was associated with
DAKAP200 (Fig. 5, compare A, lanes 2 and 5, with B, lanes
2 and 5) or distinct 200-kDa proteins that co-migrate with DAKAP200 (Fig. 5B, lane
4). These results were replicated when detergent-soluble
proteins from the particulate fraction of S2 cells were analyzed by
immunoprecipitation and overlay binding assays (data not shown). Thus,
the predicted DAKAP200 protein (Fig. 1) is produced endogenously in
Drosophila embryo cells. Authentic DAKAP200 binds RII
subunits with high affinity and appears to be the principal (perhaps
only) anchor protein in S2 cells.
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Fig. 5.
Antibodies directed against DAKAP200
efficiently precipitate the fly anchor protein and DAKAP95; DAKAP200 accounts for all of the RII
binding activity in S2 cells. A, a Western blot was
prepared, probed, and developed as described under "Experimental
Procedures." Lane 1 received 50 µg of
cytosolic proteins derived from AV12 cells transfected with the
DAKAP200 transgene (positive control). Lane 2 contained 50 µg of cytosolic proteins from S2 cells; lane 3 received 50 µg of particulate proteins from S2 cells.
Anti-DAKAP200 serum (1.5 µl) was added to 0.25 ml of S2 cytosol (300 µg of protein). After 16 h at 4 °C, antigen-antibody
complexes were harvested by the addition of protein A-Sepharose 4B and
centrifugation. Precipitated proteins were applied to lane 5. A sample of nonprecipitated proteins in the supernatant
solution (20% of total sample) was placed in lane 4. Signals from the ECL assay were recorded on x-ray film.
Only the relevant portion of the gel is shown. No other bands were
observed on the blot. B, blots were prepared as described
above. The identity and amounts of samples applied to lanes 1-5 are the same as described for A. The blot
was incubated to equilibrium with 0.3 nM
32P-labeled RII (overlay assay, see "Experimental
Procedures") to identify AKAPs. An autoradiogram is shown. Antibodies
directed against DAKAP200 precipitated essentially all of the RII
tethering activity (lane 5) originally observed
in S2 cytosol (lane 2). Depletion of the AKAP
from cytosol is documented in lane 4. Cytosol
from transfected AV12 cells contains recombinant DAKAP200 (positive
control) as well as an endogenous mammalian, RII-binding protein
(Mr ~260,000) (lane 1).
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Overall patterns of in vivo expression of DAKAP200 and
DAKAP200 were established by performing Western blot analysis on
protein samples isolated from flies at various developmental stages.
Results obtained for cytosolic (Fig. 6)
and detergent-solubilized, particulate proteins were similar. The
DAKAP200 protein cluster was enriched in pupae (3-4-fold higher than
other stages) (Fig. 6), but substantial levels of these anchor proteins
were also evident in embryos, adults, and larvae (Fig. 6, larvae not
shown). DAKAP200 is also detected at all phases of the
Drosophila life cycle (Fig. 6). This indicates that
alternative splicing of DAKAP200 gene transcripts is operative during
the progression of embryonic and postembryonic development. The
concentration of DAKAP200 in the adult head, which is enriched in
neurons, is ~7-fold higher than that observed in body parts (Fig. 6).
Therefore, alternative excision of exon 5 from DAKAP200 mRNA and/or
stability of DAKAP200 protein may be differentially regulated in a
cell/tissue specific fashion in mature Drosophila.
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Fig. 6.
DAKAP200 and DAKAP200 are expressed at all stages of
Drosophila development. Cytosol was isolated from
embryos (E), larvae (not shown), and pupae (P) as
well as the bodies (AB) and heads (AH) of adult
flies (see "Experimental Procedures"). Proteins (25 µg) were
size-fractionated by SDS-PAGE (7.5% gel), transferred to a
polyvinylidine difluoride membrane, and probed with anti-DAKAP200 serum
(1:3000). Immunoreactive proteins were visualized by indirect, enhanced
chemiluminescence methodology. The four lanes
shown on the left were replicated and incubated with
anti-DAKAP200 serum in the presence of excess antigen (3 µg). No
signals were observed (four lanes on the
right).
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A critical test of the idea that the Drosophila anchor
protein mediates physiologically relevant targeting/immobilization of
PKAII in situ involves isolation of stable RII·DAKAP200
complexes from intact cells. S2 cells accumulate DAKAP200 (Fig. 5), but contain an extremely low level of endogenous RIIDR (data
not shown). Therefore, cDNA encoding the complete amino acid
sequence of RIIDR was inserted into the multiple cloning
region of a Drosophila expression plasmid pMK33/HS. This
positions the 5'-end of the insert downstream from a
Drosophila metallothionein promoter/enhancer sequence; the
3'-end of RIIDR cDNA is followed by a poly(A) addition signal from an actin gene. Moreover, the plasmid contains a hygromycin resistance gene under the control of a strong constitutively active promoter (Copia LTR) that enables drug-based selection of
stably transfected cell lines. The fly metallothionein promoter is
tightly regulated and exhibits little activity in the absence of
Cu2+ ions. The addition of 0.5-1 mM
CuSO4 to the medium efficiently activates transactivating
factors that potently promote transcription of the chimeric gene.
Stable lines of S2 cells that carry the RIIDR transgene
were established by calcium phosphate-mediated transfection and growth
for 14 days in the presence of 0.3 mg/ml hygromycin.
Antibodies initially directed against murine RII were
affinity-purified on a column of His-tagged RIIDR-Sepharose
4B (see "Experimental Procedures"). The purified IgGs bound both
(His)6-RIIDR and glutathione
S-transferase-RIIDR recombinant fusion proteins on a Western blot (Fig. 7A,
lanes 1 and 5). Thus, the antibodies recognized and complexed epitopes within RIIDR in addition
to (or instead of) the artificial 20-residue His6 fusion
peptide that was appended to the original RII antigen. No
immunoreactive polypeptides were evident among cytosolic proteins
isolated from nontransfected S2 cells (Fig. 7A,
lane 3). In contrast, affinity-purified IgGs
bound a 54-kDa protein in cytosol prepared from S2 cells that were
transfected with the RIIDR transgene and induced with 1 mM CuSO4 (Fig. 7A, lane
4). Moreover, cytosol derived from adult Drosophila also contained a single immunoreactive protein
with an apparent Mr of 54,000 (Fig.
7A, lane 2). The
Mr observed for RIIDR in S2 cells
and intact flies is the same as that previously reported for a near
homogenous preparation of the R subunit of Drosophila PKAII
(19). Similar results were obtained for RIIDR associated
with the particulate fractions of adult Drosophila and
transfected S2 cells (data not shown). The blot shown in Fig. 7A was duplicated and probed with anti-RIIDR
IgGs in the presence of excess highly purified RIIDR. No
signals were observed under these conditions. Thus, a subset of
antibodies directed against mammalian RII selectively and
specifically complex RIIDR in extracts of
Drosophila cells and tissues.
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Fig. 7.
Affinity-purified anti-mouse
RII IgGs bind Drosophila RII;
DAKAP200 avidly binds RIIDR in intact Drosophila cells. A, a Western blot analysis was performed
with IgG (1 µg/ml) that was purified on a column of
RIIDR-Sepharose 6B. Electrophoresis, blotting, incubation
with IgG, and the ECL procedure were carried out as described under
"Experimental Procedures." Lane 1 contained
250 ng of His-tagged RIIDR; samples (30 µg) of cytosolic
proteins from adult Drosophila, S2 cells and S2 cells
expressing an RIIDR transgene were assayed in
lanes 2-4, respectively; lane 5 contained 50 ng of a glutathione
S-transferase-RIIDR fusion protein.
B, samples (50 µg) of cytosolic (lanes 1 and 3) and particulate (lanes 2 and 4) proteins from two lines of S2
transfectants (which carry the RIIDR transgene) were
size-fractionated in a denaturing (10%) gel. Cells were induced by
incubation with 1 mM CuSO4 for 16 h prior
to lysis. Accumulation of the regulatory subunit of
Drosophila PKAII was assessed by Western immunoblot
analysis. C, Western immunoblot analysis was performed as
described under "Experimental Procedures." Samples (30 µg of
protein) of cytosol from control S2 cells and S2 cells carrying the
RIIDR transgene (TFx RII) were assayed in
lanes 1 and 3, respectively.
Particulate proteins (30 µg) from control and transfected S2 cells
were applied to lanes 2 and 4,
respectively. Transfected cells were incubated with 1 mM
CuSO4 for 16 h prior to lysis. Cytosol (300 µl, 500 µg of protein) from either control S2 cells or S2 cells transfected
with RIIDR was mixed with 4 µg of affinity-purified
anti-RIIDR IgGs or an equal amount of preimmune
(PI) IgG and incubated overnight. Antigen-IgG complexes were
harvested with protein A Sepharose 6B and washed as described under
"Experimental Procedures." Proteins precipitated from control and
transfected cells by preimmune IgGs were analyzed in lanes 5 and 7, respectively. Proteins precipitated from
control and transfected S2 cells by anti-RIIDR IgGs were
analyzed in lanes 6 and 8,
respectively. The blot was probed with anti-DAKAP200 serum
(1:3000).
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In Situ Generation of DAKAP200·RIIDR
Complexes--
Formation of complexes was investigated in cloned S2
cell lines that were stably transfected with the inducible
RIIDR transgene. Similar amounts of RIIDR
accumulated in the cytosolic and particulate fractions upon incubation
of the various cloned cell lines with CuSO4. Results from
two representative S2 transfectants are presented in Fig.
7B. Expression of RIIDR subunits did not affect
the levels or intracellular distribution of the DAKAP200 protein (Fig.
7C, lanes 1-4). However, after
induction of RIIDR with Cu2+ (Fig.
7C), affinity-purified anti-RIIDR IgGs mediated
precipitation of a substantial amount of DAKAP200 (Fig. 7C,
lane 8). These antibodies failed to precipitate
the anchor protein in the absence of RIIDR (Fig.
7C, lane 6). The inability of
preimmune IgGs to bind the complex (Fig. 7C, lane
7) confirms the specificity of the methodology. DAKAP200
avidly sequesters RIIDR in the milieu of intact
Drosophila cells and produces a complex that is highly
stable during immunoisolation and extensive washing. The results
support the concept that DAKAP200 and Drosophila PKAII can
engage in physiologically significant interactions.
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DISCUSSION |
Complementary DNAs encoding a novel Drosophila AKAP
(DAKAP200) were discovered by screening expression libraries for high affinity RII-binding proteins. The predicted anchor protein (753 amino
acids) has a calculated Mr of 79,075. However,
DAKAP200 exhibits an apparent Mr of ~200,000
in SDS-polyacrylamide gels. This discrepancy is probably due to the
exceptionally acidic character (pI ~3.8) of the PKAII tethering
protein. A high level of net negative charge (116 at pH 7) apparently
reduces SDS binding and promotes retention of higher order structure
under conditions that are fully denaturing for more typical proteins.
Previously characterized AKAPs are also acidic proteins that display
aberrantly large Mr values during denaturing
electrophoresis (5, 6, 9, 10, 42). Conservation of a high level of
negative charge among Drosophila, Caenorhabditis
elegans, and mammalian AKAPs suggests that this electrostatic
property may play a role in targeting, stabilizing, or immobilizing
anchor protein·PKA complexes.
Binding of RII at tethering sites in AKAPs is governed by hydrophobic
interactions (35, 42). Structural features and RII binding assays
suggest that amino acids 511-530 in DAKAP200 constitute an RII binding
site. However, amino acid sequences of tethering domains from DAKAP200
and mammalian anchor proteins are only 20-33% identical. Ligation of
RII, RII, and RIIDR by DAKAP200 is explained by
conservation of higher order structure. Like tethering sites in
mammalian AKAPs (5, 6, 50), the candidate DAKAP200 RII binding domain
is predicted to fold into an amphipathic -helix with opposing
hydrophobic and hydrophilic surfaces (Fig. 2). Five amino acids with
bulky, aliphatic side chains (Ile511, Ile519,
Val520, Thr523, and Val530) in the
putative DAKAP200 tethering region align in register with Ile, Val,
Thr, or Leu residues in RII binding sites of mammalian AKAPs (Fig. 2).
These side chains create a large hydrophobic surface that docks with a
complementary apolar surface that is exposed at the N terminus of RII
subunits (32, 45-47). Strong evolutionary selection pressure has
evidently been exerted on RII tethering modules to retain a
configuration of hydrophobic residues that supports highly selective
complex (AKAP·RII) formation. Conservation of this structural module
and binding mechanism in organisms as diverse as insects and humans
suggests that RII tethering sites in AKAPs subserve fundamentally
important physiological functions in eukaryotes.
The physiological relevance of DAKAP200 was investigated by testing the
ability of the anchor protein to bind an endogenous ligand,
RIIDR. The RIIDR polypeptide
sequence contains the hallmarks of classical RII isoforms (55%
identical with RII) but shares little homology with RI (~28%
identity).4 DAKAP200 and AKAP75 sequestered radiolabeled
RIIDR in vitro. Thus, RIIDR contains
the hydrophobic docking region that binds the conserved PKAII tethering
module in AKAPs. In addition, anti-RIIDR IgGs precipitated
a DAKAP200·RIIDR complex from S2 cell cytosol. Therefore,
DAKAP200 encounters and tightly binds RIIDR subunits in
intact Drosophila cells.
Several domains in DAKAP200 may contribute to the targeting of tethered
PKAII. Two Pro-rich regions (residues 328-332 and 468-479) are
potential binding sites for cytoskeleton/organelle-associated proteins
that contain Src homology 3 domains (41). Amino acids 2-7 (Fig. 1)
constitute an acceptor site for N-myristoyltransferase. Myristoylation of DAKAP200 would provide a long saturated aliphatic chain that inserts into the hydrocarbon interior of phospholipid bilayers (51, 52). A segment of DAKAP200 (residues 118-148) includes a
PSD-like cluster of 13 Lys, five Ser, and five large hydrophobic
residues. Positive charge in PSD2S promotes electrostatic binding with
negatively charged head groups of membrane phospholipids (53-56).
Intercalation of PSD hydrophobic side chains into the apolar interior
of a bilayer further stabilizes association of PSD-containing proteins
with membranes. N-terminal myristate and a PSD are critical features of
MARCKS proteins, which mediate interactions between plasma membrane and
F-actin (39, 40, 51-56). The MARCKS PSD is phosphorylated in
situ by diacylglycerol-activated protein kinase C isoforms (39,
40, 51). Phosphorylation inhibits binding with membrane phospholipids,
enables translocation of MARCKS from cell surface to cytoplasm, and
promotes cytoskeleton remodeling (51-58). The nonphosphorylated PSD
sequesters calmodulin in a calcium-dependent manner (52,
56, 57). Binding of Ca2+-calmodulin diminishes the ability
of MARCKS to cross-link and bundle actin filaments (57). By analogy,
the fly anchor protein may be involved in integrating signals
propagated by three critical second messenger molecules: cAMP,
diacylglycerol, and calcium. Moreover, the PSD region of DAKAP200 may
enable phosphorylation-controlled shuttling of tethered PKA between two
or more intracellular sites.
A novel 1.9-kbp cDNA encodes a second product (DAKAP200)
of the anchor protein gene. DAKAP200 is composed of 372 amino
acids (calculated Mr = 38,000). Messenger RNA
encoding the smaller protein is derived from DAKAP200 transcripts by
alternative excision of exon 5 (381 codons). Since exon 5 encodes the
RII tethering region, DAKAP200 will not anchor PKAII to
intracellular sites. Elimination of a Pro-rich region encoded by exon 5 may also preclude Src homology 3 domain-mediated binding with protein
ligands and result in the routing of DAKAP200 and DAKAP200·RII
complexes to distinct intracellular destinations. It is possible to
speculate that a primordial DAKAP200 gene was converted to a modern
DAKAP200 gene by insertion of a new exon ("exon shuffling") that
originally encoded a distinct RII binding protein comprising 381 amino
acids. If downstream effector/substrates for PKA co-clustered at
docking sites for DAKAP200, then substantial selection pressure
would have favored retention of the "foreign" exon in the mutant
gene. The large size of exon 5 raises the possibility that it arose
from reverse transcription of fully processed mRNA.
DAKAP200 and DAKAP200 are synthesized in embryo-derived S2 cells and
at all stages of Drosophila development. Apparently, both
proteins play important physiological roles throughout the life span of
the fly. Since the sequence of DAKAP200 is included within DAKAP200,
it is probable that these proteins subserve partially overlapping
functions. However, exon 5 provides novel amino acid sequences and
domains that adapt DAKAP200 for distinct physiological roles
(e.g. PKAII targeting). The more compact DAKAP200
polypeptide may (a) adopt an alternative folding pattern in
one or more domains that mediate unique functions (e.g.
across the junction encoded by exons 4 and 6); (b) undergo
targeting to organelles or regions of cytoskeleton that differ from the
destinations reached by DAKAP200·PKAII complexes, and/or
(c) subserve functions redundant with those mediated by
DAKAP200 in order to ensure proper regulation of critical aspects of
homeostasis. Enrichment of DAKAP200 protein in adult head (brain)
relative to other body parts (Fig. 6) suggests that a
post-transcriptional mechanism might control (in part) targeting of
signals along the cAMP/PKA pathway. This mechanism would involve the
regulated enhancement or suppression of the excision of an exon during
pre-mRNA processing.
,
TOP
ABSTRACT
INTRODUCTION
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@aecom.yu.edu.
