(Received for publication, August 28, 1996, and in revised form, January 15, 1997)
From the Department of Chemistry and Biochemistry and
the Department of Pharmacology and Neuroscience Program, School
of Medicine, University of California, San Diego, La
Jolla, California 92093-0654
Compartmentalization of cAMP-dependent protein kinase is achieved in part by interaction with A-kinase anchoring proteins (AKAPs). All of the anchoring proteins identified previously target the kinase by tethering the type II regulatory subunit. Here we report the cloning and characterization of a novel anchoring protein, D-AKAP1, that interacts with the N terminus of both type I and type II regulatory subunits. A novel cDNA encoding a 125-amino acid fragment of D-AKAP1 was isolated from a two-hybrid screen and shown to interact specifically with the type I regulatory subunit. Although a single message of 3.8 kilobase pairs was detected for D-AKAP1 in all embryonic stages and in most adult tissues, cDNA cloning revealed the possibility of at least four splice variants. All four isoforms contain a core of 526 amino acids, which includes the R binding fragment, and may be expressed in a tissue-specific manner. This core sequence was homologous to S-AKAP84, including a mitochondrial signal sequence near the amino terminus (Lin, R. Y., Moss, S. B., and Rubin, C. S. (1995) J. Biol. Chem. 270, 27804-27811). D-AKAP1 and the type I regulatory subunit appeared to have overlapping expression patterns in muscle and olfactory epithelium by in situ hybridization. These results raise a novel possibility that the type I regulatory subunit may be anchored via anchoring proteins.
cAMP-dependent protein kinase (PKA),1 one of the first protein kinases discovered, mediates a variety of hormonal and neurotransmitter responses by phosphorylating different substrate proteins in the cell. Although PKA is a multifunctional enzyme with a broad substrate specificity, activation of this kinase permits preferential phosphorylation of specific target substrates (1). For example, phosphorylation of membrane-bound ion channels modulates the flow of ions into the cell (2), while phosphorylation of CREB, a nuclear transcription factor, alters the expression of cAMP-responsive genes (3). While the importance of PKA in regulating many cellular processes has long been apparent, the potential importance of compartmentalization for the function and regulation of PKA has only recently been recognized.
In the absence of its activating ligand, cAMP, PKA exists as an inactive holoenzyme of two regulatory (R) and two catalytic (C) subunits. The two classes of R subunits, RI and RII, based on their elution from the DEAE cellulose (4), define the two types of holoenzymes. A significant proportion of the type II holoenzyme associates with the particulate fraction of cell homogenates, while the type I holoenzyme appears to be mostly cytoplasmic (5). Following an increase in intracellular cAMP, the R subunits bind cAMP, resulting in the dissociation of the holoenzyme and the release of free active C subunits. The free C subunit can then either phosphorylate cytoplasmic substrates or translocate into the nucleus by passive diffusion and phosphorylate nuclear substrates (6). In addition to the C subunit migrating between compartments, the holoenzyme itself can be anchored to specific sites via interactions of its regulatory subunits with specific anchoring proteins. This may allow for activation of localized pools of the kinase (7, 8).
Both classes of R subunits contain two tandem cAMP binding sites at the
carboxyl terminus that account for approximately two-thirds of the
protein. Both R subunits also contain a site that mimics a substrate or
inhibitor and lies in the active site cleft of the C subunit in the
holoenzyme complex. RI contains a pseudosubstrate site,
while RII contains a phosphorylatable substrate site at
this region. The N terminus of each R subunit contains a very stable
dimerization domain. This domain, which is the region of the least
sequence identity between the two R subunits, is thought to be
responsible for interaction with the anchoring proteins (9). Several
AKAPs (-
inase
nchoring
roteins) have been characterized, and all bind
specifically with very high affinity to the type II regulatory subunit
(RII) (1, 10).
Recently, it was shown that anchoring proteins may also act as adapters for assembling multiprotein complexes. For example, Scott and co-workers (11, 12) showed that AKAP79, in addition to binding tightly to RII, also interacts with the calcium and calmodulin-dependent protein phosphatase 2B (calcineurin) and protein kinase C. Targeting AKAP79 to neuronal postsynaptic densities would therefore bring enzymes with opposite catalytic activities together in a single transduction complex. This adds another level of intracellular organization for PKA and also facilitates the diversity of the cAMP-mediated signal transduction pathway (13).
In most cells, the type I holoenzyme appears not to be anchored and is typically cytoplasmic; however, there are cases where RI is compartmentalized. For example, RI subunits in human erythrocytes are tightly bound to the plasma membrane (14). Type I holoenzyme is also depleted from the cytoplasm and accumulates at the "cap" site of lymphocytes when stimulated with anti-CD3 antibodies (15). Here we report the identification and characterization of a potential dual specificity protein kinase A anchoring protein, D-AKAP1, which binds to both the RI and the RII subunits of PKA.
All vectors for the yeast two-hybrid system were
from Dr. Stan Hollenberg (Vollum Institute). The following reagents
were purchased as indicated: mouse 16-day embryonic cDNA library in EXlox vector (Novagen); mouse multiple tissue northern blot and mouse embryonic Northern blot (Clontech); Ready-to-Go DNA labeling kit
(Pharmacia Biotech Inc.); Genius labeling system (Boehringer Mannheim);
Affi-Gel 15 (Bio-Rad); ECL kit (Amersham Corp.); ATP, phenylmethylsulfonyl fluoride, benzamidine, Triton X-100, and GST-agarose resin (Sigma); SSC buffer (5 Prime
3 Prime, Inc., Boulder, CO); nickel-NTA resin (Qiagen);
5-bromo-4-chloro-3-indoyl
-D-galactoside and enzymes
used for DNA manipulations (Life Technologies, Inc.); and the DNA
sequencing kit (U.S. Biochemical Corp.). Antibodies were generated in
female rabbits at Cocalico Corp. All oligonucleotides were synthesized
at the Peptide and Oligonucleotide Facility at the University of
California, San Diego.
A yeast two-hybrid screen was performed
according to Vojtek et al. (16). Briefly, cDNA coding
for the Ret/ptc2 oncogene, which consists of the N-terminal two-thirds
of RI fused to the c-Ret tyrosine kinase domain (17),
was subcloned into the pBTM116 LexA fusion vector. L40 yeast
transformed with this construct was used to screen an embryonic mouse
random-primed cDNA library (18). From approximately 2 million
transformants, 10 survived nutritional selection and were
-galactosidase-positive specifically for the RI portion
of Ret/ptc2. The library plasmids from these co-transformants were
isolated, and cDNA sequences were determined by the procedure of
Sanger et al. (19). Two of these encoded fragments of
RI
corresponding to residues 12-120 and 17-117. Four
of the remaining eight contained an identical cDNA, coding for a
protein fragment designated RPP7.
To determine whether the protein coded for by RPP7 binds
to RI in vitro, the RPP7 cDNA was subcloned
from the pVP16 plasmid into both pGEX-KG, to make a GST fusion protein,
and into pRSETb-NotI, to make a polyhistidine fusion
protein. pRSETb-NotI was constructed by ligating a linker
containing a NotI site into the NdeI and HindIII sites of pRSETb. These RPP7 fusion proteins,
designated GST-RPP7 and His6RPP7, respectively, were
expressed in Escherichia coli BL21(DE3) at 37 °C and
purified to near homogeneity using either glutathione resin for
GST-RPP7, as described previously (20), or nickel-NTA resin for
His6RPP7. In short, bacterial cell lysates containing
His6RPP7 were incubated with nickel-NTA resin in PBS (10 mM potassium phosphate, 150 mM NaCl, pH 7.4) with 0.1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 5 mM benzamidine, and 5 mM
-mercaptoethanol at 4 °C for 1 h and then washed with the
same buffer with 5 mM imidazole to remove nonspecific
proteins. His6-RPP7 was then eluted from the resin with PBS
containing 100 mM imidazole. The BL21(DE3) cell strain was
a gift from Bill Studier (Brookhaven National Laboratories).
RI
was expressed in BL21(DE3) cells and purified on a DE52 ion exchange
column (21). His6RII and
His6RI(63-379) were purified on nickel-NTA
resin as described previously. RII(
46-400) was
expressed and purified as a polyhistidine-tagged fusion protein. After
removing the polyhistidine tag with factor X (22), the R subunit was
further purified to homogeneity by gel filtration using Sephedex
75.
Bacterial cell lysates containing
GST-RPP7 were incubated with glutathione resin for 2 h. at 4 °C
in PBS with 0.1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 1 mM EDTA, 5 mM benzamidine, and 5 mM -mercaptoethanol and then washed extensively with the same buffer. Full-length and deletion mutants (100-200 µg) of RI and/or RII were added to the resin and
incubated for 2 h at 4 °C. After washing the resin extensively
with PBS, proteins associated with the GST-RPP7 were eluted by boiling
in SDS gel-loading buffer and analyzed by SDS-PAGE. All electrophoresis
was performed using Mini-Protean II electrophoresis system (Bio-Rad).
SDS-PAGE reagents were prepared according to Laemmli (23). Proteins
were visualized by Coomassie Staining.
Blots containing 2 µg of immobilized samples of mRNAs from selected adult tissues or total mRNA at different embryonic stages were probed with 32P-radiolabeled RPP7 cDNA. Nitrocellulose filters were prehybridized in 5 × SSC (750 mM sodium chloride, 75 mM sodium citrate, pH 7.0), 5 × Denhardt's reagent (0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 0.1% acetylated bovine serum albumin), 0.5% SDS, and 50% formamide for 6 h at 42 °C and then hybridized to 1.5 × 106 cpm/ml of denatured radiolabeled cDNA probe in the same buffer. Hybridization was performed at 42 °C for 16 h, and nonhybridized probe was removed with 0.1 × SSC, 0.1% SDS at 68 °C. Hybridizing mRNA signals were detected by autoradiography.
Screening of cDNA LibrariesA 16-day mouse embryonic
cDNA library in vector EXlox was screened with
32P-labeled RPP7 cDNA. The cDNA fragment RPP7 was
excised from the two-hybrid library plasmid pVP16, using the
NotI restriction endonuclease, and purified on agarose gel.
This purified cDNA was then labeled with
[
-32P]dCTP using random prime labeling. Approximately
1.6 million plaques were screened, and positive clones were
plaque-purified. Positive phage clones were converted into plasmids by
infection of hosts expressing the P1 cre recombinase, which recognizes
the loxP site on the
EXlox vectors and forms the plasmids by
site-specific recombination. Plasmids were isolated, and the cDNA
inserts were then subcloned into the EcoRI and
HindIII sites of pBluescriptII KS(+). DNA sequences were
determined by the dideoxynucleotide chain termination procedure (19).
DNA sequence analysis revealed three cDNA sequences, including one
5
sequence and two 3
sequences after composition. These cDNAs all
had identical overlapping DNA sequences and were designated N1 splice,
C1 splice, and C2 splice, respectively. The 5
545 base pairs of the 5
clone, corresponding to DNA sequence 5-549 of the N1 splice, were then
amplified by polymerase chain reaction and used as a probe to screen
the same library. This round of screening yielded a novel 5
sequence
with the proper Kozak start site and an upstream stop codon, designated N0 splice. The composite cDNA sequences revealed the possibility of
four isoforms, and the deduced amino acid sequences were named D-AKAP1a, D-AKAP1b, D-AKAP1c, and D-AKAP1d, respectively, as will be
discussed later (Fig. 4). All sequences were analyzed using PCGENE-IntelliGenetics software and the BLAST program provided by the
NCBI server at the National Library of Medicine, National Institutes of
Health.
Production and Purification of Antibodies against His6RPP7
To prepare antibodies against purified His6RPP7, the protein was expressed and purified on NTA resin to near homogeneity and then run on an SDS-PAGE gel. Fusion protein was then excised from SDS-PAGE and used as the antigen. The subsequent preparation of rabbit antibodies was carried out at Cocalico Biological Co. according to established procedures. Affinity-purified polyclonal antibodies against His6RPP7 were obtained from 4 ml of serum on a 1-ml Affi-Gel 15 antigen column (10 mg/ml resin).
Western Immunoblot AnalysisProteins from different mouse tissues were extracted, separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. Blots were blocked overnight in 5% powdered non-fat milk and then incubated with affinity-purified His6RPP7 antibodies at a 1:1000 dilution. After extensive washing with TTBS buffer (50 mM Tris, 150 mM NaCl, 0.1% Tween, pH 7.5), D-AKAP1 was visualized by 1:10,000 dilution alkaline phosphatase-conjugated secondary antibodies or enhanced chemiluminescence with a 1:2,000 dilution of horseradish peroxidase-conjugated secondary antibodies.
In Situ HybridizationRNA probes of D-AKAP1 were derived
from cDNA sequence 523-3514 of isoform D-AKAP1d. RI probes were
derived from residues 18-169. These cDNA fragments were first
subcloned into pBluescriptII KS(+), and digoxigenin-labeled riboprobes
were transcribed in the sense and antisense orientations following
template linearization. Probe synthesis employed the Genius labeling
system, and hybridization was carried out on 20-µm cryostat sections
of embryonic day 16 Balb/c mice as described previously by Chun
et al. (24). Hybridized probe was visualized by alkaline
phosphatase histochemistry.
A
yeast two-hybrid screen was used to isolate proteins that associate
with the Ret/ptc2 oncoprotein, where the C-terminal domain of the Ret
receptor tyrosine kinase is fused to the N terminus of the
RI subunit of PKA (17). Ret/ptc2 begins with the first
235 amino acids of RI
, which include the dimerization domain, the C
subunit inhibitor site, and most of the first cAMP binding domain.
Using Ret/ptc2 to screen a mouse embryonic cDNA library, several
interacting clones were isolated (18). Eight of these cDNA clones
coded for three novel protein fragments, designated RPP7, RPP8, and RPP9, that associated specifically with the RI portion of
Ret/ptc2. Four of the clones coded for the same protein, RPP7, which
was 125 residues in length.
To determine whether RPP7 bound RI in vitro, RPP7 was expressed as a fusion protein to GST in E. coli from a pGEX vector containing GST fused to the RPP7 cDNA. GST-RPP7 was then expressed and tested for its ability to bind RI in an affinity precipitation assay. The expressed protein had an apparent molecular mass on SDS-PAGE of 40 kDa, consistent with a protein of 13 kDa fused to GST. GST-RPP7 was fully soluble.
Cell lysates containing either GST or GST-RPP7 were incubated with
glutathione resin. Bacterially expressed RI was then added.
Proteins associated with the resin after stringent washing were
analyzed on SDS-PAGE. As seen in Fig. 1, this construct codes for a stable protein that can specifically pull down nearly stoichiometric amounts of RI. GST alone does not interact
with RI. These results confirmed the results of the
two-hybrid screen and established furthermore that no other factors are
required for the interaction between RI and RPP7 in
vitro and in vivo.
Specificity of RPP7
AKAPs identified until now have all bound
specifically to the type II regulatory subunit. We therefore tested
whether RPP7 could bind to RII. As seen in Fig.
2A, RPP7 also interacted with
His6RII in the assay. Thus, in contrast to
other AKAPs described so far, RPP7 is the first member of this family
demonstrated to bind both RI and RII with high
affinity. For this reason, we designate RPP7 as an active fragment of
D-AKAP1, for ual specificity
.
Localization of D-AKAP1 Binding Site
To localize more
precisely the RPP7 binding site on RI, a series of deletion
mutants, summarized in Fig. 3, were used. As seen in
Fig. 2B, GST-RPP7 associated with
His6RI(63-379), which contains only the
first 62 residues of RI. In contrast,
RI(
1-91), which lacks the N-terminal 91 residues of
RI, was not precipitated with GST-RPP7 (data not shown).
Most AKAPs have been shown to specifically bind the N terminus of
RII (17, 25, 26). To determine whether RPP7 also bound to
the N terminus of RII, several deletion mutants of
RII were tested for their ability to bind GST-RPP7. As
shown in Fig. 2B, GST-RPP7 interacted with
RII(
46-400), a construct containing only the N-terminal
45 residues of RII. GST alone does not interact with any of
the R subunits we have tested. Thus, the N terminus of RI
or RII is sufficient for the interaction with D-AKAP1.
To further characterize the interaction between the two types of R
subunits and RPP7, competition experiments were performed. When assayed
individually, a nearly stoichiometric amount of RI or
RII was pulled down by GST-RPP7 based on SDS-PAGE (Fig.
2A); however, when incubated with both R subunits, GST-RPP7
preferentially bound RII. As shown in Fig.
4, when an equal molar ratio of either full-length RI and His6RII or
His6RI(63-379) and
RII(
46-400) were added in the assay, GST-RPP7
preferentially bound to the two RII subunit constructs.
These results indicate that the binding regions of RI and
RII on D-AKAP1 are partially, if not completely,
overlapping. More detailed mapping of the binding sites on
RI, RII, and D-AKAP1 is now under way.
Preliminary results using surface plasmon resonance indicated that the
affinity between RI and GST-RPP7 is at most 25-fold lower
than that for RII and GST-RPP7 (data not shown).
To investigate the
tissue and developmental expression patterns of D-AKAP1, Northern blots
containing 2 µg of poly(A)+ RNA from different adult
mouse tissues or different embryonic stages were probed with
32P-labeled RPP7 cDNA. As shown in Fig.
5A, a 3.8-kb mRNA was detected in all
tissues except the spleen. D-AKAP1 mRNA expression is highest in
heart, liver, skeletal muscle, and kidney. In addition, a strong signal
at 3.2 kb was detected only in the testis sample. The 3.8 kb transcript
was detected in all embryonic stages with comparable intensity (Fig.
5B), indicating that the mRNA expression level in the
embryo was the same throughout different developmental stages. The
3.2-kb mRNA was not detected in the embryonic samples. Since RPP7
was isolated from a mouse embryonic library, the 3.8-kb signal was
predicted to represent the full-length message for D-AKAP1.
Cloning of D-AKAP1
To obtain a full-length clone of D-AKAP1,
a mouse embryonic cDNA library was screened using RPP7 cDNA as
a probe. After radiolabeling of the RPP7 probe, 13 positive clones
containing overlapping partial cDNA sequences were isolated from
approximately 1.6 million recombinants. After sequence analysis, three
cDNA inserts were identified. These included two, designated C1 and
C2, that differed only in their 3 region, and a third, designated N1,
in which the 3
end overlapped with the 5
ends of C1 and C2. Both C1
and C2 have identical 5
sequences of 1581 base pairs but different 3
termini. N1 has additional 5
sequence including a consensus Kozak
start site (27). The 5
end of N1 was amplified using polymerase chain reaction to generate an additional probe to rescreen the library. Three
positive clones were identified and sequenced, and they yielded 810 base pairs of a novel 5
sequence, N0, containing a consensus Kozak
sequence and an in-frame stop codon 60 base pairs upstream. This
precluded the possibility of using an alternative ATG further
upstream.
Sequence analysis of these cDNAs revealed a core open reading frame
of 526 residues. RPP7 is included within this protein and corresponds
to amino acid residues 284-408 in the core sequence (Fig.
6). In addition to this core, cDNAs coding for two
N-terminal and two C-terminal splice variants were discovered (Fig.
7). At the 5 end, one splice generated a message coding
for 33 residues before the core, designated isoform N1. These
additional 33 amino acids include a potential myristoylation site and a
potential PKA phosphorylation site. N1 begins its coding region with
its first ATG, because the 5
sequence of N1 did not contain another Kozak sequence. The other 5
variant, designated N0, has an in-frame stop codon upstream from its start site, which corresponds to the start
site of the core. The two 3
variants, designated C1 and C2, had an
additional 18 or 331 residues 3
from the core open reading frame.
Therefore, although the mRNA for D-AKAP1 appeared as a single
species of 3.8 kb in mouse embryo by Northern analysis, cDNA
cloning has identified at least four isoforms, designated as D-AKAP1a,
D-AKAP1b, D-AKAP1c, and D-AKAP1d. These potential isoforms contain 544, 577, 857, and 890 amino acid residues, respectively.
Western Blot Analysis
Antiserum against D-AKAP1 was raised in
rabbits using the His6RPP7 fusion protein as the antigen
and purified on a antigen column. This antibody recognized both
His6RPP7 and GST-RPP7 specifically in crude cell lysates,
as shown in Fig. 8A. Proteins were extracted from different mouse tissues, separated on SDS-PAGE gel, Western blotted, and probed with the antibody. As seen in Fig. 8B,
two protein bands with apparent molecular masses of 86 and 57 kDa were
detected in the brain extract, a 64-kDa protein was detected in the
muscle extract, and a doublet of 132 kDa was detected in the liver
sample. Since the message of D-AKAP1 was not found in the spleen in the
Northern analysis, extracts from the spleen were used as a negative
control. These bands were not detected in the spleen and, therefore,
were predicted to represent D-AKAP1 proteins in the tissue samples. In
addition, preincubation of the antigen with the antibody abolished
these bands, suggesting that the signal is specific for D-AKAP1. The
existence of different protein isoforms of D-AKAP1 is consistent with
the identification of various cDNA splice variants. The sizes of
132 and 86 kDa were confirmed by in vitro translation of the
cDNAs coding for the C1 and C2 splice isoforms of D-AKAP1 (data not
shown). In the tissue extracts and also the in vitro
translation, the apparent molecular masses were higher than the
calculated ones. This discrepancy has also been observed for many other
AKAPs (27, 28). Antibodies specific for each isoform are currently
being raised to further establish whether the isoforms are expressed in
a tissue-specific manner.
In Situ Hybridization
To determine the expression pattern of
D-AKAP1 in comparison with RI, in situ
hybridization was performed using probes for both D-AKAP1 and
RI in whole embryonic day 16 mouse embryo sections.
Riboprobes were derived from the C2 splice of D-AKAP1. Expression was
most prominent in brown fat surrounding the trapezius muscle. Other
skeletal muscle, intestine, olfactory epithelium, and numerous regions of the central nervous system (CNS) also showed a
significant hybridization signal (Fig. 9A).
Interestingly, in brain, the regions of early cerebral cortex and basal
ganglia that showed the greatest signal were zones containing
postmitotic neurons. Similar results have also been shown for AKAP150
(28). The sense strand control showed no hybridization.
In situ patterns were also investigated for
RI on the adjacent embryonic section. Riboprobes were
made from residues 18-169 of RI
, including the
N-terminal region and the beginning of the first cAMP binding site.
This fragment of RI was used because it has the least
similarity in DNA sequence to RII. When comparing the
in situ patterns, overlapping expression for D-AKAP1 and
RI
were found in muscle, such as the tongue, but the
most striking overlap patterns came from the olfactory bulb and
olfactory epithelium. As shown in Fig. 9, D-G, the
RI and D-AKAP1 messages appear to be localized in many of
the same regions of these structures.
Anchoring of PKA through the regulatory subunit is proposed to localize the kinase at specific subcellular sites. All AKAPs documented so far interact specifically with the type II regulatory subunit. Here we report a novel PKA anchoring protein, D-AKAP1, that binds and potentially targets both the type I and the type II regulatory subunits.
D-AKAP1, named for its potential dual specificity, was first identified
as a fragment from a yeast-two hybrid screen based on specific
interaction with the RI portion of Ret/ptc2. As
demonstrated in an affinity precipitation assay, this fragment, RPP7,
includes most, if not all, of the RI/RII
binding domain. Secondary structure predictions indicate that RPP7 has
an amphipathic -helix at its N terminus, and this is consistent with
the proposed model for other AKAPs where predicted amphipathic helices
are hallmarks for R binding domains (26, 29-31).
Using this functional R-binding fragment, RPP7, the interaction regions
on both RI and RII were localized to their N
termini. This N-terminal region corresponds to the first 62 amino acids
in RI and the first 45 amino acids in RII.
Since the N-terminal dimerization domain is also proposed to be a key
requirement for interaction between other AKAPs and RII (9,
25, 32), the amino acid sequences at the N terminus of RI
and RII were aligned to identify conserved residues. When
aligned, the N-terminal regions of RI and RII
show the least sequence identity. However, conserved Leu29
and Phe52 on RI were identified at positions
equivalent to Leu13 and Phe36 of
RII. Substitution of Ala for these two residues generates
monomeric RII subunits that cannot bind AKAP75 (32).
Leu36 in RI is located at a position that is
equivalent to Val20 in RII. An
RII
mutant where Val20-Leu21
were replaced by Ala-Ala still dimerized but was unable to bind to
AKAPs (32). Whether mutations of these corresponding residues on
RI will disrupt dimerization and/or interaction with
D-AKAP1 is still under investigation.
Since binding competition between RI and RII showed that RII can compete with RI and is actually preferentially bound by GST-RPP7, the regions on RPP7 that are responsible for binding RI or RII are likely to be partially, if not completely, overlapping. However, we cannot rule out the possibility that RI and RII bind at distinct but interacting sites. Although D-AKAP1 preferentially binds RII in vitro, since RI and RII differ in their tissue expression, subcellular localization, and temporal expression during development, the actual microenvironment for D-AKAP1 with respect to RI and/or RII is not understood at this point. These results raise the possibility that, in addition to RII, RI may also be a target for compartmentalization. Further characterization will be required to predict the interaction patterns between D-AKAP1 and both types of R subunits in vivo.
Further potential for diversity is indicated by the fact that there are multiple splice variants of D-AKAP1 resulting in a family of different isoforms. Western analysis using antibodies against RPP7 detected various protein bands of different molecular masses in extracts from various tissues. Two protein bands with apparent molecular masses of 86 and 57 kDa were detected in the brain extract, a 64-kDa protein was detected in the muscle extract, and a doublet at 132 kDa was detected in the liver sample. From two N-terminal splice variants and two C-terminal splice variants identified in cDNA cloning, four of the isoforms were identified. Each of these splice variants contains distinct features. For example, the N1 splice contains a potential myristoylation site and a potential PKA phosphorylation site, while the C1 splice variant contains several potential casein kinase II phosphorylation sites (Fig. 7). Since different protein isoforms were detected in different tissues using the antibodies against RPP7, these isoforms may be expressed in a tissue-specific manner. Whether these splice variants have distinct physiological functions in the particular tissues that express them is still unclear. This heterogeneity in the isoform expression pattern of D-AKAP1 also introduces an additional mechanism for regulating the compartmentalization of PKA.
During the cloning of D-AKAP1, data base comparison identified a new
AKAP protein, S-AKAP84, sharing homologous amino acid sequence with
D-AKAP1 (33). Unlike D-AKAP1, which is expressed in most tissues,
S-AKAP84 is a PKA anchoring protein expressed principally in the male
germ cell lineage. It is likely that the 3.2-kb transcript detected in
the testis sample is indeed the mouse homolog of S-AKAP84. For S-AKAP84
there was also evidence of multiple mRNA isoforms. A simple
S-AKAP84 genomic pattern was described by Rubin and co-workers (33)
that indicated these S-AKAP84 RNA transcripts may arise from a single
gene. It is therefore likely that the different isoforms of D-AKAP1
emerged from splicing variants as well. Interestingly, however,
S-AKAP84 was not found to interact with RI. Amino acid
sequence comparison between the two proteins revealed the greatest
similarity in the core section of the protein, which includes a
mitochondria target/signal region at the N terminus and an
RII binding domain, as described by Rubin and co-workers
(33) (Fig. 10). The putative RII binding
domain in S-AKAP84 is contained within RPP7, the R binding fragment of
D-AKAP1. The question of whether the specific segment homologous to the
RII binding domain is indeed the RI and/or
RII binding site of D-AKAP1 and the specific side chains
required for interactions are still under investigation. Since the core of D-AKAP1 contains the potential mitochondrial anchor region at the N
terminus, it is likely that at least some of the D-AKAP1 isoforms are
targeted to the mitochondria. This is consistent with results
indicating that the message of this protein was found predominantly in
tissues with high mitochondria content, such as cardiac or skeletal
muscle and liver. Results by Schwoch et al. (34) also
documented that RI holoenzyme anchored in the inner
membrane of mitochondria. Immunofluorescence microscopy will further
determine the subcellular localization of the D-AKAP1 isoforms. D-AKAP1
does not contain the leucine zipper motif that was identified in
S-AKAP84. Further investigation to identify additional partners for the
D-AKAP1·PKA or the S-AKAP84·PKA complex will give more information
on the physiological function of this family of anchoring proteins and
especially the function of the leucine zipper motif of S-AKAP84.
In situ hybridization experiments were performed to
determine the expression patterns of RI and D-AKAP1 in
embryonic day 16 mouse embryos. These data showed that RI
and D-AKAP1 have overlapping expression patterns in muscle cells and
olfactory epithelium. In the olfactory system, D-AKAP1 appeared to be
expressed in the same regions as RI. It has been
demonstrated that the odorant-induced cAMP response in the olfactory
system is attenuated by PKA, possibly through phosphorylation of the
odorant receptors (35-37). D-AKAP1 could therefore function to anchor
PKA near the target receptor.
We report here for the first time the use of the yeast two-hybrid system to identify a cAMP-dependent protein kinase anchoring protein and raise the novel possibility that like RII, RI can potentially be specifically anchored at various subcellular locations via AKAPs.
We thank Poopak Banky for providing
His6RI(63-379) and Marceen Newlon for
providing RII(
46-400). We also thank Dr. John Lew for
valuable discussions.
During the review of our manuscript, Trendelenburg and co-workers published a report of a related protein that has a C2 splice similar to D-AKAP1 (Trendelenburg, G., Hummel, M., Riecken, E., and Hanski, C. (1996) Biochem. Biophys. Res. Commun. 225, 313-319). These authors pointed out that the C2 splice region contains a KH domain which is a potential RNA-binding motif. This KH motif corresponds to residues 38-87 in the C2 splice of D-AKAP1.