From the
Differential localization of the type II cAMP-dependent protein
kinase (PKA) is achieved by interaction of the regulatory subunit (RII)
with A-kinase anchor proteins (AKAPs). Anchoring is a likely means to
adapt PKA for regulation of cAMP-responsive events through
colocalization of the kinase with preferred substrates. Using an
interaction cloning strategy with an RII
Considerable research has focused on determining the role of
cAMP in signal transduction since it was discovered as an intracellular
second messenger of hormone action
(1, 2) . Cyclic AMP
is released from distinct locations at the cell membrane by hormonal
activation of adenylate cyclase and diffuses or is transported to its
site of action. The predominant effect of cAMP is to activate a
cAMP-dependent protein kinase (PKA).
The cellular location of PKA is dictated by
the regulatory (R) subunit
(7) . Two classes of R subunit exist,
RI and RII, which form the type I and type II PKA holoenzymes,
respectively
(8, 9) . The RI isoforms (RI
In this report, we describe
the cloning and characterization of a novel AKAP, AKAP100, which is
selectively expressed in the brain, cardiac, and skeletal muscle.
Biochemical and immunochemical analyses suggest that AKAP100 and RII
associate in vivo at the sarcoplasmic reticulum.
In this report, we describe the cloning and characterization
of a novel A-kinase anchor protein, AKAP100, which is specifically
localized to the sarcoplasmic reticulum. AKAP100 is selectively
expressed in certain tissues, and high levels of the 8-kilobase message
are detected in various brain regions and cardiac and skeletal muscle.
This finding is supported by cloning studies that isolated the original
cDNA fragment from a human hippocampal library. The calculated
molecular weight of AKAP100 is 78,172, although the protein migrates
with a mobility of M
A
growing body of evidence suggests that AKAPs contain a conserved
RII-binding site responsible for interaction with PKA. Recently, we
have shown that RII binding proceeds through sites in the extreme amino
terminus of RII, and isoleucines 3 and 5 on each RII protomer are
required for interaction with an amphipathic helix on the surface of
the AKAP
(22) . Site-directed mutagenesis studies have
demonstrated that an intact
It would also appear that AKAP100 binds RII
Previous studies have proposed that the type II PKA is
associated with native sarcoplasmic reticulum vesicles
(40) .
This finding is supported by our immunocytochemical data showing that
AKAP100 and a significant proportion of the cellular RII pool appear to
be localized at the sarcoplasmic reticulum. In light of both
observations, we suggest that AKAP100 functions to adapt PKA for a role
in the phosphorylation of proteins in or surrounding the sarcoplasmic
reticulum. Although the precise identity of these target substrates
remains to be determined, anchoring of kinases close to ion channels is
an attractive hypothesis. Levitan and others
(13, 40, 41, 42, 43, 44) have proposed that targeting of kinases and phosphatases
could permit the precise regulation of ion channel phosphorylation
status. This view is supported by evidence suggesting that anchoring of
PKA by AKAPs is required for modulation of glutamate receptor ion
channels. Intracellular perfusion of cultured hippocampal neurons with
anchoring inhibitor peptides derived from the conserved kinase-binding
domain of AKAP79 or Ht31 prevented PKA-mediated regulation of the
AMPA-kainate currents
(25) . Therefore, the AKAP100-PKA complex
might interact directly with ion channels or may be targeted to
structural proteins within the sarcoplasmic reticulum. For example, the
ryanodine receptor, which forms the intracellular Ca
In
conclusion, these studies suggest that AKAP100 is a novel protein that
binds RII
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U17195.
We thank our colleagues at the Vollum Institute for
critical reading of this manuscript and Kenneth N. Fish for technical
assistance in confocal microscopy.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
protein probe, we have
identified a 655-amino acid protein (named AKAP100). Recombinant
AKAP100, expressed in Escherichia coli, binds RII
in a
solid-phase overlay assay. The cellular and subcellular distribution of
AKAP100 was analyzed by various methods. Northern blot analysis with
the AKAP100 cDNA as a probe detected an 8-kilobase message in some
human tissues including various brain regions; however, the message was
predominately expressed in cardiac and skeletal muscle. Anti-AKAP100
antibodies confirmed expression in the rat cardiac and skeletal muscle
cell lines, H9c2 and L6P, whereas immunohistochemical analysis revealed
that AKAP100 was localized to the sarcoplasmic reticulum of both cell
types. RII was also detected in these regions. AKAP100 was detected in
preparations of RII purified from L6P cell extracts by cAMP-agarose
affinity chromatography. Collectively, these results suggest that
AKAP100 functions to maintain the type II PKA at the sarcoplasmic
reticulum.
(
)
Four
molecules of cAMP bind each dormant PKA holoenzyme, activating the
kinase by releasing the catalytic (C) subunits from the regulatory (R)
subunit-cAMP complex
(2) . Active PKA potentiates the hormonal
response by phosphorylating key enzymes and structural proteins, thus
modifying their function. Although the mechanics of PKA activation are
well understood, it is still unclear how individual hormones that
utilize this ubiquitous cAMP signaling pathway are able to exert
localized and specific effects
(3, 4, 5) . To
accommodate these pleotrophic properties, PKA must be localized in a
microenvironment with the adenylate cyclase complex or in a cellular
microcompartment that preferentially concentrates cAMP. Therefore,
selective activation of compartmentalized PKA is a plausible mechanism
to ensure the selectivity of a hormonal response, and, accordingly, the
cellular location of the kinase must be highly regulated
(3, 6) .
and
RI
) are thought to be primarily cytoplasmic, although RI is
membrane bound in erythrocytes
(10) and may associate with the
T-cell receptor in activated lymphocytes
(11) . In contrast, a
significant proportion of the RII isoforms (RII
and RII
) are
particulate, and up to 75% of the cellular RII pool associates with the
plasma membrane, cytoskeletal components, endoplasmic reticulum,
secretory granules, or nuclei
(9, 12, 13, 14, 15, 16) .
The subcellular localization of RII is maintained through high affinity
interaction with a family of adapter proteins called A-kinase anchor
proteins (AKAPs)
(12, 17, 18, 19) . RII
dimerization is required for AKAP binding
(20, 21) .
More recently, the first 5 amino acids of each RII protomer have been
shown to be critical for AKAP interaction, specifically isoleucines at
position 3 and 5
(22) . The acceptor site on the AKAPs for PKA
is more defined; anchoring proteins contain a conserved amphipathic
helix of 14-18 amino acids, which must be maintained in the
correct conformation to permit binding to the kinase
(19, 23, 24) . Synthetic peptides patterned
after the amphipathic helix bind RII and the type II PKA holoenzyme
with nanomolar affinity and can block interaction with AKAPs
(24) . These ``anchoring inhibitor peptides'' have
been used to disrupt the RII-AKAP interaction in neurons and uncouple
the regulation of AMPA-kainate receptors by PKA
(25) . In
accordance with the targeting subunit hypothesis
(26) , each
AKAP must contain additional binding sites responsible for targeting to
cellular structures and/or interaction with other proteins. In an
effort to identify AKAP binding proteins, complementary DNAs that
encode proteins that associate with AKAP79, a neuronal anchor protein
(24) , were isolated using the yeast two-hybrid system
(27, 28) . A cDNA for the phosphatase 2B, calcineurin,
was isolated and is consistent with earlier observations that PKA and
calcineurin copurify through a number of chromatography steps
(29) . Biochemical and immunological studies have confirmed that
both PKA and calcineurin are targeted to subcellular sites by
association with AKAP79, possibly to regulate the phosphorylation state
of key neuronal substrates
(28) .
Cloning Strategy
Human cDNA expression
libraries were screened by direct overlay with P-labeled
RII
as a probe with modifications to the method of Lohmann et
al. (17) as described in Ref. 18. Plaques were lifted onto
nitrocellulose filters as described
(30) . Phosphorylation of
RII
by the C subunit of PKA was performed with
[
-
P]ATP as described
(31) . All
sequencing reactions were performed by the dideoxy chain termination
method of Sanger et al. (32) .
RNA Analysis
Filters containing
immobilized samples of mRNAs of selected human tissues (Clontech) and
of human brain regions (provided by Dr. Jeff Arriza, Vollum Institute,
Portland, OR) were probed with a P-radiolabeled 1400-base
pair fragment encompassing the 3`-coding region of the AKAP100 cDNA.
Radiolabeling of the AKAP100 cDNA probe was achieved by the random
priming method described in Ref. 33 using
[
-
P]dCTP. Nitrocellulose filters were
prehybridized in 400 m
M sodium phosphate, pH 6.6, 1 m
M EDTA, 5% SDS, 1 mg/ml BSA, 50% formamide for 2 h at 42 °C. The
radiolabeled cDNA probe was denatured by heating at 100 °C for 10
min and then added directly to the nitrocellulose filters.
Hybridization was performed in the same buffer at 42 °C overnight
with gentle agitation. Non-hybridized probe was removed by washing
(three times) in excess 0.1
SSC, 0.1% SDS, and 1 m
M EDTA at 53 °C for a total of 2 h.
P-Radiolabeled
-actin cDNA was used to probe both of these blots under similar
conditions. Hybridizing mRNA species were detected by autoradiography.
Expression of Recombinant
AKAP100
Construction of the AKAP100 expression vector
pET11d AKAP100kfc was performed by simultaneous ligation of two DNA
fragments into the bacterial expression vector pET11dkfc (34).
Initially, a 1398-bp EcoRI- BamHI fragment was excised
from the original cDNA clone. Second, an 821-base pair fragment
encompassing the 5`-end of the AKAP100 coding region was amplified by
polymerase chain reaction. Primers were designed to create an
NcoI site at the 5`-end
(5`-TCCCAAACCATGGCCTTTACTGGCAG-3`) and the T3 site at the 3`-end
(5`-ATTAACCCTCACTAAAG-3`). Digestion of the polymerase chain reaction
product with NcoI and EcoRI removed a 597-bp
fragment. The expression plasmid pET11d AKAP100kfc was constructed by
ligating the 1398-bp EcoRI- BamHI and 597-bp
NcoI- EcoRI fragments into an
NcoI- BamHI cut vector. The fidelity of the AKAP100
coding region was confirmed by nucleotide sequencing. Expression of
AKAP100kfc protein was achieved in Escherichia coli pLysS
cells as described
(35) . Soluble Kfc protein was purified by
affinity chromatography on calmodulin-agarose as described
(34) . Protein was concentrated by ultrafiltration (Amicon).
RII Overlays
Overlay assays were
performed by the method of Lohmann et al. (18) , and
quantitative overlays were performed as described
(22) . Binding
was detected by autoradiography and was measured by densitometry after
scanning into a computer and analyzed by the National Institutes of
Health Image 1.55 program. In control experiments,
P-labeled RII probe was pre-incubated with
Ht31-(493-515) anchoring inhibitor peptide (0.5 m
M) as
described
(36) .
Western Blot Analysis
Proteins were
separated by SDS-PAGE as described
(37, 38) and
electrotransferred to polyvinylidene difluoride membrane (Immobilon,
Millipore). Rabbit polyclonal antibodies to AKAP100 (produced by Bethyl
Laboratories, Inc., Montgomery, TX) were affinity purified using
AKAP100 protein coupled to Affi-Gel 15 (Bio-Rad) and used at 1:250
dilution. Anti-RII antibodies, raised in rabbits
(36) , were
affinity purified and used at 1:5000 dilution.
Preparation of H9c2 and L6P Cell
Extracts
Rat cardiac muscle and skeletal muscle cell lines
H9c2 and L6P were grown in 75-cmtissue culture flasks
(NUNC) in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum until confluent. The cells were washed three times
with phosphate-buffered saline (PBS), pH 7.4, and scraped from the
tissue culture flasks. The cell pellet was concentrated by
centrifugation at 3000
g for 5 min at 4 °C,
resuspended in buffer A (50 m
M Tris-HCl, pH 7.5, 0.1% Triton
X-100 0.05 m
M dithiothreitol, 0.5 m
M MgCl
, 0.125 m
M EDTA, 0.1 µ
M leupeptin, 0.1 µ
M pepstatin, 0.1 m
M benzamidine, 0.1 m
M phenylmethylsulfonyl fluoride), and
sonicated for 30 s (three bursts of 10 s) on ice. After sonication, the
cell suspension was centrifuged at 14,000
g for 20 min
at 4 °C, and the pellet was resuspended in buffer A. Protein
concentrations of the supernatant and pellet fractions were measured by
Bradford assay (Pierce). Equal concentrations of protein (100 µg),
unless otherwise stated, were loaded onto SDS-PAGE gels for subsequent
analyses.
Immunocytochemistry
Immunofluorescence
studies were performed on rat cardiac muscle H9c2 cells grown in
Dulbecco's modified Eagle's medium containing 10% fetal
calf serum on glass coverslips in 6-well tissue culture (NUNC) plates
for 72 h at 37 °C. Cells were fixed in 3.7% formalin in PBS, pH
7.4, extracted in absolute acetone at -20 °C, and rehydrated
in PBS + 0.1% BSA before incubation with affinity-purified primary
antibodies (1:100 dilution anti-goat RII and 1:250 dilution AKAP100)
for 1 h at room temperature. Control experiments with antibody to
signal sequence receptor (1:50 dilution, kindly provided by Dr. T. A.
Rapoport, Max-Delbruck center for Molecular Medicine, Germany) and
preimmune sera were performed under identical conditions. Cells were
washed with excess PBS + 0.1% BSA (3 times) before incubation with
secondary antibodies (1:100) for 1 h at room temperature. RII was
detected with fluorescein isothiocyanate-conjugated donkey anti-goat
IgG, and AKAP100 was detected with Texas Red donkey anti-rabbit IgG.
Unbound secondary antibodies were removed by washing with excess PBS
+ 0.1% BSA (three times). Coverslips were mounted with
Vectashieldmounting medium. Staining patterns were
observed on a Leitz Fluovert FU confocal photomicroscope under oil
immersion using a 63/1.4 OEL PL APO lens.
Purification of the RII-AKAP100 Complex by
cAMP-Agarose
RII-AKAP complexes were isolated from rat
skeletal muscle cell extracts as previously described
(36) .
Unbound protein was removed from the cAMP-agarose by washing with
hypotonic buffer plus 1
M NaCl and four additional washes with
hypotonic buffer. Finally, specifically bound proteins were eluted from
the affinity resin by incubation at room temperature with hypotonic
buffer containing 75 m
M cAMP.
Other Methods
Antibodies against AKAP100
peptide and recombinant AKAP100 were produced by Bethyl Laboratories,
Inc. (Montgomery, TX). Oligonucleotide sequencing primers were
purchased from the Center for Gene Research and Biotechnology, Oregon
Science University (Corvallis, OR).
Cloning of AKAP100
Clones encoding
AKAPs were isolated from a human hippocampal cDNA expression library by
the RII overlay method using RII as a probe. One positive clone
was isolated from
250,000 recombinants. This clone was called
hhBD-1 (1888 bp) and contained a partial open reading frame of 599
amino acids (Fig. 1 A). When Northern blots were screened with
a hhBD-1 probe, the only mRNA species detected in human tissues was 8
kilobases, confirming that hhBD-1 was a partial fragment of the
full-length cDNA. The human hippocampal cDNA library was rescreened
with a 1400-bp HindIII- BamHI fragment excised from
the 5`-end of hhBD-1, but this did not yield clones with any additional
information. However, a random and oligo(dT)-primed human fetal brain
cDNA library (provided by Dr. W. Michael Gallatin, ICOS Corp., Seattle,
WA) was screened with the same probe, and 12 positive clones were
identified from
500,000 recombinants. DNA from seven of the
positive clones was sequenced and indicated that one clone, called hFB
7-1 (1314 bp), overlapped with hhBD-1 and yielded 822 bp of
additional 5`-sequence. A composite sequence of 2595 is presented in
Fig. 1A and contains an open reading frame encoding a
655-amino acid protein with a predicted M
78,172.
However, the protein migrates on SDS-polyacrylamide gels with an
apparent molecular mass of 100 kDa (see below); thus, in accordance
with the nomenclature proposed for RII anchoring proteins by Hirsch and
colleagues
(39) , we have named the protein AKAP100.
Figure 1:
Sequence of AKAP100. A, the
nucleotide sequence ( upper line) and deduced amino acid
sequence ( lower line) of a cDNA encoding the protein kinase A
anchor protein AKAP100. The boxed region indicates
the putative RII-binding region, and a sequence used to raise peptide
antisera is underlined. B, sequence homology between
AKAP100 (residues 396-411) and the RII-binding regions of three
other AKAPs, MAP2, AKAP150, and Ht31. Boxed regions show amino acid identity, and conservative changes (*) are
indicated. C, helical wheel representation of AKAP100
(residues 392-408) drawn as a -helix of 3.6 amino
acids/turn. The shaded area indicates hydrophobic residues,
and the open area indicates hydrophilic residues. Amino acids
are indicated in the one-letter code. The arrow indicates the
direction of the helix.
Comparison of the AKAP100 sequence to the DNA and protein data base
did not identify any overall similarity to other known proteins.
Computer-aided analysis of the AKAP100 sequence identified a putative
amphipathic -helix located between residues 392 and 408 (Fig.
1 B). This region is compared with the RII-binding regions of
other AKAPs (Fig. 1 C). Both of these findings are
consistent with the notion that residues 392-408 of AKAP100 form
the RII-binding domain.
The Tissue Distribution of AKAP100
mRNA
To establish the tissue expression pattern of AKAP100,
filters of immobilized poly(A)RNA from several human
tissues were screened with a radiolabeled 1400-bp
HindIII- BamHI fragment excised from hhBD-1. A single
mRNA species of 8 kilobases was detected in certain human tissues and a
variety of brain regions (Fig. 2, A and C). The
AKAP100 mRNA was most highly expressed in the heart and skeletal muscle
(Fig. 2 A). When the filters were probed with a
-actin probe, equal levels of message were detected in each lane
(Fig. 2, B and D).
Figure 2:
The tissue distribution of the AKAP100
mRNA. 2 µg of poly(A)RNA from human tissues
(human MTN, Clontech) ( A) and selected brain regions (5
µg) ( C) on nitrocellulose were probed with a
P-radiolabeled 1400-bp HindIII- BamHI
fragment excised from the 5`-end of hhBD-1 as described under
``Experimental Procedures.'' Human tissues (human MTN,
Clontech) ( B) and selected brain regions ( D) were
probed with
P-radiolabeled
-actin. Hybridizing mRNA
species were detected by autoradiography. The tissue source of each RNA
is indicated above each lane. kb,
kilobases.
Expression and Characterization of
AKAP100
To confirm that AKAP100 was an RII binding protein,
the full-length cDNA was expressed in E. coli using the kfc
fusion system
(34) as described under ``Experimental
Procedures.'' A 115-kDa protein was detected by SDS-PAGE in
bacterial extracts of induced cells and was purified to homogeneity by
affinity chromatography on calmodulin-Sepharose (Fig. 3 A). The
expression of the recombinant fusion protein was monitored by Western
blot (Fig. 3 A) using anti-peptide antisera raised
against residues 188-203 of AKAP100
(Val-Lys-Arg-Val-Ser-Glu-Asn-Asn-Gly-Asn-Gly-Lys-Asn-Ser-Ser-His). The
recombinant protein bound P-RII as assessed by a direct
overlay (Fig. 3 C). Solid-phase RII binding was blocked
when overlay blots were incubated with the anchoring inhibitor
Ht31-(493-515) peptide (Fig. 3 D). This peptide has
been previously shown to block RII-AKAP interaction
(24, 36) . Nonspecific binding to a band of 80 kDa was
detected upon prolonged exposure of the control blot
(Fig. 3 D).
Figure 3:
Recombinant AKAP100 binds RII. The
entire coding region of AKAP100 was expressed using the pET11dkfc
plasmid. Expression of recombinant protein was induced by the addition
of 0.4 m
M isopropyl-1-thio-
-
D-galactopyranoside
to growing bacterial cultures. Bacterial extracts (100 µg) or
purified protein were separated by electrophoresis on 10% (w/v)
SDS-polyacrylamide gels and electrotransferred to polyvinylidene
difluoride membranes. Blots were stained with Coomassie Blue
( A) and analyzed by autoradiography ( B-D).
B, AKAP100 was detected by Western blot with anti-peptide
antibodies. RII binding proteins were detected by a solid-phase binding
assay (17) using
P-radiolabeled RII
as a probe in
absence ( C) or presence ( D) of 1 µ
M anchoring inhibitor Ht31-(493-515) peptide. Sample sources,
indicated above each lane, are uninduced bacterial
lysate ( lane 1),
isopropyl-1-thio-
-
D-galactopyranoside-induced bacterial
lysate ( lane 2), and affinity-purified protein
( lane 3). Molecular mass markers are indicated on
each panel.
The binding affinity of AKAP100 was
assessed by quantitative overlays using RII protein at a specific
activity of 2.1-1.5
10
cpm/pmol. The binding
to immobilized AKAP100 and Ht31 over a range of 0.01-10 nmol/100
µl was detected by autoradiography and was measured by
densitometry. Both proteins bound RII with high affinity, with
half-maximal binding values calculated at 10 µ
M for
AKAP100 and 2.5 µ
M for Ht31 (Fig. 4). Collectively, these
data are consistent with the notion that the AKAP100 cDNA encodes a
high affinity RII binding protein.
The Subcellular Location of AKAP100
The
tissue distribution of AKAP100 mRNA suggested that the protein may be
predominately expressed in cardiac and skeletal muscle tissues. To test
this hypothesis, affinity-purified polyclonal AKAP100 antisera, raised
against purified recombinant AKAP100, was used to probe protein
extracts from a variety of muscle cell lines including the rat cardiac
muscle (H9c2) and rat skeletal muscle (L6P) cell lines. A single
immunoreactive protein of 100 kDa was detected by Western blot for both
H9c2 (Fig. 5, lane 2) and L6P (see below) cells.
There was no immunoreactivity when identical blots were probed with
preimmune serum (Fig. 5, lane 3). Numerous RII
binding proteins ranging in size from 250 to 40 kDa were detected in
both cell lines by direct overlay (data not shown).
Figure 5:
AKAP100 is present in an H9c2 cell lysate.
A solubilized extract (250 µg) of H9c2 cells was stained with
Coomassie Blue ( lane 1) or probed for AKAP100 ( lane
2) using purified antibodies. Detection was by enhanced
chemiluminescence. Preimmune sera is shown in lane 3.
Molecular mass markers are indicated.
The subcellular
location of AKAP100 was examined using indirect immunofluorescence
techniques (Fig. 6). In quiescent H9c2 cells (Fig. 6 A),
AKAP100 staining was restricted to the perinuclear regions
(Fig. 6 C) and exhibited a similar staining pattern to
the sarcoplasmic reticulum marker protein, signal sequence receptor
(Fig. 6 B). Conversely, the staining pattern of the Golgi
marker protein, mannosidase II, was distinct from AKAP100 (data not
shown). Double immunofluorescence staining for AKAP100
(Fig. 6 C) and RII (Fig. 6 D) suggested
that both proteins had overlapping cellular distributions and were
concentrated in the same cellular compartment. This observation was
confirmed by confocal microscopy showing that AKAP100 and RII have
overlapping staining patterns in 0.1-micron-thick focal sections of
H9c2 cells (Fig. 6, C and D). Control
experiments confirmed that no staining was observed with preimmune
serum (Fig. 6, E and F) or secondary antibody
alone (data not shown). These experiments are consistent with the
localization of AKAP100 and a significant proportion of RII to the
sarcoplasmic reticulum of H9c2 cells. An indistinguishable staining
pattern for AKAP100, RII, and marker proteins was obtained when these
experiments were repeated in L6P cells (data not shown).
Figure 6:
Immunocytochemical analysis of rat cardiac
muscle cell line (H9c2). H9c2 cells were formalin-fixed ( A)
and incubated with anti-sequence-specific receptor antibodies
( B), anti-AKAP100 antibodies ( C), anti-RII antibodies
( D), or preimmune serum for AKAP100 ( E) and for RII
( F) under the conditions described under ``Experimental
Procedures.'' Fluorescein isothiocyanate-conjugated anti-rabbit
secondary antiserum was used in panels B, C,
and E). Texas red conjugated anti-goat secondary antiserum was
used in panels D and
F.
Purification of the RII-AKAP100
Complex
To examine whether AKAP100 was associated with RII
in cell lysates, we attempted to purify the RII-AKAP100 complex using
affinity chromatography on cAMP-agarose. L6P cells were used for these
experiments because they grow more rapidly and, thereby, provide more
starting material. Western blot analysis of solubilized L6P cell
lysates indicated that the lysates contained RII that was purified by
affinity chromatography on cAMP-agarose (Fig. 7 A). Identical
blots probed with anti-AKAP100 antibodies indicated that a proportion
of the AKAP100 present in the lysate copurified with RII
(Fig. 7 B). These results provide evidence that
RII-AKAP100 complexes exist and can be purified from cell lysates.
Figure 7:
AKAP100 copurifies with RII on
cAMP-agarose. Detection of proteins on Western blots is shown for
fractions isolated from cAMP-agarose affinity chromatography. Separate
filters were probed for either RII ( A) or AKAP100
( B) using purified antibodies and enhanced chemiluminescence
detection. A solubilized extract of L6P cells ( lane 1) was
incubated with cAMP-agarose. Unbound protein ( lane 2) was
separated from the affinity matrix by centrifugation, and the pellet
containing protein bound to cAMP-agarose was washed extensively in
washing buffer. After a final wash ( lane 3), proteins
remaining bound to the affinity matrix were eluted in 75 m
M cAMP ( lane 4) and boiled in sample buffer. Fractions (100
µg, lanes 1 and 2; 10 µg, lane 4)
were separated on 7.5% SDS-polyacrylamide gels and electrotransferred
to polyvinylidene difluoride membranes. Molecular mass markers are
indicated.
100,000 on SDS-polyacrylamide
gels. Apparently, anomalous migration on SDS-PAGE gels is a
characteristic of several AKAPs. For example, the bovine
neural-specific anchoring protein, AKAP75, has a calculated molecular
weight of 47,085 but migrates with an apparent mobility of 75 kDa on
SDS-PAGE gels
(24) . This has led Hircsh and colleagues
(39) to suggest that the abundance of acidic residues in the
amino-terminal portion of the protein alters its migration pattern.
Consistent with this hypothesis, AKAP100 also contains stretches of
acidic side chains between residues 80 and 110 of the protein.
-helical structure is required for
RII-AKAP interaction
(23) , and synthetic peptides encompassing
the amphipathic helix regions of three AKAPs (Ht31, AKAP79, and AKAP95)
block RII-AKAP interaction in vitro (19, 24, 36) . On the basis of these
observations, it seems likely that residues 392-408 may form the
RII-binding site on AKAP100. This region exhibits a high probability of
amphipathic
-helix formation and shares 30-40% sequence
identity with the RII-binding regions of other AKAPs
(Fig. 1 B); deletion constructs lacking residues
392-410 are unable to bind RII by the overlay assay.
(
)
Moreover, the anchoring inhibitor peptide,
Ht31-(393-415), effectively competes with AKAP100 for RII binding
in direct overlays.
with high affinity, as half-maximal binding to the immobilized
anchoring protein was measured in the micromolar range. Although it is
technically infeasible to measure precise binding constants due to
aggregation of AKAP100, it would appear that the intracellular levels
of AKAP100 and RII (assessed by Western blotting) are within a
concentration range sufficient to permit complex formation in
vivo. This notion is supported by our evidence that AKAP100 is
copurified with RII from L6P cell lysates by affinity chromatography on
cAMP-agarose. Since recombinant AKAP100 does not display any intrinsic
cAMP binding affinity, it is therefore likely that the anchoring
protein was purified as complex with RII. In addition, both RII and
AKAP100 were detected in the flow-through from the cAMP-agarose
(Fig. 7). There are two potential explanations for this
observation: the amount of RII in the cell lysate was in excess of the
capacity of the cAMP-agarose preventing retention of all RII complexed
with AKAP and an undetermined proportion of AKAP100 was not associated
with RII. This observation is consistent with similar findings for
AKAP79 and AKAP95
(24, 36) , which indicate that a
proportion of the RII-binding sites on anchoring proteins are
unoccupied. Furthermore, our findings show that only a fraction of RII
purified is associated with AKAP100 (Fig. 7 B, lane 4). This is because the cAMP-agarose purification
protocol isolates the total R subunit pool, which includes soluble RI,
soluble RII, and RII associated with other AKAPs present in the L6P
cell extract.
release channel of the sarcoplasmic and endoplasmic reticulum, is
activated by PKA
(45) , influencing Ca
mobilization within the cell
(46, 47) . Other
reports have suggested PKA activity is closely associated with a
reconstituted calcium-activated potassium channel
(41) and a
sarcoplasmic reticulum-associated chloride channel
(40) .
Moreover, recent studies suggest that voltage-dependent potentiation of
the L-type Ca
-gated channels in skeletal muscle
requires anchored PKA. Catterall and colleagues
(48) have shown
that perfusion of the Ht31 anchoring inhibitor peptide prevents
cAMP-responsive potentiation of the channel. In addition, AKAP100 may
function to target other enzymes involved in signal transduction to the
sarcoplasmic reticulum, as we have recently demonstrated that AKAP79
forms a ternary complex with PKA and the phosphatase 2B, calcineurin
(28) . These results suggest that both kinase and phosphatase
are targeted to subcellular sites by association with a common anchor
protein to regulate the phosphorylation state of key substrates.
with high affinity. The anchoring protein is selectively
expressed in certain human tissues and has been localized to the
sarcoplasmic reticulum in rat cardiac H9c2 cells. Current studies are
focusing on identifying other proteins that interact with AKAP100 to
establish those molecules responsible for targeting the entire complex
to the sarcoplasmic reticulum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.