2725
Journal of Cell Science 112, 2725-2736 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0512
mAKAP: an A-kinase anchoring protein targeted to the nuclear membrane of
differentiated myocytes
Michael S. Kapiloff1,2, Robynn V. Schillace1, Ann M. Westphal1 and John D. Scott1,*
1Howard Hughes Medical Institute,
2Department of Pediatrics, Oregon
L-474, Vollum Institute, 3181 SW Sam Jackson Park Road, Portland OR 97201-3098, USA
Health Sciences University, 3181 SW Sam Jackson Park Road, Portland OR 97201-3098, USA
*Author for correspondence (e-mail: [email protected])
Accepted 8 June; published on WWW 21 July 1999
SUMMARY
The compartmentalization of second messenger-activated
protein kinases contributes to the fidelity of hormonemediated signal transduction events. For example, the
cAMP-dependent protein kinase is tethered at specific
intracellular locations through association with A-kinase
anchoring proteins (AKAPs). We now report the cloning of
mAKAP, an anchoring protein found predominantly in
heart, skeletal muscle and brain, and whose expression is
induced in neonatal ventriculocytes by treatment with
hypertrophic stimuli. mAKAP is targeted to the nuclear
membrane of differentiated myocytes. Analysis of
mAKAP-green fluorescent protein (GFP) fusion constructs
revealed that nuclear membrane targeting is conferred by
two regions of the protein, between residues 772-915 and
915-1065, which contain spectrin-like repeat sequences.
Heterologous expression of the mAKAP targeting
sequences displaced the endogenous anchoring protein
from the nuclear membrane, demonstrating that mAKAP
targeting is saturable. Collectively, these data suggest that
a domain containing spectrin-like repeats mediates
targeting of the anchoring protein mAKAP and the cAMPdependent protein kinase holoenzyme to the nuclear
membrane in response to differentiation signals.
INTRODUCTION
cells also increases cAMP levels, but in contrast, activates only
soluble PKA, without effect on troponin or glycogen
phosphorylase. One mechanism that explains this sophisticated
level of PKA signaling is the selective activation of
compartmentalized PKA pools. This view is now supported by
increasing evidence illustrating that compartmentalization of
the cAMP-dependent protein kinase is maintained by a family
of A-kinase anchoring proteins (AKAPs) (Colledge and Scott,
1999; Dell’Acqua and Scott, 1997; Rubin, 1994).
Mammalian PKA is a heterotetramer composed of two
regulatory (R) and two catalytic (C) subunits, for which there
are ubiquitously expressed four R-subunit genes, RIα, RIβ,
RIIα and RIIβ, and two C-subunit genes, Cα and Cβ. AKAPs
bind to the regulatory subunit dimer of the PKA holoenzyme
and target the PKA/AKAP complex through protein-lipid or
protein-protein interactions with membranes of cellular
organelles (Dell’Acqua et al., 1998; Fraser et al., 1998). In
excess of 25 AKAPs have been described from diverse species,
ranging from C. elegans and Drosophila to humans, all of
which contain a conserved R-subunit binding site (Angelo and
Rubin, 1998; Huang et al., 1997a). These binding sites form
an amphipathic helix that binds to a hydrophobic pocket
formed by the N termini of the R-subunit dimer (Newlon et al.,
1999). AKAP specificity is conferred by a unique targeting
domain that directs the PKA/AKAP complex to discrete
subcellular compartments.
The phosphorylation of intracellular proteins is a universal
mechanism used to control many cellular events that occur in
response to extracellular signals. Occupancy of G-proteincoupled receptors on the plasma membrane triggers the
generation of second messengers such as cyclic AMP (cAMP)
which, upon diffusion to their sites of action, influence the
activation of protein kinases and phosphatases (Lefkowitz,
1993). In turn, the phosphorylation state of intracellular
proteins is regulated by the activation or suppression of these
enzymes. Since many kinases and phosphatases are widely
distributed and often exhibit broad substrate specificities,
additional mechanisms are in place to restrict their access to
preferred substrates. Increasing evidence suggests that the
subcellular location of the cAMP-dependent protein kinase
(PKA) is tightly controlled (Colledge and Scott, 1999;
Dell’Acqua and Scott, 1997). Moreover, activation of PKA by
different extracellular stimuli results in the preferential
activation of distinct processes within the same cell (Livesey
et al., 1982). Isoproterenol treatment of cardiac myocytes
increases cAMP levels and activates both particulate and
soluble PKA (Buxton and Brunton, 1983). The resulting
phosphorylation of the contractile protein troponin I and
activation of glycogen phosphorylase contributes to an increase
in cardiac contractility. Prostaglandin E1 treatment of the same
Key words: Protein kinase A, Heart, Spectrin repeat, Differentiation,
AKAP
2726 M. S. Kapiloff and others
By screening cDNA expression libraries with radiolabeled,
bacterially expressed RIIα-subunit in a protein overlay assay,
we had previously isolated clones for an AKAP found in heart,
skeletal muscle and brain, AKAP100 (McCartney et al., 1995).
We now report the complete cloning of the rat and human
isoforms of AKAP100, which we have renamed mAKAP for
its presence in striated muscle. We show that mAKAP is
targeted to the nuclear membrane of differentiated cardiac
ventriculocytes by a spectrin repeat homology domain and is
induced in expression by hypertrophic agents such as
phenylephrine.
MATERIALS AND METHODS
Cloning of mAKAP
The complete cloning of mAKAP was accomplished by a
modification of the Marathon Rapid Amplification of Cloned Ends
(RACE) Cloning Kit (Clontech), in which rounds of polymerase chain
reaction (PCR) were performed using pairs of nested primers that
were based initially on the 5′ sequence of the rat ROB1 clone (Fraser
et al., 1998), the overlapping human AKAP100 contig (McCartney et
al., 1995) and GenBank sequence KIAA0311. Marathon brain, heart
or skeletal muscle cDNA was purchased from Clontech or synthesized
as per the manufacturer’s protocol, except that 2.5 µg poly(A)selected RNA were annealed to 10 pmol specific primer for the reverse
transcriptase reaction, and 2 µg carrier tRNA were included during
the DNA precipitation steps. RACE-PCR was performed using the
‘touchdown’ conditions suggested by the manufacturer using Klentaq
Advantage polymerase (Clontech), nested specific primer, Marathon
AP1 primer and 10 minute extension times.
RACE-PCR products were phenol-chloroform extracted, purified
through a Centricon-100 filter (Amicon), and digested overnight with
NotI and a second restriction enzyme that recognized a site near the
5′ end of the previously determined cDNA sequence. The digested
products were ligated into pBSK− (Stratagene). β-galactosidasepositive E. coli DH5α transformants were screened by PCR with Taq
polymerase and standard universal and reverse M13 primers, such that
cloned RACE products on agarose gels large enough to encode new
5′ information were then subjected to automated, fluorescent
dideoxynucleotide sequencing. For each of the rat and human cDNAs,
final RACE reactions with multiple, different antisense
oligonucleotides yielded discrete products of lengths consistent with
a single 5′ end. 36 primers were designed to complete the cDNA
sequencing of larger PCR clones.
Using pairs of specific oligonucleotides designed to amplify large
fragments that might be used to construct full-length clones, PCR was
performed with the high fidelity enzyme PfuTurbo polymerase. A
varient pBSK−, pBNX, was constructed using a novel polylinker
(GGC CTC GTC GAC GCG GCC GCT CCG GAC CAT GGG CCC
TAG GAC GTC GCA TGC TAG CTC CGG AGA TCT AGA GGT
ACC TCG AGT AGT AC) in order that PCR products might be easily
subcloned and color-screened. PCR products were subcloned as above
into pBNX using unique sites within the rat and human cDNA and
the NotI site within the 5′ Marathon linker. Two human and two rat
subclones and pBSK-ROB-1 were then used to assemble full-length
rat and human cDNAs in pBNX. Both full-length clones were verified
by complete sequence comparison to the original contigs.
Mutagenesis and construction of mAKAP expression
vectors
Full-length mAKAP cDNAs (a NotI-XhoI human fragment and an
EagI-SalI rat fragment) were subcloned into the NotI and XhoI sites
in pCDNA3 (Invitrogen). A SalI-NsiI fragment of the rat full-length
clone was subcloned into pEGFPN1 (Clontech), yielding a vector that
expresses essentially full-length mAKAP (aa residues 1-2312) fused
to the N terminus of green fluorescent protein (GFP). Fragments of
rat or human cDNA were amplified as above using the full-length
pBNX clones, PfuTurbo polymerase, and specific primers that contain
an N-terminal BglII site and artificial translation site (CGG ACT CAG
ATC TGC CAC CAT GGC C followed by appropriate sense mAKAP
sequence) and a C-terminal SalI site (CAT ATA TAT GTC GAC CC
followed by appropriate antisense mAKAP sequence) or that contain
an N-terminal XhoI site and artificial translation site (CTA ACC AAC
TCG AGG GCT TGC CAC CAT GGC followed by appropriate sense
mAKAP sequence) and a C-terminal BamHI site (GGG CGC GGA
TCC followed by appropriate antisense mAKAP sequence), which
could be ligated into pEGFPN1 or pEGFPN3, respectively, to yield
vectors that express mAKAP fragment-GFP fusion protein. Each
pEGFP vector insert was sequenced to verify faithful construction,
and expression of each fusion protein was confirmed by western
blotting of transfected COS-7 cells using anti-GFP monoclonal
antibody (Clontech). A point mutation I2062P was introduced into the
full-length pBNX-rat mAKAP cDNA by the Quickchange method
(Stratagene) using PfuTurbo polymerase and paired oligonucleotides
(sense: CAG TAC ATG ACT TTG TTA AGG AAC CAA TTG ACA
TGG CAT CAA CAG CCC).
Production of affinity-purified antibodies
Plasmids that express in bacteria His6-tagged mAKAP C-terminal
fragments, human aa residues 1666-2319 or rat aa residues 14012314, were constructed by insertion of the appropriate mAKAP cDNA
sequences into pET30a (Novagen). After expression in E. coli BL21
DE3, recombinant proteins were purified by FPLC with a Ni-column
as recommended by the manufacturer. Rabbits were injected with
purified recombinant protein, rabbit VO54 with rat mAKAP 14012314 and rabbit VO55 and VO56 with human mAKAP 1666-2319.
Bleeds were collected for antisera (Covance, Inc.). Purified rat
recombinant protein was coupled to activated agarose, and all antisera,
VO54, VO55 and VO56, were purified on the rat mAKAP column by
batch elution with 50 mM glycine, pH 2.4.
Immunoblotting with mAKAP antibodies
Human tissues (Clontech) or rat tissues homogenized briefly
by Polytron in 50 mM Hepes, pH 7.4, 150 mM NaCl, 0.1%
Triton X-100, 1 mM dithiothreitol (DTT), 1 mM 4-(2aminoethyl)benzenesulfonyl fluoride (AEBSF), 2 µg/ml pepstatin,
2 µg/ml leupeptin, 1 mM benzamidine and 5 mM
ethylenediaminetetraacetic acid (EDTA) were assayed by the Lowry
method (Lowry et al., 1951), and 50-300 µg total protein were boiled
in Laemmli sample buffer (Laemmli, 1970) and fractionated on SDSPAGE gels with 3% acrylamide stacking and 5% resolving phases.
Western blotting was performed using semi-dry transfer to
nitrocellulose with a Biorad Transfer Unit, incubation with primary
mAKAP and horseradish peroxidase-conjugated anti-rabbit secondary
antibodies (Jackson Laboratories) in TTBS (Tris-buffered saline: 10
mM Tris, pH 7.5, 150 mM NaCl, with 0.03% Tween) buffer followed
by chemiluminescent detection (Supersignal, Pierce). Transfected
COS-7 cell extracts were similarly analyzed. For detection of native
mAKAP, affinity-purified antibody diluted 1:100 in buffer was
overlaid overnight, and chemiluminescence exposure to film was for
at least 30 minutes. On western blots all purified antibodies, including
the previously reported AKAP100 antibody (McCartney et al., 1995),
recognize recombinant human mAKAP; however, only VO54 and
VO56 (and weakly VO55) recognize recombinant rat mAKAP by
western blotting. Size markers were (unstained) Biorad SDS-PAGE
High Range Molecular Weight Standards, which include myosin, βgalactosidase, phosphorylase b, BSA and ovalbumin.
Cell culture and transfection
Rat neonatal ventriculocytes (RNV) were prepared according to the
protocol of Thorburn et al. (1995). Briefly, 1- to 3-day-old rat pups
A nuclear membrane AKAP in myocytes 2727
were killed under ether anesthesia by decapitation, and the hearts were
removed through a sternotomy. The ventricles were trimmed free of
atria, fat and connective tissue, while immersed in a neutral buffer.
Myocytes were dissociated by several 20 minute cycles of
collagenase/pancreatin treatment and serum neutralization. After
dissociation, RNV cells were collected by centrifugation, passed
through a 149 mm mesh cell filter to remove clumps, and pre-plated
in culture flasks to remove fibroblasts. After 1.5 hours, the medium
containing the unattached RNV cells was removed, cells were
collected by centrifugation and were plated again on dual-well
chamberslides previously coated with 1% gelatin and 1 mg/ml laminin
solution at density varying from 13,000 to 125,000 myocytes/cm2.
Plating medium was Dulbecco’s Modified Eagle Medium (DMEM)
with 17% Media 199, 1% penicillin/streptomycin solution (GibcoBRL), 10% horse serum (HS) and 5% fetal bovine serum (FBS).
After 1 day in culture, cells were transfected by the method of Chen
and Okayama (1987), as follows: 2-3 hours before transfection, the
medium was changed to maintenance medium (80% DMEM, 20%
Media 199, 1% penicillin/streptomycin) with 4% HS. 2.5 µg total
plasmid in 50 µl 0.25 M CaCl2 in water was added to 50 µl 2× BS
(50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4, pH 6.95), mixed,
and left at room temperature for 20 minutes. The mixture was added
to a single well containing 1 ml medium and left overnight in a 5%
CO2, 37°C incubator. The following day the cells were washed three
times with maintenance medium and then left in maintenance medium
with or without agonists for 2 days before harvesting. 100 µM
phenylephrine was added to the medium in order to induce the
hypertrophic response.
COS-7 and L6 rhabdomyosarcoma cells were passaged at low
density and plated in 6-well dishes containing sterile coverslips at 1075% confluence and transfected as above or with lipofectamine as per
the manufacturer’s protocol (Gibco-BRL). Cells were cultured in
DMEM with 10% FBS, 1% penicillin/streptomycin, and L6 were
differentiated in DMEM with 4% HS for a minimum of 2 days.
Immunocytochemistry
2 days after cells were transfected, cells were rinsed twice with
phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde in
PBS for 10 minutes, and washed again with PBS. Cells were extracted
for 1 minute with cold methanol or 10 minutes with 0.3% Triton X100 in PBS, washed once with PBS, and blocked with 0.2% bovine
serum albumin in PBS (BSA/PBS) for 30 minutes. Cells were
incubated with primary antibody diluted as required in 150 µl
BSA/PBS for 1 hour and then washed three times with BSA/PBS for
5 minutes. Cells were then incubated with appropriate fluorescent
secondary antibodies in the same manner (FITC, Texas Red and Cy5
conjugated-secondary donkey antibodies; Jackson Laboratories).
After three PBS washes, the cells were rinsed with water and mounted
on slides (for coverslips) or with glass coverslips (for chamberslides)
with Slowfade Antifade Kit (Molecular Probes). All mAKAP staining
was repeated with both the VO54 and VO56 antibodies. Rhodaminephalloidin (Molecular Probes) was used to visualize actin fibers.
Hoechst 33258 stain (10 µg/ml) was included in the last PBS wash for
detection of nuclei. Either the specific indirect immunofluorescence
staining or intrinsic GFP fluorescence was detected in successive focal
planes by laser-scanning confocal microscopy on an MRC1024
Biorad UV/Vis system. (Dell’Acqua et al., 1998). Frozen tissue
sections (Medico, Inc.) were stained as above, except that sections
were permeabilized three times for 1 minute with cold methanol or
for 10 minutes with 0.3% Triton X-100 in PBS.
Immunoprecipitation of mAKAP and PKA filter assay
L6 cells were washed twice with PBS and scraped into 2 ml cold PBS.
Cells were collected by centrifugation at 1000 rpm for 5 minutes and
lysed in 2.5 ml of fresh TBS with 0.5% Triton X-100, 1 mM DTT,
1 mM AEBSF, 2 µg/ml pepstatin, 2 µg/ml leupeptin, 1 mM
benzamidine and 5 mM EDTA. Extracts were clarified by
centrifugation at 2000 g for 15 minutes at 4°C. 5 µl whole V054 or
preimmune serum were added to 1.2 ml supernatant and incubated at
4°C for 1 hour. 100 µl pre-rinsed 20% (v/v) protein G-sepharose
(Gamma-bind Plus, Pharmacia) was added and the mixture was
incubated at 4°C for 2 hours, with rotation. Beads were collected by
brief centrifugation at 2500 g. Beads were washed twice for 5 minutes
with TBS with 0.5% Triton X-100, 1 mM DTT, 1 mg/ml BSA and
once with the same buffer containing 1 M NaCl. PKA C-subunit was
eluted with 150 µl 1 mM adenosine 3′,5′cyclic monophosphate
(cAMP) in water for 5 minutes at room temperature and assayed by
the filter binding method (Corbin and Reimann, 1974). Specific PKA
activity was determined by subtracting background activity found in
the presence of 20 µM protein kinase inhibitor (PKI).
α overlay assay
RIIα
Overlay assay was performed as previously described (Carr and Scott,
1992). Briefly, COS-7 cells were transfected with pCDNA3 or
pEGFP-expression vectors and were lysed into Laemmli buffer or
immunoprecipitated prior to SDS-PAGE and blotting as above. Blots
were blocked with Blotto (5% non-fat dried milk, 0.1% BSA in Trisbuffered saline) for 30 minutes and then incubated overnight at room
temperature with 100,000 cpm/ml 32P-RIIα in Blotto. Blots were
washed in TTBS four times for 5 minutes and then exposed to X-ray
film at −70°C with intensifier screens for 1-7 days.
RESULTS
Cloning of mAKAP
We have previously isolated cDNA clones for several A-kinase
anchoring proteins (AKAPs) using an interaction cloning
strategy with radiolabelled RIIα-subunit as a probe. One clone,
originally called AKAP100, represented a sole human mRNA
of approx. 10 kb, which was enriched in heart and skeletal
muscle (McCartney et al., 1995). Additional cloning
experiments isolated a rat AKAP100 homologue with an
extended open reading frame at the 5′ end. Further analysis
revealed that the 5′ end of the original AKAP100 cDNA was
rearranged, thereby introducing an in-frame stop codon. The
complete cloning of human and rat AKAP100 cDNAs was
achieved by multiple rounds of a modified 5′-Rapid
Amplification of cDNA Ends (RACE) procedure, and new
sequence has been entered into the GenBank database
(Accession numbers AF139518 and U17195). Sequence from
45 human and 47 rat RACE clones contributed to complete the
double-stranded sequence for each cDNA. Although we cannot
completely exclude the possibility of alternative splicing, all of
the clones yielded sequences that may be attributed to a single
gene product, whether derived from heart, skeletal muscle or
brain cDNA. We acquired 8.8 kb of rat and 10.3 kb of human
cDNA sequence, encoding proteins of 2314 and 2319 residues,
respectively (Fig. 1). The predicted rat and human amino acid
sequences are 79% identical. Because AKAP100 is enriched
in striated muscle, we now refer to it as mAKAP (muscle
AKAP).
BLAST search of the National Center for Biotechnology
Information (NCBI) with mAKAP sequences found four STSmarkers with identical matches to the human mAKAP cDNA
sequence: WI-9732 (308 bp of identity to the human cDNA),
WI-14096 (367 bp), N39150 (239 bp) and SHGC-37333 (250
bp). These markers have all have been mapped (with different
resolution) to 28 cM and 60.4 cR on the genetic maps of
chromosome 14q, according to information provided in the
2728 M. S. Kapiloff and others
human
rat
1
1
human
rat
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76
human
rat
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L L W H Q L R V S V L V L R E R I L Q G L Q D A N G N Y T R Q T D I L Q A F S E E T K E G R L D S L T E V D D S G Q L T I K C S Q N Y L S L D C G I T
L L W H Q L R V S V L V L R E R I L Q G L Q D A N G N Y T R Q T D I L Q A F S E E T T E G R L D S L T E V D D S G Q L T I K C S Q D Y L S L D C G I T
223
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human
rat
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A F E L S D Y S P S E D L L S G L G D M T S S Q V K T K P F D S W S Y S E M E K E F P E L I R S V G L L T V A A D S I S T N G S E A V T E E V S Q V S
A F E L S D Y S P S E D L L G G L G D M T T S Q A K T K S F D S W S Y S E M E K E F P E L I R S V G L L T V A T E P V P S S C G E A N - E D S S Q A S
298
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human
rat
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300
L S V D D K G G C E E D N A S A V E E Q P G L T L G V S S S S G E A L T N A A Q P S S E T V Q Q E S S S S S H H D A K N Q Q P V P C E N A T P K R T I
L S D D H K G E H G E D G A P V P G Q Q L D S T V G M S S L D G - T L A N A A E H P S E T A K Q D S T S S P Q L G A K K T Q P G P C E I T T P K R S I
373
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human
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R D C F N Y N E D S P T Q P T L P K R G L F L K E E T F K N D L K G N G G K R Q M V D L K P E M S R S T P S L V D P P D R S K L C L V L Q S S Y P N S
R D C F N Y N E D S P T Q P T L P K R G L F L K E T Q - K N E R K G S D R K G Q V V D L K P E L S R S T P S L V D P P D R S K L C L V L Q S S Y P S S
448
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human
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P S A A S Q S Y E C L H K V G N G N L E N T V K F H I K E I S S S L G R L N D C Y K E K S R L K K P H K T S E E V P P C R T P K R G T G S G K Q A K N 523
P S A A S Q S Y E C L H K V G L G N L E N I V R S H I K E I S S S L G R L T D C H K E K L R L K K P H K T L A E V S L C R I P K Q G G G S G K R S E S 522
human
rat
524
523
T K S S A V P N G E L S Y T S K A I E G P Q T N S A S T S S L E P C N Q R S W N A K L Q L Q S E T S S S P A F T Q S S E S S V G S D N I M S P V P L L
T G S S A G P S M V S P G A P K A T M R P E T D S A S T A S G G L C H Q R N R S G Q L P V Q S K A S S S P P C S H S S E S S L G S D S I K S P V P L L
598
597
human
rat
599
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S K H K S K K G Q A S S P S H V T R N G E V V E A W Y G S D E Y L A L P S H L K Q T E V L A L K L E N L T K L L P Q K P R G E T I Q N I D D W E L S E
S K N K S Q K S S P P A P C H A T Q N G Q V V E A W Y G S D E Y L A L P S H L K Q T E V L A L K L E S L T K L L P Q K P R G E T I Q D I D D W E L S E
673
672
human
rat
674
673
M N S D S E I Y P T Y H V K K K H T R L G R V S P S S S S D I A S S L G E S I E S G P L S D I L S D E E S S M P L A G M K K Y A D E K S E R A S S S E
M N S D S E I Y P T Y H I K K K H T R L G T V S P S S S S D I A S S L G E S I E S G P L S D I L S D E D L C L P L S S V K K F T D E K S E R P S S S E
748
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human
rat
749
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K N E S H S A T K S A L I Q K L M Q D I Q H Q D N Y E A I W E K I E G F V N K L D E F I Q W L N E A M E T T E N W T P P K A E M D D L K L Y L E T H L
K N E S H S A T R S A L I Q K L M H D I Q H Q E N Y E A I W E R I E G F V N K L D E F I Q W L N E A M E T T E N W T P P K A E T D S L R L Y L E T H L
823
822
human
rat
824
823
S F K L N V D S H C A L K E A V E E E G H Q L L E L I A S H K A G L K D M L R M I A S Q W K E L Q R Q I K R Q H S W I L R A L D T I K A E I L A T D V 898
S F K L N V D S H C A L K E A V E E E G H Q L L E L V V S H K A G L K D T L R M I A S Q W K E L Q R Q I K R Q H S W I L R A L D T I K A E I L A T D V 897
human
rat
899
898
S V E D E E G T G S P K A E V Q L C Y L E A Q R D A V E Q M S L K L Y S E Q Y T S S S K R K E E F A D M S K V H S V G S N G L L D F D S E Y Q E L W D 973
S V E D E E G T G S P K A E V Q L C H L E T Q R D A V E Q M S L K L Y S E Q Y T S G S K R K E E F A N M S K A H A E G S N G L L D F D S E Y Q E L W D 972
human
rat
974
973
C L I D M E S L V M D S H D L MM S E E Q Q Q H L Y K R Y S V E M S I R H L K K T E L L S K V E A L K K G G V L L P N D L L E K V D S I N E K W E L L
W L I D M E S L V M D S H D L MM S E E Q Q Q H L Y K R Y S V E M S I R H L K K S E L L S K V E A L K K G G L S L P D D I L E K V D S I N E K W E L L
1048
1047
human
rat
1049
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G K T L G E K I Q D T M A G H S G S S P R D L L S P E S G S L V R Q L E V R I K E L K G W L R D T E L F I F N S C L R Q E K E G T M N T E K Q L Q Y F
G K T L R E K I Q D T I A G H S G S G P R D L L S P E S G S L V R Q L E V R I K E L K R W L R D T E L F I F N S C L R Q E K E G T S - A E K Q L Q Y F
1123
1121
human
rat
1124
1122
K S L C R E I K Q R R R G V A S I L R L C Q H L L D D R E T C N L N A D H Q P M Q L I I V N L E R R W E A I V M Q A V Q W Q T R L Q K K M G K E S E T
K S L C R E I K Q R R R G V A S I L R L C Q H L L D D R D T C N L N A D H Q P M Q L I I V N L E R R W E A I V M Q A V Q W Q T R L Q K K M G K E S E T
1198
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human
rat
1199
1197
L N V I D P G L M D L N G M S E D A L E W D E M D I S N K L I S L N E E S N D L D Q E L Q P V I P S L K L G E T S N E D P G Y D E E A D N H G G S Q Y 1273
L N V I D P G L M D L N G M S E D A L E W D E T D I S N K L I S V H E E S N D L D Q D P E P M L P A V K L E E T H H K D S G Y E E E A G D C G G S P Y 1271
human
rat
1274
1272
A S N I T A P S S P H I Y Q V Y S L H N V E L Y E D N H M P F L K N N P K V T G M T Q P N V L T K S L S K D S S F S S T K S L P D L L G G S N L V K P
T S N I T A P S S P H I Y Q V Y S L H N V E L H E D S H T P F L K S S P K F T G T T Q P T V L T K S L S K D S S F S S T K S L P D L L G G S G L V R P
1348
1346
human
rat
1349
1347
C A C H G G D M S Q N S G S E S G I V S E G D T E T T T N S E M C L L N A V D G S P S N L E T E H L D P Q M G D A V N V L K Q K F T D E G E S I K L P
Y S C H S G D L S Q N S G S E S G I V S E G D N E M P T N S D M S L F S M V D G S P S N P E T E H P D P Q M G D A A N V L E Q K F K D N G E S I K L S
1423
1421
human
rat
1424
1422
N S S Q S S I S P V G C V N G K V G D L N S I T K H T P D C L G E E L Q G K H D V F T F Y D Y S Y L Q G S K L K L P M I M K Q S Q S E K V H V E D P L
S V S R A S V S P V G C V N G K A G D L N S V T K H T A D C L G E E L Q G K H D V F T F Y D Y S Y L Q G S K L K L P M I M K Q P Q S E K A H V E D P L
1498
1496
human
rat
1499
1497
L R G F Y F D K K S C K S K H Q T T E L Q P D V P P H E R I L A S A S H E M D R I S Y K S G N I E K T F T G M Q N A K Q L S L L S H S S S I E S L S P
L G G F Y F D K K S C K A K H Q A S E S Q P D A P P H E R I L A S A P H E M G R S A Y K S S D I E K T F T G I Q S A R Q L S L L S R S S S V E S L S P
1573
1571
human
rat
1574
1572
G G D L F G L G I F K N G S D S L Q R S T S L E S W L T S Y K S N E D L F S C H S S G D I S V S S G S V G E L S K R T L D L L N R L E N I Q S P S E Q 1648
G G D L F G L G I F K N G S D S L Q R S T S L E S W L T S Y K S N E D L F S C H S S G D I S V S S G S V G E L S K R T L D L L N R L E N I Q S P S E Q 1646
human
rat
1649
1647
K I K R S V S D I T L Q S S S Q K M S F T G Q M S L D I A S S I N E D S A A S L T E L S S S D E L S L C S E D I V L H K N K I P E S N A S F R K R L T 1723
K I K R S V S D M T L Q S S S Q K M P F A G Q M S L D V A S S I N E D S P A S L T E L S S S D E L S L C S E D I V L H K N K I P E S N A S F R K R L N 1721
human
rat
1724
1722
R S V A D E S D V N V S M I V N V S C T S A C T D D E D D S D L L S S S T L T L T E E E L C I K D E D D D S S I A T D D E I Y E D C T L M S G L D Y I
R S V A D E S D V N V S M I V N V S C T S A C T D D E D D S D L L S S S T L T L T E E E L C L K D E D D D S S I A T D D E I Y E E S N L M S G L D Y I
human
rat
1799
1797
K N E L Q T W I R P K L S L T R D K K R C N V S D E M K G S K D I S S S E M T N P S D T L N I E T L L N G S V K R V S E N N G N G K N S S H T H E L G 1873
K N E L Q T W I R P K L S L T R E K K R S G V T D E I K V N K D G G G N E K A N P S D T L D I E A L L N G S I R C L S E N N G N G K T P P R T H G S G 1871
human
rat
1874
1872
T K R E N K K T I F K V N K D P Y V A D M E N G N I E G I P E R Q K G K P N V T S K V S E N L G S H G K E I S E S E H C K C K A L M D S L D D S N T A 1948
T K G E N K K S T Y D V S K D P H V A D M E N G N I E S T P E R E R E K P Q G L P E V S E N L A S N V K T I S E S E L S E Y E A V M D G S E D S S V A 1946
human
rat
1949
1947
G K E F V S Q D V R H L P K K C P N H H H F E N Q S T A S T P T E K S F S E L A L E T R F N N R Q D S D A L K S S - D D A P S M A G K S A G C C L A L 2022
R K E F C P P N D R H P P Q M G P K L Q H P E N Q S G D C K P V Q N P C P G L L S E A G V G S R Q D S N G L K S L P N D A P S G A R K P A G C C L - L 2020
human
rat
2023
2021
E Q N G T E E N A S I S - N I S C C N C E P D V F H Q K D A E D C S V H N F V K E I I D M A S T A L K S K S Q P E N E V A A P T S L T Q I K E K V L E
E Q N E T E E S A S I S S N A S C C N C K P D V F H Q K D D E D C S V H D F V K E I I D M A S T A L K S K S Q P E S E V A A P T S L T Q I K E K V L E
human
rat
2097
2096
H S H R P I Q L R K G D F Y S Y L S L S S H D S D C G E V T N Y I E E K S S T P L P L D T T D S G L D D K E D I E C F F E A C V E G D S D G E E P C F 2171
H S H R P I H L R K G D F Y S Y L S L S S H D S D C G E V T N Y I D E K S S T P L P P D A V D S G L D D K E D M D C F F E A C V E D E P V N E E A G L 2170
human
rat
2172
2171
S S A P P N E S A V P S E A A M P L Q A T A C S S E F S D S S L S A D D A D T V A L S S P S S Q E R A E V G K E V N G L P Q T S S G C A E N L E F T P
P G A L P N E S A I E D G A E Q - - - - - K S E Q K T A S S P V L S D K T D L V P L S G L S P Q K G A D D A K E G D D V S H T S Q G C A E S T E P T T
human
rat
2247
2241
S K L D S E K E S - S G K P G E S G M P E E H N A A S A K S K V Q D L S L K A N Q P T D K A A L H - P S P K T L T C E E N L L N L H E K R H R N M H R 2319
P S G K A N A E G R S R M Q G V S A T P E E - N A A S A K P K I Q A F S L N A K Q P K G K V A M R Y P S P Q T L T C K E K L V N F H E D R H S N M H R 2314
M L T M S V T L S P L R S Q D L D P M A T D A S P M A I N M T P T V E Q - - G E G E E A M K D M D S D Q Q Y E K P P P L H T G A D W K I V L H L P E I
M L T M S V T L S P L R S Q G P D P M A T D A S P M A I N M T P T V E Q E E G E G E E A V K A I D A E Q Q Y G K P P P L H T A A D W K I V L H L P E I
73
75
E T W L R M T S E R V R D L T Y S V Q Q D S D S K H V D V H L V Q L K D I C E D I S D H V E Q I H A L L E T E F S L K L L S Y S V N V I V D I H A V Q 148
E T W L R M T S E R V R D L T Y S V Q Q D A D S K H V D V H L V Q L K D I C E D I S D H V E Q I H A L L E T E F S L K L L S Y S V N V I V D I H A V Q 150
1798
1796
2096
2095
2246
2240
A nuclear membrane AKAP in myocytes 2729
Fig. 1. The primary structure of human and rat mAKAP. Human and
rat amino acid sequences are aligned using the MacVector gene
analysis program. Identical residues are in gray and conservative
substitutions are in light gray. The putative RIIα-binding site in rat
mAKAP is outlined with brackets.
National Center for Biotechnology Information UniGene GB4
database.
Detection of mAKAP protein
Recombinant fragments corresponding to residues 1666-2319
of human mAKAP or residues 1401-2314 of rat mAKAP were
used as antigens for the production of polyclonal antibodies.
Antisera were affinity purified and used in immunoblotting of
whole tissue extracts. Two antibodies recognized a single band
of approximately 300 kDa in rat heart; there was less intense
recognition in skeletal muscle, but none in colon (Fig. 2A). A
slightly higher band was detected in brain, which migrated
with the same mobility as the full-length, recombinant rat
mAKAP protein expressed in COS-7 cells (Fig. 2B). Muscle
bands at 120 and 135 kDa were consistently detected with
horseradish peroxidase-conjugated secondary antibody alone
(not shown), whereas the detection of bands at 200-250 kDa
in skeletal muscle and at 70 kDa in brain were variable and
probably represent proteolytic degradation products. Similar
results were obtained for human tissue extracts (data not
shown).
Fig. 2. Detection and expression of mAKAP. Rat tissue extracts (50
µg) were separated on 5% SDS-PAGE gels, electrotransferred to
nitrocellulose and probed for mAKAP by western blot using affinitypurified VO56 (A) or VO54 (B) antibody. (A) Detection of
endogenous mAKAP. The tissue source is indicated above each lane.
(B) Comparison of the recombinant protein and brain mAKAP (300
µg). Whole COS-7 cell extracts were prepared from cells expressing
either the pCDNA3 parent vector alone or pCDNA3-mAKAP and
treated as described above. Sample sources are indicated above each
lane. Relative molecular mass markers (kDa) are indicated. Blots are
representative of three independent experiments.
mAKAP binds PKA holoenzyme
In order to establish that mAKAP functions as an A-kinase
anchoring protein inside cells, endogenous mAKAP was
immunoprecipitated from L6 rhabdomyosarcoma cells (Fig. 3).
mAKAP protein of approximately 300 kDa was detected in
samples immunoprecipitated with specific antibody, while
no immunoblot signal was detected in control samples
immunoprecipitated with non-immune antibodies (Fig. 3;
insert). Kinase activity toward the heptapeptide substrate
kemptide was enriched 2.6±0.6-fold (n=3, P<0.03, t-test) in
mAKAP immunoprecipitates compared to that with the
preimmune sera (Fig. 3). The inhibition of the mAKAPassociated kinase activity by the protein kinase inhibitor (PKI)
5-24 peptide, which is a specific inhibitor of the catalytic
subunit of PKA, confirms that mAKAP can be isolated in a
complex with the PKA holoenzyme from cells.
Mapping of the mAKAP PKA-binding domain
We have previously shown that the C-terminal third of mAKAP
binds RIIα in vitro (McCartney et al., 1995). In order to map
the RIIα-binding domain further, a series of smaller fragments
fused to GFP were expressed in COS-7 cells (Fig. 4A). All
constructs spanning residues 2013-2083 bound RIIα, as
determined by binding in an overlay assay (Fig. 4C, right
panel), whereas mAKAP fragments lacking this region were
unable to interact with RIIα. As expected, several RIIαbinding proteins which are endogenous to COS-7 cells were
detected in the overlay assay (Fig. 4C, right panel, data for GFP
alone); therefore, the migration position of each recombinant
mAKAP fragment was determined by detection with an
antibody against the GFP tag (Fig. 4C, left panel).
Sequences that are predicted to form an amphipathic helix
comprise the principal determinants for R-subunit binding in
all AKAPs studied so far (Carr et al., 1991). A sequence
between residues 2055-2072 of mAKAP fulfills this criterion
(Fig. 4B). Substitution of proline for isoleucine at position
Fig. 3. mAKAP binds PKA in cells. mAKAP was
immunoprecipitated from extracts of L6 rhabdomyosarcoma cells
using VO54 (Anti-mAKAP) or control antisera (Preimmune).
Detection of the protein was confirmed by western blot (inset). PKA
activity was detected using kemptide filter assays in the presence or
absence of the specific PKA inhibitor PKI. Relative kinase activity is
presented as the fold increase over control ± s.e.m. Data is an
amalgam of three independent experiments. Statistical significance
was calculated by Student’s t-test.
2730 M. S. Kapiloff and others
Fig. 4. Mapping the RIIα-binding site on mAKAP. (A) Schematic diagram presenting a family of epitope-tagged human mAKAP protein
fragments that were expressed in COS-7 cells to map the RIIα-binding domain. The first and last residues of each fragment are indicated. RIIαbinding fragments (filled boxes) and non-binding fragments (open boxes) are indicated. (B) Helical wheel representation of residues 2055-2072
showing hydrophobic (thick line) and hydrophilic faces. (C) COS-7 cells were transfected with vectors expressing GFP-tagged human mAKAP
fragments. Whole cell extracts were fractionated on 10% SDS-PAGE gels, electrotransferred to nitrocellulose, and the expressed proteins were
detected by monoclonal antibodies to the green fluorescent protein (GFP) epitope tag (left panel) or by 32P-RIIα overlay assay (right panel). A
representative of five individual experiments is presented. The sample source is indicated above each lane. (D) Full-length mAKAP or the
I2062P mutant were immunoprecipitated from COS-7 cell extracts using the VO54 antibody. Proteins were detected by western blot (left panel)
and RIIα overlay (right panel) as described above. Sample sources are indicated above each lane in C and D, and relative molecular mass
markers are indicated. Blots are representative of three independent experiments.
2062 is predicted to disrupt secondary structure within the
putative helical region. The involvement of this region in
RIIα-binding was confirmed by site-directed mutagenesis
experiments (Fig. 4D). To test this hypothesis a full-length
mAKAP and I2062P mutant were expressed in COS-7 cells.
Both proteins were immunoprecipitated with anti-mAKAP
antibodies and tested for interaction with RIIα by the overlay
assay (Fig. 4D, right panel). Only the wild-type mAKAP
bound RIIα, confirming that the residues surrounding Ile 2062
are determinants for PKA anchoring.
Intracellular localization of mAKAP
The intracellular location of mAKAP was examined in a
variety of cell types by immunofluorescence techniques.
Consistent with a previous report (McCartney et al., 1995),
mAKAP was targeted to the endoplasmic reticulum in COS-7
cells (data not shown). However, the mAKAP signal was
concentrated at the nuclear membrane in frozen, tissue sections
of rat cardiac ventricle (Fig. 5A-C). A composite image that
merges mAKAP, DNA and actin staining shows that the
anchoring protein is concentrated at the nuclear membrane of
some, but not all cells (Fig. 5D). Additional controls
demonstrated that no staining was observed with secondary
antibodies alone (Fig. 5E). Identical results were obtained with
two affinity-purified mAKAP antibodies, and a similar staining
pattern was detected for rat thigh skeletal muscle (data not
shown). In light of the differences we observed in mAKAP
localization between transformed cell lines and in tissue, we
decided to examine mAKAP expression and location in
primary cardiac myocyte cultures.
Rat neonatal ventriculocytes (RNV) revert to an
undifferentiated phenotype when dissociated and grown at low
density in low serum (Schaub et al., 1997; Sugden and Clerk,
1998). However, the cells adopt a more differentiated,
hypertrophic phenotype when grown at high density or in the
presence of phenylephrine, an α-adrenergic agonist. The
differentiated cells are mono- or binucleate, undergo myofibrillar
organization and exhibit spontaneous contractions (Thorburn et
al., 1995). RNV were cultured at a range of densities to yield
cells in diverse states of differentiation (Fig. 6A-F, low density;
A nuclear membrane AKAP in myocytes 2731
Fig. 5. The subcellular distribution of
mAKAP in heart tissue. Frozen
longitudinal sections of rat heart tissue
were stained with anti-mAKAP VO54 and
FITC-conjugated fluorescent secondary
antibody (A, green), Hoechst DNA stain
for nuclei (B, blue), and rhodaminephalloidin, which detects F-actin in
myofibrils (C, red). A composite is shown
in (D). An FITC-channel image is shown
for a control section stained identically as
above, but with no mAKAP primary
antibody (E). Each image is at the same
scale; bar, 20 µm. All images were
acquired by confocal microscopy and are
representative of four independent
experiments with each of the VO56 and
VO54 antibodies.
H-L, high density). The differentiation state of individual cells
was monitored by F-actin staining to establish the extent of
myofibrillar organization (Fig. 6C,I). There was less staining of
actin fibers in undifferentiated RNV, which were plated at low
density and exhibited a more extended appearance (Fig. 6C).
mAKAP was not detected in undifferentiated RNV
permeabilized with Triton X-100 (Fig. 6A), and the intracellular
distribution of RII was reticular and cytoplasmic (Fig. 6B). In
contrast, differentiated RNV contained striated myofibrils that
were heavily stained by rhodamine-phalloidin (Fig. 6I).
Furthermore, mAKAP staining was readily apparent at the
nuclear membrane and at low levels within the nucleus (Fig. 6G).
Although widely distributed, RII was also more concentrated at
the nuclear membrane in differentiated cells (Fig. 6H). Nuclei
were detected with Hoechst 33258 (Fig. 6E,K), and control
FITC-channel images are shown in Fig. 6F,L. Collectively, these
results suggest that mAKAP expression is upregulated in
differentiated RNV and that the net effect of these events is to
concentrate PKA at the nuclear membrane.
Mapping of the mAKAP targeting domain
On the basis of our immunofluorescence studies, it was apparent
that endogenous mAKAP was targeted to the nuclear membrane
in heart tissue and differentiated RNV. This provided us with
an assay to map a targeting domain on mAKAP that maintained
the protein at the nuclear membrane. Therefore, a family of
mAKAP fragments fused to GFP was constructed and
expressed in RNV (Fig. 7A). Control experiments confirmed
that the endogenous protein (Fig. 7B) and the full-length
mAKAP-GFP-fusion protein were concentrated at the nuclear
membrane (Fig. 7D). In contrast, GFP alone was distributed
throughout the cell (Fig. 7C). These studies established that
fusion of the GFP fluorescent tag to the C terminus of mAKAP
did not affect the correct intracellular targeting of the protein.
Further expression of mAKAP-GFP fragments in RNV
demonstrated that GFP-fusion proteins containing residues 5851286 were properly targeted to the nuclear membrane (Fig.
7D,G,H). In contrast, mAKAP fragments flanking this region
were broadly distributed in the cell (Fig. 7E,F). Interestingly,
residues 585-1286 of the anchoring protein encompass a series
of three spectrin repeat-like sequences (Fig. 8). Thus, an
additional series of deletion fragments were produced to assess
the contribution of individual spectrin repeats in mAKAP
targeting (Fig. 7A). Fragments of mAKAP encompassing the
first and second spectrin repeats were correctly targeted to the
nuclear membrane (Fig. 7J,L), whereas fragments encompassing
the third spectrin repeat or flanking regions were not targeted
(Fig. 7I,K,M). These experiments illustrate that residues 772915 and 915-1065 of mAKAP represent individual regions that
are sufficient for nuclear membrane targeting and that the
anchoring protein contains more than one targeting signal.
In order to avoid overexpression artifacts, only those RNV
exhibiting low GFP-fusion protein fluorescence were selected
for study in the above mapping experiments. Indeed, high level
expression of these mAKAP fragments resulted in
accumulation of the signal in the cytoplasm (Fig. 9A). This
suggests that mAKAP targeting to the nuclear membrane is
saturable. This conclusion is supported by evidence that
overexpression of mAKAP targeting fragment 585-1286-GFP
results in the concomitant displacement of the endogenous
anchoring protein from the nuclear membrane (Fig. 9B,C). In
contrast, overexpression of control GFP constructs did not
displace mAKAP (Fig. 9D-F). Non-transfected cells in each
field (two shown) served as internal positive controls for
endogenous mAKAP targeting (Fig. 9B,E). These results
suggest that the mAKAP spectrin repeats interact with a
targeting locus at the nuclear membrane and can displace the
endogenous anchoring protein from targeting sites inside cells.
DISCUSSION
This work describes the cloning and characterization of
mAKAP, an A-kinase anchoring protein that is targeted to the
nuclear membrane. Targeting is mediated by a region of the
AKAP containing spectrin-like repeats and appears to be
2732 M. S. Kapiloff and others
Fig. 6. Localization of mAKAP in primary cultures
of rat neonatal ventriculocytes (RNV) is dependent
upon state of RNV differentiation. RNV were
cultured and grown on chamberslides as described in
Materials and methods. (A-F) RNV cultured at low
density were stained with rabbit anti-mAKAP VO54
and FITC-conjugated fluorescent secondary antibody
(A, green), goat anti-PKA-RIIα and -RIIβ and Cy5conjugated fluorescent secondary antibody (B, blue),
rhodamine-phalloidin, which detects F-actin in
myofibrils (C, red), and Hoechst DNA stain for nuclei
(E, grayscale). A three-color composite is shown in
(D). An FITC-channel image is shown for a control
slide identically stained, but with no mAKAP
primary antibody (F). (G-L) Primary RNV cultured at
high density in the presence of 100 µM
phenylephrine to induce differentiation were labeled
as above: mAKAP VO54 antibodies (G), anti-PKARIIα and -RIIβ (H), rhodamine-phalloidin (I),
composite (J), Hoechst stain (K) and FITC control
(L). Each image is at the same scale; bars, 20 µm. All
images were acquired by confocal microscopy and
are representative of more than three independent
experiments with each of the VO56 and VO54
antibodies.
related to the differentiation state of the cell. The full-length
mAKAP protein is a 255 kDa, striated muscle- and brainselective A-kinase anchoring protein, which is highly
conserved between the rat and human species. Immunoblotting
of whole tissue extracts revealed a single band in striated
muscle that migrated in SDS-PAGE slightly faster than the
single band found in brain. Analysis of the muscle and brain
mAKAP mRNA species revealed no differences. Recombinant
full-length mAKAP migrated with the native brain form. Thus,
the difference in migration in SDS-PAGE between these forms
suggests that mAKAP may be post-translationally modified. At
this time it is unclear what forms of post-translational
modification are responsible for the differences in mobility.
Characterization of mAKAP/ PKA interaction
AKAPs are a group of 50 or so functionally related proteins
that have been classified on the basis of their ability to associate
with the PKA holoenzyme inside cells (Colledge and Scott,
1999). Several lines of evidence imply that mAKAP represents
a conventional anchoring protein. First, immunoprecipitation
of mAKAP from L6 rhabdomyosarcoma cell extracts results in
the copurification of the PKA holoenzyme and an enrichment
of cAMP-dependent kinase activity. This classifies the protein
as an AKAP. Second, overlay experiments detect a 255 kDa
RIIα-binding band upon heterologous expression of the fulllength recombinant protein. Third, we have mapped the RIIαbinding site to include residues 2013-2083 of the protein.
Within this region there is a sequence (residues 2055-2072)
that is predicted to form an amphipathic helix. This is another
hallmark of the AKAPs, as numerous studies have suggested
that an R-subunit binding site forms an amphipathic helix
that participates in protein-protein interactions with a
complimentary surface on the R-subunit dimer (Hausken et al.,
1994, 1996; Li and Rubin, 1995). A model for the RIIα/AKAP
A nuclear membrane AKAP in myocytes 2733
Nuclear
Membrane
Targeting
A.
GFP Constructions
D. 1-2312
E. 1286-2312
F. 1-587
G. 1-1286
+
+
H. 585-1286
I. 585-771
J. 772-915
K. 883-915
L. 915-1065
+
+
+
-
M. 1065-1286
Spectrin Repeats (615-1231)
RII-binding site (2055-2072)
Fig. 7. Spectrin repeat-like
sequences are required for
mAKAP targeting.
(A) Schematic diagram
presenting a family of GFPtagged human mAKAP protein
fragments that were expressed in
RNV to map the targeting
domain. The first and last
residues of each fragment are
indicated. Fragments that target
(filled boxes) are distinguished
from those that do not (open
boxes). The locations of spectrin
repeats and the RIIα binding
domain are indicated. (B) Control
cell showing the
immunofluorescence detection of
endogenous mAKAP. (C) Control
cell showing fluorescence
detection of GFP (D-M)
Detection of mAKAP-GFP
fragments expressed in RNV. The
first and last residues of each
fragment are indicated.
Representatives are shown of the
multiple cells studied in each of
three experiments for each
construct.
spectrin
772-882
952-1059
1071-1187
A
N
A
L
K
Y
H
S
L
E
A
P
N
A
E
E
E
I
G
S
S
W
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G
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N
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R
R
G
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L
I
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spectrin
772-882
952-1059
1071-1187
D
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A
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A
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spectrin
772-882
952-1059
1071-1187
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Fig. 8. Alignment of
mAKAP amino acid residue
sequences with the chicken
brain α-spectrin 16th repeat.
Dots designate residues
involved in inter-helix
bonding. The three helices
are overlaid by bars.
Structural data were derived
from Pascual et al. (1996).
2734 M. S. Kapiloff and others
Fig. 9. Displacement of
endogenous mAKAP from the
nuclear membrane by
overexpression of mAKAP aa
585-1286-GFP. mAKAP aa 5851286 or GFP alone were
overexpressed at high levels in
phenylephrine-treated RNV (A,D).
Endogenous mAKAP was
detected by staining with VO54
primary and Texas-red conjugated
secondary antibody (B,E). Nuclei
of non-transfected cells are
indicated by empty arrowheads,
and nuclei of transfected cells by
filled arrowheads. Composite
images are shown in (C,F).
Multiple cells were studied in each
of eight experiments.
complex has been recently proposed based on the NMR
structure of the dimeric RIIα anchoring domain. A
hydrophobic face on the AKAP amphipathic helix packs onto
a reciprocal surface formed by the two chains of each RIIα
protomer (Newlon et al., 1997, 1999). It is likely that mAKAP
binds RIIα through a similar mechanism, because substitution
of isoleucine 2062 with a proline residue abolished PKA
binding. It is presumed that the isoleucine-to-proline
substitution perturbed the helical conformation of the RIIαbinding site on mAKAP, thereby altering the organization of
hydrophobic residues.
Intracellular location of mAKAP
Each AKAP contains a unique targeting motif that directs the
anchored PKA holoenzyme to precise intracellular locations
(Colledge and Scott, 1999). Although AKAPs have now been
identified in most cellular compartments and associated with a
variety of organelles, this is the first report of an anchoring
protein that can direct PKA to the nuclear membrane. The
nuclear membrane is composed of two lipid bilayers separated
by the perinuclear space, which is continuous with the lumen
of the endo/sarcoplasmic reticulum (ER/SR). Although our
immunofluorescence data of heart tissue and primary
ventriculocyte cultures suggests that mAKAP is at the nuclear
membrane, we cannot exclude the possibility that low levels of
mAKAP are present in the SR. A recent report has also
detected mAKAP in a number of myocyte compartments
including the SR and the nucleus (Yang et al., 1998).
Furthermore, different permeabilization conditions in
immunohistochemistry can lead to varying results. For
example, in undifferentiated RNV permeabilized with
methanol, we found minimal levels of specific mAKAP
staining within the nucleus (data not shown). Also, it is
important to note that the localization of mAKAP in RNV and
tissue differs from that in tumor cell lines, including embryonic
cardiac H9c2, transfected COS-7 and L6 cell lines, in which
mAKAP is detected at the ER/SR (McCartney et al., 1995; M.
S. K., unpublished observations). This leaves open the
possibility that other compartmentalized pools of mAKAP
exist in certain cell types.
Another important observation from Figs 5 and 6 is that
mAKAP targeting to the nuclear membrane occurs in a
differentiation-dependent manner. Furthermore, our results
suggest not only that the expression levels of mAKAP depend
upon the state of cellular differentiation, but that these events
may promote a redistribution of the PKA holoenzyme to the
nuclear membranes of RNV. The induction of mAKAP
expression appears coincident with an upregulation of RII
protein. This would be consistent with other reports where
hormones such as FSH and TSH promote selective expression
of AKAPs, upregulation of RII and a redistribution of the PKA
holoenzyme (Carr et al., 1993; Hunzicker-Dunn et al., 1998).
However, at this stage it is not clear what differentiation signals
stimulate mAKAP expression or whether the enhanced RII
signal represents a stabilization of the protein through
interaction with mAKAP or increased protein synthesis.
Mechanism of mAKAP targeting
Our mapping studies suggest that at least two regions of
mAKAP are necessary and sufficient for targeting to the
nuclear membrane. The participation of multiple targeting
signals is a common theme in directing AKAPs to their correct
intracellular sites. For example, the neuronal anchoring protein
AKAP79 is targeted to the plasma membrane by two or three
repeated motifs rich in basic and hydrophobic amino acids that
can bind acidic phospholipid (Dell’Acqua et al., 1998; Glantz
et al., 1993; Li et al., 1996). Likewise, AKAP18 is targeted also
to the plasma membrane by the combined action of
myristoylation and dual palmitoylation signals (Fraser et al.,
1998; Gray et al., 1998). Alternately spliced targeting cassettes
A nuclear membrane AKAP in myocytes 2735
in the amino terminus of S-AKAP84/D-AKAP-1 confer
targeting to the mitochondria or endoplasmic reticulum,
respectively (Chen et al., 1997; Huang et al., 1997b; Lin et al.,
1995). However, a distinct feature of mAKAP targeting is
highlighted by the displacement studies which demonstrate
that mAKAP targeting is saturable (Fig. 9). These findings are
consistent with the idea that the targeting locus or acceptor site
may be another protein expressed at limiting concentrations in
the nuclear membrane. This view is further supported by our
evidence that spectrin repeats are determinants for mAKAP
targeting. Spectrin-repeat sequences are found in the
cytoskeletal proteins spectrin, dystrophin and α-actinin and
participate in protein-protein interactions with cytoskeletal
components such as actin and/or adducin and PDZ domaincontaining proteins (Brown, 1997; Li and Bennett, 1996; Xia
et al., 1997). Future studies are planned to use the spectrinrepeat region as a probe to identify the mAKAP targeting
protein.
Linkage to ARVD
Although we have not yet ascribed a functional consequence
of PKA targeting to the nuclear membrane of differentiated
cardiac myocytes, we are intrigued by the chromosomal
location of the mAKAP gene. Interestingly, a genetic locus on
chromosome 14q for the autosomal dominant disorder
arrhythmogenic right ventricular dysplasia (ARVD) is bounded
by markers D14S262 and D14S69, which surround the human
mAKAP gene (Severini et al., 1996). Familial ARVD is an
autosomal dominant disorder with reduced penetrance, which
has an estimated prevalence as high as 6 per 10,000 in the
general population and is due to mutations at one of several
genetic loci (Rampazzo et al., 1997). Although ARVD was
initially described as a rare cause of right-sided heart failure,
it is now recognized as an important cause of ventricular
arrhythmia and sudden death in adolescents and adults. ARVD
is defined pathologically as fibrofatty atrophy of the right
ventricular myocardium with progressive involvement of the
septum and the left ventricle (McKenna et al., 1994). mAKAP
binds a protein kinase involved in signal transduction and is
found at the nuclear membrane of the heart. Current studies are
focused on analysis of the human mAKAP gene as a candidate
gene for the Severini ARVD kinship.
We thank Andrew Thorburn, Matt Thayer, M. Geoffrey Rosenfeld,
Mark Dell-Acqua, Ryan Westphal and Lorene Langeberg for
important intellectual and technical discussions. We thank Iain Fraser
for the ROB-1 clone and other reagents and Kim Sandstrom and Adam
Bishop for technical assistance. Supported in part by NIH grant K08
CA69137 to M.S.K. and NIH grant DK44239 to J.D.S. and the
Howard Hughes Medical Institute.
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