1 Institute of Cell Biology, ETH Hönggerberg, 8093 Zurich,
Switzerland
2 Division of Clinical Chemistry and Biochemistry, Department of Pediatrics,
University of Zurich, 8032 Zurich, Switzerland
* Present address: Dualsystems Biotech AG, Winterthurerstrasse 190, 8057
Zürich, Switzerland
Author for correspondence (e-mail:
jcp{at}cell.biol.ethz.ch)
Accepted 23 September 2002
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Summary |
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Key words: LIM domain, Sarcomere, Cardiac cytoarchitecture
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Introduction |
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In skeletal muscles and in the heart several LIM domain proteins have been
discovered that participate in muscle differentiation and play important roles
in linking the myofibrils to the surrounding cytoskeleton
(Arber et al., 1994;
Luo et al., 1997
;
Pomies et al., 1999
;
Zhou et al., 2001
). Mice
deficient in the muscle LIM protein MLP display severe alterations in
intercalated disc structure and ultimately develop dilated cardiomyopathy
(Arber et al., 1997
;
Ehler et al., 2001
). Mice
lacking the
-actinin associated LIM protein Alp show a specific
cardiomyopathy of the right ventricle
(Pashmforoush et al., 2001
).
Ablation of cypher, a striated muscle specific LIM-PDZ domain protein, leads
to congenital myopathy and ventricular dilation in mice
(Zhou et al., 2001
). Elevated
expression levels of N-RAP, an intercalated disc associated LIM-domain
protein, serve as one of the earliest indicators for dilated cardiomyopathy
(Ehler et al., 2001
). These
findings suggest that LIM domain proteins act as adaptors in striated muscle
in general and the heart especially and help to maintain the cytoarchitecture
during contraction. In their absence, decreased power output results, often
followed by dilated cardiomyopathy (reviewed by
Chien, 1999
;
Chien, 2000
).
DRAL/FHL-2 is a member of the four and a half LIM domain protein subfamily
which possesses an N-terminal half LIM domain followed by four complete LIM
domains. Members of this family include FHL-1, DRAL also called FHL-2, FHL-3,
FHL-4 and ACT (Morgan and Madgwick,
1996; Genini et al.,
1997
; Fimia et al.,
1999
; Morgan and Madgwick,
1999a
). A characteristic property of all FHL proteins is their
tissue-specific expression pattern (Chu et
al., 2000a
). Only FHL-1 is expressed in a broad range of tissues,
ranging from skeletal and heart muscle to kidney, lung and brain
(Fimia et al., 2000
).
DRAL/FHL-2 was discovered in a screen for cancer-related genes that are
downregulated in a rhabdomyosarcoma cell line (hence the name DRAL, for
downregulated rhabdomyosarcoma LIM protein) and was subsequently shown to be
highly expressed in the heart (Genini et
al., 1997
; Chan et al.,
1998
; Chu et al.,
2000a
; Müller et al.,
2000
; Scholl et al.,
2000
; Li et al.,
2001a
). FHL-3 is strongly expressed in skeletal muscle, where it
may serve similar functions as DRAL/FHL-2 in the heart
(Fimia et al., 2000
;
Morgan and Madgwick, 1999b
).
FHL-4 and ACT are different from the other members of the family since they
are not expressed in either heart or skeletal muscles, but in the testis
(Fimia et al., 2000
).
To date, several binding partners of DRAL/FHL-2 have been identified. The
N-terminal fragment of the Alzheimer's disease-associated protein presenilin-2
interacts with DRAL/FHL-2 via its hydrophilic loop region
(Tanahashi and Tabira, 2000).
The cytoplasmic domains of several
- and ß-integrins also interact
with DRAL/FHL-2, implicating DRAL/FHL-2 as an adaptor/docking protein involved
in integrin signalling (Wixler et al.,
2000
). DRAL/FHL-2 can form homodimers as well as heterodimers
together with its family member FHL-3
(Wixler et al., 2000
;
Li et al., 2001b
). DRAL/FHL-2
has been identified as a tissue-specific coactivator of the androgen receptor
(Müller et al., 2000
),
and also interacts with insulin-like growth factor-binding protein 5
(Amaar et al., 2002
), the
DNA-binding nuclear protein hNP220 (Ng et
al., 2002
) and the transcription factor CREB
(Fimia et al., 1999
;
Fimia et al., 2000
). CREB is
induced in cardiac cells after ß-adrenergic stimulation
(Goldspink and Russell, 1996
)
and mice bearing a dominant negative isoform of CREB have been shown to
develop dilated cardiomyopathy (Fentzke et
al., 1998
). DRAL/FHL-2 knockout experiments also lend support to a
role for DRAL/FHL-2 as an adaptor protein involved in cardiac stress
management and signalling. Mice lacking DRAL/FHL-2 develop normally but show a
hypertrophic response following ß-adrenergic stimulation, indicating an
involvement of DRAL/FHL-2 in the remodelling mechanisms employed by
cardiomyocytes in response to stress (Kong
et al., 2001
).
In cardiomyocytes, DRAL/FHL-2 is associated with sarcomeres where it is
found in a cross-striated pattern (Scholl
et al., 2000). Sarcomeres are the contractile units of muscle and
are composed of highly regular, interdigitating arrays of thick and thin
filaments. Two transverse structures, the M-band and the Z-disc, serve as
primary anchor points for the thick and thin filaments, respectively.
Sarcomere assembly and integrity are controlled by the elastic filament system
composed of the giant protein titin. Titin molecules form elastic scaffolding
structures spanning more than 1 µm from a Z-disc to the M-band
(Obermann et al., 1996
;
Fürst et al., 1988
;
Trinick and Tskhovrebova,
1999
). The distinct localisation of DRAL/FHL-2 at the M-band and,
more prominently, around the Z-disc region in cardiomyocytes suggests that it
may link other proteins to the sarcomere. To investigate a potential function
of DRAL/FHL-2 as a sarcomeric adaptor molecule in the heart we searched for
interaction partners that mediate the binding of DRAL/FHL-2 to the sarcomere.
We have identified two different regions in the sarcomeric ruler molecule
titin that act as binding sites for DRAL/FHL-2. In addition, we show an
interaction of DRAL/FHL-2 with the metabolic enzymes creatine kinase,
phosphofructokinase and adenylate kinase. We propose that DRAL/FHL-2 plays a
crucial role in the recruitment of metabolic enzymes to sites of high energy
consumption in the cardiac sarcomere.
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Materials and Methods |
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Whole mount staining of rat papillary muscle
Papillary muscle was dissected from adult rat hearts, incubated for 30
minutes in relaxation buffer (7 mM EGTA, 20 mM Imidazol, 1 mM
MgCl2, 14.5 mM creatine phosphate, 4 mM MgATP, 100 mM KCl, pH7.0,
0.1% saponin), tied onto plastic strips at slightly extended length and fixed
in 4% PFA/PBS for 1 hour. Permeabilisation with 0.2% Triton X-100 was carried
out for 30 minutes, subsequently the muscle strips were cut into smaller
pieces and stained as a whole mount as described previously for embryonic
heart whole mount preparations (Ehler et
al., 1999).
Eukaryotic expression constructs
The eukaryotic expression vector pMCS-HA was constructed by removal of GFP
in pEGFP (Clontech) with BamHI and NotI and ligation of a
linker encoding the HA epitope followed by a stop codon into the
BamHI and NotI sites. The constructs NF-DRAL/FHL-2 and
DRAL/FHL-2-CF, encoding N- and C-terminally FLAG-tagged DRAL/FHL-2 in the
pcDNA3.1 vector (Invitrogen, Basel, Switzerland) have been described
(Scholl et al., 2000). The
plasmid pGFP-DRAL/FHL-2 was constructed by excising DRAL/FHL-2 from
DRAL/FHL-2-CF using BamHI and XhoI and subcloning into the
BglII and SalI sites of pEGFP. The constructs GFP-is2 and
MM-CK-HA were made by subcloning is2 and MM-CK from the original yeast
two-hybrid library plasmids into pEGFP and pMCS-HA using EcoRI and
XhoI sites. To create GFP-N2B, the sequence encoding amino acids 3455
to 4427 in the primary sequence of human cardiac titin
(Labeit and Kolmerer, 1995
)
was amplified from embryonic human cardiac RNA with the Qiagen one step RT-PCR
kit (Qiagen, Basel, Switzerland) using the primers n2bfw
(5'gaagatctatggaaggcactggcccaattttcatcaaagaa3') and n2brv
(5'ccgctcgagcactgtcacagttagtgtggctgtacagct3'). Reverse
transcription was carried out using a three step protocol: 50°C for 30
minutes, 60°C for 30 minutes and 95°C for 15 minutes. This was
followed by the amplification step: 35 cycles of 30 seconds at 94°C, 45
seconds at 65°C and 3 minutes at 72°C and a final incubation for 10
minutes at 72°C. The PCR product was subcloned into the BglII and
SalI sites of pEGFP. To construct GFP-tagged adenylate kinase and
phosphofructokinase, the entire open reading frame was amplified using the
primers akfw (5'gaagatctaccatggaagagaagctgaagaaggcc3'), akrv
(5'ccgctcgagcttcagggagtcaagataggtgca3'), pfkfw
(5'gaagatctaccatgacccatgaagagcatcatgc3') and pfkrv
(5'ccgctcgaggacggcggcttctccagaccg3') and subcloned into the Bgl II
and Sal I sites in pEGFP. To construct GFP-tagged Smpx/Csl, the entire open
reading frame was amplified by PCR using the primers cslfw
(5'gaagatctaccatgtcgaagcagccaatttccaat3') and cslrv
(5'ccgctcgagctgttcacctttggggacaaattt3') and subcloned into the
BglII and SalI sites of pEGFP. GFP-N2B deletion mutants were
constructed by removing the fragments indicated in
Fig. 5 using internal
restriction sites. All constructs derived by PCR were fully sequenced on both
strands.
|
Yeast two-hybrid assays
The entire coding region of human DRAL/FHL-2 was amplified by PCR and
cloned into the EcoRI and NotI sites of pGilda
(Gyuris et al., 1993),
Clontech, Palo Alto, CA). Yeast two-hybrid screening procedures and
ß-galactosidase filter assays were carried out as described by the
manufacturer. The plasmid was transferred into the yeast strain EGY48
(Estojak et al., 1995
), which
was then transformed with an adult human cardiac cDNA library (Clontech).
1x107 primary transformants were screened, resulting in 200
clones that grew on selection plates lacking the amino acids histidine,
tryptophan and leucine and were positive in a ß-galactosidase filter
assay. Library plasmids from positive clones were isolated and sequenced. To
confirm the interaction between DRAL/FHL-2 and either is2 or MM-CK, bait and
library inserts were switched using the EcoRI and XhoI sites
in the bait and prey vectors, cotransformed into EGY48 and assayed for growth
on selective plates and ß-galactosidase activity. To assay the
interaction between DRAL/FHL-2 and N2B, the coding sequence of DRAL/FHL-2 was
subcloned into the EcoRI and XhoI sites of the pLexA bait
vector (Stenmark et al.,
1995
). The sequence encoding N2B was excised from GFP-N2B by
cutting with XhoI, filling of 5' overhangs using Klenow enzyme
and cutting with BamHI and cloned into the BglII and
SmaI sites of the vector pACT2 (Clontech). The two plasmids were
cotransformed into the yeast strain L40
(Vojtek et al., 1993
) and
assayed for growth on selection plates lacking the amino acids tryptophan,
leucine and histidine as well as for ß-galactosidase activity.
Immunofluorescent staining of NRC and sections
Primary cultures of neonatal rat cardiomyocytes were isolated and cultured
as previously described (Auerbach et al.,
1999). Cells were cultured in Maintenance medium (20% medium M199,
75% DBSS-K, 4% horse serum, 4 mM glutamine, 1% penicillin/streptomycin, 0.1 mM
phenylephrine; DBSS-K: 6.8 g/l NaCl, 0.14 mM NaH2PO4,
0.2 mM CaCl2, 0.2 mM MgSO4, 1 mM dextrose, 2.7 mM
NaHCO3) and transient transfections were carried out after 1 day in
culture using the reagent Escort III (Sigma) according to the manufacturer.
24-48 hours after transfection, cells were fixed in 4% paraformaldehyde/PBS
for 10 minutes and stained with different antibodies as previously described
(Auerbach et al., 1999
).
Confocal microscopy
The specimen were analysed using confocal microscopy on an inverted
microscope DM IRB/E equipped with a true confocal scanner TCS NT, a PL APO
63x/1.32 oil and a PL APO 100x/1.40 immersion objective (Leica) as
well as an argon-krypton mixed gas laser. Image processing was done on a
Silicon Graphics workstation using Imaris® (Bitplane AG), a 3D
multichannel image processing software specialised for confocal microscopy
data sets (Messerli et al.,
1993).
Preparation of COS-1 total lysates
COS-1 cells (Gluzman, 1981)
were cultured in maintenance medium consisting of DMEM, 10% fetal calf serum
and 4 mM glutamine (Gibco, Basel, Switzerland). For transfection, the cells
were grown to 80% confluency in 10 cm dishes, 107 cells were
pelleted, washed twice with PBS and resuspended in 800 µl PBS. 10 µg of
plasmid were added to a 4 mm gap cuvette (BTX, Axon Lab, Baden, Switzerland)
on ice, mixed with 800 µl of cells by tapping and incubated for 5 minutes
on ice. Cells were electroporated in a BTX Electro cell manipulator 600 (BTX)
using the following conditions: capacitance setting 125 µF, voltage 300 V,
resistance 72
. After electroporation, cells were left on ice for 5
minutes, transferred to 10 cm dishes with maintenance medium and incubated for
48-72 hours. Lysates were prepared by washing cells twice with ice-cold PBS,
adding 1 ml of lysis buffer (10 mM HEPES pH 7.9, 100 mM KCl, 5 mM
MgSO4, 50 µM ZnSO4, 1.5% Triton X-100, 1 mM DTT,
protease inhibitors) and incubation for 10 minutes on ice. Cells were scraped
off the plate, transferred to eppendorf tubes and incubated for 10 minutes on
ice. Lysates were sonicated in an ice/water bath (ELMA sonicator, Transsonic
Digitals, Singen, Germany) at maximum setting for 10 minutes and centrifuged
for 5 minutes at 5000 g. The supernatants were transferred to fresh
tubes and stored on ice.
Pull-down assays
A plasmid encoding GST-DRAL/FHL-2 fusion protein
(Genini et al., 1997) was
transformed into E. coli BL21 Star cells (Invitrogen). The cells were
grown at 37°C in LB supplemented with 50 µM ZnSO4 to an
OD600 of 1.0, induced with 0.2 mM IPTG and grown for another 30
minutes at 37°C. Cells were pelleted and resuspended in 1/25 volume of
lysis buffer (10 mM HEPES pH 7.9, 100 mM KCl, 50 µM ZnSO4, 1%
Triton X-100, 1 mM DTT) supplemented with 0.5 mg/ml lysozyme (Sigma),
incubated for 30 minutes on ice and sonicated 5 times 10 seconds (Branson
Sonifier 250, output setting 6, 60% duty cycle). Cellular debris were removed
by centrifugation for 20 minutes at 15,000 g and GST-fusion protein
was absorbed on beads by incubation with Glutathione-Sepharose 4B beads
(Pharmacia, Uppsala, Sweden) for 30 minutes on ice. The beads were washed four
times in ST buffer and stored on ice. For pull-down assays, defined amounts of
bead-coupled GST-DRAL/FHL-2 or GST alone were washed once in IP buffer (10 mM
HEPES pH 7.9, 100 mM KCl, 5 mM MgSO4, 0.5% NP-40, 50 µM
ZnSO4, 1 mM DTT) supplemented with a protease inhibitor cocktail
(Roche Diagnostics, Rotkreuz, Switzerland) and incubated for 3 hours on ice
with precleared lysates from COS-1 cells expressing the respective binding
partners or controls. Following incubation, the complexes were pelleted by
centrifugation, washed four times in IP buffer, resuspended in SDS-PAGE sample
buffer, boiled and loaded on 12.5% SDS-PAGE gels. The proteins were
transferred to nitrocellulose membranes according to standard procedures and
visualised by immunoblotting as described above.
Co-immunoprecipitation
COS cells were co-transfected with GFP-N2B5 and DRAL/FHL-2-FLAG and
lysed as described above. After preclearing by incubation with Protein G
Plus/Protein A agarose (Oncogene) diluted in IP buffer (see above), the
lysates were incubated with the monoclonal mouse anti-FLAG antibody at 4°C
overnight. Then Protein G Plus/Protein A agarose was added and incubated for 3
hours at 4°C. Following several washes with IP buffer, the beads were
processed for an SDS sample and immunoblotting with the Horse radish
peroxidase-conjugated polyclonal rabbit anti-GFP antibody (Clontech) was
carried out as described above.
Antibodies
Monoclonal mouse antibodies against -cardiac actin were obtained
from Progen (Heidelberg, Germany), monoclonal mouse antibodies against FLAG M2
were from Sigma (Buchs, Switzerland), monoclonal mouse anti-GFP (clones 7.1
and 13.1) and monoclonal rat anti-HA tag (clone 3F10) antibodies were from
Roche Diagnostics (Rotkreuz, Switzerland) and polyclonal Horse radish
peroxidase-conjugated rabbit anti-GFP antibodies were from Clontech.
Polyclonal rabbit antibodies against DRAL/FHL-2 were produced in the lab
(Scholl et al., 2000
),
polyclonal rabbit antibodies against the titin m8 epitope and monoclonal mouse
antibodies against the titin N2B epitope (clone I19) were generously donated
by Mathias Gautel (King's College London, UK). Polyclonal chicken antibodies
against N2A were a kind gift from Carol Gregorio (University of Arizona,
Tucson, AZ) and monoclonal mouse anti-titin T12 epitope antibodies were
generously provided by Dieter Fürst (University of Potsdam, Germany). The
monoclonal mouse anti-titin 9D10 epitope antibody was obtained from the
Developmental Studies Hybridoma Bank, University of Iowa, USA.
As secondary antibodies FITC-conjugated anti-rabbit and anti-mouse Igs were employed (Cappel, via ICN Germany). Cy3-conjugated anti-mouse Igs, Cy3-conjugated anti-rat Igs (mouse Ig absorbed), Cy5-conjugated anti-rabbit and anti-mouse Igs, Cy5-conjugated anti-mouse Igs (rat Ig absorbed) were all purchased from Jackson Immunochemicals via Milan (La Roche, Switzerland). FITC anti-mouse IgM was from Sigma, FITC anti-chicken IgY was from The Binding Site Ltd (University of Birmingham, UK). Horse radish peroxidase-conjugated anti-rabbit Igs were from Calbiochem (Luzern, Switzerland), horse radish peroxidase-conjugated anti-mouse and anti-rat Igs were from DAKO (Zug, Switzerland).
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Results |
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|
DRAL/FHL-2 localises to specific regions in the cardiac
sarcomere
Initial studies have shown that ectopically expressed DRAL/FHL-2 as well as
endogenous DRAL/FHL-2 is localised in a cross-striated pattern at two distinct
sites in sarcomeres of cultured neonatal rat cardiomyocytes. DRAL/FHL-2 is
found in broad striations at the Z-disc and in fainter striations at the
M-band region (Scholl et al.,
2000). In order to precisely identify the region near the Z-disc
where DRAL/FHL-2 is bound, double labeling experiments with antibodies
directed against DRAL/FHL-2 together with antibodies directed against
different epitopes of the sarcomeric ruler titin were performed
(Fig. 2). We employed
antibodies against the T12 epitope, which is located at the outer edge of the
Z-disc region of titin, against the I-band epitopes N2B and N2A and against
the 9D10 epitope located in the extensible PEVK region
(Fig. 2A). The signal for
DRAL/FHL-2 was compared with the signals of the different titin epitopes
obtained with myofibrils in different states of contraction
(Fig. 2B). In relaxed
sarcomeres, DRAL/FHL-2 was consistently found as a narrow doublet flanking the
Z-disc and in very faint striations at the M-band
(Fig. 2Bc'). In contrast,
the T12 epitope, which denotes the border of the Z-disc, could not be resolved
into a doublet. This finding suggests that DRAL/FHL-2 is located outside of
the Z-disc. Comparison of the other two titin epitopes with DRAL/FHL-2 showed
that DRAL/FHL-2 colocalised with the N2B epitope independently of the
contraction state, whereas the N2A and PEVK epitopes were always located
further towards the M-band than DRAL/FHL-2
(Fig. 2B). These results
suggest that DRAL/FHL-2 is bound at or very near the N2B region of cardiac
titin.
|
Identification of DRAL/FHL-2 binding partners by yeast two hybrid
assay
The sarcomeric localisation of DRAL/FHL-2 at the Z-disc and at the M-band
cannot be explained by any of its interaction partners that have been
identified so far. For this reason, a yeast two-hybrid screen was carried out
to identify sarcomeric proteins that bind to DRAL/FHL-2 and that may serve to
anchor it to the sarcomere. Full-length DRAL/FHL-2 was used to screen an adult
human cardiac cDNA library. Out of 1x107 primary
transformants, 200 clones were isolated that survived selection and were
positive in a ß-galactosidase assay. Of these, one clone encoded a region
of titin termed is2 (Labeit and Kolmerer,
1995) and five clones encoded the entire open reading frame of the
muscle isoform of creatine kinase (MM-CK)
(Ordahl et al., 1984
;
Wallimann et al., 1992
). The
interaction between DRAL/FHL-2 and either is2 or MM-CK was reproducible
regardless of whether DRAL/FHL-2 or its interacting partners were fused to the
DNA-binding domain or to the activation domain (data not shown). The is2
region is situated in the M-band region of titin
(Labeit and Kolmerer, 1995
)
and MM-CK, despite being a cytosolic enzyme, is found at distinct sites near
the M-band and near the Z-disc (Wallimann
et al., 1992
). For this reason, is2 and MM-CK represent potential
binding partners for DRAL/FHL-2.
No clones were isolated in our yeast two-hybrid screen that would explain the localisation of DRAL/FHL-2 in the Z-disc region. Since DRAL/FHL-2 colocalises exactly with the N2B titin epitope, we reasoned that it may be bound to this region of titin. The direct interaction of DRAL/FHL-2 and N2B was investigated in a yeast two-hybrid assay. As shown in Fig. 3, the entire N2B region of titin interacts strongly and specifically with DRAL/FHL-2. N2B does not bind the LexA DNA-binding domain alone, and it does not interact with an unrelated bait (Fig. 3). In summary, the yeast two-hybrid assays demonstrate an interaction of DRAL/FHL-2 with the is2 and N2B regions of titin, and with the metabolic enzyme MM-CK. The interaction with two distinct regions of titin may thus serve to anchor DRAL/FHL-2 to the sarcomere.
|
Confirmation of the interactions by colocalisation and pull-down
assays
Next, we sought to confirm the interaction of DRAL/FHL-2 with titin in an
in vivo environment. Primary cultures of neonatal rat cardiomyocytes were
cotransfected with constructs encoding FLAG-tagged DRAL/FHL-2 in combination
with GFP-tagged is2 or GFP-tagged N2B segments of titin. In cotransfected
cardiomyocytes, DRAL/FHL-2 was found in a doublet flanking the Z-disc and in a
faint striation in the M-band (Fig.
4A), colocalising exactly with transfected N2B. An identical
pattern was observed in cells cotransfected with DRAL/FHL-2 and is2: both
proteins were found in a doublet flanking the Z-disc and in a weaker striation
at the M-band (Fig. 4A). The
finding that exogenously expressed N2B was also localised at the M-band,
whereas is2 was also found in a doublet flanking the Z-disc strongly suggests
that these fragments are targeted to the sites by means of their interaction
with DRAL/FHL-2.
|
The direct interaction of DRAL/FHL-2 with the is2 and N2B regions of titin was further confirmed by an in vitro binding assay (Fig. 4B). Recombinant GST-DRAL/FHL-2 was incubated with total lysates from COS-1 cells that had been transfected with eukaryotic expression constructs encoding GFP-tagged is2 or N2B, respectively. As a control, GST alone was incubated in parallel with the same lysates. Both is2 and N2B were found in the GST-DRAL/FHL-2 fraction, whereas they were absent from the GST fraction. This result confirms the observed colocalisation in the transient transfection experiments and suggests that DRAL/FHL-2 is targeted to these sites in the sarcomere by a direct interaction with the N2B and the is2 regions of titin, respectively.
The N2B region of titin contains an N-terminal and a C-terminal block of
two Ig-like domains interrupted by a large stretch of unique sequence
(Fig. 5A)
(Labeit and Kolmerer, 1995).
In order to determine whether DRAL/FHL-2 binds to the blocks of the Ig-like
domains or to the unique sequence that connects them, we constructed several
deletion mutants of N2B and assayed these for interaction with GST-DRAL/FHL-2
in pull-down assays (Fig. 5B).
GFP-N2B
1, which lacks the two C-terminal Ig-like domains, was still
capable of interacting with DRAL/FHL-2. Additional deletion of the C-terminal
half of the unique sequence in GFP-N2B
2 did not affect its binding to
DRAL/FHL-2, either. However, deletion of another 270 amino acids from the
central part of the unique sequence in GFP-N2B
4 completely abolished
the interaction of DRAL/FHL-2 and N2B. Likewise, GFP-N2B
3, which
contains the C-terminal end of the unique sequence followed by the two
C-terminal Ig-like domains, did not retain any affinity for DRAL/FHL-2. These
results suggest that the interaction with DRAL/FHL-2 is mediated by a binding
site located within the central 270 amino acids of the N2B region. To
investigate this putative binding site more closely, we created an additional
construct, GFP-N2B
5, which comprises just this sequence. This minimal
binding domain shows targeting that is indistinguishable from the full-length
titin N2B segment (Fig. 5C).
Additional proof for an interaction between DRAL/FHL-2 and this region in
titin N2B was provided by assaying COS cells that were transiently transfected
with DRAL/FHL-2-FLAG and GFP-N2B
5. Following immunoprecipitation with
an anti-FLAG antibody, GFP-N2B
5 could be visualised on a immunoblot
with anti-GFP antibodies, confirming the stabile interaction of these protein
domains in vivo (Fig. 5D).
In summary, the colocalisation and pull-down experiments confirm the interaction between DRAL/FHL-2 and two distinct regions in the giant protein titin and strongly suggest that DRAL/FHL-2 is targeted to two sarcomeric sites in cardiomyocytes by virtue of those interactions. Furthermore, the DRAL/FHL-2-binding site on N2B is located in the central 270 amino acids of the unique insertion connecting the Ig-like domains.
Interaction of DRAL/FHL-2 with the enzymes creatine kinase, adenylate
kinase and phosphofructokinase
The metabolic enzyme MM-CK (EC 2.7.3.2) was isolated five times in our
yeast two-hybrid screen. MM-CK is a predominantly cytosolic enzyme, but a
minor fraction is located at two distinct sites in the sarcomere near the
M-band and flanking the Z-disc
(Schäfer and Perriard,
1988; Wallimann et al.,
1992
; Wegmann et al.,
1992
). In order to investigate whether exogenous DRAL/FHL-2 and
MM-CK colocalise in the sarcomere, we cotransfected primary cultures of rat
cardiomyocytes with constructs encoding FLAG-tagged DRAL/FHL-2 and GFP-tagged
MM-CK. Both DRAL/FHL-2 and MM-CK were found in a doublet pattern flanking the
Z-disc and in a single striation at the M-band
(Fig. 6A). However, while there
was less DRAL/FHL-2 bound to the M-band than to the Z-disc region, the signals
for MM-CK flanking the Z-disc and the M-band appeared equally intense (compare
insets in Fig. 6Aa and
a'), indicating that MM-CK may target to the M-band by
interaction with other proteins apart from DRAL/FHL-2
(Hornemann et al., 2000
).
|
Two other metabolic enzymes, adenylate kinase (EC 2.7.4.3) and
phosphofructokinase (EC 2.7.1.11), are also found in the M-band and, depending
on the buffer conditions used for fixation, in the I-band as well
(Dolken et al., 1975;
Kraft et al., 2000
). We
therefore asked whether those proteins may achieve their localisation in the
sarcomere via an interaction with DRAL/FHL-2, too. First, we compared the
sarcomeric localisation of FLAG-tagged DRAL/FHL-2 and either adenylate kinase
or phosphofructokinase tagged with GFP by cotransfection into rat
cardiomyocytes (Fig. 6A).
Adenylate kinase and phosphofructokinase were found in a doublet flanking the
Z-disc and in a single striation at the M-band
(Fig. 6A). Superimpositions
showed that both proteins colocalised exactly with DRAL/FHL-2. Thus, MM-CK,
adenylate kinase and phosphofructokinase are all located at the same
sarcomeric sites flanking the Z-disc and the M-band together with
DRAL/FHL-2.
To investigate wether the colocalisation of the three metabolic enzymes is
due to their direct interaction with DRAL/FHL-2, we performed in vitro
pull-down assays. GST-DRAL/FHL-2 was incubated with lysates of COS-1 cells
expressing MM-CK, adenylate kinase or phosphofructokinase. As a control for
non-specific binding, GST alone was used. As shown in
Fig. 6B, all three enzymes
interacted with GST-DRAL/FHL-2 but not with GST alone. A recently identified
muscle protein, Smpx/Csl (Kemp et al.,
2001; Palmer et al.,
2001
), which shows a localisation pattern similar to that of
DRAL/FHL-2 (Fig. 6A), was also
evaluated for its interaction with DRAL/FHL-2. Smpx/Csl interacted neither
with DRAL/FHL-2-GST nor with GST alone, confirming the specificity of the
pull-down assay. These results strongly suggest that in cardiomyocytes the
three metabolic enzymes MM-CK, adenylate kinase and phosphofructokinase are
bound to two distinct sarcomeric sites near the Z-disc and the M-band by their
interaction with DRAL/FHL-2. In contrast to the interactions between
DRAL/FHL-2 and titin, attempts to co-immunoprecipitate the metabolic enzymes
with DRAL/FHL-2 were not successful (data not shown). The failure to detect
the interaction of DRAL/FHL2 and the metabolic enzymes may indicate that their
association is transient in nature and may serve as a means for dynamic
compartmentalisation of these enzymes, rather than a strict immobilisation to
the sarcomere.
DRAL/FHL-2 interacts with FHL-1 but not with the LIM domain protein
MLP
Recently, it has been shown that DRAL/FHL-2 and FHL-3 associate to form
heterodimers (Li et al.,
2001b). We wanted to investigate whether DRAL/FHL-2 also forms
dimers with the other four and a half LIM-only family members. We focused on
FHL-1, since it is the only other family member apart from DRAL/FHL-2 that is
also expressed in the heart (Fimia et al.,
2000
). First, we cotransfected rat cardiomyocytes with constructs
encoding GFP-tagged DRAL/FHL-2 and FLAG-tagged FHL-1 to compare the
localisation of the two proteins. DRAL/FHL-2 and FHL-1 colocalised exactly in
two striations flanking the Z-disc and in a single striation in the M-band
(data not shown). Next, we tested whether the colocalisation of DRAL/FHL-2 and
FHL-1 in the sarcomere is due to a direct interaction using a pull-down assay.
As shown in Fig. 7, FHL-1 bound
to GST-DRAL/FHL-2 but not to GST alone. The interaction was abolished by the
addition of 10 mM EDTA to the incubation buffer. EDTA complexes and removes
the two Zn2+ ions that are bound to histidine and cysteine residues
of a zinc finger, thereby unfolding it. Thus, the proper conformation of the
LIM domain is needed for the interaction of DRAL/FHL-2 with FHL-1.
|
To exclude non-specific interactions between LIM domains, we also tested an
unrelated LIM protein, MLP, for binding to DRAL/FHL-2. MLP is expressed in the
heart but shows a different subcellular localisation than DRAL/FHL-2
(Arber et al., 1997), making a
direct interaction of the two proteins extremely unlikely. For this reason,
MLP serves as a good control to assess non-specific interactions between LIM
domains. As expected, MLP interacted neither with GST-DRAL/FHL-2 nor with GST
alone (Fig. 7). The fact that
DRAL/FHL-2 does not bind to MLP in our pull-down assays suggests that no
non-specific interaction between LIM domains occurs under our assay
conditions. Furthermore, the loss of interaction between DRAL/FHL-2 and FHL-1
in the presence of EDTA shows that the binding between the two proteins is
dependent on the proper folding of the LIM domains and is not mediated by the
non-specific sticking of LIM domains to each other.
![]() |
Discussion |
---|
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---|
|
DRAL/FHL-2 localises to the N2B region of titin, which is present only in
cardiac-specific isoforms of titin (Gautel
et al., 1996), with DRAL/FHL-2 being at the same time
predominantly expressed in cardiac muscle. The importance of the N2B region
for cardiomyocyte structure was demonstrated by the observation that
overexpression of N2B fragments in cardiomyocytes can lead to a disruption of
thin filaments and it has been suggested that the N2B region might be
important for the binding of several sarcomeric components
(Linke et al., 1999
). In our
transfection assays no deleterious effects of expression of N2B could be
observed; however, we analysed the cardiomyocytes already 1-2 days after
transfection, when the disruption of thin filament structures is probably not
yet visible and the cross-striated pattern of ectopically expressed N2B in
I-band and M-band can still be detected. A possible explanation of why
ectopically expressed N2B fragments target to the I-band in transiently
transfected NRC could be that DRAL/FHL-2 exerts its function as a homodimer,
which has been strongly suggested by in vitro observations
(Wixler et al., 2000
). In
contrast to the PEVK region, which is situated further towards the C-terminus
of titin, no direct binding of N2B to actin could be found
(Gutierrez-Cruz et al., 2001
;
Kulke et al., 2001
;
Yamasaki et al., 2001
).
DRAL/FHL-2 interaction with metabolic enzymes in the heart
Targeting of metabolic enzymes to the I-band has been shown previously
(Arnold and Pette, 1970;
Dolken et al., 1975
;
Wegmann et al., 1992
;
Kraft et al., 2000
) and is
thought to be necessary for energy provision during contraction
(Wallimann and Eppenberger,
1985
). The I-band association seems to be dependent on the buffer
conditions used for the sample preparation, since it is primarily observed if
bivalent cation chelators such as EDTA are omitted
(Wegmann et al., 1992
). Since
our experiments indicated that the presence of EDTA abolished the in vitro
interaction between DRAL/FHL-2 and FHL-1, it seems very likely that the
association of metabolic enzymes is also dependent on a conserved conformation
of LIM domains in DRAL/FHL-2. DRAL/FHL-2 expression is upregulated in the
heart only rather late during embryonic development. This is similar to
results obtained for MM-CK expression levels in developing muscle and might
explain the delay in sarcomeric compartmentalisation of this enzyme that is
observed during development (Carlsson et
al., 1982
; Carlsson et al.,
1990
; Ventura-Clapier et al.,
1998
). Animals that are homozygous null for DRAL/FHL-2 seem normal
and display no obvious abnormalities in myofibrils
(Chu et al., 2000b
). However,
they seem to be more sensitive to stress situations such as ß-adrenergic
stimulation (Kong et al.,
2001
), which might suggest an impaired ability to cope with
abnormal requirements on the cardiac energy metabolism.
DRAL/FHL-2 interactions with non-sarcomeric proteins
Previous investigations in non-cardiac cells have identified hCDC47,
presenilin-2, the androgen receptor and several - and ß-integrins
as potential interaction partners of DRAL/FHL-2
(Chan et al., 2000
;
Tanahashi and Tabira, 2000
;
Müller et al., 2000
;
Wixler et al., 2000
); however,
the biological significance of these interactions in the heart is still
unclear. It has been suggested that the androgen receptor might be involved in
the development of hypertrophy in the heart
(Marsh et al., 1998
) and a
decrease in DRAL/FHL-2 expression seems to occur in patients with dilated
cardiomyopathy (Müller et al.,
2000
), again underlining the importance of LIM-domain-containing
proteins for the maintenance of cardiac function
(Arber et al., 1997
;
Pashmforoush et al., 2001
).
Despite the reported binding of DRAL/FHL-2 to integrins we were unable to see
the typical costameric localisation pattern as expected for an
integrin-associated protein in cardiomyocytes. Several other
LIM-domain-containing proteins target to focal adhesions, such as paxillin
(Brown et al., 1996
) and zyxin
(Sadler et al., 1992
), or bind
to integrin in vitro, such as N-RAP (Luo
et al., 1999
). DRAL/FHL-2 is indeed localised at focal adhesions
in non-muscle cells (Scholl et al.,
2000
) and also in not yet differentiated muscle cells
(Li et al., 2001b
). However,
as soon as myofibrils are formed, the preferential association of DRAL/FHL-2
seems to occur in the region of the I-band
[(Li et al., 2001b
) and this
study] and no costameric association can be observed any longer. This might
suggest that in cardiomyocytes the binding affinity of DRAL/FHL-2 for titin is
so strong that it competes completely for the interaction with other potential
binding partners. The localisation pattern of DRAL/FHL-2 or other
LIM-domain-containing proteins could therefore be dependent on the molecular
expression profile in different cell types.
In summary, subcellular localisation experiments and in vitro binding assays suggest that the localisation of DRAL/FHL-2 at two defined sites in the cardiac sarcomere is mediated by its interaction with the N2B and is2 regions of titin. In turn, DRAL/FHL-2 binds to the metabolic enzymes MM-CK, adenylate kinase and phosphofructokinase, thus serving as an adaptor to couple them to the N2B and is2 regions in titin. The results presented here point towards a crucial role for DRAL/FHL-2 in the compartmentalisation of metabolic enzymes in the heart. The participation of DRAL/FHL-2 in signalling and response to extracellular stimuli such as growth factors and mechanical stress remains to be established.
![]() |
Acknowledgments |
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Footnotes |
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