From the Department of Life Science, Kwangju
Institute of Science and Technology, Kwangju 500-712, the
Department of Microbiology, Changwon
National University, Changwon 641-773, and the ¶ School of
Biological Sciences, Seoul National University,
Seoul 151-742, Korea
Received for publication, October 16, 2000, and in revised form, January 19, 2001
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ABSTRACT |
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In the yeast two-hybrid screening, we have
isolated a cDNA clone from a human heart library using Nck Src
homology 3 (SH3) domains as bait. The full-length cDNA, which
encoded 722 amino acids, was identified as a VIP54-related gene
containing an SH3 domain, proline-rich motifs, a serine/threonine-rich
region, and a long C-terminal hydrophobic region. We refer to this
protein as SPIN90 (SH3 Protein
Interacting with Nck, 90 kDa). The
amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human
tissues; in particular, it was highly expressed in heart, brain, and
skeletal muscle, and its expression was developmentally regulated
during cardiac myocyte differentiation. SPIN90 is able to bind to the
first and third SH3 domains of Nck, in vitro, and is
colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important
role in the maintenance of sarcomere structure and/or in the
assembly of myofibrils into sarcomeres.
Integrins are a large family of heterodimeric transmembrane
receptors that play key roles in mediating interactions between cells
and ECM1 proteins and between
intercellular proteins. As such, they are crucially involved in such
biological processes as embryonic development, cell migration, and cell
growth and differentiation (1).
The development of cultured cardiac myocytes is characterized by
myofibrillogenesis, during which myofibrils are organized into
sarcomeric structures. Interactions between integrins and ECM take
place continuously throughout sarcomere assembly and underlie the
progressive assembly of thick and thin filaments into sarcomeres with
appropriately spaced Z-discs (2). Nck, which contains three Src homology 3 (SH3) domains and one SH2
domain, is ubiquitously expressed in a variety of tissues and cells
(8). As it lacks catalytic activity, Nck is referred to as an adaptor
molecule, a group of proteins that also includes Grb2 and Crk. SH2 and
SH3 domains are peptide motifs found in a wide variety of molecules
implicated in the regulation of cell growth (9). SH2 domain-containing
proteins capable of tightly associating with catalytic molecules
function as adaptors linking the latter to phosphotyrosine-containing
proteins (10). In addition to recruiting such signaling enzymes as
phosphoinositide 3-kinase, phospholipase C- Over the past couple of years, several effector molecules that interact
with the SH3 domains of Nck have been identified. Nck binds to PRK2, a
serine/threonine kinase, closely related to the putative Rho effector,
and also to NIK, which activates the mitogen-activated protein
kinase kinase kinase/mitogen-activated protein kinase
kinase/stress-activated protein kinase pathway (18, 19). However, the
biological significance of these interactions is not yet known.
Furthermore, although we have previously reported on the involvement of
Nck in a To better understand the Materials
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum,
antibiotic/antimycotics, complete Freund's adjuvant, incomplete Freund's adjuvant, and trypsin were obtained from Life Technologies, Inc. Tissue culture dishes were from Falcon. Monoclonal
anti- Cardiac Myocyte Cultures
Neonatal rat ventricular myocytes were isolated according to the
method of Borg et al. (4) and Simpson et al. (20)
with major modification. Briefly, hearts from 12-25 neonatal rats
(2-3 days old) were excised and placed in Ads buffer (116 mM NaCl, 20 mM HEPES, 10 mM
NaH2PO4, 5.5 mM glucose, 5 mM KCl, 0.8 mM MgSO4, pH 7.4),
after which they were trimmed of their atria and transferred to fresh
Ads buffer. The retained ventricles were then minced, placed in a 50-ml
flask containing enzyme digestion solution (Ads buffer containing 65 units/ml collagenase type II and 0.6 mg/ml pancreatin) and incubated at
37 °C for a series of 20-min intervals. After each interval, the
solution was centrifuged, the cell-containing supernatant was
collected, and the pellet was resuspended in 10% fetal bovine serum.
To enrich the myocyte suspension, the supernatant from the first
dissociation interval was discarded. The cells from all of the
digestions were pooled, washed, and then subjected to centrifugation
through a discontinuous Percoll gradient of 1.050, 1.062, and 1.082 g/ml. The interface band between 1.062 and 1.082 was collected and used
as the sole source of purified myocytes. These myocytes were
resuspended in DMEM supplemented with 10% fetal bovine serum, plated
at a density of 200 cells/mm2 on collagen-coated dishes (50 µg/ml), and maintained at 37 °C under a humidified atmosphere
containing 5% CO2.
Yeast Two-hybrid Screening
The yeast two-hybrid screening system was used as described by
Fields et al. (21). A cDNA encoding the three SH3
domains of Nck (amino acids 1-251) was amplified via PCR and cloned
into pGBT9 vector containing the GAL4 DNA-binding domain. The Nck
construct was cotransformed into the yeast strain HF7c with human heart Matchmaker cDNAs containing the activation domain of GAL4 in the pGAD10 vector using the lithium acetate (LiAc) method. Approximately 3 × 106 yeast transformants were pooled and subjected
to selections as described below. The transformants were selected at
30 °C on synthetic complete medium plates lacking tryptophan,
leucine, and histidine and containing 4 mM 3-amino-
(1,2,4)-triazole. After 8-10 days, prey plasmids exhibiting
Cloning the Full-length spin90 cDNA: Phage Library
Screening
The clone identified in the yeast two-hybrid screening did not
contain the 3' end of the gene. To obtain a full-length cDNA, the
Northern Analysis
The human multiple tissue poly(A)+ RNA blot (2 µg/lane) was purchased from CLONTECH. The
cDNA inserts from clone spin90 and Construction of Expression Vectors and in Vitro Binding of SPIN90
with Nck
Expression of GST Fusion Proteins for in Vitro Binding Assay--
cDNAs encoding the full-length of Nck (full Nck; amino acids
1-337), the three SH3 domains (total SH3; amino acids 1-251), the SH2
domain (SH2; amino acids 268-377), the first SH3 domain (SH3-1; amino
acids 1-96), the second SH3 domain (SH3-2; amino acids 68-183), and
the third SH3 domain (SH3-3; amino acids 170-251) were amplified by
PCR and cloned, in frame, into pGEX4T-1 vector (Amersham Pharmacia
Biotech). GST fusion proteins were overexpressed in bacteria and
purified on glutathione-Sepharose columns according to the
manufacturer's protocol.
Pull-down Assay--
To assess in vitro binding, the
clone spin90 insert was subcloned into pRSET, a bacterial
expression vector, and in vitro translated using a TNT
T7-coupled reticulocyte lysate system (Promega). The radiolabeled
products were incubated with purified GST or GST fusion proteins bound
to glutathione beads. All incubations were performed in
phosphate-buffered saline (PBS) containing 1% bovine serum albumin at
4 °C for 6 h. The glutathione beads were then washed four times
in PBS containing 1% Triton X-100, and the radiolabeled proteins bound
to the beads were solubilized by addition of sodium dodecyl sulfate
(SDS) sample buffer in the presence of a reducing agent and subjected
to 8% SDS-polyacrylamide gel electrophoresis (PAGE). Radioactivity was
detected by autoradiography of the dried gels.
Purification of GST Fusion Proteins and Generation of Antibodies
against SPIN90
To generate anti-SPIN90 antibodies, the cDNA corresponding
to full-length SPIN90 was amplified by PCR and subcloned, in frame, into pGEX4T-1 vector for GST fusion protein expression. GST-SPIN90 fusion proteins were overexpressed in bacteria and purified according to the method of Merilainen et al. with some modification
(22). The cells were suspended in a lysis buffer (140 mM
NaCl, 2.7 mM KCl, 10 mM
Na2PO4, 1.8 mM
KH2PO4, 1% Triton X-100) and lysed by sonication. SDS sample buffer was added to the lysates, which were then
resolved by 8% SDS-PAGE. Thereafter, the GST-SPIN90 fusion protein was
electroeluted from the gel using a Bio-Rad model 422 electroeluter, and
the purified GST fusion protein was dialyzed and used for
immunizations. After the fifth injection, the specificity of the serum
was tested by immunoblot analysis, and then further purified by
affinity chromatography.
Western Blotting
For immunoblot analysis, selected tissues from rat were minced,
rinsed once with extraction buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate,
0.1% SDS, 1 mM EDTA), and homogenized in extraction buffer
containing 1 mM phenylmethylsulfonyl fluoride, 10 mM leupeptin, 1.5 mM pepstatin, 1 mM aprotinin, and 50 mM sodium fluoride (NaF)
using a Dounce homogenizer. The homogenates were incubated for 1 h
at 4 °C with gentle agitation and then centrifuged at 12,000 rpm for
15 min at 4 °C. The dissociated cells obtained were lysed by boiling in a lysis buffer (1% SDS, 1 mM sodium orthovanadate, 10 mM Tris-HCl, pH 7.4, 1 mM phenylmethylsulfonyl
fluoride, 10 mM leupeptin, 1.5 mM pepstatin,
and 1 mM aprotinin), and then centrifuged for 10 min to
remove insoluble material. Protein concentrations in the soluble
fraction were measured using a BCA protein assay reagent kit (Pierce),
after which equal amounts of protein were separated by 8% SDS-PAGE.
The electrophoretically separated polypeptides were transferred onto a
polyvinylidene difluoride membrane (Bio-Rad), which was then blocked
with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween
20 (TBST) for 1 h and incubated first with anti-GST-SPIN90
antibodies and then with horseradish peroxidase-conjugated anti-rabbit
IgG. The antigen-antibody complexes were detected using enhanced
chemiluminescence (ECL) (Amersham Pharmacia Biotech.). In some cases,
blots were stripped by heating them to 55 °C for 30 min in stripping
buffer (100 mM Coimmunoprecipitation
Cardiac myocytes grown on collagen-coated culture dishes were
washed three times with cold PBS and extracted for 1 h at 4 °C
in extraction buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 10%
glycerol, 1 mM CaCl2, 1 mM
MgCl2) supplemented with protease inhibitors. The extracts
were clarified by centrifugation for 10 min at 10,000 × g, after which the protein concentrations of supernatants
were determined using the BCA method, and samples containing 1 mg of
total protein were taken for subsequent immunoprecipitation. Cell
lysates were immunoprecipitated using preimmune serum, anti-GST-Nck, or
anti-GST-SPIN90 antibody as a probe and then incubated for an
additional 4 h at 4 °C with protein A-Sepharose beads. Immune
complexes were extensively washed with the same extraction buffer,
after which the immunoprecipitates were boiled and subjected to 8%
SDS-PAGE, and the proteins transferred to a polyvinylidene difluoride
membrane. The resultant blots were blocked and incubated first with
anti-GST-Nck or anti-GST-SPIN90 antibody and then with horseradish
peroxidase-conjugated goat anti-rabbit IgG diluted in TBST. After
washing three times with TBST, the blots were developed using ECL reagents.
Detection of SPIN90 within Cardiac Myocytes by
Immunofluorescence
Cardiac myocytes grown on collagen-coated (50 µg/ml) glass
coverslips were briefly washed with PBS and then fixed with 5% paraformaldehyde in PBS for 10 min at room temperature. After washing
in PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS for
5 min at room temperature and then incubated for 45 min at room
temperature, first with the appropriate primary antibodies diluted with
PBS containing 1% bovine serum albumin and then with TRITC-conjugated
goat anti-IgG. For double staining, these cells were incubated again
with the appropriate antibodies and FITC-conjugated goat anti-IgG for
45 min. Thereafter, the coverslips were washed with PBS and mounted
with 90% glycerol and 0.1% O-phenylenediamine in PBS. The
cells were viewed under a Leica DMRBE fluorescence microscope equipped
with a 63× objective lens and appropriate filters. Fluorescence
micrographs were taken on T-max P3200 film (Eastman Kodak Co.).
Cell Permeabilization and Antibody Internalization
Cardiac myocytes grown on collagen-coated dishes for 8 days were
washed twice with DMEM and exposed to streptolysin O (80 µg/ml) for
30 min at 37 °C (7, 23). The cells were then washed three times with
DMEM, and incubated with growth medium containing normal rabbit IgG
(NRIgG), or with affinity-purified anti-Nck or anti-SPIN90 antibody
(200 µg/ml). After incubation for 1, 12, 24, or 48 h at
37 °C, the cells were extensively washed with DMEM and subjected to
immunofluorescence assays using anti- Isolation of a cDNA Clone Encoding SPIN90--
In a search to
identify the proteins with which the Nck SH3 domains interact, yeast
two-hybrid screening analysis was performed. The three SH3 domains of
Nck (amino acids residues 1-251) was fused to the GAL4 DNA-binding
domain and used to screen a human heart Matchmaker cDNA library. Of
3 × 106 independent clones plated, 15 positive clones
were identified that specifically interacted with Nck. Sequence
analysis showed that two clones encoded the same protein, which
contained a proline-rich motif that matched consensus motifs previously
shown to be critical for interaction of SH3 domains with target
proteins (13). Of these, clone 12 contained a 2.1-kb gene insert
and retained its
Analysis of the full-length cDNA showed the clone to encode 722 amino acids, including several regions homologous with other genes in
the data base. The N terminus contains an SH3 domain that had the
highest homology (35% identity and 50% similarity) with the SH3
domains of Fyn, Yes, and c-Src (Fig.
2A) (24-26). The middle
portion of the cDNA contained three proline-rich motifs: a type I
motif (RXXPXXP) at amino acids 176-182 and two
type II motifs (PPXPX(R/K)) at amino acids
170-176 and 242-249, which are matched with consensus sequences
important in mediating SH3 domain binding to protein targets. These
motifs seem to be responsible for the interaction between SPIN90 and
Nck in the yeast two-hybrid system. The C terminus is very hydrophobic
and lacks any obvious sequence motifs. An amino acid sequence
comparison revealed that the C terminus of SPIN90 is, in part,
identical with VIP54, a VacA-interacting protein colocalized with
vimentin- and desmin-containing intermediate filaments in human
parietal cells (Fig. 2B) (27). To verify whether these
proteins are alternative spliced-variants, RT-PCR was performed using
the specific primers for SPIN90 or VIP54. The SPIN90 was detected in
all the samples including heart and HeLa cells and heart and HeLa
cDNA libraries but VIP54 was not (Fig. 2C). However, to
address whether these proteins are the alternative spliced variants,
detailed biochemical study will be necessary.
Taken together, these results strongly suggest that the cDNA we
have isolated encodes a VIP54-related protein expressed in human heart;
therefore, we have designated it SPIN90 (SH3
Protein Interacting with Nck,
90 kDa).
In Vitro Association between SPIN90 and the SH3 Domains of
Nck--
To further investigate the specificity of the interaction
between SPIN90 protein and Nck SH3 domains, spin90 was
translated in vitro using TNT T7-coupled reticulocyte
lysate. In addition, cDNAs encoding Nck protein fragments were
cloned into pGEX4T-1 vector and expressed as GST fusion proteins in
Escherichia coli. [35S]Met-translated
spin90 products were mainly detected at ~90 kDa following
SDS-PAGE (Fig. 3A) and were
coprecipitated with GST-full Nck, GST-Nck total SH3 (containing three
SH3 domains), GST-Nck SH3-1, and GST-Nck SH3-3 (containing the first
and third SH3 domains, respectively), but not with purified GST protein
(Fig. 3B). The translated spin90 product also did
not coprecipitate with GST-Nck SH2 (containing the SH2 domain) or with
GST-Nck SH3-2 (containing the second SH3 domain). Thus, SPIN90 appears
to bind exclusively to the first and third SH3 domains of Nck, in
vitro.
Tissue Distribution of SPIN90--
Northern blot analysis of
various human tissues revealed spin90 expression to be
fairly ubiquitous. A single transcript about 3.4 kb in length was
expressed in all of the adult tissues examined, except intestine (Fig.
4). The highest levels of expression were found in brain, heart, skeletal muscle, kidney, and liver. Intermediate levels of expressions were detectable in placenta, lung, and leukocyte, and low levels were found in colon, thymus, and spleen.
SPIN90 Is Ubiquitously Expressed in Rat Tissues--
To obtain a
GST fusion protein using clone spin90, the gene insert from
clone spin90 was subcloned into pGEX 4T-1 vector, and the
encoded GST-SPIN90 fusion protein was expressed in bacterial cells.
After SDS-PAGE, the resultant protein band (115 kDa) was purified by
electro-elution. Rabbits were then immunized with the eluted fusion
protein to generate a polyclonal antiserum, which recognized species of
approximately 115 kDa in bacterial lysates and of about 90 kDa in whole
cell extracts from various tissues (Fig.
5A). In particular, a protein
highly expressed in heart, skeletal muscle, and two skeletal muscle
cell lines (C2C12 and L8E63 cells) was recognized, which is consistent
with the Northern blots shown in Fig. 4. SPIN90 was found to be the
same size in adult rat heart and in cultured neonatal rat cardiac
myocytes, and expression of both SPIN90 and Nck was up-regulated during cardiac myocyte differentiation (Fig. 5B).
Cellular Localization of SPIN90 during Cardiac Myocyte
Differentiation--
To better understand the function of SPIN90 in
cultured cardiac myocytes, immunohistochemical analysis was used to
determine its location within the cells. Double immunofluorescent
labeling using antibodies against SPIN90 and Nck revealed that, in
9-day cultured cardiac myocytes, Nck and SPIN90 were both distributed in a striated pattern characteristic of the Z-discs (Fig.
6A, c and
d); the same pattern of distribution was also observed using Specific Antibodies against Nck or SPIN90 Disrupt Sarcomere
Structure in Cardiac Myocytes--
The localization of SPIN90 in
sarcolemmal regions associated with Z-discs suggested a potential role
for SPIN90 in the organization of sarcomere structure. To further
clarify its function, anti-SPIN90 antiserum was introduced into 8-day
cultured cardiac myocytes that had been previously permeabilized with
streptolysin O, and its effect on sarcomere structure was examined by
analyzing the distribution of anti-
The percentage of sarcomeres disrupted by antibody treatment was
calculated from the mean number of cardiac myocytes containing disrupted sarcomeres (Fig. 7B). Streptolysin O and NRIgG had
little effect on sarcomere structure; in the absence of antibodies,
~6% of cells exhibited disrupted sarcomeres. On the other hand,
cells treated with anti-Nck or anti-SPIN90 antibodies exhibited
substantially greater numbers of disrupted sarcomeres within 1 h
of exposure (Fig. 7B, white bar), and
within 24 h, ~40-50% of cells showed disrupted sarcomeres
(Fig. 7B, hatched bar). Thus, Nck and
SPIN90 may be crucial for the maintenance of sarcomere structure and/or the assembly of myofibrils into sarcomeres.
Specific protein-protein interactions are essential in many
biological processes, including gene replication, transcription, metabolism, and signal transduction. Over the past several years, it
has become apparent that, although different signaling molecules may
contain distinct catalytic activities, most share several conserved
protein domains (10, 11). Among such signaling molecules, Nck is a
ubiquitously expressed protein containing one SH2 and three SH3
domains. As Nck lacks any known catalytic domains, it was classified as
an adaptor molecule (28). We previously reported that Nck is localized
at sarcomeres within cardiac myocytes and takes part in
In the present study, we used the yeast two-hybrid screening system and
phage library screening to identify a VIP54-related protein that
interacts with two of Nck's SH3 domains. As determined by SDS-PAGE and
amino acid sequence analysis, the positive clone encodes for a 90-kDa
protein containing an SH3 domain; hence, it was designated SPIN90
(SH3 Protein Interacting with
Nck, 90 kDa).
When the amino acid sequences were compared, the N-terminal SH3 domain
of SPIN90 was found to have the highest homology with those of Fyn,
Yes, and c-Src. Like most other SH3 domains, that of SPIN90 contains a
well conserved two-tryptophan (WW) sequence within the domain. It is
interesting that the amino acid sequences within the ligand-binding
site are completely conserved among the SH3 domains of Fyn, Yes, c-Src,
and SPIN90, suggesting that SPIN90 may interact with a variety of
proteins containing proline-rich motifs, including signaling molecules,
enzymes, and structural proteins.
SPIN90 also contains three proline-rich motifs: a type I motif
(RXXPXXP) at amino acids 176-182 and two type II
motifs (PPXPX(R/K)) at amino acids 170-176 and
242-249. These are important in mediating SH3-domain binding to
protein targets and are likely to be responsible for the interaction
between SPIN90 and Nck. At the SH3-binding motif, two conserved proline
residues would protrude from the proline-rich motif and intercalate
between the aromatic residues in the ligand-binding site, perhaps
forming the long, shallow groove of the SH3 ligand-binding site.
Although we do not provide specific evidence that the proline-rich
motifs of SPIN90 interact with the SH3 domains of Nck, we clearly
confirmed that SPIN90 binds to two of the three SH3 domains of Nck,
both in vitro and in vivo.
In addition, serine/threonine-rich sequences within the proline-rich
motifs would seem to be candidate sites for phosphorylation by
serine/threonine kinases. In fact, although SPIN90 is detected at ~90
kDa by Western blot analysis of several tissues and cell lines, the
predicted molecular mass of the protein is ~80 kDa. It therefore
seems likely that SPIN90 is phosphorylated by serine/threonine kinases.
The C-terminal region of SPIN90 is very hydrophobic and lacks any known
sequence motifs. To understand the function of this region, additional
detailed studies will be required. Recently, Sano et al.
(29) identified the gene AF3p21 (ALL-1 fused gene from
chromosome 3p21) as a mixed lineage leukemia (MLL) fusion partner gene
whose product is expressed as a fusion protein with MLL. This protein
has never been observed as an individual molecule, even though its
sequence is completely identical with SPIN90. The SH3 domain-deleted
form of AF3p21 can be fused with MLL, and the resultant fusion protein
(MLL-AF3p21) seems to be essential for leukemogenesis. Unlike most
known MLL fusion partner genes (e.g. EEN and ABI-1), which
have an SH3 domain in their C terminus, AF3p21 contains an SH3 domain
in the N terminus, and MLL-AF3p21 contains no SH3 domains. For this
reason, we would expect the downstream region, which includes the
proline-rich, serine/threonine-rich, and C-terminal regions of SPIN90,
to play an essential role in leukemogenesis.
Comparison of the amino acid sequences revealed that the SPIN90
has a high degree of similarity to VIP54 (VacA-interacting protein),
which colocalizes with vimentin- and desmin-containing intermediate
filaments in human parietal cells (27). Although both SPIN90 and VIP54
appeared to have the same size of transcripts and show the same tissue
distribution pattern, immunoblot analysis revealed that they encode
different molecular size of protein. In RT-PCR, SPIN90 was detected in
both heart and HeLa cells, respectively, but VIP54 was not. To address
whether these two proteins are alternatively spliced variants, further
biochemical studies will be conducted. However, it seems that these two
proteins are closely related to each other. VIP54 was found to be
associated with vimentin, and SPIN90 also seems to be able to associate
with cytoskeletal proteins. For example, immunofluorescent labeling
showed SPIN90 to be localized at Z-discs of cardiac myocytes, and
introduction of SPIN90 antiserum into cells disrupted sarcomere
structure. This suggests that SPIN90 may participate in the maintenance
of sarcomere structure and/or the assembly of myofibrils into
sarcomeres via association with cytoskeletal proteins. In addition, we
reported previously that Nck participates in downstream signaling
triggered by In conclusion, we have described the sequence of SPIN90 and the binding
of it to Nck, and have provided evidence of its function in cardiac
myocytes. Together, these findings suggest that SPIN90, which contains
an SH3 domain, three proline-rich motifs, and serine/threonine-rich sequences, may be an adaptor protein that acts in concert with Nck to
mediate sarcomere development during cardiac myocyte differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 integrin is
expressed in the costameres of cardiac myocytes, where the cells attach
to the collagen network; it forms the strong attachment sites required
by highly contractile cells (3, 4). The organization of sarcomere
structure within cardiac myocytes is thus crucially dependent on the
presence of
1 integrin (5). We previously reported that
3 integrin is also localized at costameres and is
associated with
1 integrin during myofibrillogenesis
(6). Moreover, Nck appears to be distributed in a sarcomeric banding pattern in cardiac myocytes, colocalizing at the Z-discs with
-actinin, and participating in a
1A integrin-mediated
signaling pathway (7).
, and p125GAP, adaptor
molecules are able to bind to receptor tyrosine kinases via their SH2
domains (11). SH3 domains are mostly found in kinase-mediated signal
transduction molecules and in cytoskeletal components (12); the latter
possess SH3 domains that bind to proline-rich motifs (minimal consensus motif: PXXP) in other signaling molecules (13). For example, they bind to SOS, dynamin, Cbl, and Wiskott-Aldrich syndrome protein (WASP), which are responsible for mediating the effects of CDC42 on
reorganization of actin cytoskeleton (14-17).
1A integrin-mediated signal pathway, the
functions of Nck and the signal molecules with which it interacts in
cardiac myocytes are also not yet known.
1A integrin-mediated signal
pathway, we have used yeast two-hybrid screening to find proteins that interact with Nck. By screening a human heart cDNA library using Nck SH3 domains as bait, we isolated a positive cDNA clone,
spin90, which encodes a VIP54-related protein containing an
SH3 domain, proline-rich motifs and serine/threonine-rich sequences.
Here, we describe its association with Nck and provide evidence of its function in the heart.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin antibody (clone EA-53) was purchased from Sigma.
Monoclonal anti-Nck antibody was purchased from Transduction
Laboratory. Horseradish peroxidase-labeled anti-mouse immunoglobulin
(IgG) and fluorescein isothiocyanate (FITC)- or tetramethyl rhodamine
isothiocyanate (TRITC)-conjugated goat anti-mouse IgG were from Jackson
Immunoresearch Laboratory. Protein A-Sepharose and
glutathione-Sepharose 4B fast flow were purchased from Amersham
Pharmacia Biotech. Human heart Matchmaker cDNA library and
TriplEX human heart cDNA library were obtained from
CLONTECH. The primers used for polymerase chain reaction (PCR) and DNA sequencing were synthesized by Genotech. TNT
T7-coupled reticulocyte lysate system was from Promega.
[32P]dCTP and [35S]methionine
([35S]Met) were from PerkinElmer Life Sciences.
-galactosidase activity were recovered from His+
colonies and were tested in a cotransformation assay with the bait
construct, pGBT9/Nck SH3 or with control pGBT9 vector. To assay for
-galactosidase activity, individual yeast transformants were patched
onto filter paper and frozen in liquid nitrogen, after which the
filters were stained with buffer (consisting of 0.01%
5-bromo-4-chloro-3-indolyl
-galactopyranoside, 60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0, 50 mM
-mercaptoethanol) at 30 °C.
TriplEX human heart cDNA library was plated and transferred to a
Protran BA 85 nitrocellulose membrane (Schleicher & Schuell) according
to the manufacturer's protocol. The library was screened by
hybridization using a 32P-labeled insert as a probe
(obtained from clone spin90). After an additional two rounds
of purification, three positive pTriplEX plasmids were obtained. The
cDNA inserts were sequenced using an ABI PRISM 377 DNA sequencer
and PRISMTM Ready Reaction DideoxyTM Terminator cycle sequencing kit
(PerkinElmer Life Sciences). The sequences were then confirmed manually
using a T7 Sequenase version 2.0 DNA sequencing kit (U. S. Biochemical
Corp.).
-actin, which
served as a control, were labeled with [32P]dCTP by
random-primer extension using the oligonucleotide labeling kit
(Amersham Pharmacia Biotech). These probes were then used for
hybridization according to the manufacturer's protocol, after which
the blot was exposed to x-ray film (Fuji Co.) for 12-24 h at
70 °C.
-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.7) and reprobed with anti-
-tubulin antibody, which served as a control.
-actinin antibodies to show the
sarcomere structure. Cells with disrupted sarcomeres were counted; data
from three independent experiments are presented graphically.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity in secondary screening.
This clone, however, seemed to contain only a partial cDNA insert,
as a stop codon was not found within the 3' region of the insert. To
obtain a full-length cDNA clone, a
TriplEX human heart cDNA
library was screened using the 2.1-kb cDNA gene insert from clone
12; three clones were isolated, with the size of the largest gene
insert being 1.2 kb (clone 1-1). The full-length cDNA was then
completed with the contribution of sequences from two clones (12 and
1-1). A schematic diagram of the full-length cDNA shows the
overlapping region between clone 12 and the newly obtained 3' region
from clone 1-1 (Fig. 1A).
This clone contained a single open reading frame from an ATG start
codon to a TAG stop codon followed by a stretch of 762 nucleotides
comprising the 3'-untranslated region that contains the polyadenylation
signal (AATAAA, 2940-2945) and the poly(A) tail (Fig.
1B).
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Fig. 1.
Cloning of spin90.
A, strategy for cloning a full-length spin90
cDNA. Clone 12 (~2.1 kb) was obtained by two-hybrid screening of
a human heart cDNA library using Nck SH3 domains as bait. Clone
1-1 (~1.2 kb) was obtained from human heart phage library by
screening with the gene insert of clone 12. The overlapping region is
shaded. B, nucleotide and deduced amino acid
sequences of spin90. Overlapping clones 12 and 1-1 complete
the full-length sequence. The open reading frame begins at position 94, which corresponds to the putative ATG start codon. An
asterisk at position 2260 indicates the TAG termination
codon. The polyadenylation recognition sequence (AATAAA, 2940-2945) is
in bold. The SH3 domain is shaded,
serine/threonine-rich sequences are underlined, and
proline-rich motifs are boxed. The accession number of
SPIN90 in GenBankTM is AF303581.
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Fig. 2.
Alignment and sequence comparison of
SPIN90. A, alignment and sequence comparison of the SH3
domains of SPIN90 and other related proteins. The SH3 domain of SPIN90
shows high homology with those of Fyn, Yes, and c-Src. Black
boxes indicate the conserved amino acids. B,
alignment of the amino acid sequences of SPIN90 and VIP54.
Black boxes indicate the conserved amino acids.
C, RNAs (5 µg) from heart and HeLa cells and DNAs (4 µg)
from human heart tissue and HeLa cell libraries were subjected to
RT-PCR using the specific primers for SPIN90 or VIP54. The 5' primer
(5'-TGCAGGACGTGTTTGGCAG-3', 1947-1966) was picked from the homology
region of SPIN90 and VIP54, and the 3' primers were from
3'-untranslated region of SPIN90 (5'-GCTTTCTCAGTGCCATCATCC-3',
2817-2836) or VIP54 (5'-CGTTGCTCCTGGTGCTGAAC-3', 3140-3159). SPIN90
(889 base pairs) was detected in all the samples but VIP54 (1212 base
pairs) was not. The accession number of VIP54 in GenBankTM is
AJ242655. S.M., size marker.
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Fig. 3.
In vitro association of SPIN90
with Nck. All recombinant GST-Nck fusion proteins overexpressed in
E. coli were immobilized on glutathione-Sepharose beads,
after which GST pull-down assays were performed by incubating the beads
with [35S]Met-labeled, in vitro translated
SPIN90 for 6 h at 4 °C. Bound proteins were eluted, analyzed by
SDS-PAGE, and visualized by autoradiography. SPIN90 binds to GST-full
Nck, GST-total SH3, GST-SH3-1, and GST-SH3-3, but not to GST-SH2,
GST-SH3-2, or GST alone (control).
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Fig. 4.
Expression of SPIN90 mRNA in various
human adult tissues. Poly(A)+ RNAs isolated from the
indicated human tissues (CLONTECH) were hybridized
with the 32P-labeled cDNA fragment corresponding to the
nucleotides 1-2145 of spin90. Molecular size makers are
indicated on the left. The SPIN90 mRNA (~3.4 kb)
appears to be ubiquitously expressed.
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Fig. 5.
Expression of SPIN90 protein in various rat
tissues. A, antiserum raised against SPIN90 recognized
a 90-kDa protein in various rat tissues and cell lines. Highest levels
of SPIN90 expression were found in heart, skeletal muscle, and two
skeletal muscle cell lines (C2C12 and L8E63). B, expression
of SPIN90 in neonatal rat cardiac myocytes. Expression of SPIN90 and
Nck are up-regulated during cardiac myocyte differentiation.
-Tubulin was used as a control.
-actinin as a Z-disc marker (Fig. 6A, a). In
contrast, anti-GST antibody, serving as a control, showed no specific
labeling pattern (Fig. 6A, b). Nck and SPIN90 are
thus apparently colocalized at Z-discs in mature cardiac myocytes. To
assess whether SPIN90 binds to Nck at the Z-discs, lysates obtained
from 9-day cultured cardiac myocytes were immunoprecipitated with
anti-Nck antiserum and immunoblotted with anti-SPIN90 antiserum. As
shown in Fig. 6B, SPIN90 was indeed immunoprecipitated with
Nck, and may thus participate in the
1A integrin-mediated signaling pathway via interaction with Nck.
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Fig. 6.
Intracellular localization of SPIN90 in
cultured cardiac myocytes. A, after 9 days in culture,
cardiac myocytes were fixed, permeabilized, and labeled first with
anti- -actinin (a) or anti-GST antibodies (b)
and then with TRITC-conjugated goat anti-IgGs. For double labeling, the
fixed cells were first incubated with monoclonal anti-Nck antibodies
and FITC-conjugated goat anti-mouse IgG (c) and then
incubated again with anti-SPIN90 antiserum and TRITC-conjugated goat
anti-rabbit IgG (d). Nck (c) and SPIN90
(d) localized at the Z-discs in a pattern characteristic of
sarcomeric banding, a pattern not detected when cells were labeled with
-GST antibody (b).
-Actinin served as a Z-disc marker
(a). B, in vivo association between
SPIN90 and Nck SH3 domains in cardiac myocytes. To confirm the
interaction between Nck and SPIN90 in vivo, lysates of
cardiac myocytes (9-day cultures) were immunoprecipitated with
anti-GST-Nck antiserum, and the precipitants were immunoblotted with
anti-SPIN90 antiserum. Protein A and anti-GST antiserum were used as
controls. Bar = 10 µm.
-actinin antibody. Normal 8-day
cultured myocytes exhibited typical sarcomere structure (Fig.
7A, a);
streptolysin O itself had no effect on cell viability, which is
consistent with our earlier reports (7). Similarly, myocytes exposed to normal rabbit IgG (NRIgG) and then immunolabeled with anti-
-actinin antibodies also exhibited typical sarcomeric patterns (Fig.
7A, b). On the other hand, exposing the cells to
anti-Nck or anti-SPIN90 antisera and then immunolabeling with
anti-
-actinin antibodies revealed severely disrupted sarcomere
structures (Fig. 7A, c and d).
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Fig. 7.
Disruption of sarcomere structure by anti-Nck
and anti-SPIN90 antibodies. A, myocytes cultured for 8 days were treated with streptolysin O for 30 min to permeabilize their
cell membranes. To visualize the changes in sarcomere structure,
following antibody treatment for 24 h, the cells were labeled with
anti- -actinin antibodies and FITC-conjugated goat anti-mouse IgG.
Untreated cells exhibited typical sarcomeric structures (a),
as did cells treated with NRIgG, which served as a control
(b). In contrast, anti-Nck (c) and anti-SPIN90
(d) antibodies caused clear disruptions of sarcomeric
structure. B, the percentage of disrupted sarcomeres was
counted from the mean number of myocytes in which disrupted sarcomeres
were observed. In the presence of streptolysin O and NRIgG, the
incidence of sarcomeric disruption remained at basal levels (~6%).
In contrast, anti-Nck and anti-SPIN90 antibodies disrupted ~50% of
sarcomeres. White bar, 1 h; black
bar, 12 h; hatched bar, 24 h; dotted bar, 48 h after internalization of
NRIgG, anti-Nck, or anti-SPIN90. Bar = 10 µ m.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1A integrin-mediated signal transduction (7); however,
Nck's function during differentiation of cardiac myocytes, as well as
the functions of the downstream signaling molecules regulated by Nck,
remain unknown.
1A but not
1D integrin.
Thus, the association of SPIN90 with Nck in sarcomeres of cardiac
myocytes supports the notion that SPIN90 is involved in a
1A integrin-mediated signal transduction pathway
initiated by the interaction of integrin and ECM during cardiac myocyte
differentiation, though additional detailed studies will be required to
determine specifically how this protein is involved.
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FOOTNOTES |
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* This work was supported by in part by grants from the Protein Network Research Center and the Life Phenomena and Function Research Group (Korean Ministry of Science and Technology).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF303581.
§ Supported by the Brain Korea 21 program.
** Supported by a grant from the Korean National Research Laboratory.
§§ To whom correspondence should be addressed: Dept. of Life Science, Kwangju Inst. of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, South Korea. Tel.: 82-62-970-2487; Fax: 82-62-970-2484; E-mail: wksong@pia.kjist.ac.kr.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M009411200
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; SPIN90, SH3 Protein Interacting with Nck, 90 kDa; SH, Src homology; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate; GST, glutathione S-transferase; RT, reverse transcription; PCR, polymerase chain reaction; kb, kilobase pair(s); TBST, Tris-buffered saline with Tween 20; MLL, mixed lineage leukemia; NRIgG, normal rabbit IgG.
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REFERENCES |
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