* Department of Biology, University of Utah, Salt Lake City, Utah 84112-0840
Members of the cysteine-rich protein (CRP)
family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding
partners. In order to identify proteins that interact with
CRP1, a prominent protein in fibroblasts and smooth
muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A
100-kD protein bound to the CRP1 column and could
be eluted with a high salt buffer; Western immunoblot
analysis confirmed that the 100-kD protein is -actinin.
We have shown that the CRP1-
-actinin interaction is
direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1-
-actinin interaction is 1.8 ± 0.3 µM. The results of the in vitro
protein binding studies are supported by double-label
indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and
-actinin along the
actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that
-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth
muscle cells. By in vitro domain mapping studies, we
have determined that CRP1 associates with the 27-kD
actin-binding domain of
-actinin. In reciprocal mapping studies, we showed that
-actinin interacts with
CRP1-LIM1, a deletion fragment that contains the
NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the
-actinin binding domain of CRP1
would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2
(aa 108-192) were microinjected into cells. By indirect
immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin
stress fibers whereas CRP1-LIM2 fails to associate with
the cytoskeleton. Collectively these data demonstrate
that the NH2-terminal part of CRP1 that contains the
-actinin-binding site is sufficient to localize CRP1 to
the actin cytoskeleton. The association of CRP1 with
-actinin may be critical for its role in muscle differentiation.
MYOGENESIS is a complex multistep process that
involves the specification of muscle progenitor
cells, the determination of a subset of these cells
to become myoblasts, the proliferation of these determined
cells, and ultimately the differentiation of these cells into
fully functional muscle. A variety of growth factors and
transcription factors, including members of the MyoD family of basic helix-loop-helix proteins and the MEF2
family, contribute to the coordinated control of muscle cell
differentiation. These myogenic factors regulate both the
exit of myoblasts from the cell cycle as well as the initiation of muscle-specific gene transcription (Cossu et al.,
1996 Recently, members of a family of proteins called the cysteine-rich protein (CRP)1 family have been shown to be
involved in a late stage in muscle differentiation. In vertebrates, the CRP family is comprised of three closely related
proteins, CRP1, CRP2, and the muscle LIM protein (MLP),
also referred to as CRP3 (Weiskirchen et al., 1995 Members of the CRP family exhibit a conserved molecular architecture (Weiskirchen et al., 1995 In addition to their common structural features, CRP
family members are functionally related as well. CRP1 was
initially identified as a binding partner for zyxin, a low
abundance phosphoprotein that is concentrated at adhesion plaques and in association with actin filament arrays
(Sadler et al., 1992 To understand the mechanism by which CRP1 affects
muscle differentiation, we have initiated an effort to identify CRP1-binding proteins in chicken smooth muscle, the
source from which CRP1 was originally purified (Crawford et al., 1994 Isolation of Avian Smooth Muscle Proteins
Avian smooth muscle proteins were extracted from frozen chicken gizzards as described previously (Crawford and Beckerle, 1991 Purification and Radioiodination of Purified Purification of Bacterially Expressed CRP1,
CRP1-LIM1, and CRP1-LIM2
CRP1-LIM1 corresponds to the NH2-terminal part of chicken CRP1
(cCRP1) (amino acids [aa] 1-107) including the NH2-terminal LIM domain followed by the first glycine-rich repeat of the protein. CRP1-LIM2
corresponds to the COOH-terminal portion of cCRP1 (aa 108-192) containing the COOH-terminal LIM domain and the second glycine-rich repeat of the protein. Techniques for the purification of the bacterially expressed full-length CRP1 and CRP1-LIM2 were described previously
(Michelsen et al., 1993 hCRP1 Expression, Isolation, and Radiolabeling
A plasmid engineered for the bacterial expression of human CRP1
(hCRP1) was generously provided by S.A. Liebhaber. The methods for
expression, purification and radiolabeling of the glutathione-S-transferase (GST)-hCRP1 fusion protein were described previously (Feuerstein et al.,
1994 Affinity Chromatography
Bacterially expressed CRP1 or BSA was covalently coupled to Affi-gel 10 (Bio-Rad Laboratories, Hercules, CA) in coupling buffer (0.1 M Hepes,
pH 7.8, 0.1% 2-mercaptoethanol, 0.1 mM EDTA) for 4 h at 4°C. The
affinity resins were transferred to two different columns, washed with
coupling buffer, and then equilibrated with the column buffer (20 mM
Tris-acetate, pH 7.6, 0.1% 2-mercaptoethanol, 0.1 mM EDTA). A 27-34%
ammonium sulfate precipitate from avian smooth muscle was loaded onto
each column. The columns were washed with 0.1 M NaCl in column buffer.
Proteins eluted with 1 M NaCl in column buffer were collected in 300-µl
fractions and 15 µl of each fraction were resolved by electrophoresis on
12.5% SDS-polyacrylamide gels. Gel Electrophoresis and Western
Immunoblot Analysis
SDS-PAGE was performed according to the method of Laemmli (1970) Solution Binding Assay
GST-hCRP1 or GST agarose beads were incubated at 20°C with purified
In competition experiments, GST-hCRP1 agarose beads were incubated at 20°C with 100 µl of [125I] Blot Overlay Assay
Blot overlay assays were performed as previously described (Crawford et
al., 1992 Solid-phase Binding Assay
Removable microtiter wells (Dynatech Laboratories, Inc., Chantilly, VA)
were coated overnight at 4°C with 120 µl of bacterially expressed CRP1 at
0.1 mg/ml. The wells were washed three times with Hepes binding buffer
(HBB) (20 mM Hepes, pH 7.4, 10 mM NaCl, 0.1 mM EGTA, 0.1% 2-mercaptoethanol) and blocked with 300 µl 2% BSA in HBB. After a 120-min
incubation at 37°C, the blocking solution was removed and the wells were
washed with HBB plus 0.2% BSA. The wells were next incubated for 2.5 h
at 37°C with [125I] Confocal Immunofluorescence Microscopy
Chicken embryo fibroblasts (CEF) were cultured on glass coverslips in
DME supplemented with 10% FBS. Smooth muscle cells were prepared
from gizzards taken from 16-d-old chicken embryos as previously described, except that trypsin was used instead of collagenase (Gimona et
al., 1990 Cell Labeling and Immunoprecipitation
CEF cells were radiolabeled with [35S]methionine-cysteine (Tran35S-label;
ICN Biomedicals Inc., Irvine, CA). Metabolic labeling was carried out
with adherent cells that were washed twice with PBS at 37°C and incubated in one part DME, plus nine parts DME without methionine and
cysteine supplemented with 10% FBS in the presence of 200 µCi of
[35S]methionine-cysteine for 18 h. After three washes with PBS, the cells
were lysed in Laemmli sample buffer with protease inhibitors (0.1 mM
PMSF, 0.1 mM benzamidine, 1 µg/ml pepstatin A, 1 µg/ml phenantholine), and scraped off the dish. Cell lysates were boiled for 5 min. Immunoprecipitation was then performed as described below. In immunoprecipitation experiments using nonlabeled cells, smooth muscle cells from
adult chicken gizzards were lysed in 10 mM Tris, pH 8, 140 mM NaCl, 1%
Triton X-100, 0.2% deoxycholate, 0.02% SDS, 0.1 mM PMSF, 0.1 mM
benzamidine, 1 µg/ml pepstatin A, 1 µg/ml phenantholine, and scraped off
the dish. After incubation on ice for 30 min, the lysate was centrifuged at
10,000 rpm for 10 min, and the soluble material was recovered in the supernatant. The supernatant was then incubated with protein A-agarose
beads (Sigma Chemical Co.) for 1 h at 4°C under gentle agitation. After a
2-min centrifugation at 2,000 rpm, the supernatant was incubated for 1 h
at 4°C with either 3 µl of the polyclonal antibody B37 raised against CRP1
or 3 µl of the corresponding preimmune serum, followed by a 1.5-h incubation with protein A-agarose beads. At the end of the incubation period,
the beads were washed twice with the lysis buffer to remove the unbound
proteins; more extensive washing resulted in a loss of our ability to detect
protein that coimmunoprecipitated with CRP1. 40 µl of 2× Laemmli sample buffer were then added to the pelleted beads and boiled for 5 min. The immunoprecipitated proteins were resolved by SDS-PAGE. Gels containing metabolically labeled, immunoprecipitated proteins were dried and
subjected to autoradiography, while nonlabeled proteins were transferred
to nitrocellulose for immunoblotting as described above. CRP1 was detected using the polyclonal antibody B37, while Heterologous Expression and Immunofluorescence
Expression vector construction involved amplifying coding regions from
full-length cCRP1 cDNAs by PCR using Pfu Polymerase (Stratagene, La
Jolla, CA). Primers encoded EcoRV (5 Recovery of Affinity chromatography was used to identify CRP1-binding proteins in an avian smooth muscle extract. Briefly,
proteins extracted from smooth muscle preparations were
fractionated by precipitation with increasing amounts of
ammonium sulfate (27-34, 34-43, and 43-61% saturation).
Each of the ammonium sulfate precipitates was subjected
to affinity chromatography on a CRP1 column. The CRP1
column was prepared from bacterially expressed avian
smooth muscle CRP1. We have shown previously that bacterially expressed CRP1 exhibits a native structure (Michelsen et al., 1993
A Direct Interaction between CRP1 and To examine whether CRP1 can interact directly with
To examine the specificity of the CRP1- A Direct Interaction between CRP1 and We have further characterized the CRP1-
To analyze further the specificity of the CRP1-
The The affinity of the association between
CRP1 and The work described above reports the ability of CRP1 and
An In Vivo Interaction between CRP1 and We performed a coimmunoprecipitation experiment to
evaluate the ability of CRP1 to interact with
Mapping the Domains of CRP1 and To map the binding site for CRP1 on
CRP1 displays two LIM domains separated by 56 amino
acids (Crawford et al., 1994
Colocalization of CRP1 and CRP1-LIM1
with Actin Filaments
Given the fact that
In this study, we have identified the actin-binding protein,
Prior to this report it was known that CRP family members appear to play a role in muscle
differentiation; however, the mechanism by which they
might act has not been clarified. The ability of CRP1 to interact in vivo with In summary, using in vitro and in vivo biochemical studies and immunochemistry we have demonstrated a direct
and specific interaction between the LIM domain protein
CRP1 and the cytoskeletal protein, ; Molkentin and Olson, 1996
). The ultimate product
of the muscle differentiation program is the ordered assembly of an actomyosin-rich contractile machinery.
). CRP1
expression is prominent in smooth muscle derivatives and is correlated with muscle development in avian embryos
(Crawford et al., 1994
). Furthermore, overexpression of
either CRP1 or MLP/CRP3 potentiates the differentiation
of myoblasts in culture (Arber et al., 1994
). Perturbation
of MLP/CRP3 expression by anti-sense RNA technology
results in a failure of muscle differentiation (Arber et al.,
1994
). Similarly, elimination of MLP/CRP3 function in the
mouse by targeted gene disruption results in dramatic disorganization of myofibrils (Arber et al., 1997
). Recently,
two Drosophila CRP family members, Mlp60A and Mlp84B,
have also been described (Arber et al., 1994
; Stronach et
al., 1996
). Both proteins exhibit muscle-specific expression
in developing embryos and a cytoskeletal localization when expressed in vertebrate cells (Stronach et al., 1996
).
Collectively, although the specific mechanism of action of
CRP family members is unknown, the available data suggest a role for CRPs as essential positive regulators of
muscle differentiation.
). CRPs exhibit
two tandemly arrayed LIM domains, each of which is
flanked by a conserved glycine-rich repeat (Weiskirchen
et al., 1995
). The LIM domain is a cysteine-rich sequence
(CX2CX16-23HX2CX2CX2CX16-21CX2[C,H,D]) (Freyd et al.,
1990
; Sadler et al., 1992
) that coordinates two zinc atoms (Michelsen et al., 1993
) and mediates specific protein-protein interactions (Schmeichel and Beckerle, 1994
). LIM
domains are found in a number of proteins that are involved in control of gene expression and cell differentiation. The LIM motif was first identified in three developmentally regulated transcription factors, Caenorhabditis
elegans Lin-11, rat Isl-1, and C. elegans Mec-3, from which
the term LIM is derived (Freyd et al., 1990
; Karlsson et al.,
1990
). LIM domains can be found in association with functional domains such as kinase domains, transcriptional activation domains, or DNA-binding homeodomains. Alternatively, LIM domains sometimes represent the primary
sequence information in a protein.
; Crawford et al., 1994
). All three CRP
family members have now been shown to bind directly to
zyxin (Louis et al., 1997
). Moreover, all three proteins are prominently associated with the actin cytoskeleton (this
report; Louis et al., 1997
).
). Here we report that CRP1 interacts directly with the actin-binding protein,
-actinin. Moreover,
we demonstrate that the two proteins are substantially colocalized along the actin stress fibers. The findings reported here suggest that CRPs may function as regulators
of myogenesis by virtue of their ability to interact directly
with
-actinin, an essential structural element in the myofibril.
MATERIALS AND METHODS
; Crawford et
al., 1994
). The resulting extract was sequentially precipitated with 27-34,
34-43, and 43-61% saturated ammonium sulfate. These ammonium sulfate precipitates were dialyzed against the column buffer (20 mM Tris-
acetate, pH 7.6, 0.1% 2-mercaptoethanol, 0.1 mM EDTA) before loading
onto affinity columns. The 27-34% ammonium sulfate precipitate contains
-actinin whereas the 34-43% ammonium sulfate precipitate contains CRP1.
-Actinin from
Avian Smooth Muscle
-Actinin was purified from the 27-34% ammonium sulfate precipitate as
described previously (Crawford et al., 1992
). Cleavage of
-actinin by the
proteolytic enzyme thermolysin (Sigma Chemical Co., St Louis, MO) was
performed in 40 mM ammonium acetate, 1 mM CaCl2 for 5 h at 20°C with
an enzyme to substrate ratio of 1:25. The
-actinin concentration was 3.2 mg/ml.
-actinin was radioiodinated as described previously (Crawford et al., 1992
), except that the incubation period of
-actinin with
[125I]Na was reduced to 2.5 min. The purity of the labeled
-actinin was ascertained by SDS-PAGE followed by autoradiography.
; Kosa et al., 1994
). CRP1-LIM1 was purified in the
same manner as CRP1-LIM2 except that the CM-52 cation-exchange column was equilibrated in 5 mM potassium phosphate and 0.01% 2-mercaptoethanol.
). The GST-hCRP1 fusion protein was used in blot overlay assays
and in the solution binding assay whereas cCRP1 was used in all the other
experiments. There is a high degree of sequence similarity between human and chicken forms of CRP1 (91% identity; Crawford et al., 1994
),
and the two proteins appear to be functionally interchangeable.
-Actinin was detected by Western immunoblot analysis using a polyclonal antibody raised against chicken
-actinin that was generously provided by K. Burridge.
except with 0.13% bisacrylamide. 12.5% polyacrylamide gels were used
routinely, however 17.5% gels were employed to resolve low mol wt proteins such as CRP1-LIM1 and CRP1-LIM2. Western immunoblot analysis
was performed using horseradish peroxidase linked to protein A (Amersham Life Science Inc., Cleveland, OH) as a second reagent and enhanced
chemiluminescent detection (Amersham Life Science Inc.).
-actinin or a 27-34% ammonium sulfate precipitate from avian smooth
muscle for 1.5 h on an orbital shaker. The agarose beads were washed
three times with PBS and three times with buffer B10 (20 mM Tris-acetate, pH 7.6, 10 mM NaCl, 0.1 mM EDTA, 0.1% 2-mercaptoethanol). The
beads were then mixed in 40 µl 2× Laemmli sample buffer (Laemmli,
1970
), boiled, and the supernatants were analyzed by SDS-PAGE and
Western immunoblot using a polyclonal antibody raised against chicken
-actinin.
-actinin (500,000 cpm) for 1.5 h on an
orbital shaker in the absence of competing protein or in the presence of
unlabeled
-actinin or BSA. The agarose beads were washed three times
with PBS, centrifuged, and the counts bound to the agarose beads were
analyzed using a Packard Multi-Prias 1
counter (Packard Instrument
Co., Inc., Meriden, CT).
). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose strips were incubated in the presence of
[32P]GST or [32P]GST-hCRP1 fusion protein probes (600,000 cpm/ml), or
an [125I]
-actinin probe (250,000 cpm/ml). For competition experiments, unlabeled competing proteins were added into the blot overlay buffer immediately before the introduction of the labeled probe. Autoradiography
was performed at
80°C with an intensification screen.
-actinin, in the presence of competing proteins in HBB.
The final volume was 120 µl. At the end of the incubation period, the radioactive material was removed from the wells and they were washed six
times with HBB plus 0.2% BSA followed by a final rinse in HBB. The
wells were air dried and bound counts were determined using a
counter.
For these solid-phase binding studies, the
-actinin was radioiodinated to
a specific activity between 5.8 × 106 and 14.4 × 106 cpm/µg.
). Primary cultures derived from smooth muscle contained both fibroblasts and smooth muscle cells. We previously showed that differentiated smooth muscle cells, which express the smooth muscle marker calponin, also exhibit dramatically higher levels of CRP expression than
fibroblasts and undifferentiated smooth muscle cells in the culture (Crawford et al., 1994
). Based on these observations, one can unequivocally identify differentiated smooth muscle cells in the population based on
their CRP levels. Double-label indirect immunofluorescence (Beckerle,
1986
) was performed using an anti-
-actinin primary monoclonal antibody (ICN Biomedicals Inc., Irvine, CA) followed by an FITC-conjugated
goat anti-mouse secondary antibody, and an anti-cCRP1 primary polyclonal antibody (B37) raised against the eleven carboxy-terminal amino acids GQGAGALIHSQ of cCRP1 followed by a Texas red-conjugated goat
anti-rabbit secondary antibody. The B37 antibody was generated by K. Shepard and J.D. Pino in the Beckerle laboratory (University of Utah,
Salt Lake City, UT). All fluorochrome-labeled secondary antibodies were
obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove,
PA). Cells were viewed on a confocal laser scanning microscope (Bio-Rad
Laboratories, Hercules, CA) with an optical section height of 1 µm.
-actinin was detected using the polyclonal antibody raised against chicken
-actinin provided by
K. Burridge.
end) or NotI (3
end) restriction
sites. Amplified fragments were digested and ligated into a pcDNA1/
NEO vector (Invitrogen, Carlsbad, CA) that was modified by inserting sequences encoding the myc epitope (EQKLISEEDLL) downstream from
the NotI site. Ligation at this site generated in-frame CRP1, CRP1-LIM1,
and CRP1-LIM2 fusions with myc. Constructs were sequenced prior to
use. Plasmid DNAs were isolated using a polyethyleneglycol precipitation
procedure (Sambrook et al., 1989
) and were ultimately resuspended in
PBS for microinjection. Rat embryo fibroblast (REF52) cells were grown to 50-70% confluence on coverslips in a 1:3 mixture of Ham's F-12 and
DME containing 10% FBS, and microinjected with plasmid DNA at 250 ng/µl by a previously described technique in Beckerle and Porter (1983)
,
except an inverted microscope was used. Cells were fixed 24 h later and
processed for fluorescence microscopy with rhodamine-phalloidin (Molecular Probes, Inc., Eugene, OR) and indirect immunofluorescence
(Beckerle, 1986
) with anti-myc primary monoclonal antibody and FITC-conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA).
RESULTS
-Actinin from a CRP1-affinity Column
, 1994
) and retains the ability to bind zyxin
(Schmeichel and Beckerle, 1994
). When a 27-34% ammonium sulfate precipitate from the avian smooth muscle extract (Fig. 1 A, lane 2) is loaded on a CRP1 column, four
proteins of ~115, 100, 41, and 35 kD elute from the column with a high salt buffer as detected by silver staining (Fig. 1 B, lane 2). By Western immunoblot analysis using specific
antibodies, we determined that the 100-kD protein that
binds to the CRP1 column is
-actinin (Fig. 1 B, lane 3).
No protein was detected using antibodies against the two
cytoskeletal proteins, talin and vinculin (data not shown).
Because zyxin has previously been shown to interact with
both
-actinin and CRP1 (Crawford et al., 1992
; Sadler et al., 1992
), zyxin could theoretically have been responsible
for linking
-actinin to CRP1 in this experiment. However, no zyxin is detected in the 27-34% ammonium sulfate precipitate (Crawford and Beckerle, 1991
), and therefore the CRP1-
-actinin interaction can not be mediated
by zyxin. In control experiments, the 27-34% ammonium
sulfate precipitate was loaded on a BSA column. In this case,
no
-actinin was recovered after a high salt buffer elution as monitored by silver staining and Western immunoblot
(Fig. 1 B, lanes 4 and 5). Collectively, the results of these
experiments suggest that CRP1 can interact either directly
or indirectly with the actin binding protein
-actinin.
Fig. 1.
Specificity of the -actinin-CRP1 interaction under
nondenaturing conditions. (A) A Coomassie blue-stained gel
showing molecular mass markers M, purified
-actinin (lane 1),
and the 27-34% ammonium sulfate precipitate from avian
smooth muscle extract (lane 2) that was loaded onto the affinity
columns and used in the affinity resin binding assay. (B) Lane 1,
Western immunoblot analysis of the 27-34% ammonium sulfate
precipitate that was loaded onto the affinity columns using a
polyclonal antibody raised against chicken
-actinin; lane 2, silver-stained gel showing the proteins eluted from the CRP1 column; lane 3, Western immunoblot analysis of the proteins shown
in lane 2 using a polyclonal antibody raised against
-actinin; lane 4,
silver-stained gel showing the material eluted from the BSA column; lane 5, Western immunoblot revealed that no
-actinin
bound to the BSA column (
-a,
-actinin). (C) Coomassie blue-
stained gel showing the purified GST (lane 1) and GST-CRP1 (lane 2) proteins that were used to generate the affinity resins. (D) Western immunoblot analysis to detect chicken
-actinin.
The gel was loaded with
-actinin (lane 1) or a 27-34% ammonium sulfate precipitate from a smooth muscle cell extract (lane
2). Purified
-actinin or proteins found in the 27-34% ammonium sulfate precipitate were incubated with GST agarose (lanes
3 and 5) or GST-CRP1 agarose (lanes 4 and 6).
-Actinin binds
to the GST-CRP1 affinity resin. A mock affinity resin binding assay was performed with GST-CRP1 agarose beads in the absence
of
-actinin; no immunoreactive product is observed (lane 7). (E)
[125I]
-actinin was incubated with GST-CRP1 (left) or GST agarose beads (right) in the absence of competing proteins (+ buffer), in the presence of a 2,000-fold molar excess of unlabeled
-actinin (+ unlabeled
-actinin), or in the presence of an equivalent molar amount of BSA (+ BSA). The counts bound to the
agarose beads were analyzed using a
counter and expressed as a
percentage of bound [125I]
-actinin in absence of competing proteins. Mean and SEM from three experiments are shown.
[View Larger Versions of these Images (47 + 32K GIF file)]
-Actinin Is
Detected under Nondenaturing Conditions
-actinin, we used an affinity resin-binding assay. Purified
-actinin or a 27-34% ammonium sulfate precipitate containing
-actinin (Fig. 1 A) was incubated with GST-CRP1
or GST (Fig. 1 C) coupled to glutathione-agarose beads.
After washing the beads, the proteins that remained
bound to the GST or GST-CRP1 affinity resins were resolved by SDS-PAGE. Western immunoblot analysis revealed that GST-CRP1 (Fig. 1 D, lanes 4 and 6), but not
GST alone (Fig. 1 D, lanes 3 and 5), extracts
-actinin
from a solution of purified protein or from a complex mixture of proteins. No band corresponding to
-actinin is detected when GST-CRP1 agarose beads are incubated with
a solution that lacks
-actinin (Fig. 1 D, lane 7); this control confirms that detection of the immunoreactive 100-kD
protein is dependent on the addition of
-actinin and cannot be due to crossreactivity of the antibody with fusion
protein dimers that migrate at a similar molecular mass.
These experiments show a direct interaction between CRP1
and
-actinin under nondenaturing conditions.
-actinin interaction observed by the affinity resin-binding assay, we performed a competition experiment (Fig. 1 E). [125I]
-Actinin
was incubated with GST-CRP1 or GST agarose beads in the absence of competing proteins or in the presence of a
2,000-fold molar excess of either unlabeled
-actinin or
unlabeled BSA. The counts bound to the agarose beads
were measured using a
counter. The GST-CRP1 agarose
beads bind the radioiodinated
-actinin in the absence of
competing proteins or in the presence of BSA. In the presence of an excess of unlabeled
-actinin, binding of the radiolabeled
-actinin to GST-CRP1 is reduced to the level
obtained with GST agarose beads. These results demonstrate that the interaction between CRP1 and
-actinin is
direct, specific, and saturable under nondenaturing conditions.
-Actinin Is
Detected by the Blot Overlay Assay
-actinin interaction using a blot overlay assay that has been used previously to study many protein-protein interactions (Belkin
and Koteliansky, 1987; Crawford et al., 1992
; Sadler et al.,
1992
). We evaluated the ability of [125I]
-actinin to bind
directly to CRP1 present in fractions derived from an
avian smooth muscle extract. Three different ammonium sulfate precipitates that include a diverse collection of
smooth muscle-derived proteins were resolved by SDS-PAGE (Fig. 2 A) and transferred to nitrocellulose. CRP1
is found in the 34-43% ammonium sulfate (Fig. 2, lane 2)
but not in the 27-34 and the 43-61% ammonium sulfate
precipitates. Purified
-actinin was radioiodinated and used as a probe to examine its ability to interact with CRP1
that was immobilized on nitrocellulose (Fig. 2 B). The purity of the [125I]
-actinin used in this experiment is shown
in Fig. 2 C. Among the proteins that are precipitated from
the smooth muscle extract, [125I]
-actinin recognizes a protein that exhibits an apparent molecular mass of 23 kD,
corresponding to the molecular mass of CRP1. A number
of other abundant proteins present on the nitrocellulose membrane fail to interact with the radioiodinated
-actinin
showing the selectivity of the radiolabeled probe.
Fig. 2.
Demonstration of a direct interaction between CRP1
and [125I]-actinin using a blot overlay assay. (A) Coomassie
blue-stained gel showing a 27-34 (lane 1), a 34-43 (lane 2), and a
43-61% (lane 3) ammonium sulfate precipitates from an avian
smooth muscle extract. Proteins from a parallel gel were transferred to nitrocellulose and the nitrocellulose strip was probed
with [125I]
-actinin. The resulting autoradiograph shown in B illustrates [125I]
-actinin binding to CRP1. (C) Autoradiograph
demonstrating the purity of the radioiodinated
-actinin probe.
The position of the molecular mass markers is indicated on the
left, in kD.
[View Larger Version of this Image (73K GIF file)]
-actinin
interaction, a competition experiment was performed using the blot overlay assay. Purified CRP1 was resolved by
SDS-PAGE (Fig. 3 A) and transferred to nitrocellulose.
The immobilized CRP1 was probed with radioiodinated
-actinin in the absence or presence of a 2,000-fold molar
excess of unlabeled
-actinin or BSA (Fig. 3, B-D). The
radioiodinated probe interacts with the purified CRP1
confirming that, under the conditions of this experiment, [125I]
-actinin interacts directly with CRP1. Moreover, in
the presence of unlabeled
-actinin, but not in the presence of an equimolar amount of unlabeled BSA, the binding of the radioiodinated
-actinin to CRP1 was dramatically reduced. Collectively, these experiments demonstrate
that in the blot overlay assay, the association between CRP1 and
-actinin is direct, specific and saturable.
Fig. 3.
Specificity of the [125I]-actinin-CRP1 interaction. (A)
Coomassie blue-stained gel showing molecular mass markers and
the purified recombinant CRP1. Autoradiograph of parallel nitrocellulose strips probed with [125I]
-actinin in the absence of
competing protein (B), or in the presence of either a 2,000-fold
molar excess of unlabeled
-actinin (C), or a 2,000-fold molar excess of unlabeled BSA (D).
[View Larger Version of this Image (50K GIF file)]
-Actinin-CRP1 Interaction Displays a
Dissociation Constant in the Micromolar Range
-actinin and
CRP1 was characterized by a solid-phase binding assay.
Purified bacterially expressed CRP1 (Fig. 3 A) was adsorbed to microtiter wells, unoccupied sites on the plastic
wells were blocked with BSA, and the immobilized CRP1
was incubated with [125I]
-actinin. The amount of bound
[125I]
-actinin was determined by
counting. The specificity of the CRP1-
-actinin interaction in this solid-phase
binding assay was evaluated by comparing the ability of
unlabeled
-actinin or BSA to compete with radiolabeled
-actinin for binding to CRP1 (Fig. 4 A). A constant amount
of [125I]
-actinin was incubated in CRP1-coated wells in
the presence of increasing concentrations of competing
proteins. The interaction between CRP1 and [125I]
-actinin
is inhibited by the unlabeled
-actinin but not by an equivalent molar excess of BSA, demonstrating the specificity of the interaction between CRP1 and
-actinin in the
solid-phase binding assay. A typical curve predicted by the
simple binding reaction: CRP1 +
-A
CRP1·
-A, was
obtained by plotting the moles of
-actinin bound to CRP1
against the concentration of free
-actinin (Fig. 4 B). Half
maximum binding in this experiment occurs at 1.9 µM free
ligand. From the average of three different experiments
using two different probes we calculate an average Kd of
1.8 ± 0.3 µM (mean ± SEM) for the CRP1-
-actinin interaction.
Fig. 4.
Binding of [125I]-actinin to CRP1 in a solid-phase binding assay. (A) The
specificity of the association
between CRP1 and [125I]
-actinin in a solid-phase binding assay was analyzed in a
competition experiment. A
constant amount of [125I]
-actinin (0.26 pmoles in 120 µl) was incubated in CRP1-coated wells with increasing
concentrations of unlabeled
-actinin (+
-actinin) or
BSA (+ BSA). In this experiment, a total of 3,076 cpm
were bound specifically to
CRP1 when no competing
unlabeled
-actinin was
added. The data are expressed as a percentage of the maximum binding obtained when the [125I]
-actinin is incubated with the CRP1-coated wells in the absence of competing
protein. (B) From the competition experiment shown in A, the
moles of
-actinin bound to CRP1 were plotted as a function of
the free
-actinin concentration. In this particular experiment, the
-actinin was radioiodinated to a specific activity of 5.8 × 106
cpm/µg; assuming a mol wt of 200,000 g/mol for
-actinin. The calculated dissociation constant (Kd) was 1.9 µM. The mean dissociation constant determined from three different experiments
using two different probes is 1.8 ± 0.3 µM (mean ± SEM).
[View Larger Version of this Image (20K GIF file)]
-Actinin Display Overlapping Subcellular
Distributions in CEF and Smooth Muscle Cells
-actinin to associate with each other in vitro. If this interaction also occurs in vivo, one might expect CRP1 and
-actinin to be colocalized in cells. To examine this possibility, we performed double-label immunofluorescence
microscopy using an anti-peptide antibody (B37) raised
against a sequence in cCRP1. By Western blot analysis of
a CEF lysate, the B37 antibody recognizes a single band
that exhibits an apparent molecular mass of 23 kD (Fig. 5 B) and comigrates with CRP1 (data not shown); no protein is detected using the preimmune serum (Fig. 5 B).
Similarly, a single band that migrates at an apparent molecular mass of 23 kD is immunoprecipitated from a detergent extract of [35S]methionine-labeled CEF under denaturing conditions, whereas no immunoprecipitated band is
detected under the same conditions when the preimmune
serum is used (Fig. 5 C). The B37 antibody was used to
compare the subcellular distributions of CRP1 and
-actinin using double-label indirect immunofluorescence in
CEF cells and in a primary culture of smooth muscle cells
from chicken gizzard (Fig. 6). By this approach, we observe that CRP1 and
-actinin are extensively colocalized
in cells along the actin stress fibers (Fig. 6, C and F), in accordance with the idea that they could interact in vivo. We
also observed that both proteins are present at the leading
edge of the cells, and in the adhesion plaques (Fig. 6 F).
However, in some adhesion plaques, where
-actinin is
present, CRP1 is not detected (data not shown). This observation is consistent with a previous report showing that
CRP1 is present in some adhesion plaques of CEF cells
but not in others (Crawford et al., 1994
).
Fig. 5.
Characterization of an anti-peptide antibody (B37) directed against cCRP1. (A) A Coomassie blue-stained gel showing molecular mass markers M and total CEF proteins L. (B) A
parallel gel was transferred to nitrocellulose and probed with the
anti-CRP1 antibody B37 or its corresponding preimmune serum
pre. A single polypeptide of 23 kD is recognized by the antibody.
(C) Autoradiograph of a gel loaded with a CEF lysate prepared
from [35S]methionine-cysteine-labeled cells L, the proteins immunoprecipitated from this lysate with the polyclonal antibody
raised against CRP1 B37, and with its corresponding preimmune
serum pre. A single protein of 23 kD is specifically immunoprecipitated with the antibody against CRP1.
[View Larger Version of this Image (66K GIF file)]
Fig. 6.
CRP1 and -actinin are extensively codistributed in CEF and in smooth muscle cells. CEF cells (A-C) and smooth muscle
cells (D-F), prepared for confocal indirect immunofluorescence microscopy, were double-labeled with a polyclonal antibody raised
against CRP1 (A and D), and a monoclonal antibody raised against
-actinin (B and E). C and F are composite images of CRP1 (green)
and
-actinin (red) staining; the overlapping regions appear in yellow. Confocal microscopy reveals that CRP1 and
-actinin are extensively colocalized along the actin stress fibers. Both
-actinin and CRP1 are detected at the leading edges of the cells (arrows) and in the
adhesion plaques (arrowheads and data not shown). Bars, 30 µm.
[View Larger Version of this Image (30K GIF file)]
-Actinin in
Smooth Muscle Cells
-actinin in
vivo. CRP1 can be immunoprecipitated from a smooth
muscle cell extract of smooth muscle cells under nondenaturing conditions using the B37 anti-CRP1 antibody (Fig.
7 A). Under these conditions,
-actinin is detected in the
immunoprecipitate with CRP1 (Fig. 7 B), whereas another cytoskeletal protein, vinculin, is not detected (data not
shown). Neither CRP1 nor
-actinin is detected when the
preimmune serum is used in the immunoprecipitation assay (Fig. 7, A and B). These data provide evidence that
-actinin and CRP1 can be recovered as a complex from
smooth muscle cells.
Fig. 7.
An in vivo interaction between CRP1 and
-actinin in smooth muscle
cells. Proteins were immunoprecipitated from a chicken gizzard smooth muscle lysate L with the polyclonal antibody raised against CRP1
B37 and with the corresponding preimmune serum pre. The immunoprecipitated proteins were resolved by SDS-PAGE and were transferred
to nitrocellulose and probed
with polyclonal antibodies
raised against CRP1 (A) or
-actinin (B).
-actinin is immunoprecipitated under nondenaturing conditions with the anti-CRP1 antibody, but not with the preimmune serum. The position of the molecular mass markers is indicated on the left in kD.
[View Larger Version of this Image (49K GIF file)]
-Actinin That
Participate in the Interaction of the Two Proteins
-actinin, we performed a blot overlay experiment using labeled CRP1.
-Actinin can be separated into two well-characterized
proteolytic products of 53 and 27 kD by cleavage with
thermolysin. The 27-kD fragment has been shown to interact with zyxin, vinculin, and actin, whereas the 53-kD fragment is essential for dimerization of the protein and for
interacting with the cytoplasmic domain of
1 integrin receptors for extracellular matrix (Mimura and Asano, 1986
;
Otey et al., 1990
; Pavalko and Burridge, 1991
; Crawford et
al., 1992
). Fig. 8 A shows a Coomassie blue-stained gel of
purified
-actinin and the products of partial proteolytic
cleavage with thermolysin. Proteins from a parallel gel
were transferred to nitrocellulose and the resulting blot
was incubated with [32P]GST-CRP1 (Fig. 8 B). By this blot
overlay approach, [32P]GST-CRP1 associates directly with
-actinin and also prominently with the 27-kD actin-binding domain of
-actinin; no interaction of CRP1 with the
53-kD fragment is observed. When [32P]GST is used as a
probe, no detectable protein binding is observed (Fig. 8
C). The purity of the 32P-labeled probes used in this experiment is shown in Fig. 8 D. These results demonstrate that
the binding site for CRP1 on
-actinin is in the 27-kD actin-binding domain of
-actinin.
Fig. 8.
CRP1 interacts with the 27-kD
actin-binding site of -actinin. (A) A Coomassie blue-stained gel showing molecular mass markers M, purified
-actinin
(lane 1), and the 53- and 27-kD proteolytic products of
-actinin generated by thermolysin cleavage (lane 2). Autoradiograph of overlay assay performed on parallel nitrocellulose strips containing purified
-actinin (lanes 1
and 1
) and the proteolytic fragments of
-actinin (lanes 2
and 2
) probed with [32P]GST-CRP1 (B),
or [32P]GST (C). Note that in the experiment shown, thermolysin cleavage of
-actinin was not complete, therefore
products other than the 53- and 27-kD
fragments are also detected. (D) Autoradiograph illustrating the quality of the
bacterially expressed, purified, 32P-labeled
probes, [32P]GST-CRP1 and [32P]GST.
[View Larger Version of this Image (38K GIF file)]
). To characterize the binding
site for
-actinin on CRP1, we compared the ability of
-actinin to interact with full-length CRP1 and two peptides, CRP1-LIM1 and CRP1-LIM2, derived from the intact protein. CRP1-LIM1 corresponds to the NH2-terminal part of CRP1 (aa 1-107) containing the NH2-terminal
LIM domain followed by the first glycine-rich repeat of
the protein, and CRP1-LIM2 corresponds to the COOH-terminal part of the protein (aa 108-192) containing the
COOH-terminal LIM domain and the second glycine-rich
repeat. CRP1, CRP1-LIM1, and CRP1-LIM2 were resolved by SDS-PAGE (Fig. 9 A), were transferred to nitrocellulose and were probed for their ability to interact
with [125I]
-actinin in a blot overlay assay (Fig. 9 B). The
radioiodinated
-actinin interacts with the bacterially expressed purified CRP1 and with CRP1-LIM1. The molar
amounts of the two single LIM peptides, CRP1-LIM1 and
CRP1-LIM2, loaded on the gel was twice the amount loaded for the double LIM protein, CRP1. Although the
[125I]
-actinin bound only to intact CRP1 and the CRP1-LIM1 peptide, the binding to the deletion construct
reached only about 50% of the binding observed with full-length CRP1, as measured by PhosphorImager analysis
(data not shown). No interaction is detected between the
[125I]
-actinin and CRP1-LIM2. Thus it appears that the
CRP1-LIM1 peptide contains sequence information that
establishes a docking site for
-actinin; the generation of
the CRP-LIM1 truncation may have rendered the
-actinin
binding site suboptimal. We cannot rule out the possibility
that other low affinity binding sites for
-actinin exist in
CRP1, however, the only site we have been able to map is
within the CRP1-LIM1 region.
Fig. 9.
The binding site for -actinin on CRP1 is contained
within the CRP1-LIM1 fragment. (A) Coomassie blue-stained
gel showing the purified CRP1 (lane 1), the purified CRP1-LIM1
fragment (lane 2) and the purified CRP1-LIM2 fragment (lane
3). 100 pmoles of CRP1, 200 pmoles of CRP1-LIM1, and 200 pmoles of CRP1-LIM2 were loaded on the gel. The positions of
CRP1, CRP1-LIM1, and CRP1-LIM2 are marked (CRP1, LIM1,
and LIM2, respectively). The corresponding blot overlay assay
probed with [125I]
-actinin is shown in B. (C) Autoradiograph illustrating the purity of the radioiodinated
-actinin probe. The
position of the molecular mass markers is indicated on the left
in kD.
[View Larger Version of this Image (55K GIF file)]
-actinin interacts with CRP1-LIM1,
the NH2-terminal part of CRP1, we examined the possibility that the CRP1-LIM1 peptide contains sequence information involved in targeting the protein to the actin
cytoskeleton. Eukaryotic expression constructs encoding
epitope-tagged full-length CRP1, CRP1-LIM1, and CRP1-LIM2 were microinjected into cells. We used double-label
indirect immunofluorescence to compare the subcellular
distributions of the expressed portions of CRP1 and the
actin stress fibers. The expressed CRP1 is associated with
the actin cytoskeleton (Fig. 10, A and B); this localization corresponds to the typical distribution of endogenous
CRP1 in fibroblasts (Sadler et al., 1992
; Crawford et al.,
1994
). The CRP1-LIM1 peptide also localizes with F-actin
(Fig. 10, C and D) whereas expressed CRP1-LIM2 fails to
associate with the actin cytoskeleton (Fig. 10, E and F).
We detected some nuclear localization of the two deletion
constructs, CRP1-LIM1 and CRP1-LIM2; however, the
significance of this finding is not clear. Some expressed protein is found in a punctate pattern that does not correspond to the distribution of filamentous actin; because we
do not observe such a prominent punctate pattern when
we visualize endogenous CRP1 by indirect immunofluorescence, the physiological relevance of this distribution is
questionable. These heterologous expression studies in rat
embryo fibroblasts reveal that deletion of aa 1-107 from
CRP1 eliminates the protein's ability to localize to the actin cytoskeleton. The NH2-terminal 107 aa of CRP1 retains the capacity to localize to the cytoskeleton illustrating that this region is both necessary and sufficient to
support the cytoskeletal association of CRP1.
Fig. 10.
CRP1-LIM1 is targeted to actin stress fibers. Expression constructs encoding myc-tagged CRP1 (A and B), CRP1-LIM1 (C
and D), or CRP1-LIM2 (E and F) were microinjected into rat embryo fibroblast (REF52) cells. Double-label immunofluorescence was
used to compare the subcellular distributions of the expressed CRP1 polypeptides (A, C, and E) and the actin stress fibers (B, D, and F).
The expressed proteins were visualized using an anti-myc monoclonal antibody whereas the actin stress fibers were visualized with phalloidin. Bar, 30 µm.
[View Larger Version of this Image (105K GIF file)]
DISCUSSION
-actinin, as a new binding partner for CRP1, a LIM domain protein that has been implicated in the process of
muscle differentiation. We have used a variety of solution
and solid-phase binding assays to demonstrate and characterize an association between
-actinin and CRP1. By
these approaches we have shown a direct, specific, and saturable interaction between
-actinin and CRP1. Because both smooth muscle and bacterially expressed CRP1 interact with
-actinin, eukaryotic posttranslational modification of CRP1 is not required for binding of these two proteins. From our solid-phase binding studies, the interaction
between
-actinin and CRP1 appears to occur at a single
site with a ~1.8 µM Kd, corresponding to a moderate affinity interaction between the two proteins. The dissociation constant values calculated for the interactions between
-actinin and its other binding partners are in the
same range (Fig. 11 A). The biochemical studies that provide evidence for an interaction between
-actinin and
CRP1 are supported by immunocytochemical studies that
demonstrate that the primary distributions of
-actinin and CRP1 in CEF and smooth muscle cells are very similar, with both proteins prominently concentrated along the
actin cytoskeleton and to a more limited extent within
adhesion plaques. Although some immunostaining of cell
nuclei is evident with anti-CRP antibodies and some accumulation of the proteins within nuclei is observed in overexpression studies, the physiological relevance of the nuclear CRPs is not clear. CRPs are relatively small proteins that would not be excluded from nuclei based on size.
If CRP did diffuse into the nucleus of a cell, it might be
passively trapped there as a result of its basic nature; the
isoelectric point of CRP1 is 8.5 (Crawford et al., 1994
).
That said, it is not possible to rule out a nuclear function
for CRP1; this possibility remains intriguing since the
three-dimensional conformation of a LIM domain derived
from CRP1 exhibits features that would be expected to be
compatible with nucleic acid binding (Perez-Alvarado et
al., 1994
). Moreover, Drosophila CRPs are not excluded
from cell nuclei in the developing musculature, in contrast
with myosin which is clearly excluded from the nuclear
compartment in the same cells (Stronach et al., 1996
).
Fig. 11.
-Actinin and its
binding partners. (A) A summary of the interactions between
-actinin and its binding partners: integrin, zyxin,
actin, vinculin, and CRP1.
References for the dissociation constant values are as
follows:
-actinin-integrin,
(Otey et al., 1990
);
-actinin-
zyxin, (Crawford et al.,
1992
);
-actinin-vinculin, (Wachsstock et al., 1987
);
-actinin-actin, (Wachsstock
et al., 1993
);
-actinin-CRP1,
this report; vinculin-actin,
(Menkel et al., 1994
). (B) A
schematic representation of
an adhesion plaque showing
a model for the association of
-actinin with its known binding partners within a cell.
-Actinin and CRP1 may cooperate to localize a complex
of zyxin, Ena/VASP, and profilin and thus could participate in
the regulation of actin assembly dynamics. (Z) zyxin; (P) profilin;
(V) vinculin; (
-A)
-actinin; (X) other CRP1-binding partners;
(PM) plasma membrane; (ECM) extracellular matrix.
[View Larger Version of this Image (29K GIF file)]
-Actinin has been extensively studied and much is understood about its biochemical properties.
-Actinin forms
antiparallel homodimers (Wallraff et al., 1986
; Imamura et
al., 1988
) that cross-link actin filaments into parallel arrays
(Maruyama and Ebashi, 1965
; Podlubnaya et al., 1975
). In
nonmuscle cells such as cultured fibroblasts,
-actinin is
found along the stress fibers and in the adhesion plaques
where actin filament bundles associate with the plasma
membrane (Lazarides and Burridge, 1975
). In striated and
smooth muscle,
-actinin is most prominently concentrated in the Z discs and dense bodies and plaques, respectively (Blanchard et al., 1989
). Mutation of the gene encoding
-actinin in Drosophila results in disorganized
myofibrillar arrays and reduced muscle function (Fyrberg
et al., 1990
; Roulier et al., 1992
).
-actinin has the
capacity to interact with four different adhesion plaque
and cytoskeletal proteins: integrin, vinculin, zyxin, and actin. A model for the associations among these proteins,
based on what has been learned from protein binding
studies, is shown in Fig. 11 B. Binding studies using the
proteolytic fragments of
-actinin digested by thermolysin have shown that the
1 integrin subunit interacts with the
53-kD rod-like domain of
-actinin (Otey et al., 1990
),
whereas vinculin, zyxin, and actin interact with the 27-kD
globular head of
-actinin (Mimura and Asano, 1986
;
Pavalko and Burridge, 1991
; Crawford et al., 1992
). Here
we have demonstrated an interaction between CRP1 and
the 27-kD globular head domain of
-actinin. We have also shown that
-actinin interacts with the NH2-terminal
region of CRP1 (CRP1-LIM1) which contains one LIM
domain followed by a glycine-rich repeat. Moreover, by
heterologous protein expression experiments, we have
shown that the full-length CRP1 and the CRP1-LIM1 peptide have the capacity to localize along the actin cytoskeleton whereas the COOH-terminal part of the protein does
not. These results illustrate that the sequence information
required both for
-actinin binding and the cytoskeletal
localization of CRP1 is contained within the NH2-terminal
107 aa of the protein. Based on these observations, we
speculate that it is CRP1's ability to bind to
-actinin that
targets it to the actin cytoskeleton; however, additional
work will be necessary to demonstrate whether this is indeed the case. The primary features of the NH2-terminal 107 aa of CRP1 are the presence of a single LIM domain
and a glycine-rich repeat. Since LIM domains have been
shown to function in specific protein-protein interactions,
it seems likely that the COOH-terminal LIM domain of
CRP1 will also interact with a specific binding partner.
CRP1 could thus serve as an adaptor protein that is targeted to the actin cytoskeleton by virtue of an interaction
with
-actinin. The COOH-terminal LIM domain of CRP1
may function as a ligand for a factor whose function depends on a cytoskeletal localization (Fig. 11 B).
-actinin in smooth muscle cells raises
the possibility that
-actinin and CRP1 cooperate to perform an essential function in smooth muscle differentiation. One intriguing possibility is that the two proteins collaborate to localize protein machineries involved in actin assembly or dynamics (Fig. 11 B). It is interesting in this
regard that both
-actinin and CRPs bind zyxin, a protein
that has been implicated in the spatial control of actin assembly by virtue of its ability to bind Ena/VASP family
members that associate with profilin (Reinhard et al., 1995 a, b; Gertler et al., 1996
). In future work, it will be very important to perform immunocytochemical studies to characterize the subcellular distributions of CRP1 and
-actinin
within intact tissues. Likewise, functional studies will be
essential in order to assess the physiological significance of
the CRP1-
-actinin interaction in vivo.
-actinin. Our data are
consistent with the possibility that the localization of
CRP1 along the actin cytoskeleton is due to the interaction between the NH2-terminal LIM domain of CRP1 and
the actin-binding protein,
-actinin. CRP1 has been implicated as a key regulator in the control of muscle differentiation. The appropriate targeting of CRP1 to the actin cytoskeleton is likely to be important for the function of the
protein during myogenesis. Given the finding that loss of
one CRP family member, MLP/CRP3, results in dramatic
disorganization of myofibrils (Arber et al., 1997
), it is reasonable to speculate that the appropriate localization and
function of CRPs at subcellular domains that are enriched in
-actinin may be required in order for a cell to build or
maintain the semicrystalline arrays of actin and myosin filaments that constitute the contractile machinery.
Received for publication 6 December 1996 and in revised form 2 July 1997.
Address all correspondence to Mary Beckerle, Department of Biology, 201 South Biology Building, University of Utah, Salt Lake City, UT 84112-0840. Tel.: (801) 581-4485. FAX: (801) 581-4668. E-mail: beckerle{at}bioscience.utah.eduThe authors thank all members of the Beckerle laboratory for helpful discussions (University of Utah, Salt Lake City, UT). We are particularly
grateful to K.L. Schmeichel who generously provided the CRP1-LIM1
and CRP1-LIM2 expression constructs used in this study. Thanks go to E. King for assistance and helpful discussions with confocal immunofluorescence microscopy. We thank K. Shepard and J.D. Pino for generating the
B37 antibody, K. Burridge (University of North Carolina, Chapel Hill,
NC) for his generous gift of -actinin antibodies, and S.A. Liebhaber
(University of Pennsylvania School of Medicine, Philadelphia, PA) for
providing the GST-hCRP1 expression construct.
aa, amino acids; CEF, chicken embryo fibroblasts; CRP, cysteine-rich protein; HBB, Hepes binding buffer; GST, glutathione-S-transferase.
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