Báo cáo khoa học: Tyrosine phosphorylation of calponins Inhibition of the interaction with F-actin pot

Tyrosine phosphorylation of calponins
Inhibition of the interaction with F-actin
Julien Abouzaglou
1
, Christine Be
´
nistant
1
, Mario Gimona
2
, Claude Roustan
3
, Rhida Kassab
1
and Abdellatif Fattoum
1
1
Centre de Recherches de Biochimie Macromole
´
culaire du CNRS, Montpellier, France;
2
Institute of Molecular Biology,
Salzburg, Austria;
3
UMR 5539, CNRS, UM2, EPHE, Montpellier, France
The phosphorylation-dephosphorylation of serine and
threonine residues of calponin is known to modulate in vitro
its interaction with F-actin and is thought to regulate several
biological processes in cells, involving either of the calponin
isoforms. Here, we identify, for the first time, tyrosine-
phosphorylated calponin h3 within COS 7 cells, before and

after their transfection with the pSV vector containing
cDNA encoding the cytoplasmic, Src-related, tyrosine kin-
ase, Fyn. We then describe the specific tyrosine phosphory-
lation in vitro of calponin h1 and calponin h3 by this kinase.
32
P-labeling of tyrosine residues was monitored by combined
autoradiography, immunoblotting with a specific phospho-
tyrosine monoclonal antibody and dephosphorylation with
the phosphotyrosine-specific protein phosphatase, YOP.
PhosphorImager analyses showed the incorporation of
maximally 1.4 and 2.0 mol of
32
P per mol of calponin h3 and
calponin h1, respectively. As a result, 75% and 68%,
respectively, of binding to F-actin was lost by the phos-
phorylated calponins. Furthermore, F-actin, added at a two-
or 10-fold molar excess, did not protect, but rather increased,
the extent of
32
P-labeling in both calponins. Structural
analysis of the tryptic phosphopeptides from each
32
P-labe-
led calponin revealed a single, major
32
P-peptide in calponin
h3, with Tyr261 as the phosphorylation site. Tyr261 was also
phosphorylated in calponin h1, together with Tyr182. Col-
lectively, the data point to the potential involvement, at least
in living nonmuscle cells, of tyrosine protein kinases and the

conserved Tyr261, located in the third repeat motif of the
calponin molecule, in a new level of regulation of the actin–
calponin interaction.
Keywords: actin; calponin h1; calponin h3; tyrosine phos-
phorylation.
Calponin belongs to a family of actin-binding proteins,
which includes the basic and neutral calponin isoforms,
designated h1 and h2, respectively, both of which are
present in smooth muscle [1,2], and the acidic calponin, h3,
expressed in smooth muscle as well as in nonmuscle cells [3].
They are thought to be involved in a variety of biological
processes, such as the regulation of smooth muscle
contraction, by inhibiting the actomyosin ATPase activity
[4,5], the organization of the actin cytoskeleton in smooth
muscle and nonmuscle cells by stabilizing actin networks
[6–8], and cell signaling at the surface membrane of vascular
smooth muscle [9,10]. All calponin isoforms are composed
of a conserved N-terminal calponin homology domain,
followed by a primary actin-binding consensus sequence
and three 30-residue tandem repeats harboring a secondary
actin-binding site and associated with a C-terminal tail of
variable length and primary structure [11–13]. Their actin-
binding properties are believed to be regulated by two main
factors, revealed by in vitro studies, namely the interaction
of calponin with calcium-binding proteins, such as calmod-
ulin [5], and the phosphorylation of specific serine/threonine
residues by protein kinase C and Ca
2+
calmodulin-
dependent protein kinase II [14,15]. Both regulatory events

lead to the dissociation of the calponin–F-actin complex
in vitro. Given the relatively low affinity of calponin for
calmodulin [5], the second process would be the most active
effector in vivo. On the other hand, tyrosine phosphoryla-
tion of calponin could represent an additional mechanism
involved in the regulation of the calponin–actin interaction.
In this regard, in recent years several actin-binding proteins,
including villin [16,17], gelsolin [18,19] and cortactin [20],
have been shown to be tyrosine phosphorylated with
concomitant changes in their actin-binding properties, and
most are, like calponin, proteins participating in the
reorganization of the actin cytoskeleton. Less well charac-
terized is the tyrosine phosphorylation of calponins and its
effects on the actin-binding capacity of the different
calponin isoforms. To gain further insight into the mech-
anisms regulating the calponin–actin interaction, we
assessed in this study, for the first time, the production of
tyrosine-phosphorylated calponin h3 in COS 7 cells and
investigated the tyrosine phosphorylation in vitro of both
calponin h3 and calponin h1 by the cytoplasmic, Src-related
protein tyrosine kinase, Fyn. Our quantitative data illustrate
the specific incorporation of
32
P into each calponin, which
takes place both in the absence and in the presence of
F-actin, together with the resulting significant decrease of
F-actin binding to either phosphorylated calponin. More-
over, structural studies, using radio-Edman sequencing, and
Correspondence to A. Fattoum, Centre de Recherches de Biochimie
Macromole

´
culaire, CNRS UPR 1086, 1919, route de Mende,
F-34293, Montpellier Cedex 5, France. Fax: + 33 467 521559,
Tel.: + 33 467 613338, E-mail:
Note: a website is available at l
(Received 4 February 2004, accepted 28 April 2004)
Eur. J. Biochem. 271, 2615–2623 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04190.x
direct amino acid sequencing of
32
P-labeled peptides from
each calponin, allowed identification of the conserved
Tyr261, located in the third repeat motif of the calponin
molecule, as the major target of the kinase reaction.
Together, the results suggest that the enzymatic tyrosine
phosporylation of calponin represents a biologically rele-
vant process which can occur both in vivo and in vitro,with
Tyr261 of the protein forming a novel site involved in the
regulation of interactions between calponins and F-actin.
Materials and methods
Chemicals
Monoclonal anti-phosphotyrosine Ig (clone 4G10) was
purchased from Upstate Biotechnology Inc., and
[
32
P]dATP[cP] (5000 CiÆmmol
)1
) was from Amersham
Biosciences. The recombinant full-length human protein
tyrosine kinase, Fyn, had a specific activity of 1035 UÆmg
)1

and was obtained from Upstate Biotechnology Inc. The
recombinant protein tyrosine phosphatase, YOP, from
Yersinia enterocolitica was supplied by New England Bio-
Labs Inc. at a specific activity of 100 000 UÆmg
)1
.Trypsin
was from Worthington and ATP was from Boehringer.
Protein preparations
F-actin from rabbit skeletal muscle was prepared as
described previously [21]. Basic calponin h1 was isolated
from fresh turkey gizzards, as described previously [21].
Recombinant rat acidic calponin h3 was expressed as
described previously [13]. The protein concentrations were
measured spectrophotometrically using A
1%
280
¼ 11.7 for
actin, 7.6 for calponin h1, and 8.9 for calponin h3 [22].
Tyrosine phosphorylation of calponins with Fyn
Calponin h1 or calponin h3 (0.16 lg) were phosphorylated
in vitro with increasing concentrations of Fyn
(0–0.20 UÆlg
)1
of calponin) in an assay mixture (30 lL)
containing 25 m
M
Hepes, pH 7.5, 1 m
M
dithiothreitol,
12 m

M
MnCl
2
,0.16lCi of [
32
P]dATP[cP] and 5 l
M
ATP.
The reactions were carried out for 60 min at 30 °Cand
stopped by the addition of Laemmli sample buffer. Tyro-
sine-phosphorylated calponins were separated by SDS–
PAGE and the radioactivity incorporated into each protein
was detected by autoradiography and quantified by Phos-
phorImager analysis (Typhon 9200; Amersham Bioscienc-
es) or by densitometric measurements of the
autoradiograms using the Scan Analysis software of Biosoft
(Cambridge, UK). The calponin phosphorylation in the
presence of F-actin was conducted under the same experi-
mental conditions using 0.20 U of Fyn per lg of calponin, a
calponin/actin molar ratio of 1 : 2 or 1 : 10, and an
incubation period of 10 min. A phosphorylation control
of F-actin, without calponin, was carried out in parallel.
After gel electrophoresis, the amounts of radioactivity
associated with calponin and actin were measured.
The dephosphorylation of calponins was achieved using
the final 30 lL of Fyn kinase mixture, which was adjusted
to 50 m
M
Tris/HCl, pH 7.0, 100 m
M

NaCl, 2 m
M
EDTA,
5m
M
dithiothreitol, 0.01% Brij 35 and supplemented with
1mgÆmL
)1
BSA and 50 U of YOP protein tyrosine
phosphatase. After 60 min at 30 °C, the reaction was
terminated by the addition of Laemmli sample buffer. The
extent of tyrosine dephosphorylation was assessed by SDS/
PAGE and autoradiography.
Cell culture and transfections
COS 7 cells were grown to 60–80% confluence in DMEM
(Dulbecco’s modified Eagle’s medium) supplemented with
10% fetal bovine serum and penicillin/streptomycin
(10 UÆmL
)1
/10 lgÆmL
)1
)at37°Cand5%CO
2
. Cells
were transfected with the pSV vector containing the
cDNA encoding Fyn kinase [23], or with the empty pSV
vector. The transfection was conducted by using the
lipofectamin system (Gibco) according to the protocol of
the manufacturer. The transfected cells were incubated for
36 h and, after the addition of 1 m

M
Na vanadate,
incubated for a further 30 min. They were then washed
twiceincoldNaCl/P
i
(PBS), scraped into 400 lLof
RIPA buffer [20 m
M
Tris/HCl, pH 7.5, 150 m
M
NaCl,
1% (v/v) Triton-X-100, 1% Na deoxycholate, 0.1% SDS
containing 1 m
M
dithiothreitol, 1 m
M
Na vanadate, 1 m
M
NaF, 1 m
M
phenylmethanesulfonyl fluoride, 10 lgÆmL
)1
aprotinin and 20 l
M
leupeptin] and then incubated at
4 °C for 10 min. The overall cell medium was subse-
quently potterized 10· in a Dounce potter with a small
clearance to achieve an efficient extraction of proteins,
and then centrifuged at 20 000 g for 5 min at 4 °C. The
supernatants were collected and subjected to immuno-

precipitation with affinity-purified anti-(calponin h3) Ig
(20 lg). The immunocomplexes were recovered over
Protein A–Sepharose beads (Pharmacia), which were
washed once in RIPA buffer and twice in WLB buffer
(20 m
M
Tris/HCl, pH 7.5, 150 m
M
NaCl, 1% Nonidet
P-40 containing 1 m
M
dithiothreitol and 1 m
M
Na
vanadate). Beads were resuspended in Laemmli sample
buffer and centrifuged. The supernatants were submitted
to 10% acrylamide gel electrophoresis and Western
blotting analyses using anti-phosphotyrosine, anti-
(calponin h3) or anti-Fyn [23] Ig.
Electrophoresis and immunoblots
Unless stated otherwise, SDS–PAGE was carried out in
5–18% gradient acrylamide gels [24]. The running buffer
was 50 m
M
Tris, 100 m
M
boric acid, pH 8.0. The gels
were stained with Coomassie Blue R-250 and destained
with 7% acetic acid. Densitometric analysis of protein
bands on electrophoretic gels was carried out using the

SCAN ANALYSIS
software of Biosoft. Following electro-
phoresis, phosphorylated calponins were then transferred
to nitrocellulose membranes and subjected to immuno-
blotting with phosphotyrosine-specific monoclonal anti-
body (diluted 1 : 1000) and secondary anti-mouse
immunoglobulin G (IgG) conjugated to horseradish
peroxidase (diluted 1 : 5000). The reaction was revealed
by the enhanced chemiluminescence technique, using the
kit from Amersham Biosciences [25]. Immunoblots were
prepared in a similar manner using affinity-purified anti-
(calponin h3) Ig directed to the specific C-terminal tail of
the protein (E 311-Q 326) [26].
2616 J. Abouzaglou et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Co-sedimentation assays
Calponin h1 or h3 (5 lg), in a final volume of 50 lL, was
first phosphorylated with nonradioactive ATP (5 l
M
)and
3.4UofFynin25 m
M
Hepes, pH 7.5, under the conditions
described above. F-actin was then added at a twofold molar
excess over calponin. After incubation for 30 min at 30 °C,
the protein mixtures were centrifuged, in a Beckman
Airfuge, at 90 000 g for 30 min at 25 °C. The same
experiments, carried out in the absence of Fyn, were used
as controls. The supernatants and pellets that were homo-
genized in the same buffer, were subjected to gel electro-
phoresis.

Structural analyses of tryptic phosphopeptides
32
P-labeled calponin h1 or calponin h3 (7 lg) were first
purified by SDS–PAGE in 10% gels. After staining with
Coomassie Blue R-250, the corresponding bands were
excised and subjected to in-gel digestion with trypsin
according to Rosenfeld et al. [27]. The resulting digests
were extracted from the gel slices with 50% acetonitrile and
concentrated in vacuo. Each peptide mixture was fraction-
ated by reverse-phase HPLC on a 2· 100 mm, Aquapore
C8 column (Applied Biosystems), eluted with a linear
acetonitrile gradient (5–30%) in 0.1% trifluoroacetic acid
for 60 min, at a flow rate of 0.18 mLÆmin
)1
, and monitored
by Cerenkov counting. The radioactive fractions were
separated and radiosequenced with a Procise sequencer
(Applied Biosystems) using a program which allows detec-
tion of the position of
32
P-labeled amino acids by counting
the radioactivity of the anilino-thiazolinone derivative
released at each Edman degradation cycle. For direct amino
acid sequencing, each radioactive peak was further purified
by HPLC on a 1· 100 mm Aquapore C 18 column eluted
with a linear gradient of 9–33% acetonitrile. Sequence
determination of each pure phosphopeptide was performed
with the same Procise sequencer using the standard pulsed-
liquid program.
Results

Tyrosine phosphorylation of calponin h3 in COS 7 cells
We initiated our work by experiments aiming to determine
the ability of calponin h3 to be phosphorylated on tyrosine
in cultured COS 7 cells by cytoplasmic, Src-related protein
tyrosine kinases, such as Fyn. Our attention was focused
towards this enzyme because it is an ubiquitous kinase
expressed in most tissues and it exhibits the functional
properties of all the other Src kinases [28]. Moreover,
transfection of COS 7 with the pSV vector carrying Fyn
cDNA takes place with a high yield.
To assess tyrosine phosphorylation of the endogenous
calponin h3 present in COS 7 cells, we employed protein
extracts derived from cells transfected either with the pSV
vector carrying the Fyn cDNA, or with the empty pSV
vector. Na vanadate was added to all extraction buffers to
inhibit protein phosphotyrosine phosphatases. Calponin h3
was then immunoprecipitated with the affinity-purified anti-
(calponin h3) Ig. The immune complexes were separated by
SDS gel electrophoresis and probed with primary antibodies
directed to phosphotyrosine or to calponin h3. COS 7 cells
transfected with the empty pSV vector gave rise to a
detectable band, migrating at 36 kDa, which was identified
as calponin h3 by immunoblotting with the anti-(calponin
h3) Ig (Fig. 1A, lane 1, panel c). This band showed a faint
intensity when probed with anti-phosphotyrosine Ig
(Fig. 1A, lane 1, panel b). The cells transfected with the
pSV vector containing Fyn cDNA yielded an identical
calponin h3 species (Fig. 1A, lane 2, panel c) but with a
significantly increased intensity when probed with the
phosphotyrosine antibody (Fig. 1A, lane 2, panel b). The

densitometric measurements of the blots, presented in
Fig. 1B, show a threefold higher intensity of the phos-
phorylated calponin in the Fyn cDNA-transfected cells
Fig. 1. Tyrosine phosphorylation of calponin h3 in COS 7 cells. (A) The
lysates prepared from COS 7 cells transfected with pSV vector con-
taining Fyn cDNA (lane 2) or with the empty pSV vector (lane 1) were
treated with anti-(calponin h3) Ig, as indicated in the Materials and
methods; the resulting immunoprecipitates were subjected to acryla-
mide gel electrophoresis (10% gel) and Western blot analyses with anti-
(Fyn kinase) Ig (a), anti-(phosphotyrosine) Ig (b), or anti-(calponin h3)
Ig (c). Lane 3: control immunoblots of purified rat calponin h3
(120 ng) using any of the three antibodies. (B) Densitometric meas-
urements of the Western blots corresponding to COS 7 transfected
with the empty pSV vector (a) or the pSV vector containing Fyn
cDNA (b). Hatched bars, calponin h3 blots; dark bars, tyrosine-
phosphorylated calponin h3 blots; white bars, Fyn kinase blots.
Ó FEBS 2004 Tyrosine phosphorylated calponin–actin interaction (Eur. J. Biochem. 271) 2617
(Figs 1B, b) than in the control cells transfected with the
empty pSV vector (Fig. 1B, a), whereas the level of total
calponin was unchanged. Moreover, the immunoprecipitate
from the former cells displayed an additional band reactive
to Fyn kinase antibodies (Fig. 1A, line 2, panel a, and
Fig. 1B, b), suggesting that the production in these cells of a
tight complex between the expressed kinase and the
endogenous calponin h3, was resistant to the immunopre-
cipitation of the latter protein. A much smaller amount of
this band was also obtained from the control cells (Fig. 1A,
line 1, panel a; and Fig. 1B, a). These findings clearly
indicated that in normal COS 7 cells, a fraction of the
endogenous calponin h3 was tyrosine phosphorylated, and

the amount of this fraction was noticeably enhanced upon
transfection of the cells with Fyn cDNA. They suggest that,
in vivo, under physiological conditions, calponin probably
represents a good substrate for Src family protein tyrosine
kinases. This proposal is supported by the data, reported
below, describing the tyrosine phosphorylation in solution
of the calponin isoforms h1 and h3 by Fyn.
In vitro
phosphorylation of calponin h3 and calponin h1
by Fyn tyrosine kinase
Incubation of purified calponin h3 or calponin h1, in the
presence of increasing concentrations of Fyn kinase and
[
32
P]dATP[cP], resulted in the progressive and efficient
incorporation of radioactive
32
P in both calponins. How-
ever, the maximum extent of phosphorylation was different
for the two calponins. At saturation, the stoichiometry of
the phosphorylation reaction reproducibly plateaued at
1.4 mol of
32
P per mol of calponin h3 (Fig. 2A) and 2.0 mol
of
32
P per mol of calponin h1 (Fig. 2B). These results
suggest that the two calponin isoforms in solution are
adequate substrates for in vitro phosphorylation with Fyn.
Upon SDS gel electrophoresis, each

32
P-labeled calponin
displayed an electrophoretic band migrating identically to
the unlabeled protein (Fig. 3A,B). To determine whether
the calponins were phosphorylated on tyrosine residues, we
subjected each radioactively phosphorylated protein to
dephosphorylation by the phosphotyrosine-specific protein
phosphatase YOP and to immunoblotting with monoclonal
anti(phosphotyrosine) Ig. The treatment of calponin h1 with
YOP for 30 min caused a complete loss of the incorporated
32
P (Fig. 3C, lanes a and b); similarly, incubation of YOP
with calponin h3 resulted in a total dephosphorylation of
the protein (Fig. 3C, lanes c and d). These results demon-
strate that the Fyn-catalyzed phosphorylation of calponins
was also a reversible reaction. On the other hand, the
Western blot analyses presented in Fig. 3D clearly indicate
that the phosphorylated calponins were reactive to the
anti(phosphotyrosine) Ig (Fig. 3D, lanes b and d) but not
the unphosphorylated proteins (Fig. 3D, lanes a and c).
Together, the findings support the conclusion that the
phosphorylation of calponin h1 and calponin h3 takes place
on tyrosine residues.
Inhibition of actin binding to tyrosine-phosphorylated
calponins
To investigate the effects of tyrosine phosphorylation on the
actin-binding activity of the two calponins, we analyzed, by
quantitative co-sedimentation experiments, the interaction
of F-actin with each modified calponin isoform. Calponin h3
or calponin h1, both exhibiting the maximum level of

tyrosine phosphorylation, were mixed with F-actin at a
molar ratio of 1 : 2 and then subjected to high-speed
centrifugation. The partitioning of each phosphorylated
calponin, and of actin, between the supernatant and pellet
fractions, was determined by densitometry of Coomassie
Blue-stained electrophoretic gels (Fig. 4B), and the data were
compared with those obtained in the control co-sedimenta-
tion experiments carried out with calponins incubated in the
phosphorylation medium devoid of Fyn (Fig. 4A). The
densitometric analyses depicted in Fig. 4C show that  98%
of the control unphosphorylated calponin h1 (Fig. 4C, lanes
a) and calponin h3 (Fig. 4C, lanes c) are associated with the
F-actin pellet. In contrast,  68% of phosphorylated calpo-
nin h1 (Fig. 4C, lanes b) and 78% of phosphorylated
calponin h3 (Fig. 4C, lanes d) failed to bind to F-actin and
remained in the supernatant fractions. These data strongly
indicate that tyrosine phosphorylation of calponin h1 or h3
decreases the amounts of calponin bound to F-actin and
imply that the affinity of each calponin can be modulated
along the actin filament by tyrosine phosphorylation.
Fig. 2. In vitr o phosphorylation of calponin h3 (A) and calponin h1 (B)
by Fyn tyrosine kinase. Calponins (0.16 lg) in 30 lL of a kinase assay
consisting of 25 m
M
Hepes, pH 7.5, 1 m
M
dithiothreitol, 12 m
M
MnCl
2

,0.16lCi of [
32
P]dATP[cP] and 5 l
M
ATP were treated for
60 min at 30 °C with the indicated concentrations of Fyn kinase. The
phosphorylation level of each calponin was monitored by autoradio-
graphy followed by PhosphorImager analyses. Data representing at
least five independent experiments are shown.
2618 J. Abouzaglou et al. (Eur. J. Biochem. 271) Ó FEBS 2004
We did not extend the present study to calponin h2
because this particular calponin isoform includes a mutated
inactive primary actin-binding site [5], which precludes any
comparison of the functional effects of its phosphorylation
relative to those found with the two other calponins.
Next, we examined the influence of F-actin on the extent
of phosphorylation of calponin h1 and calponin h3 by Fyn.
In order to ensure detection of any blocking effect of
F-actin, the phosphorylation reaction with [
32
P]dATP[cP]
was performed for 15 min instead of 30 min, and with the
use of two molar ratios of calponin/actin corresponding to
1 : 2 or 1 : 10. After gel electrophoresis, the radioactivity
associated with each protein was revealed by autoradiog-
raphy (Fig. 5) and quantified by PhosphorImager analysis
(Table 1). The phosphorylation of the complexes between
F-actin and calponin h1 (Fig. 5, lane c) or calponin h3
(Fig. 5, lane d) led to the incorporation of c
32

P, not only
into each calponin but also into actin. The radiolabeling of
actin by Fyn kinase was not unexpected because, previously,
actin has been shown to be phosphorylated in vivo in
Dictyostelium [29–31] and plant cells [32] by nonidentified
tyrosine protein kinases with a unique target site, identified
as the tyrosine residue at position 53 [31]. Our quantitative
radioactivity measurements, depicted in Table 1, show that
nearly 1 mol of
32
P was incorporated per mol of actin, both
in the absence and presence of calponins. We conclude that
F-actin in solution is also an excellent substrate for Fyn
kinase, which probably phosphorylates the protein exclu-
sively on Tyr53. Furthermore, this efficient actin phos-
phorylation can be considered as an internal marker
pinpointing the good experimental conditions of our Fyn
kinase assays. On the other hand, the data of Table 1
further show that actin not only failed to hinder the
phosphorylation of both calponins, but even caused an
increase in the phosphorylation levels of each calponin
isoform as compared to the level of the control calponins,
especially when a calponin/actin molar ratio of 1 : 10 was
employed. The observed shift, from 0.7 to 2.0 mol of
32
Pper
mol of calponin h1, and from 1.4 to 3.0 mol of
32
Ppermol
of calponin h3, probably results from an actin-induced

conformational change in the calponin molecule, which
enhances the exposure of phosphorylatable tyrosine resi-
dues to Fyn kinase. This conclusion is supported by control
cosedimentation experiments (data not shown) using phos-
phorylated actin and native calponin h1 or calponin h3,
which did not show a noticeable change in the binding of
either calponin to the modified actin. Thus, the actin-
stimulated phosphorylation of calponins we observed was
not the result of a nonspecific alteration of the actin
structure caused by its co-phosphorylation.
Identification of the tyrosine phosphorylation sites
of calponin h3 and calponin h1
To determine the major sites of calponin h3 and calponin
h1 that are phosphorylated by Fyn, we first subjected the
tryptic in-gel digest of each
32
P-labeled protein to
fractionation by HPLC. By radioactivity counting of the
eluted fractions, we identified a single major radioactive
peak in the digest of calponin h3 (Fig. 6A) and two
radioactive peaks, designated a and b, in the digest of
calponin h1 (Fig. 6B). Each peptide peak was subse-
quently processed for radio-Edman sequencing and, after
further HPLC purification, each posphopeptide was
submitted to N-terminal sequencing. In the peak from
Fig. 3. Electrophoretic profiles on a 5–18% gradient polyacrylamide gel
of
32
P-labeled calponin h3 and calponin h1 after autoradiography (B).
These profiles were compared with the patterns of control calponins on

the same gel stained with Coomassie Blue R-250 (A). (C) Autoradio-
grams of phosphorylated calponin h1 and calponin h3 before (lanes a
and c, respectively) and after treatment with YOP protein tyrosine
phosphatase, as described in the Materials and methods (lanes b and d,
respectively). (D) Anti-(phosphotyrosine) probed Western blots of
phosphorylated (lanes b and d) and unphosphorylated (lanes a and c)
calponins. The experimental conditions of the immunoblots were as
reported in the Materials and methods.
Ó FEBS 2004 Tyrosine phosphorylated calponin–actin interaction (Eur. J. Biochem. 271) 2619
calponin h3, a single phosphotyrosine was found in cycle
5 and the N-terminal segment of the corresponding pure
peptide was identified as GMSV. The only amino acid
sequence of rat calponin h3 [5] accounting for these results
is G257-R265, with the phosphorylation site on Tyr261.
In the peak a of calponin h1, one
32
P-labeled tyrosine was
identified in cycle 10 and the N-terminal end of the
purified peptide was determined as GASQQG. Conse-
quently, the corresponding calponin h1 sequence should
be G252-R265 and the phosphorylated tyrosine is also
localized at Tyr261, a position which is conserved in all
the calponin sequences. Finally, the same analyses carried
out on peak b led to the identification of the calponin h1
sequence, F173-R185, with Tyr182 as the phosphorylated
residue. The presence of one and two phosphopeptides in
calponin h3 and calponin h1, respectively, is essentially in
agreement with the observed stoichiometry of
32
P incor-

poration in each protein.
Fig. 4. Tyrosine phosphorylation of calponins inhibits their binding to F-actin. Samples (5 lg) of calponin h1 or calponin h3, either unphosphorylated
(A) or phosphorylated with nonradioactive ATP (B), were incubated with F-actin (molar ratio 1 : 2) in 25 m
M
Hepes, pH 7.5, 1 m
M
dithiothreitol,
12 m
M
MnCl
2
for 30 min at 30 °C and then subjected to high-speed centrifugation. The supernatant and pellet fractions were separated by gel
electrophoresis and the partioning of calponin h1 (lanes 1) and calponin h3 (lanes 2) between the supernatant and pellet fractions was determined
after staining with Coomassie Blue R-250. (C) The gels were analyzed by densitometry and the percentage of the control calponin h1 (lanes a) or
calponin h3 (lanes c), together with the percentage of phosphorylated calponin h1 (lanes b) or calponin h3 (lanes d) in either fraction were
calculated. The data represent the mean of three experiments. Hatched bars, calponin h1; dark bars, calponin h3.
2620 J. Abouzaglou et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Discussion
In this study, we first investigated the enzymatic tyrosine
phosphorylation of the calponin h3 isoform in COS 7 cells
before and after transfection with the pSV vector containing
the cDNA encoding the tyrosine kinase, Fyn. Tyrosine
phosphorylated calponin was detected in both cases, with a
marked increase of the phosphotyrosine content of the
endogenous calponin upon transfection of the cells. These
results strongly suggest that tyrosine phosphorylation of
calponin h3 by Src kinases can occur in vivo and its
biological effects remain to be elucidated. To assess the
functional consequences of this phosphorylation on the
F-actin–calponin interaction, purified calponin h3 and

calponin h1 in solution were phosphorylated by Fyn kinase.
Our data revealed that both calponin isoforms are conveni-
ent substrates for specific tyrosine phosphorylation by this
kinase, with concomitant inhibition of their binding to
F-actin. However, although a stoichiometric quantity of
phosphotyrosine was formed in each calponin, the complete
inhibition of actin binding to any phosphorylated calponin
did not occur, as the maximal reduction of actin binding
observed was 75%. It seems likely that tyrosine phosphory-
lation modulates the amount of calponin bound to actin,
probably by lowering the affinity of the phosphocalponin–
F-actin complex, rather than by fully dissociating the
Fig. 5. Tyrosine phosphorylation of calponins is enhanced by F-actin.
Calponin h1 or calponin h3 were phosphorylated in the absence (lanes
a and b, respectively) or presence (lanes c and d, respectively) of F-actin
at a molar ratio of 1 : 2. The experimental conditions for the phos-
phorylation reaction were as reported in the Materials and methods.
After gel electrophoresis and autoradiography, the radioactivity was
measured by PhosphorImager analysis. The data are presented in
Table 1.
Table 1. Compared phosphorylation of calponins by Fyn kinase per-
formed in the absence or presence of varying concentrations of F-actin.
The experimental conditions for the phosphorylation reactions and
radioactivity measurements were as described in the Materials and
methods.
Proteins
Moles of
32
P
incorporated

per mol of protein
Calponin h1 0.7
Calponin h3 1.4
Calponin h1
(+ F-actin at a
molar ratio of 1 : 2)
1.0
Calponin h1
(+ F-actin at a
molar ratio of 1 : 10)
2.0
Calponin h3
(+ F-actin at a
molar ratio of 1 : 2)
1.8
Calponin h3
(+ F-actin at a
molar ratio of 1 : 10)
3.0
Actin 0.9
a
a
Value found both for the control actin alone and for actin
complexed to either calponin.
Fig. 6. Separation of
32
P-labeled tryptic peptides of calponin h3 (A) and
calponin h1 (B) by reverse-phase HPLC. After in-gel digestion of each
phosphorylated protein with trypsin, the corresponding tryptic peptide
mixtures were extracted and applied to an Aquapore C8 reverse-phase

column, which was then eluted with a linear gradient of 0–30%
acetonitrile for 60 min. The radioactive peptides were separated and
their amino acid sequences were determined by combining their radio-
Edman degradation with their direct N-terminal sequencing. Phos-
phorylated Tyr261 was identified in the phosphopeptide representing
the major peak of calponin h3 and in the phosphopeptide corres-
ponding to peak a of calponin h1.
Ó FEBS 2004 Tyrosine phosphorylated calponin–actin interaction (Eur. J. Biochem. 271) 2621
complex. An essentially similar modulation of the interac-
tion of tyrosine phosphorylated villin with F-actin has been
reported previously [16]. The association of F-actin with
either calponin h1 or h3 during the kinase reaction did not
hinder the phosphorylation process of the calponins;
however, it increased their phosphotyrosine content. The
lack of F-actin protection against tyrosine phosphorylation
of calponins contrasts with the F-actin-induced blocking
effect on the enzymatic Ser/Thr phosphorylation of calpo-
nin h1 in vitro, reported previously [33]. On the other hand,
the observed extent of actin-induced phosphorylation was
different for the two calponins as, for calponin h1 it reached
the same level as found in the absence of actin, whereas for
calponin h3 it significantly exceeded the level obtained
without actin. Thus, the actin-bound conformations of the
two calponins are probably different. The F-actin-depend-
ent tyrosine phosphorylation of calponin h3 could be of
biological significance as the additional phosphorylated sites
may also contribute to modulate actin binding. Further
investigations are currently underway for assessing its
influence on the actin–calponin interaction and for identi-
fying the sites specifically stimulated by actin binding.

In the work presented here, we have localized the
tyrosines phosphorylated without actin to Tyr261 in
calponin h3 and to Tyr261 and Tyr182 in calponin h1.
The latter residue is within the first calponin repeat and
represents a chymotrypsin cleavage site residing at the
C-terminus of the central, protease-ensitive segment of
calponin h1 that starts at Tyr144. Although being conserved
in calponin h3, Tyr182 did not undergo phosphorylation in
this isoform. Probably, the protein structure around this
particular residue is different in calponin h3 from that of
calponin h1. This suggestion is supported by the differences
observed in the chymotryptic cleavage patterns of the two
calponin isoforms. It should be noted that its phosphory-
lation did not have any effect on F-actin binding as the
extent of inhibition of the calponin–actin interaction was
quite similar for the two phosphorylated isoforms. Thus,
Tyr261 in calponin h3 or calponin h1 represents the only site
whose phosphorylation promotes the observed inhibition of
the protein binding to F-actin. Tyr261 is positioned one
amino acid apart from Thr259, which was shown to be
phosphorylated in calponin h1 by Rho-kinase [34]. The
amino acid sequence around these two residues is highly
conserved, being GMTVY261GL in chicken gizzard calpo-
nin h1 and GMSVY261GL in rat calponin h3, the latter
containing a serine residue instead of a threonine at position
259 [5]. Furthermore, Tyr261 is located in a strategic region
of the calponin molecule, namely the conserved third repeat
motif in the C-terminal portion of the protein. It is also a
specific residue of calponins, as in the thin filament
associated Unc87 protein from Caenorhabditis elegans,

which contains seven copies of the calponin repeat, phenyl-
alanine replaces the tyrosine residue in all repeats [35]. In the
reported 3D reconstruction of the F-actin–calponin com-
plex, the position of the calponin repeats relative to the
actin–calponin interface has not been determined [36].
However, the Unc87 repeats were shown to directly bind to
F-actin, both in vitro and in vivo [37]. In addition, the direct
involvement of the calponin repeats in the stabilization of
the actin filaments was recently described [38]. Together,
these findings support the conclusion that the third calponin
repeat motif, comprising Tyr261, may directly contribute to
the building of a stable calponin–actin interface. Conse-
quently, phosphorylation of the critical Tyr261 would cause
a conformational change in the repeat domain with a
marked reduction of the binding strength at the calponin–
actin interface, thereby promoting the observed partial
dissociation of the complex between F-actin and phosphor-
ylated calponins. Previously, the association of F-actin with
calponin repeats was found to be regulated by the tail region
of calponin in an isoform-specific manner [39]. The present
investigation indicates that it could be further modulated by
direct tyrosine phosphorylation of the repeats.
In conclusion, our studies open the way for the
characterization of tyrosine phosphorylated calponins in
other cell types using the Fyn cDNA transfection approach
and the determination of their roles in the regulation
in vivo of the structural dynamics and the interactions of
F-actin.
Acknowledgements
This research was supported by grants from the Centre National de la

Recherche Scientifique, the Institut National de la Sante
´
et de la
Recherche Me
´
dicale, and the Association Franc¸ aise contre les
myopathies. We thank J. L. Derancourt for kindly performing the
structural analyses of tryptic phosphopeptides and Dr S. Roche for
providing us with the PSV–Fyn construct and the Fyn2 antibody.
References
1.Gimona,M.,Herzog,M.,Vandekerckhove,J.&Small,J.V.
(1990) Smooth muscle specific expression of calponin. FEBS Lett.
274, 159–162.
2. Strasser,P.,Gimona,M.,Moessler,H.,Herzog,M.&Small,J.V.
(1993) Mammalian calponin. Identification and expression of
genetic variants. FEBS Lett. 330, 13–18.
3. Applegate, D., Feng, W., Green, R.S. & Taubman, M.B. (1994)
Cloning and expression of a novel acidic calponin isoform
from rat aortic vascular smooth muscle. J. Biol. Chem. 269,
10683–10690.
4. Winder, S.J. & Walsh, M.P. (1990) Smooth muscle calponin.
Inhibition of actomyosin MgATPase and regulation by phos-
phorylation. J. Biol. Chem. 265, 10148–10155.
5. Gimona, M. & Small, J.V. (1996) Calponin. In Biochemistry of
Smooth Muscle Contraction, Vol 7 (Barany, M., ed), pp. 91–103.
Academic Press, New York.
6. North, A., Gimona, M., Cross, R.A. & Small, J.V. (1994) Cal-
ponin is localised in both the contractile apparatus and the
cytoskeleton of smooth muscle cells. J. Cell Sci. 107, 437–444.
7. Kake, T., Kimura, S., Takahashi, K. & Maruyama, K. (1995)

Calponin induces actin polymerization at low ionic strength and
inhibits depolymerization of actin filaments. Biochem. J. 312,
587–592.
8. Leinweber, B., Tang, J.X., Stafford, W.F. & Chalovich, J.M.
(1999) Calponin interaction with alpha-actinin-actin: evidence for
a structural role for calponin. Biophys. J. 77, 3208–3217.
9.Menice,C.B.,Hulvershorn,J.,Adam,L.P.,Wang,C L.A.&
Morgan, K.G. (1997) Calponin and mitogen-activated protein
kinase signaling in differentiated vascular smooth muscle. J. Biol.
Chem. 272, 25157–25161.
10. Leinweber, B., Parissenti, A., Gallant, C., Gangopadhyay, S.S.,
Kirwan-Rhude, A., Leavis, P.C. & Morgan, K.G. (2000) Reg-
ulation of protein kinase C by the cytoskeletal protein calponin.
J. Biol. Chem. 257, 40329–40336.
2622 J. Abouzaglou et al. (Eur. J. Biochem. 271) Ó FEBS 2004
11. Gimona, M. & Mital, R. (1998) The single CH domain of calponin
is neither sufficient nor necessary for F-actin binding. J. Cell Sci.
111, 1813–1821.
12. Mezgueldi,M.,Mendre,C.,Calas,B.,Kassab,R.&Fattoum,A.
(1995) Characterization of the regulatory domain of gizzard
calponin. Interaction of the 145–163 region with F-actin, calcium-
binding protein, and tropomyosin. J. Biol. Chem. 270, 8867–8876.
13. Burgstaller, G., Kranewitter, W.J. & Gimona, M. (2002) The
molecular basis for the autoregulation of calponin by isoform-
specific C-terminal tail sequences. J. Cell Sci. 115, 2021–2029.
14. Nakamura,F.,Mino,T.,Yamamoto,J.,Naka,M.&Tanaka,T.
(1993) Identification of the regulatory site in smooth muscle cal-
ponin that is phosphorylated by protein kinase C. J. Biol. Chem.
268, 6194–6201.
15. Tang, D C., Kang, H M., Jin, J P., Fraser, E.D. & Walsh,

M.P. (1996) Structure–function relations of smooth
muscle calponin. The critical role of serine 175. J. Biol. Chem. 271,
8605–8611.
16. Zhai, L., Zhao, P., Panebra, A., Guerrerio, A.L. & Khurana, S.
(2001) Tyrosine phosphorylation of villin regulates the organiza-
tion of the actin-cytoskeleton. J. Biol. Chem. 276, 36163–36167.
17.Zhai,L.,Kumar,N.,Panebra,A.,Zhao,P.,Parryl,A.L.&
Khurana, S. (2002) Regulation of actin dynamics by tyrosine-
phosphorylation: identification of tyrosine phosphorylation sites
within the actin-severing domain of villin. Biochemistry 41,
11750–11760.
18. DeCorte,V.,Gettemans,J.&Vandekerckhove,J.(1997)Phos-
phatidylinositol 4,5-bisphosphate specifically stimulates PP60
(c-src) catalyzed phosphorylation of gelsolin and related actin-
binding proteins. FEBS Lett. 401, 191–196.
19.Wang,Q.,Xie,Y.,Du,Q S.,Wu,X J.,Feng,X.,Mei,L.,
McDonald, J.M. & Xiong, W C. (2003) Regulation of the for-
mation of osteoclastic actin rings by proline-rich tyrosine kinase 2
interacting with gelsolin. J. Cell Biol. 160, 565–575.
20. Huang,C.,Ni,Y.,Wang,T.,Gao,Y.,Haudenschild,C.C.&
Zhan, X. (1997) Down-regulation of the filamentous actin cross-
linking activity of cortactin by src-mediated tyrosine phosphor-
ylation. J. Biol. Chem. 272, 13911–13915.
21. Mezgueldi, M., Fattoum, A., Derancourt, J. & Kassab, R. (1992)
Mapping of the functional domains in the amino-terminal region
of calponin. J. Biol. Chem. 267, 15943–15951.
22. Fattoum, A., Roustan, C., Smyczynski, C., Der Terrossian, E.
& Kassab, R. (2003) Mapping the microtubule binding regions of
calponin. Biochemistry 42, 1274–1282.
23. Roche, S., Koegl, M., Barone, M.V., Roussel, M.F. & Court-

neidge, S.A. (1995) DNA synthesis induced by some but not all
growth factors requires Src family protein tyrosine kinases. Mol.
Cell. Biol. 15, 1102–1109.
24. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227,680–
685.
25. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D.,
Seidman, J.G., Smith, J.A. & Struhl, K. (1994) Current Proto-
cols in Molecular Biology. Greene Publishing Associates/Wiley,
New York.
26. Plantier, M., Fattoum, A., Menn, A., Ben-Ari, Y., Der Terrossian,
E. & Represa, A. (1999) Acidic calponin immunoreactivity in
postnatal rat brain and cultures: subcellular localization in growth
cones, under the plasma membrane and along actin and glial
filaments. Eur. J. Neurosci. 11, 2801–2812.
27. Rosenfeld, J., Capdevielle, J., Guillemot, J.C. & Ferrara, P. (1992)
In-gel digestion of proteins for internal sequence analysis after one
or two-dimentional gel electrophoresis. Anal. Biochem. 203,
173–179.
28. Thomas, S.M. & Brugge, J.S. (1997) Cellular functions regulated
by Src family kinases. Annu. Rev. Cell Dev. Biol. 13, 513–609.
29. Schweiger, A., Mihalache, O., Ecke, M. & Gerisch, G. (1992)
Stage-specific tyrosine phosphorylation of actin in Dictyostelium
discoideum cells. J. Cell Sci. 102, 601–609.
30. Howard, P.K., Sefton, B.M. & Firtel, R.A. (1993) Tyrosine
phoshorylation of actin in Dictyostelium associated with cell-shape
changes. Science 259, 241–244.
31. Jungbluth, A., Eckerskorn, C., Gerish, G., Lottspeich, F., Stocker,
S. & Schweiger, A. (1995) Stress-induced tyrosine phosphorylation
of actin in Dictyostelium cells and localization of the phosphor-

ylation site to tyrosine-53 adjacent to the DnaseI binding loop.
FEBS Lett. 375, 87–90.
32. Kameyama, K., Kishi, Y., Yoshimura, M., Kanzawa, N., Same-
shima, M. & Fsuchiya, T. (2000) Tyrosine phosphorylation in
plant bending. Nature 407, 37–40.
33. Nagumo, H., Sakurada, K., Seto, M. & Sasaki, Y. (1994) Phos-
phorylation of calponin by PKC is blocked by F-actin in vitro.
Biochem. Biophys. Res. Commun. 203, 1502–1507.
34. Kaneko, T., Amano, M., Maeda, A., Goto, H., Takahashi, M.I. &
Kaibuchi, K. (2000) Identification of calponin as a novel substrate
of Rho-kinase. Biochem. Biophys. Res. Commun. 273, 110–116.
35. Goetinck, S. & Waterson, R.H. (1994) The Caenorhabditis elegans
muscle-affecting gene, unc-87, encodes a novel thin filament-
associated protein. J. Cell Biol. 127, 79–93.
36. Bramham, J., Hodgkinson, J.L., Smith, B.O., Uhrin, D., Barlow,
P.N. & Winder, S.J. (2002) Solution structure of the Calponin CH
domain and fitting to the 3D-helical reconstruction of F-actin
calponin. Structure 10, 249–258.
37. Kranewitter, W.J., Ylanne, J. & Gimona, M. (2001) Unc-87 is an
actin bundling protein. J. Biol. Chem. 276, 6306–6312.
38. Gimona, M., Kaverina, I., Resch, G.P., Vignal, E. & Burgstaller,
G. (2003) Calponin repeats regulate actin filament stability and
formation of Podosomes in smooth muscle cells. Mol. Biol. Cell
14, 2482–2491.
39. Burgstaller, G., Kranewitter, W.J. & Gimona, M. (2002) The
molecular basis for the autoregulation of calponin by isoform-
specific C-terminal tail sequences. J. Cell Sci. 115, 2021–2029.
Ó FEBS 2004 Tyrosine phosphorylated calponin–actin interaction (Eur. J. Biochem. 271) 2623