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

Tyrosine phosphorylation of calponinsInhibition of the interaction with F-actin Julien Abouzaglou1, Christine Be´nistant1, Mario Gimona2, Claude Roustan3, Rhida Kassab1 and Abdellatif Fa

Tyrosine phosphorylation of calponins

Inhibition of the interaction with F-actin

Julien Abouzaglou1, Christine Be´nistant1, Mario Gimona2, Claude Roustan3, Rhida Kassab1

and Abdellatif Fattoum1

1

Centre de Recherches de Biochimie Macromole´culaire du CNRS, Montpellier, France;2Institute of Molecular Biology,

Salzburg, Austria;3UMR 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

32P-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 of32P 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 32P-labeling in both calponins Structural analysis of the tryptic phosphopeptides from each32 P-labe-led calponin reveaP-labe-led a single, major32P-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 Ca2+ 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 addiphosphoryla-tional 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 of32P 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: fattoum@crbm.cnrs-mop.fr

Note: a website is available at http://www.crbm.html

(Received 4 February 2004, accepted 28 April 2004)

direct amino acid sequencing of32P-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

[32P]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 enterocoliticawas 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 A1%280¼ 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)1of calponin) in an assay mixture (30 lL)

containing 25 mM Hepes, pH 7.5, 1 mM dithiothreitol,

12 mMMnCl2, 0.16 lCi of [32P]dATP[cP] and 5 lMATP

The reactions were carried out for 60 min at 30C and

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 mMTris/HCl, pH 7.0, 100 mMNaCl, 2 mMEDTA,

5 m dithiothreitol, 0.01% Brij 35 and supplemented with

1 mgÆmL)1 BSA and 50 U of YOP protein tyrosine phosphatase After 60 min at 30C, 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) at 37C and 5% CO2 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 mM Na vanadate, incubated for a further 30 min They were then washed twice in cold NaCl/Pi (PBS), scraped into 400 lL of RIPA buffer [20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% (v/v) Triton-X-100, 1% Na deoxycholate, 0.1% SDS containing 1 mMdithiothreitol, 1 mMNa vanadate, 1 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 10 lgÆmL)1 aprotinin and 20 lM leupeptin] and then incubated at

4C 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 4C 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 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40 containing 1 mM dithiothreitol and 1 mM 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 mM Tris, 100 mM 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]

Co-sedimentation assays

Calponin h1 or h3 (5 lg), in a final volume of 50 lL, was

first phosphorylated with nonradioactive ATP (5 lM) and

3.4 U of Fyn in 25 mMHepes, 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 30C,

the protein mixtures were centrifuged, in a Beckman

Airfuge, at 90 000 g for 30 min at 25C 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

32P-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 posidetec-tion of32P-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.

(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

[32P]dATP[cP], resulted in the progressive and efficient

incorporation of radioactive32P 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 of32P per mol of calponin h3 (Fig 2A) and 2.0 mol

of 32P 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, each32P-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

32P (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 vitro 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 , 0.16 lCi of [32P]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.

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 [32P]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 c32P, 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 of32P 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 of32P per mol of calponin h1, and from 1.4 to 3.0 mol of32P per mol

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 32P-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.

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, one32P-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 32P 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.

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 of32P incorporated per mol of protein

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

a

Value found both for the control actin alone and for actin

complexed to either calponin.

Fig 6 Separation of32P-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.

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 calpocalpo-nin 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 vivoof 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.

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