This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

Author's personal copy

Filamin isoforms in molluscan smooth muscle

Lucía Méndez-López a, Ulf Hellman b, Izaskun Ibarguren a, J. Antonio Villamarín a,⁎a Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spainb Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden

a b s t r a c ta r t i c l e i n f o

Article history:Received 9 May 2012Received in revised form 28 June 2012Accepted 23 July 2012Available online 29 July 2012

Keywords:Filamin isoformsCatch muscleProteolysisMass spectrometryMolluscMytilus

The role of filamin in molluscan catch muscles is unknown. In this work three proteins isolated from the pos-terior adductor muscle of the sea mussel Mytilus galloprovincialis were identified by MALDI-TOF/TOF MS ashomologous to mammalian filamin. They were named FLN-270, FLN-230 and FLN-105, according to theirapparent molecular weight determined by SDS-PAGE: 270 kDa, 230 kDa and 105 kDa, respectively. BothFLN-270 and FLN-230 contain the C-terminal dimerization domain and the N-terminal actin-binding domaintypical of filamins. These findings, together with the data from peptide mass fingerprints, indicate thatFLN-270 and FLN-230 are different isoforms of mussel filamin, with FLN-230 being the predominant isoformin the mussel catch muscle. De novo sequencing data revealed structural differences between both filaminisoforms at the rod 2 segment, the one responsible for the interaction of filamin with the most of its bindingpartners. FLN270 but not FLN230 was phosphorylated in vitro by cAMP-dependent protein kinase. As forthe FLN-105, it would be an N-terminal proteolytic fragment generated from the FLN-270 isoform or aC-terminally truncated variant of filamin. On the other hand, a 45-kDa protein that copurifies with musselcatch muscle filamins was identified as the mussel calponin-like protein. The fact that this protein coeluteswith the FLN-270 isoform from a gel filtration chromatography suggests a specific interaction betweenboth proteins.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Molluscan catch muscles are specialized smooth muscles that cansustain high tension for very long periods with a low energetic cost(for review see [1]). On the basis of experimental data collected sofar, various speculative models have been proposed to explain themolecular mechanism of catch (for review see [2]). All of thesemodels are based on the formation of passive linkages betweenthick and thin filaments, mediated, directly or indirectly, by twitchin,a member of the titin/connectin family [3]. Despite the fact that themolecular mechanism of catch has yet to be completely elucidated,it has been demonstrated that the phosphorylation state of twitchinplays a crucial role in the regulation of catch. Briefly, Ca+2- stimulateddephosphorylation of twitchin by protein phosphatase 2B (calcineurin)allows this protein to bind F-actin, initiating the catch state [4]. In con-trast, phosphorylation of twitchin by cAMP-dependent protein kinase(PKA), at the named D2 site, nullifies its ability to interact with F-actinand thus releases the catch state [5–7].

In order to know the functional properties of PKA from catchmuscles,we have purified and characterized both the regulatory (R) and catalytic(C) subunits of PKA from the posterior adductormuscle (PAM) of the seamusselMytilus galloprovincialis. Two different isoforms of the R-subunit,named Rmyt1 and Rmyt2, were isolated frommussel PAM; however, Rmyt1,which is homologous to the mammalian type I R-subunit, was thepredominant isoform in that tissue [8,9]. Furthermore, the results fromimmunohistochemical studies showed that Rmyt1 appears to be uniform-ly distributed inside the muscle fibers, whereas Rmyt2 was only observedat the cell periphery; this suggests that the PKA containing Rmyt1 mightbe the isoform involved in the regulation of catch [10].

The PKAC-subunitwas also purified from themolluscan PAMmuscle.The enzyme can mainly phosphorylate in vitro two PAM proteins:twitchin and actin [11,12]. Recently we used a cation-exchange chroma-tography (Mono S-FPLC) to separate three different isoforms of theC-subunit from PAM, named C1, C2 and C3; they appear to be generatedby alternate splicing from a sole gene [12]. The purification procedureof C-isoforms includes a DEAE-chromatography step, where theC-subunit is dissociated from the R-subunit, and thus specificallyeluted, by using cAMP in the washing buffer. We observed that thispreparation of C-subunit contains a relatively large amount of a pro-tein with a high molecular weight (>200 kDa) that is different fromthe main PAM contractile proteins: twitchin, paramyosin, myosinand catchin. This protein was identified byMS/MS analysis as homol-ogous to filamin (unpublished results).

Biochimica et Biophysica Acta 1824 (2012) 1334–1341

Abbreviations: PAM, posterior adductormuscle; PKA, cAMP-dependent protein kinase;C, catalytic subunit of PKA; R, regulatory subunit of PKA⁎ Corresponding author at: Departamento de Bioquímica e Bioloxía Molecular,

Facultade de Veterinaria, Universidade de Santiago de Compostela, Campus de Lugo,27002 Lugo, Spain. Tel.: +34 82 822 210; fax: +34 82 252 195.

E-mail address: antonio.villamarin@usc.es (J.A. Villamarín).

1570-9639/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bbapap.2012.07.011

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbapap

Author's personal copy

Filamins are a family of actin-binding proteins. In mammals, thefilamin family consists of three homologous proteins (filamin A, B andC) encoded by three different genes, and their mRNA splice variants[13]. They are elongated homodimeric proteins with a V-shaped struc-ture. Each monomeric chain contains an actin-binding domain (ABD)at the N-terminus, followed by a rod segment, and a dimerizationdomain at the C-terminus. The rod segment consists of 24 highly ho-mologous repeats of ~96 amino acids that adopt an immunoglobulin(Ig)-like anti-parallel β-sheet fold. Two calpain sensitive hinge domainsseparate these repeats into rod 1 (repeats 1–15), rod 2 (repeats 16–23)and the dimerization domain (repeat 24) [14].

Filaminswere initially considered only to be cytoskeleton organizersas they cross-link the F-actin filaments into dynamic networks, and linkthem to cell membrane [13]. However, the recent findings revealed thatmammalian filamins can interact with several cell proteins other thanF-actin, includingmembrane receptors, ionic channels, enzymes, signal-ling pathway proteins and transcription factors [14,15]. Given its abilityto interact with multiple proteins, filamin acts as an essential scaffoldprotein, integrating multiple cellular functions such as cell motility,cell signalling, transcription and organ development [16]. Interestingly,most of the protein-filamin interactions have been mapped to regionsbetween repeats 16 and 23 of the rod 2 domain [15].

Whereas filamins are relatively well studied in both muscle andnonmuscle cells of mammals, very little information is currentlyavailable about the structure and functions of filamin in invertebratesources. The aimof the presentworkwas to identify and characterize thefilamin(s) from a catchmuscle (an invertebrate smoothmuscle) in orderto better understand its role in the regulation of catch contraction.

2. Material and methods

2.1. Molluscs

Sea mussels of the species M. galloprovincialis Lmk. were collectedfrom a sea farm located at Ría de Betanzos (Galicia, NW Spain). Molluscswere placed in tanks containing seawater and transported to the labora-tory. PAM tissue was dissected out and immediately frozen at −80 °Cuntil use.

2.2. Isolation of filamin from mussel PAM

PAM (15–20 g) was homogenized in 6 vol of ice-cold buffer A(30 mM potassium phosphate, 2 mM EDTA, 1 mM dithiothreitol,1 mM phenylmethanesulfonyl fluoride, 1.0 mg/l leupeptin, 1.0 mg/lpepstatin, pH 7.0) using a blade homogenizer. Homogenized muscleswere extracted by agitation for 20 min in an ice bath, and the extractwas centrifuged at 35,000 g for 30 min at 4 °C. The precipitatebetween 35–50% saturation ammonium sulfate of the PAM extractwas resuspended in buffer B (45 mM potassium phosphate, 1 mMdithiothreitol, pH 7.0), exhaustively dialyzed against buffer B, andcentrifugued at 35,000 g for 15 min at 4 °C. A fraction of the superna-tant (4 mL; ~20 mg protein) was filtered through a 0.45 μm filter,and then applied to a Mono-S HR 5/5 column–FPLC (GE HealthcareLife Sciences, Uppsala, Sweden), previously equilibrated with buffer B(45 mM potassium phosphate buffer, 0.1 mM DTT, pH 7.0). The columnwas extensively washed with buffer B, and the retained proteins wereeluted by applying a continuous NaCl gradient (0–0.4 M in buffer B).The collected fractions of 1 ml were analysed by 10% SDS-PAGE.

2.3. Preparation of F-actin from mussel PAM

Mussel F-actin was isolated from the 0–33% saturated ammoniumsulphate fraction of a high ionic strength extract from mussel PAM,obtained as described by Shelud´ko et al. [7]. The precipitate between0 and 33% saturation of ammonium sulfate was resuspended in25 mL of polymerization buffer (10 mM Tris–HCl, pH 7.4, 1 mM ATP,

4 mM MgCl2, 1 mM EGTA, 50 mM KCl, 1 mM dithiothreitol, 1 mMphenylmethanesulfonyl fluoride), and the resulting suspension waskept at 4 °C for 12 h under slow stirring. Polymerized actinwas clarifiedat 20,000 g for 30 min at 4 °C and then centrifugued at 100,000 g for90 min at 4 °C. The pellet was resuspended in 20 mL of actin depoly-merization buffer (5 mM Tris–HCl, pH 7.4, 0.2 mM ATP, 0.2 mM CaCl2,1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride), exhaus-tively dialyzed against this buffer for 72 h at 4 °C, and centrifugued at100,000 g for 90 min at 4 °C. The supernanant was concentrated byultrafiltration through a PM-30 membrane to a final volume of7–8 mL. Finally, actin polymerization was induced by adding ATP to1 mM, MgCl2 to 4 mM, EGTA to 1 mM and KCl to 50 mM. PurifiedF-actin was stored on ice for up two months with 1 mM sodium azide.

2.4. SDS-PAGE and Western blotting

SDS-PAGE was carried out according to Laemmli [17] using 7.5% or10% polyacrylamide slab-gels of size 16×16 cm (Protean II xi cell) or8.2×6.2 cm (Mini Protean 3 cell) (Bio-Rad Laboratories, Hercules, CA,USA). To perform Western blot analysis, the proteins were trans-ferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P;Millipore, Billerica, MA, USA) by applying a 400 mA current for 3 h at4 °C. In order to increase transfer efficiency of filamin, 0.05% (p/v)SDS was added to the transfer buffer. The membranes were blockedfor 6 h at room temperature with 5% non-fat dry milk in 20 mMTTBS (Tris–HCl, pH 7.5, 0.15 M NaCl, 0.1% Tween 20), washed withTTBS, and then incubated overnight at 4 °C with a polyclonal antibodyanti-chicken filamin (Ab11074, Abcam plc, Cambridge, UK) diluted1:1,000 in TTBS. After washing with TTBS, the blots were incubatedfor 1 h at room temperature with anti-goat secondary antibodies,diluted 1:50,000, conjugated to horseradish peroxidase (Sigma-AldrichQuímica, Madrid, Spain). Next, the blots were extensively washed,developed with the chemiluminiscent HRP substrate (Millipore), andexposed to X-ray film (Curix RP2 Plus; Agfa-Gevaert, Mortsel, Belgium)for a few seconds.

2.5. Determination of native molecular weight by gel filtrationchromatography

A Superdex 200 HR 10/30–FPLC column (GEHealthcare Life Sciences)was equilibrated with 45 mM potassium phosphate buffer, 1.0 mMdithiothreitol, 0.1 MKCl, pH 7.0 (buffer A). The columnwas then calibrat-ed as follows: a sample (100 μL) containing a mixture of molecularweight protein standards (in mg/mL: 3.0 thyroglobulin, 0.7 ferritin, 12.0aldolase, 7.0 ovoalbumin and 1.5 chymotrypsinogen) was filteredthrough a 0.45 μm Millex HV filter (Millipore), applied to the columnand eluted with buffer A at a flow rate of 0.4 mL/min. Absorbance at280 nm was monitored and the elution volume (Ve) was determinedfor each molecular weight standard. Next, a sample (0.5 ml, 0.6 mgprotein) from amixture of the fractions 10–12 of theMono S chromatog-raphy was applied to the column and eluted under identical conditions.The apparent molecular weight of PAM filamins was determined fromthe plot of log molecular weight of standards vs Ve/Vo, where Vo is thevoid volume, determined by applying to the column a sample of a bluedextran solution.

2.6. Filamin phosphorylation assay

Aliquots from the Sephadex S-200 chromatography, containing amixture of FLN-270, FLN-230 and FLN-105, were incubated in thepresence of MgCl2 and labelled ATP with or without PKA C-subunit.The reaction mixture contained, in a total volume of 25 μl,25 mM Hepes-KOH, pH 7.0, 5.0 mM MgCl2, 0.2 mM [γ-32P]ATP(600 cpm/pmol) (Hartmann Analytic, Braunschweig, Germany),6.0 μg filamins and 0.1 μg C subunit (isoform C2) purified from mus-sel PAM as previously described [12]. After incubation for 15 min at

1335L. Méndez-López et al. / Biochimica et Biophysica Acta 1824 (2012) 1334–1341

Author's personal copy

30 °C, the reactions were stopped by adding SDS-sample buffer andboiling for 5 min. Samples were analyzed by 7.5% SDS-PAGE andthe gel was stained with Coomassie brilliant blue, destained, driedand exposed for autoradiography at −80 °C.

2.7. Actin cosedimentation assay

A sample from the Sephadex S-200 chromatography, containing amixture of FLN-270, FLN-230 and FLN-105, was incubated with orwithout 15 μM F-actin from catch muscle for 1 h at 25 °C. TheFLN-270/actin molar ratio was 1:10. F-actin was precipitated bycentrifugation at 100,000 g for 1 h at 25 °C and the pellet and super-natant fractions were diluted to equivalent volumes in SDS-PAGEsample buffer and analyzed by SDS-PAGE and Coomassie staining.

2.8. Mass spectrometry for protein identification

Proteins isolated from mussel PAM were first reduced by addingSDS-sample buffer supplemented with DTT to a final concentration10 mM, and then alkylatedwith 20 mM iodoacetamide (Sigma-AldrichQuímica) for 30 min in the dark. Next, proteins were separated bySDS-PAGE, and the protein bands were excised from the gel andsubjected to in-gel digestion procedurewithmodified trypsin, sequencegrade (Promega). Digested sample was analysed by MALDI-TOF MS,using a MALDI-TOF/TOF Ultraflex instrument (Bruker Daltonics,Bremen, Germany) in reflector mode, to obtain PMF spectra. The se-quences of tryptic peptides from PAM filamins were obtained by denovo sequencing using the CAF-PSD methodology [18]. PSD analysiswas performed according to the manufacturer and the generated spec-tra,mainly consisting of a clean y-ion series, were interpretedmanually.Sequence homology search was performed using the FASTS algorithm(http://fasta.bioch.virginia.edu/fasta_www/cgi/) against the NCBI/BLASTnon-redundant protein database with BLOSUM 50 as search matrix [19].

3. Results

3.1. Purification of filamin from mussel PAM

We had previously observed that a high molecular weight proteinfrom mussel PAM, identified as homologous of filamin, coeluted withPKA C-isoforms from a cation-exchange chromatography [12]. Toinvestigate if this protein is homologous to filamin, we developed aprocedure for filamin purification based on a Mono S-FPLC chroma-tography. A mussel PAM extract was first fractionated by ammoniumsulphate precipitation, and the 35–50% saturated ammonium sul-phate fraction was applied to a Mono S-FPLC column. The protein elu-tion profile is shown in Fig. 1A. The analysis of the collected fractionby SDS-PAGE and Coomassie staining (Fig. 1B) showed that most ofthe applied proteins (lane b) were not retained by the resin and elut-ed in the wash step (fraction 2). Four major proteins were retained bythe resin and coeluted as a broad peak by applying the salt gradient(fraction 10, bands 1–4). The protein band 2 migrated on thepolyacrylamide gel with apparent identical mobility to that of theabove-mentioned protein that copurify with PKA C-isoforms [12] (in-dicated with an arrowhead in the lane a).

The apparent molecular weight of the proteins 1–4, determined by10% SDS-PAGE, was, respectively, 272±5 kDa (p270), 230±6 kDa(p230), 105±5 kDa (p105) and 45±2 kDa (p45) (mean±SD ofthree determinations) (Fig. 2).

3.2. Identification of mussel PAM proteins by MS

In order to confirm the identity of the p230 protein and to identifythe p270, p105 and p45 proteins, a MS analysis was performed. Theprotein bands 1–4 were cut out from the polyacrylamide gel,subjected to in gel tryptic digestion, and the peptide mixtures were

analyzed by MALDI-TOF MS. The corresponding peptide mass finger-print (PMF) spectra are shown in Fig. 3.

A peptide mass database search allowed us to identify p45 as themussel calponin-like protein (GenBank ID: BAB60813) [20] with 15matched peptides representing 31% sequence coverage. Unlike p45,the remaining mussel proteins (p270, p230 and p105) could not beidentified by PMF, as their sequence is not available in the databases.

Fig. 1. Purification of filamin from mussel catch muscle. (A) Elution profile from theMono S-FPLC chromatography. (B) Analysis of the collected fractions by SDS-PAGE:aliquots (20 μL) from the indicated fractions were analyzed by 10% SDS-PAGE andCoomassie staining. Four major proteins, indicated by arrows, were retained by thecolumn and eluted with the salt gradient in the fractions 10–12. Lane M, molecularweight protein standards. Lane a, sample containing the high molecular weight proteinthat coeluteswith PKAC-subunit from aMono S chromatography (indicated by arrowhead);this proteinwas obtainedwhen the C-subunitwas purified as previously described [12]. Laneb, aliquot of the protein sample applied to the Mono S column (50 μg protein).

Fig. 2. Determination of the apparent molecular weight of mussel PAM proteins bySDS-PAGE. (A) Aliquots of 5 μg protein (lane 1), 10 μg protein (lane 2) and 15 μg protein(lane 3) from the fraction 10 of Mono S chromatography were analyzed by 10% SDS-PAGE.Lane M, molecular weight protein standards. (B) The experimental data points from themolecular weight standards were fitted to a third-order polynomial (cubic) function, andthe apparent molecular weight of catch muscle proteins was determined by extrapolationfrom the obtained curve.

1336 L. Méndez-López et al. / Biochimica et Biophysica Acta 1824 (2012) 1334–1341

Author's personal copy

However, a detailed analysis of the peptide mass spectra allowed usto draw useful information. First, p270 and p230 shared most of them/z peaks, although several peaks were unique to p270 and a fewwere unique to p230 (Fig. 3A and B). This observation suggests thatp270 and p230 are different but structurally related proteins. Second,all the m/z peaks from p105 were found in the PMF from p270(Fig. 3A and C), which suggests that p105 would be a proteolytic frag-ment derived from the p270 protein.

On the other hand, the PMF spectrum of p230 protein was identi-cal to that of the high molecular weight protein that co-purified withPKA C-subunit from PAM (Fig. 1B, lane a) [12], indicating that they arethe same protein (see supplementary figure).

As the identification of p270, p230 andp105by PMFwasunsuccessful,some of the peaks in the PMFs were selected for MS/MS analysis. Thede novo determined peptide sequences are shown in Table 1. Peptides1–6 were submitted to a homology search using the FASTS (unorderedpeptides vs protein) algorithm,which allowed us to unequivocally identi-fy the three PAMproteins as homologous tomammalian and invertebrate

filamins; the best score was obtained for filamin from Schistosomamansoni (NCBI RefSeq XP_002571464) with E-value: 5.8e-17.

Peptides 4 and 2 match two contiguous sequences located at theN-terminal actin-binding domain of human filamin A (residues 63–87)(Fig. 4). The m/z peaks corresponding to these peptides (1565.24 and1141.75, respectively; see Table 1) were found in the mass spectra ofp270, p230 andp105,which suggests that all threemussel PAMproteinshave the same N-terminal actin-binding domain. On the other hand,peptides 6 and 5 match two contiguous sequences within the Ig-likerepeat 18 of humanfilamin A (residues 2002–2025) (Fig. 4). Interesting-ly, them/z peaks corresponding to these peptides (1382.79 and 1284.74,respectively) were found in the spectra from p270 and p105, but not inthat of p230. This finding suggests structural differences at this regionbetween p270 and p230.

The identity ofmussel PAMfilaminswas confirmed byWestern blot-ting analysis. As observed in Fig. 5, the p270, p230 and p105 proteinswere recognized by a polyclonal antibody against chicken filamin A.They were named FLN-270, FLN-230 and FLN-105, respectively. Unlike

Fig. 3. Peptide mass fingerprints of mussel PAM proteins. Protein bands corresponding to p270 (A), p230 (B), p105 (C) and p45 (D) were subjected to in gel tryptic digestion andanalyzed by MALDI-TOF MS. Arrows indicated two m/z peaks present in the spectra of p270 and p105 but absent in that of p230, that were subjected to de novo sequencing.Arrowheads in panel B indicated m/z peaks that were unique to p230.

1337L. Méndez-López et al. / Biochimica et Biophysica Acta 1824 (2012) 1334–1341

Author's personal copy

these, the p45 protein present in the sample, identified as the calponin-like protein, was not recognized by this antibody.

The purification procedure yielded an average of 0.21 mg filaminper g of PAM. The relative amounts of FLN-270, FLN-230 and FLN-105,determined by densitometric analysis, were 16.4%, 66.5%, and 16.9%,respectively.

3.3. Determination of the molecular weight of mussel filamins

In order to determine the nativemolecularweight ofmusselfilamins,a sample from the Mono-S chromatography (Fig. 1B, fractions 10–12)was applied to a Sephadex S-200-FPLC column, previously calibrated asdescribed in Methods. The protein elution profile is shown in Fig. 6A.As observed, mussel filamins apparently coeluted from the S-200column as a single broad peak. However, the analysis of the collectedfractions (0.5 ml) by SDS-PAGE and Coomassie staining showed a slightbut significant difference in the elution volumes of FLN-270 (9 ml),FLN-230 (9.75 ml) and FLN-105 (11.5 ml) (Fig. 6B). The correspondingmolecular weights, determined by interpolation from the calibrationcurve (Fig. 6D) were 557±12 kDa, 442±15 kDa, and 170±8 kDa,respectively for FLN-270, FLN-230 and FLN-105 (mean±SD of threedeterminations). Taken together, the data of the molecular weight ofnative and denatured proteins indicate that both FLN-270 and FLN-230are dimers comprising two identical subunits, whereas FLN-105 appearsto have a monomeric structure. It is likely that the molecular weight ofFLN-105 is overestimated by gel filtration due to the extended rod-shaped structure of the filamin monomer, as has been previouslyreported for other rod-shaped proteins [21].

On the other hand, the calponin-like protein coeluted from the gelfiltration column with FLN-270 (fractions 18–19; Fig. 6B), which sug-gests that both proteins were associated through direct or indirectinteraction.

Fractions from the gel-filtration chromatography were kept at 4 °Cfor different periods of time and analyzed again by SDS-PAGE. As ob-served, the storage at 4 °C led to the appearance of a 105-kDa bandjust in the fractions containing FLN-270 (fractions 17–20; Fig. 6C).

3.4. Phosphorylation by cAMP-dependent protein kinase

We had previously observed that FLN-230 coeluted with PKAC-subunit isoforms from a cation-exchange chromatography, whichsuggests that it would be a PKA substrate [12]. In order to knowwheth-er mussel filamins might be phosphorylated in vitro by PKA, an aliquotfrom the fraction 21 of the Sephadex S-200 chromatography, containinga mixture of FLN-270, FLN-230 and FLN-105 was incubated with thePKA C-subunit, purified from mussel PAM, in the presence of MgCl2and [γ-32P]ATP. Results indicate that both FLN-270 and FLN-105, butnot FLN-230, were phosphorylated in vitro by PKA (Fig. 7).

3.5. F-actin binding ability of mussel filamins

The actin-binding ability of mussel filamins was evaluated by aco-sedimentation assay. A sample from the fraction 21 of the Sephadex

Table 1Peptide sequences obtained from the mussel PAM proteins by MS/MS analysis.

Peptide Measured m/z (Da) Present in De novo peptide sequence

1 1094.52 p270, p230, p105 WAEFTVDAR2 1141.75 p270, p230, p105 [I/L]VA[I/L][I/L]EV[I/L]SGK3 1475.83 p270, p230, p105 V[I/L]EF[I/L]ERDEG[I/L]R4 1565.79 p270, p230, p105 T[I/L]AD[I/L]ENDFSDG[I/L]R5 1284.74 p270, p105 EVGVH[I/L]VNVYR6 1382.79 p270, p105 [I/L]ANGH[I/L] G[I/L]SFTPR

Fig. 4. Alignment of the amino acid sequences from mussel PAM filamins with homologous regions of human filamin A (GenBank ID: BAJ83965). Vertical bars and colons indicateidentical and similar residues, respectively. The location of the homologous sequences is indicated on the schematic diagram of a filamin dimer showing the primary structuralmotifs (right).

Fig. 5. Western blot analysis of the mussel catch muscle proteins. Aliquots of 0.5 μgprotein (lane 1), 1.0 μg protein (lane 2) and 1.5 μg protein (lane 3) from the fraction10 of the Mono S chromatography (Fig. 1) were analyzed by Western blotting analysiswith a polyclonal antibody anti-chicken filamin. The positions of molecular weightstandards are indicated.

1338 L. Méndez-López et al. / Biochimica et Biophysica Acta 1824 (2012) 1334–1341

Author's personal copy

S-200 chromatography, containing a mixture of FLN-270, FLN-230 andFLN-105, was incubated in the presence and absence of F-actin, andthe supernatant and pellet fractions obtained by ultracentrifugationwere analyzed by SDS-PAGE and Coomassie staining. As observed inFig. 8, a fraction of FLN-270, FLN-230 and FLN-105 sedimented in thepresecence of F-actin and can be observed in the pellet fraction. Thisresult indicated that all three mussel filamins have the ability to bindF-actin.

4. Discussion

In this work we have purified three proteins from a molluscancatch muscle that were unambiguously identified as homologous tomammalian filamins. They were named FLN-270, FLN-230 andFLN-105 according to their apparent molecular weight determinedby SDS-PAGE: 270 kDa, 230 kDa and 105 kDa, respectively. TheFLN-230 was identified as the high molecular weight protein thatcopurified with the PKA C-isoforms from the mussel PAM muscle[12].

The purification procedure of mussel filamins was based on a cationexchange (Mono S) chromatography that allowed us to obtain a mix-ture of the three filamins together with a 45 kDa protein identified as

the mussel calponin-like protein, previously isolated by Funabara et al.from the anterior byssus retractor muscle of M. galloprovincialis [20]. Itshould be noted that all three mussel filamins can bindwithout distinc-tion to a cation- and anion-exchange resin, under identical conditions ofpH (pH 7.0) and ionic strength. This rare behaviour might be due to thepresence, within the large structure of the protein, of discrete regionswith a different net charge, positive and negative, that allows the pro-tein bind to the Mono S and the Mono Q resin, respectively.

4.1. FLN-270 and FLN-230 are different isoforms of filamin

As demonstrated by co-sedimentation assays, all three musselfilamins retain the ability to bind F-actin, indicating that they have theN-terminal actin binding-domain. The presence of N-terminus wasalso confirmed by MS analysis, given that two peptides present inthe three mussel proteins match a sequence belonging to the ABD of

Fig. 8. Actin-binding ability of mussel PAM filamins. A sample from the fraction 21 ofthe Sephadex S-200 chromatography (Fig. 6), containing a mixture of FLN-270,FLN-230 and FLN-105 was incubated with or without 15 μM F-actin isolated fromPAM for 1 h at 25 °C. The FLN-270/actin molar ratio was 1:10. F-actin was precipitatedby ultracentrifugation, and the pellet (P) and supernatant (S) fractions were dilutedto equivalent volumes in SDS-PAGE sample buffer and analyzed by SDS-PAGE andCoomassie staining.

Fig. 6. Determination of nativemolecular weight of mussel PAM filamins. (A) Elution profilefrom the Sephadex S-200 chromatography. A sample (0.5 mL) fromamixture of the fractions10–12 of theMono S chromatography (Fig. 1)was applied to a Sephadex S-200-FPLC columnpreviously calibrated. Fractions of 0.5 mLwere collected. Arrows indicate the elution volume(Ve) of molecular weight protein standards: thyroglobulin (669 kDa), ferritin (440 kDa),aldolase (158 kDa), ovoalbumin (43 kDa) and chymotrypsinogen (25 kDa). (B) Aliquots ofthe collected fractionswere analyzedby SDS-PAGE andCoomassie staining. Asterisks indicatethe fraction(s) containing the highest concentration of each mussel PAM protein. Note thedifferent elution volume of FLN-270, FLN-230 and FLN-105 and the coelution of thep45-protein with FLN-270. (C) Fractions were kept at 4 °C for 4 days, and aliquots wereanalyzed again by SDS-PAGE and Coomassie staining. Note the appearance of the 105-kDaband (indicated by arrow) in the fractions containing FLN270. (D) The molecular weight ofmussel filamins was determined from the plot log molecular weight vs Ve/Vo of the proteinmarkers (open circles).

Fig. 7. Phosphorylation of mussel PAM filamins by PKA. Aliquots (6 μg protein) fromthe fraction 21 of the Sephadex S-200 chromatography (Fig. 6), containing a mixtureof FLN-270, FLN-230 and FLN-105, were incubated in the presence of MgCl2 and[γ-32P]ATP with (+) or without (−) purified PKA C-subunit (0.1 μg protein) at 30 °Cfor 15 min. Reactions were stopped by adding SDS-sample buffer and boiling for5 min. Samples were then analyzed by SDS-PAGE and the gel was stained, destained,dried, and exposed for autoradiography at −20 °C. The positions of molecular weightstandards are indicated.

1339L. Méndez-López et al. / Biochimica et Biophysica Acta 1824 (2012) 1334–1341

Author's personal copy

mammalian filamins. On the other hand, both FLN-270 and FLN-230 aredimers, indicating that they must contain the C-terminal dimerizationdomain typical of filamins. Furthermore, results from MS analysisrevealed that most of peptidemasses were found to be shared betweenFLN-270 and FLN-230, but also that several peak masses were uniqueto FLN-270 and a few were unique to FLN-230. Taken together, theseresults rule out the possibility that FLN-230 is a proteolytic fragmentof FLN-270, and therefore they should be considered as differentisoforms of filamin, either encoded by two different genes or generatedby alternative splicing from a single gene. As judging from the purifica-tion results, FLN-230 was found to be 4–5-fold more abundant thanFLN-270 and hence the predominant filamin isoform in mussel catchmuscle.

In mammals there are three filamin isoforms (A, B and C) encodedby different genes. Filamin A and B are themost ubiquitously expressedisoforms, whereas filamin C expression is predominantly restricted toskeletal and cardiac muscle [13,16,22]. The diversity of the filamin fam-ily is increased by alternative splicing events. It has been reported theexistence of (1) splice variants of filamin B and C lacking the hinge 1domain [23,24]; (2) truncated splice variants of filamin B lacking thefour C-terminal Ig-like repeats, including the dimerization domain[25]; and (3) splice variants of filamin A and B lacking a segment of 41aminoacids between Ig-repeats 19 and 20 within the rod-2. This seg-ment contains the PKA-phosphorylation site of filamin (Ser 2151 inhuman filamin A [26]), so that these splice variants were not phosphor-ylated by PKA [25,27]. Interestingly, our results revealed structuraldifferences at the rod 2 segment between FLN-270 and FLN-230, giventhat two peptide sequences from FLN-270, which match a sequencelocated at the Ig-like repeat 18 of human filamin A, were not found inthe FLN-230 (Fig. 4); furthermore, FLN-230, unlike FLN-270 and its pro-teolytic fragment FLN-105, failed to be phosphorylated in vitro by mus-sel PKA. These data, in accordance with the apparent molecular weightvalues, suggest that FLN-230 would be a splice variant of FLN-270lacking a number of the Ig-like repeats of the rod 2 segment.

Since the rod-2 segment is responsible formost of the protein–filamininteractions, the structural differences between FLN-270 and FLN-230 atthe rod 2 segment level suggest that both mussel filamins differ in theirability to interact with protein partners [14,15].

4.2. FLN-105: a proteolytic fragment of FLN-270 or a truncated variant?

The comparative analysis of peptide mass spectra revealed that allthe tryptic peptide masses from FLN-105 are also found in the PMF ofFLN-270, but not in that of FLN-230 (Fig. 3); hence, it suggests thatFLN-105 would be a proteolytic fragment of FLN-270. This hypothesisis consistent with the observation that the storage at 4 °C of a samplecontaining purified FLN-270 leads to the gradual increasing of a105-kDa band that can be observed by SDS-PAGE and Coomassiestaining analysis. The partial sequences demonstrate that FLN-105contains the N-terminal actin-binding domain, and accordingly, itretains the ability to bind F-actin, indicating that FLN-105 would bean N-terminal proteolytic fragment.

It is known that the mammalian filamins are highly susceptible toproteolysis by calpain, within the hinge domains [28]. Several studiesindicated that limited proteolysis modulates the ability of filamin tointeract with its protein partners, and thus proteolysis was consid-ered as a regulatory mechanism of the filamin functions. For instance,it has been recently reported that the calpain-mediated cleavage offilamin reduces the cytoskeleton tension in vascular smooth musclecells and allows shortening of the contractile apparatus [29].

The hypothesis that FLN-105 is a proteolytic fragment disagrees,however, with the data of molecular weight, because the theoreticalfragment generated by proteolytic cleavage at the level of the hinge 2of filamin should be greater than 105 kDa. Another possibility is thatFLN-105 is a truncated splice variant of filamin lacking the C-terminaldomain and other internal repeats, but retaining the N-terminal

actin-binding domain. The existence of C-terminally truncated splicevariants of filamin has been reported both in mammals [25] and inC. elegans [30].

4.3. Co-elution of FLN-270 and calponin-like protein

We have identified a 45 kDa-protein that copurifies with filaminsas the calponin-like protein; this protein has previously been isolatedfrom the mussel catch muscle by Funabara et al. [20]. Vertebratesmooth muscle calponin appears to be involved in the regulation ofthe named latch contraction of vertebrate smooth muscles [31]. Mus-sel calponin, like its vertebrate counterparts, inhibits actomyosinMg-ATPase activity and decreases the sliding of actin filaments invitro [20]; however, its role in the regulation of molluscan catch con-traction has not been yet clarified.

Our results indicate that, despite the fact that the calponin-like pro-tein is a monomer of 45 kDa [20], it co-eluted from the gel-filtrationchromatographywith the FLN270 isoform. The latter suggests a specificinteraction between both proteins. This result contrasts with the onereported by Panasenko et al. [32]who found that, when amixture of pu-rified filamin and calponin from vertebrate smoothmuscle was appliedto a gel filtration chromatography, both proteins were eluted separate-ly. They concluded that filamin and calponin from vertebrate smoothmuscle do not directly interact. This different behaviour between verte-brate and molluscan smooth muscle proteins (filamin and calponin)might be due to the different experimental conditions used in the chro-matographic processes or a consequence of different functional proper-ties between vertebrate andmolluscan smoothmuscle proteins. Indeed,although mussel calponin-like protein and vertebrate calponin showremarkable similarities at the calponin-homology (CH) domain andthe calponin-like repeats, mussel protein is longer (45 kDa vs. 32 kDa)and highly divergent (26.3% identity) in comparison with its vertebratecounterparts [20,33]. These structural differences would account forthe different ability of mussel and vertebrate calponin to interact withfilamin.

In summary, we have identified and characterized two differentisoforms of filamin from molluscan catch muscle. One of these isofomsappears to interact with the calponin-like protein. Now, further investi-gations are needed to understand the possible role of these proteins inthe molecular regulation of the catch contraction.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbapap.2012.07.011.

Acknowledgements

This work was supported by grant PGIDIT02RMA26101PR fromthe Autonomous Government of Galicia (Xunta de Galicia), Spain.We would also like to thank Dr. JoDee Anderson for the linguistic sup-port she provided.

References

[1] S.L. Hopper, K.H. Hobbs, J.B. Thuma, Invertebrate muscles: Thin and thick filamentstructure; molecular basis of contraction and its regulation, catch and asynchro-nous muscle, Prog. Neurobiol. 86 (2008) 72–127.

[2] S. Galler, Molecular basis of the catch state in molluscan smooth muscles: a catchychallenge, J. Muscle Res. Cell Motil. 29 (2008) 73–99.

[3] M.J. Siegman, D. Funabara, S. Kinoshita, S.Watabe, D.J. Hartshorne, T.M. Butler, Phos-phorylation of a twitchin-related protein controls catch and calcium sensitivity offorce production in invertebrate smooth muscle, Proc. Natl. Acad. Sci. U. S. A. 95(1998) 5383–5388.

[4] A. Yamada, M. Yoshio, A. Nakamura, K. Kohama, K. Oiwa, Protein phosphatase 2Bdephosphorylates twitchin, initiating the catch state of invertebrate smooth mus-cle, J. Biol. Chem. 279 (2004) 40762–40768.

[5] D. Funabara, S. Watabe, S.U. Mooers, S. Narayan, C. Dudas, D.J. Hartshorne, M.J.Siegman, T.M. Butler, Twitchin from molluscan catch muscle: primary structureand relationship between site-specific phosphorylation and mechanical function,J. Biol. Chem. 278 (2003) 29308–29316.

[6] D. Funabara, C. Hamamoto, K. Yamamoto, A. Inoue, M. Ueda, R. Osawa, S. Kanoh, D.J.Hartshorne, S. Suzuki, S. Watabe, Unphosphorylated twitchin forms a complex with

1340 L. Méndez-López et al. / Biochimica et Biophysica Acta 1824 (2012) 1334–1341

Author's personal copy

actin and myosin that may contribute to tension maintenance in catch, J. Exp. Biol.210 (2007) 4399–4410.

[7] N.S. Shelud'ko, G.G. Matusovskaya, T.V. Pemyakova, O.S. Matusovsky, Twitchin, athick-filament protein from molluscan catch muscle, interacts with F-actin in aphosphorylation-dependent way, Arch. Biochem. Biophys. 432 (2004) 269–277.

[8] J.L. Rodríguez, R. Barcia, J.I. Ramos-MartínezI, J.A. Villamarín, Purification of a novelisoform of the regulatory subunit of cAMP-dependent protein kinase from the bi-valve mollusk Mytilus galloprovincialis, Arch. Biochem. Biophys. 359 (1998) 57–62.

[9] M.J. Díaz-Enrich, I. Ibarguren, U. Hellman, J.A. Villamarín, Characterization of atype I regulatory subunit of cAMP-dependent protein kinase from the bivalvemollusk Mytilus galloprovincialis, Arch. Biochem. Biophys. 416 (2003) 119–127.

[10] J.R. Bardales, J.L. Cascallana, J.A. Villamarín, Differential distribution of cAMP-dependent protein kinase isoforms in various tissues of the bivalve molluscMytilus galloprovincialis, Acta Histochem. 113 (2011) 743–748.

[11] P. Béjar, J.A. Villamarín, Catalytic subunit of cAMP-dependent protein kinase froma catch muscle of the bivalve mollusc Mytilus galloprovincialis: Purification, char-acterization and phosphorylation of muscle proteins, Arch. Biochem. Biophys. 450(2006) 133–140.

[12] J.R. Bardales, U. Hellman, J.A. Vilamarín, Identification of multiple isoforms of thecAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilusgalloprovincialis, FEBS J. 275 (2008) 4479–4489.

[13] A. Van der Flier, A. Sonnenberg, Structural and functional aspects of filamins,Biochim. Biophys. Acta 1538 (2001) 99–117.

[14] F. Nakamura, T.P. Stossel, J.H. Hartwig, The filamins: organizers of cell structureand function, Cell Adh. Migr. 5 (2011) 160–169.

[15] G.M. Popowicz, M. Schleicher, A.A. Noegel, T.A. Holak, Filamins: promiscuousorganizers of the cytoskeleton, Trends Biochem. Sci. 31 (2006) 411–419.

[16] A.X. Zhou, J.H. Hartwig, L.M. Akyürek, Filamins in cell signalling, transcription andorgan development, Trends Cell Biol. 20 (2010) 113–123.

[17] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head ofbacteriophage T4, Nature 277 (1970) 680–685.

[18] U. Hellman, R. Bhikhabhai, Easy amino acid sequencing of sulfonated peptides usingpost-source decay on a matrix-assisted laser desorption/ionization time-of-flightmass spectrometer equipped with a variable voltage reflector, Mass Spectrom. 16(2002) 1851–1859.

[19] A.J. Mackey, T.A.J. Haystead, W.R. Pearson, Getting more from less: Algorithms forrapid protein identification with multiple short peptide sequences, Mol. Cell. Pro-teomics 1 (2002) 139–147.

[20] D. Funabara, M. Nakaya, S. Watabe, Isolation and characterization of a novel 45kDa calponin-like protein from anterior byssus retractor muscle of the musselMytilus galloprovincialis, Fish. Sci. 67 (2001) 511–517.

[21] K.J. Sweadner, Size, shape, and solubility of a class of releasable cell surface proteinsof sympathetic neurons, J. Neurosci. 3 (1983) 2518–2524.

[22] X. Zhou, J. Borén, L.M. Akyürek, Filamins in cardiovascular development, TrendsCardiovasc. Med. 17 (2007) 222–229.

[23] Z. Xie, W. Xu, E.W. Davie, D.W. Chung, Molecular cloning of human ABPL, anactin-binding protein homologue, Biochem. Biophys. Res. Commun. 251 (1998)914–919.

[24] W. Xu, Z. Xie, D.W. Chung, E.W. Davie, A novel human actin-binding proteinhomologue that binds platelet glycoprotein Ibα, Blood 92 (1998) 1268–1276.

[25] A. Van der Flier, I. Kuikman, D. Kramer, D. Geerts, M. Kreft, T. Takafuta, S.S.Shapiro, A. Sonnenberg, Different splice variants of filamin-B affect myogenesis,subcellular distribution, and determine binding to integrin β subunits, J. CellBiol. 156 (2002) 361–376.

[26] D. Jay, E.J. García, M.L. Ibarra, In situ determination of a PKA phosphorylation sitein the C-terminal region of filamin, Mol. Cell. Biochem. 260 (2004) 49–53.

[27] M.A. Travis, A. Van der Flier, R.A. Kammerer, A.P. Mould, A. Sonnenberg, M.J.Humphries, Interaction of filamin A with the integrin β 7 cytoplasmic domain:role of alternative splicing and phophorylation, FEBS Lett. 569 (2004) 185–190.

[28] J.B. Gorlin, R. Yamin, S. Egan, M. Stewart, T.P. Stossel, D.J. Kwiatkowski, J.H. Hartwig,Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecu-lar leaf spring, J. Cell Biol. 111 (1990) 1089–1105.

[29] M. Uchida, I. Ishii, K. Hirata, F. Yamamoto, K. Tashiro, T. Suzuki, Y. Nakayama, N.Ariyoshi, M. Kitada, Degradation of filamin induces contraction of vascular smoothmuscle cells in type-I collagen matrix honeycombs, Cell. Physiol. Biochem. 27 (2011)669–680.

[30] I. Kovacevic, E.J. Cram, FLN-1/Filamin is required for maintenance of actin and exitof fertilized oocytes from the spermatheca in C. elegans, Dev. Biol. 347 (2010)247–257.

[31] P.T. Szymanski, Calponin (CaP) as a latch-bridge protein – a new concept in reg-ulation of contractility in smooth muscles, J. Muscle Res. Cell Motil. 25 (2004)7–19.

[32] O.O. Panasenko, N.B. Gusev, Mutual effects of α-actinin, calponin and filamin onactin binding, Biochim. Biophys. Acta 1544 (2001) 393–405.

[33] K. Takahashi, B. Nadal-Ginard, Molecular cloning and sequence analysis of smoothmuscle calponin, J. Biol. Chem. 266 (1991) 13284–13288.

1341L. Méndez-López et al. / Biochimica et Biophysica Acta 1824 (2012) 1334–1341