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Characterization of Human Skeletal Muscle Ankrd2
Biochemical and Biophysical Research Communications 285, 378 –386 (2001) doi:10.1006/bbrc.2001.5131, available online at http://www.idealibrary.com on
Characterization of Human Skeletal Muscle Ankrd2 Alberto Pallavicini,* Snezana Kojic´,† Camilla Bean,* Mariz Vainzof,‡ Michela Salamon,* Chiara Ievolella,* Gladis Bortoletto,* Beniamima Pacchioni,* Mayana Zatz,‡ Gerolamo Lanfranchi,* Georgine Faulkner,† and Giorgio Valle* ,1 *CRIBI Biotechnology Centre, Universita` degli Studi di Padova, via Ugo Bassi 58b, I-35121 Padua, Italy; †International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy; and ‡Centro de Estudos do Genoma Humano, IB-USB, Rua do Mata˜o 106, CEP 05508-900, Sao Paulo, Brazil
Received June 4, 2001
Human Ankrd2 transcript encodes a 37-kDa protein that is similar to mouse Ankrd2 recently shown to be involved in hypertrophy of skeletal muscle. These novel ankyrin-rich proteins are related to C-193/ CARP/MARP, a cardiac protein involved in the control of cardiac hypertrophy. A human genomic region of 14,300 bp was sequenced revealing a gene organization similar to mouse Ankrd2 with nine exons, four of which encode ankyrin repeats. The intracellular localization of Ankrd2 was unknown since no protein studies had been reported. In this paper we studied the intracellular localization of the protein and its expression on differentiation using polyclonal and monoclonal antibodies produced to human Ankrd2. In adult skeletal muscle Ankrd2 is found in slow fibers; however, not all of the slow fibers express Ankrd2 at the same level. This is particularly evident in dystrophic muscles, where the expression of Ankrd2 in slow fibers seems to be severely reduced. © 2001 Academic Press Key Words: muscle proteins; muscle plasticity; hypertrophy; ankyrin; signaling pathways.
It is generally known that people can perform physical work much better after appropriate training and exercising. Both skeletal muscle and heart can increase or decrease their mass to adapt to the tasks that The cDNA sequences reported in this paper have been submitted to the GenBank/EMBL/DDJB databases with Accession Nos. AJ304805 (human Ankrd2) and AJ011118 (mouse Ankrd2). The human genomic sequence has been submitted with Accession No. AJ304804. Abbreviations used: BAC, bacterial artificial chromosome; BSA, bovine serum albumin; EST, expressed sequence tag; GFP, green fluorescent protein; FITC, fluorescein isothiocyanate; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RTPCR, reverse transcriptase PCR; SDS, sodium dodecyl sulfate. 1 To whom correspondence should be addressed. Fax: ⫹39-0498276280. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
they usually perform, but muscle plasticity is not just a matter of mass. The quality of a muscle depends to a great extent on the type of fibers of which it is composed. Muscle fibers are characterized by different subsets of specialized proteins. For instance, there are different isoforms of myosin heavy chain: isoform I is found in type I fibers which contract more slowly than type II fibers (containing type II isoforms), thus the latter are known as fast fibers and the former as slow fibers. The difference in muscle performance depends not only on the mechanics of contractile proteins, but also on the type of metabolism used, that in slow fibers is aerobic, whereas in fast fibers is anaerobic (1). Many aspects of the molecular biology of fast and slow fibers are now understood; however, very little is known about the mechanisms responsible for sensing, signaling and adjusting the mass and type of fibers of any given muscle. In this respect, we searched our database of human skeletal muscle transcripts (2) accessible at http://muscle.cribi.unipd.it, for possible transcription factors with a muscle specific pattern of expression. Transcript HSPD02860 (now called Ankrd2) revealed some very interesting features such as a nuclear sorting signal, four tandem repeated ankyrin motifs, which are typically found in several transcription factors, and a good similarity to protein C-193, a putative transcription factor found in endothelial cells (3). While the work presented in this paper was in progress some very interesting results were published on the rat and mouse orthologs of human C-193 (called respectively CARP and MARP), indicating that they are mainly expressed in heart (4 – 6) and probably involved in the control of cardiac hypertrophy (7). Recently, a paper was published by Kemp et al. (8) on mouse Ankrd2, the closest gene related to C-193/ CARP/MARP that is expressed mainly in skeletal muscle. Thus, it is possible that Ankrd2 may play a similar
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FIG. 1. Genomic organization and promoter analysis of Ankrd2. (a) Schematic representation of the genomic organization of human (AJ304804) and mouse (AJ249346) Ankrd2 genes. The lines are connecting the corresponding exons. (b) Sequence comparison of the mouse and human promoter of Ankrd2 genes. Putative binding motifs for E47, MyoD, NF-B, and the TATA box are in bold. The predicted transcription start and the first two ATG codons are also marked. The two arrows delimit the sequence region responsible for muscle expression (see Fig. 2).
role to C-193/CARP/MARP, but in skeletal muscle rather than heart. Furthermore, mouse Ankrd2 is expressed at a higher level after muscle stretching, giving a direct indication of a possible involvement of this protein in controlling muscle hypertrophy (8). In this paper we present our work on human Ankrd2 including a detailed analysis of its pattern of expression and cellular localization, both at the mRNA and protein levels. MATERIALS AND METHODS Cell culture. Cell culture conditions for primary human (CHQ5B) and mouse (C2C12) myoblast cells were as previously described (9). Differentiation medium for these cells was Dulbecco’s modified Eagle medium without serum but supplemented with 10 g/ml of insulin (Sigma I-5500, Sigma–Aldrich, St. Louis, MO) and 100 g/ml of transferrin (Sigma T-2036) or 0.4% Ultroser G (Life Technologies, Grand Island, NY). Expression studies of the Ankrd2 mRNA. The Northern blot analysis was done using mRNA filters obtained from Clontech (Clontech). Specific radiolabeled probes corresponding to the 5⬘- or 3⬘-portions of the Ankrd2 transcript were generated by PCR amplification of the cDNA clone with specific primers. Radioactive PCRs were purified and then used for hybridization utilizing the protocol suggested by
the manufacturer (NorthernMax, Ambion, Austin, TX). The signal was detected using the Cyclone Storage Phosphor System (Packard Instruments Co., Meriden, CT). Reverse transcriptase PCR (RT-PCR) assays were performed on a panel of 16 different human tissue cDNAs (Human MTC panel I and II; Clontech) using primers specific for the 3⬘ end of the Ankrd2 mRNA. Genomic mapping. Genomic mapping was performed by PCR with specific primers designed for the 3⬘ portion of the Ankrd2 transcript using the GeneBridge 4 whole-Genome Radiation Hybrid Panel (10) (Research Genetics, Huntsville, AL). The results were processed using the RHMAPPER software program (Whitehead Institute/MIT Center for Genomic Research, Cambridge, MA). Genomic sequencing. Bacterial artificial chromosome (BAC) clones containing Ankrd2 DNA (H100F12, H443C8 and H443C9) were obtained by PCR screening at the “Fondation Jean Dausset”-CEPH (Centre d’Etu`de du Polymorphisme Humaine, Paris; http://landru.cephb.fr/ services), using Ankrd2 transcript-specific primers. The H443C9 clone was used for shotgun library construction. BAC DNA was sheared using a GeneMachine Hydroshear. The fragments obtained were size selected on agarose gels and cloned into a modified pZero vector (Invitrogen). A chromosome walking approach was applied to the 768 arrayed shotgun clones and the sequences were assembled using the program SeqMan 3.61 (DNASTAR Inc., Madison, WI). The Ankrd2 genomic sequence was analyzed to determine the transcription initiation site using the program “Promoter Prediction” by Neural Network at http://www.fruitfly.org/seq_tools/promoter.html. Finally the putative
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Ankrd2 binding transcription factors were identified by the TFSearch (http://pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html) and matSearch (http://transfac.gbf.de/cgi-bin/matSearch/matsearch.pl) programs. Immunoblot analysis. Proteins from different human tissues were obtained from Clontech (Cat. Nos. 7800 to 7808, Cat. No. 7813). The protein extracts and a pre-stained molecular weight marker (Sigma C3437) were separated on 12 or 15% SDS–polyacrylamide gels and then blotted onto immobilon P membrane (Millipore, Bedford, MA). The Ankrd2 monoclonal (ascites fluid) and polyclonal antibodies were used at the same dilutions (1/200-1/400). MF-20, myosin antibody was used at a 1/10,000 dilution. This antibody (developed by Dr. D. A. Fischman) was obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Goat antimouse immunoglobulin conjugated with alkaline phosphatase (Sigma A3562) was used as the second antibody in all Western blot experiments. Primary human and mouse muscle cells were harvested and resuspended in a urea buffer (8 M urea; 0.1 M NaH 2PO 4; 0.01 M Tris, pH 8.0), sonicated, centrifuged and the supernatants separated on 12 or 15% SDS–polyacrylamide gels. Immunofluorescence. Undifferentiated and differentiated primary human muscle cells grown on collagen-coated coverslips were fixed with paraformaldehyde (4%), then treated with 0.1 M glycine and permeabilized with 0.05% Tween 20 for 30 min. All wash and dilution buffers contained both bovine serum albumin (BSA) 1% and 0.05% Tween-20 to block any nonspecific binding. For these experiments the secondary antibody was fluorescein isothiocyanate (FITC)conjugated anti-mouse immunoglobulin (Sigma F4018) or goat antirabbit Texas red (Calbiochem, La Jolla, CA). All commercial immunochemicals were diluted as recommended by the suppliers. For confocal microscopy the nuclei were stained with propidium iodide (3.5 g/ml, Sigma P4170). A LSM 510 laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany) was used for confocal microscopy both at 40⫻ and 100⫻ magnification. For muscle biopsies sections of 5 m were cut using a cryostat and air-dried for 1 h and then incubated with 10% horse serum in PBS to prevent nonspecific binding. The anti-Ankrd2 antibodies monoclonal C-terminal and polyclonal N-terminal (1/40 dilution) and the anti-actinin3 antibody (1/100 dilution) obtained from Alan Beggs were used for immunocytochemistry. For double immunofluorescence experiments anti-ankrd2 and anti-actinin3 antibodies were mixed and incubated simultaneously overnight. After washing, the sections were incubated for 1 h with secondary antibodies (dilution 1/100): FITC-conjugated anti-rabbit-Ig (Sigma) and CY3-conjugated anti-mouse-Ig (Jackson Immunochemicals, West Grove, PA). An Axiophoto microscope (Carl Zeiss Microscopy), with epifluorescence, and filters for FITC and rhodamine was used for microscopy. Immunoprecipitation. Polyadenylated mRNA of adult skeletal muscle was in vitro translated using a reticulocyte lysate system (Promega, Madison, WI) and labeled with [ 35S]methionine (Amersham Pharmacia Biotech, Rainham, UK). Equal amounts of labeled proteins were mixed with the appropriate antibody and immunoprecipitated using protein A–Sepharose (Amersham Pharmacia Biotech) in a buffer containing 50 mM Hepes, pH 8.0, 250 mM NaCl, 0.1% Nonidet P40. The samples were separated by SDS–PAGE, dried, exposed using a super resolution phosphor screen and analyzed on a Packard Cyclone phosphor imager (Packard Instrument Co.). Polyclonal anti-Ankrd2 antibody, pre-immune sera and MF 20 were used at dilutions of 1/75 for these experiments. Promoter analysis. Three luciferase reporter constructs were produced to enable the identification of the minimal region involved in Ankrd2 skeletal muscle transcriptional activity. The reverse primer PROHindIII ⫹ 10 (⫹10, ⫺10) combined with PROXhoI-915 (⫺915, ⫺895), PROXhoI-616 (⫺616, ⫺596) and PROXhoI-280 (⫺280, ⫺260) were used for PCR amplification with the Ankrd2 genome as template. The PCR products were HindII/
FIG. 2. Transient expression assays to identify the minimal promoter region that confers muscle specificity. (a) Schematic diagrams of a series of luciferase constructs containing nested deletions of the Ankrd2 promoter. The construct name refers to the length of the genomic region cloned. (b) A histogram showing the luciferase activity of the different constructs in C2C12 cells at different stages of differentiation. All transfections were performed in triplicate.
XhoI digested and cloned into the pGL3-basic vector (Promega). The luciferase reporter constructs were co-transfected with the pCMV vector in mouse C2C12 cells and in mouse 3T3 cells using the lipofectamine 2000 reagent (Life Technologies). The luciferase activity, measured with the Luciferase Assay System (Promega) on the Jade luminometre (Anthos Labtec Instruments, Wals, Austria), was normalized for gal activity to account for variations in the efficiency of transfection. Since luciferase can be unstable during culture incubation the values of the luciferase activity were normalized to the pRL-SV40 vector (Promega) which was cotransfected with the pCMV vector.
RESULTS Sequencing of Human and Mouse Ankrd2 cDNA The systematic sequencing of human skeletal muscle ESTs carried out in our laboratory (2) has led to the discovery of many new genes expressed in muscle. To date, some 5300 transcripts have been identified from over 30,000 ESTs. Amongst this collection, transcript HSPD02860 (now called Ankrd2) was recognized to be particularly interesting as it shows a 43% identity at the amino acid level to C-193/CARP/MARP, a 319 amino acid nuclear protein expressed in endothelial cells (3), heart (6) and to a lesser extent in skeletal muscle (4), which is believed to play a role as a cofactor in local signaling pathways ranging from muscle morphogenesis (4) to cardiac hypertrophy (5).
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Ankyrin repeats are ubiquitous motifs involved in macromolecular interactions (12). There are at least four ankyrin motifs in detected in C-193/CARP/ MARP, mouse and human Ankrd2 proteins with possibly a fifth ankyrin repeat present only in the human protein. The Ankrd2 protein would appear to have several potential phosphorylation sites as noted
FIG. 3. Ankrd2 mRNA in human tissues detected by Northern blot and RT-PCR analysis. (a) Northern blot analysis of human tissues demonstrating patterns of expression of Ankrd2 mRNAs. A panel of mRNAs from 12 different human adult tissues: brain (br), heart (he), skeletal muscle (sk), colon (co), thymus (th), spleen (sp), kidney (ki), liver (li), small intestine (si), placenta (pl), lung (lu), and peripheral blood leukocytes (le) were probed with radioactive PCR fragments derived from either the 3⬘ untranslated region of Ankrd2 or with the 5⬘ region as indicated. The numbers on the side indicate the size (kb). (b) RT-PCR for different human tissues. The assays were performed on a panel of 16 different human tissue cDNAs (Human MTC panel I and II, Clontech) using primers specific for the 3⬘ portion of the Ankrd2 mRNA. The different tissues are indicated as in section (a), with the addition of pancreas (pa), ovary (ov), prostate (pr), testes (te), and control sample (cn). The number of cycles of PCR 26, 29, and 32 is given at the side of the figure.
The relative abundance of human Ankrd2 mRNA was predicted to be 0.055%, i.e., about 1/2000 of the polyadenylated mRNAs, as calculated from the percentage of Ankrd2 ESTs present in our muscle EST database. The strategies used for the construction of our libraries resulted in cDNA clones that reflect the concentration of each mRNA in the muscle tissue (2, 11). Our complete cDNA sequences of human and mouse Ankrd2 are available from EMBL under the Accession Nos. AJ304805 and AJ011118, respectively. The human transcript is 1159 nucleotides, whereas the mouse is slightly shorter in the 3⬘ untranslated region (1101 bp). Human and mouse sequences are very similar, the identity being 85% at the nucleotide level and 89% at the amino acid level. The nucleotide substitutions between the two species are evenly distributed along the sequences, with the only exception of a stretch of five contiguous amino acids near the N-terminal that are different between human and mouse. The ORFs of human and mouse Ankrd2 are 999 and 996 bases, respectively, encoding putative proteins of 333 and 332 amino acid residues with calculated molecular weights of 37,150 and 37,124 daltons, respectively.
FIG. 4. Expression pattern of Ankrd2 protein in human tissues and primary muscle cells. (a) Tissue distribution of Ankrd2 as demonstrated by Western blot analysis. Protein extracts from human heart and skeletal muscle (10 g) as well as from brain, placenta, testis, spleen, kidney, liver, and lung (60 g) were loaded in each lane, run on a 15% SDS–polyacrylamide gel and then blotted. The Immobilon P membrane (Millipore) was probed with mouse preimmune sera, polyclonal N- and C-terminal Ankrd2 antibodies as well as antibody to full-length Ankrd2 and a monoclonal antibody specific for an epitope from the C-terminal region of Ankrd2. All sera were used at a 1/200 dilution. (b) Immunoprecipitation of human skeletal muscle proteins obtained from in vitro translation of adult skeletal muscle mRNA using the rabbit reticulocyte lysate system (Promega). Equal amounts of [ 35S]methionine-labeled proteins were mixed with the appropriate antibody and immunoprecipitated using protein A–Sepharose and separated by SDS–PAGE. The muscle proteins were immunoprecipitated with mouse preimmune sera, anti-Ankrd2 C-terminal antibody and anti-myosin antibody (MF20 ascites fluid). Numbers on the left of the figures indicate molecular weights (kD); the rainbow [ 14C]methylated protein molecular weight marker (Amersham) was used. (c) Western blot analysis of protein extracts from mouse C2C12 cells at 0, 3, 6, and 8 days after the addition of differentiation medium. The blot was probed with mouse polyclonal antibody to N-terminal Ankrd2.
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FIG. 5. Time course of Ankrd2 expression in primary human muscle cells during differentiation as detected by indirect immunofluorescence. The mouse monoclonal C-terminal and polyclonal N-terminal Ankrd2 antibodies were used at a dilution of 1/40; myosin monoclonal antibody (MF 20) was used at 1/100 dilution. FITC-conjugated anti-mouse Ig (Sigma) was used as the second antibody at the recommended dilution. The days after the addition of differentiating medium are noted at the side of the figure. A preimmune mouse serum was used as a control giving a negative signal at all the time points (data not shown).
by computer analysis; six cAMP-dependent protein kinase phosphorylation sites, three casein kinase II sites, two calmodulin-dependent protein kinase II sites, one cGMP-dependent protein kinase site and one protein kinase C site. Ankrd2 can be phosphorylated in vitro by casein kinase II (data not shown), thus confirming the phosphorylation data, at least for casein kinase II. Another interesting motif found both in human and mouse Ankrd2, as well as C-193/CARP/MARP is the PEST sequence which serves as proteolytic signal that targets proteins for rapid destruction (13, 14). Both human and murine Ankrd2 proteins have nuclear localization motifs, however these proteins are predicted to be cytoplasmic by PSORT. Also C-193/CARP/MARP was classified as a cytoplasmic protein by PSORT despite having a nuclear localization signal and being actually localized in the nucleus both as an endogenous protein (6) and after expression of the protein in transfected cells (3–5).
Genome Organization and Promoter Analysis The Ankrd2 gene was mapped on the human chromosome region 10q23.31–23.32 using the radiation hybrid technique, as described in the experimental procedures. The closest marker is WI-3011, at an estimated distance of 4.29 centiRay with very significant lod score of 21. In order to characterize the structure of the human Ankrd2 gene, a CEPH human BAC library was screened by PCR. The positive BAC clone H443C9 was used to produce a shotgun library and a chromosome walking approach with hierarchical pooling strategies allowed the sequencing of a genomic region of 14,300 bp. The genomic DNA and cDNA comparison revealed that the gene is divided into 9 exons as shown in Fig. 1, and each of the exons 5, 6, 7 and 8 encode one ankyrin repeat, displaying a gene organization very similar to mouse Ankrd2 (8). A bioinformatic analysis of the transcription initiation site indicates an alternative ATG starting codon, twelve bases upstream from the ATG
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FIG. 6. The expression pattern of Ankrd2 in normal and dystrophic skeletal muscle as detected by immunofluorescence. (a) A normal skeletal muscle sections stained with three antibodies to Ankrd2 and an antibody to ␣-actinin3. The type II fibers are stained with ␣-actinin 3 (green) and some of the type I fibers are stained with the three anti-mouse Ankrd2 (red) antibodies. Clear sarcomeric striations can be seen with the ␣-actinin 3 antibody, whereas only weak striations can be seen with the C-terminal and full-length anti-Ankrd2 antibodies. The asterisk indicates a fiber that is negative both to ␣-actinin 3 and to Ankrd2. (b) A dystrophic muscle from a patient with an as yet unclassified form of merosin-positive congenital muscular dystrophy was labeled with the three antibodies to Ankrd2 and an antibody to ␣-actinin 3. Only the full-length Ankrd2 antibody (red) can be seen to stain some type II fibers; neither anti-Ankrd2 N- nor C-terminal antibodies (red) stained the type II fibers; ␣-actinin 3 (green) staining was used as a positive control for type II fiber staining.
codon identified in the cDNA clone, but maintaining the same reading frame. A similar analysis was carried out on the mouse genomic sequence indicating the presence of the additional twelve coding bases also in mouse, although in this case the cDNA sequences terminated at the second ATG. The hypothesis that this is the genuine start of transcription is further supported by the primer extension analysis performed on the CARP genomic sequence (15), that places the transcription initiation site in agreement with the bioinformatic analysis (8, 16). The 1157-bp 5⬘ flanking region of Ankrd2 shows several putative recognition sequences for transcription factors however interestingly the human and mouse Ankrd2 promoter sequence alignment shows only three boxes containing cis-elements specific for muscle transcription factors (see Fig. 1). In addition to the canonical muscle specific transcription factors MyoD and E47, the NF-B binding sequence was found, indicating that Ankrd2 gene expression may be regulated by the NF-B pathway as suggested for C-193 (3), the human orthologue of C-193/CARP/ MARP. To identify the minimal promoter region that confers spatial and temporal expression specificity to the Ankrd2 gene we made three luciferase reporter constructs. The longest promoter region tested contained 920 bp of the Ankrd2 5⬘-flanking sequence, while the constructs with the medium and the short region contained respectively 616 and 280 bp, as shown in Fig. 2. These Ankrd2 promoter/luciferase reporter constructs were transfected into mouse myoblasts (C2C12) and fibroblasts (3T3). The results showed that during dif-
ferentiation of myoblasts into multinucleated myotubes the level of expression of Ankrd2 is significantly increased, whereas in undifferentiated myoblasts and 3T3 cells the level of expression was barely detectable. Both cell lines were also co-transfected with a CMV promoter/lacZ reporter construct to normalize the efficiency of transfection. Interestingly, the three different constructs produced a very similar pattern of expression (Fig. 2), thus indicating that the 280 bp upstream region is sufficient to confer muscle-specific and temporal specific gene expression. However, the presence of other boxes that are specific targets for transcription factors indicates that the Ankrd2 promoter may extend beyond this 280 bp region. Expression of Ankrd2 in Different Human Tissues Northern blot analysis on 12 different human tissues using either the 3⬘ or 5⬘ untranslated regions of Ankrd2 as probes confirmed that skeletal muscle is the major site of expression of this gene and that a fainter signal is visible in hear (Figs. 3a and 3b). Since a single band is visible after hybridization with either probe this would suggest the presence of only a single Ankrd2 splicing product in muscle cells. RT-PCR of 16 different human tissue cDNAs confirmed the preferential expression of Ankrd2 mRNA in muscle tissues (Fig. 3b). Samples were taken after 26, 29 and 32 cycles of amplification and separated on agarose gels containing ethidium bromide. Lower amounts of the Ankrd2 transcript was also demonstrated in heart, kidney and prostate after 29 PCR cycles.
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Detection of Ankrd2 Protein in Muscle Cells The four different antiAnkrd2 antibodies were tested by Western blot analysis against protein extracts from different tissues, showing a very similar pattern and a high specificity for a 42 kD band found mainly in skeletal muscle (Fig. 4a). From these experiments it can be seen that Ankrd2 is present mainly in skeletal muscle and to a lesser extent in heart and kidney, confirming the results from Northern blot and PCR experiments. The antibody against the C-terminal region of Ankrd2 was assayed by immunoprecipitation (Fig. 4b) confirming the high specificity and the size of the Ankrd2 band, which is running a little faster than actin in Fig. 4b. Actin can be seen as a non-specific band (42 kDa) immunoprecipitated also with the preimmune serum control. In general, for our experiments we used the C-terminal monoclonal antibody; however, on mouse cells we used the N-terminal Ankrd2 polyclonal antibody as the monoclonal Ankrd2 antibody did not detect any signal in mouse cells (C2C12) either by Western blot or immunofluorescence, although this antibody functions normally on human cells (CHQ5B). The epitope seen by the monoclonal antibody could be absent in mouse Ankrd2, this is possible as the C-terminal region where the epitope has been mapped has some amino acid differences between the mouse and human Ankrd2 protein. During muscle cell differentiation there is an increase of Ankrd2 signal as detected by Western blot analysis of both mouse C2C12 (Fig. 4c) and human CHQ5B (data not shown) cell extracts. This pattern of up-regulation on differentiation has also been seen for MARP (4) and CARP (5). Immunofluorescence experiments were undertaken in primary human myoblasts and myotubes as well as skeletal and heart muscle tissues with the scope of pinpointing the intracellular localization of Ankrd2 protein. In general during differentiation both the number of fluorescing cells and the intensity fluorescence increases (Fig. 5). However, the pattern of fluorescence produced with the different antibodies is not identical. This is not too surprising since it is quite common that an antibody that works very well on the isolated and denatured proteins of a Western blot does not work as well on immunofluorescence where the proteins are still included in their cellular context, and vice versa. It is particularly interesting that the N-terminal antibody seems to work much better in immunofluorescence, as shown in Fig. 5, where Ankrd2 is evident before the beginning of cell differentiation and increases during this process thus reflecting the results obtained by Western blot analysis. An accurate analysis of the immunofluorescence obtained either with the monoclonal or polyclonal full-length or C-terminal
Ankrd2 antibody revealed that a limited number (5– 10%) of myoblasts show a pattern of nuclear speckles (Fig. 5). This pattern has never been detected with the N-terminal antibodies. Therefore, it is possible that the cytoplasmic Ankrd2 has the C-terminal region of the protein inaccessible, thus it can be recognized only by the anti N-terminal antibodies, while the nuclear Ankrd2 seems to have the N-terminal part inaccessible and is recognized only by the C-terminal antibodies. Localization of Ankrd2 Protein in Adult Skeletal Muscle Adult skeletal muscle from normal and dystrophic patients was studied by immunofluorescence assays, using three different anti-Ankrd2 antibodies, respectively against the N-terminal, the entire protein and the C-terminal. In normal tissue the pattern of labeling was apparently the same with the three antibodies, although the antibody against the entire protein gave a stronger reaction (Fig. 6a). An antibody against ␣-actinin 3 was used as a marker for fast fibers (17, 18), indicating that in a normal muscle Ankrd2 seems to be expressed in slow fibers (Fig. 6a). These results agree with the Ankrd2 Northern blot analyses on different types of mouse skeletal muscle, reported by Kemp et al. (8), showing that muscles rich in slow fibers, such as soleus, are expressing higher levels of Ankrd2. In our study, using anti-Ankrd2 antibodies, we can produce direct evidence to show that Ankrd2 is preferentially expressed in slow fibers of normal human skeletal muscle. However, a closer analysis of Fig. 6a reveals that some fibers, such as the one indicated with an asterisk in Fig. 6a, do not express either ␣-actinin 3 or Ankrd2. Therefore, not all slow fibers express similar levels of Ankrd2. A very different pattern of fluorescence is observed in dystrophic muscles. In the example of Fig. 6b the results from a patient with an unclassified form of merosin-positive congenital muscular dystrophy are shown. It can be seen that both N-terminal and C-terminal antibodies give a practically negative signal, whereas the antibody against the full-length protein shows a pattern “concordant” with that of ␣-actinin 3. Other altered muscles have been tested on a preliminary screen (data not shown), including Spinal Muscular Atrophy and dystrophic muscles with predominance of fast and slow fibers, with similar results. DISCUSSION Our study on Ankrd2 started a few years ago, as a part of a systematic investigation of genes expressed in human skeletal muscle. One of our newly discovered transcripts, HSPD02860 (now Ankrd2) showed some interesting features being expressed mainly in skeletal
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muscle and coding for a protein similar to the human protein C-193, sharing 43% identical amino acids. The C-193 protein was described by Chu et al. (3) as a possible regulatory factor involved in the activation of endothelial cells. Further independent studies (4 – 6) led to the discovery in mouse, rat and rabbit of CARP (Cardiac Ankyrin Repeat Protein, or Cardiac Adriamycin-Responsive Protein) and MARP (Muscle Ankyrin Repeat Protein), that are very similar to C-193, sharing about 90% identity at the amino acid level; thus, it is most likely that C-193, CARP and MARP are orthologs forms of the same protein. A 90% identity at the amino acid level is also shared between human Ankrd2 (described in this paper) and mouse Ankrd2 (8, 16). In this case, the assumption that mouse and human Ankrd2 are true orthologs is further supported by the identical genomic structure of the two genes (Fig. 1). Not only C-193/CARP/MARP is the nearest known sequence to Ankrd2, but also Ankrd2 is the nearest sequence to C-193/CARP/MARP. Given the high similarity between Ankrd2 and C-193/CARP/MARP, it is likely that they may be functionally related. This hypothesis is further supported by a strict preservation of all the recognizable structural and functional domains between the two proteins, in particular the ankyrin repeats and the PEST motifs. Since C-193/CARP/ MARP is found essentially in heart and Ankrd2 is found primarily in skeletal muscle, we could envisage that these two proteins may have parallel functions, with respective specializations for the tissue in which they are expressed. There is a strong evidence that C193/CARP/MARP is involved in transcriptional regulation, which if further supported by the recent finding (6) that in mouse C-193/CARP/MARP binds the ubiquitous transcription factor YB-1, however, the mechanism of action on specific promoters or signaling pathways remains unclear. It has been suggested that C-193 (3) like other primary response genes could be regulated by the NF-B pathway, especially since its expression was induced by IL-1, TNF-␣ and LPS, all of which activate transcription of NF-B. In this paper we show that the Ankrd2 promoter has indeed a NF-B box, both in human and mouse (Fig. 3). The possibility that Ankrd2 and C-193/CARP/MARP could play a role in the NK-B pathway is particularly interesting since in the C2C12 myoblast cell line NF-B functions as an inhibitor of myogenic differentiation, and myoblasts generated lacking NF-B activity displayed defects in cellular proliferation and cell cycle exit on differentiation (19). Interestingly, not only Ankrd2 and C-193/CARP/MARP could be regulated by the NF-B pathway, but they also share several striking similarities both with NF-B and with its inhibitor protein I-B (3). In particular, they all have multiple ankyrin repeats and phosphorylation sites. Further-
more, both Ankrd2 and C-193/CARP/MARP share with I-B putative PEST motifs, in similar positions (8). Therefore, it is possible that Ankrd2 and C-193/CARP/ MARP may function as I-B-like factors, and it would be interesting to investigate whether, at the protein level, they could interact with the canonical NF-B pathway, thus establishing a loop-back control, or whether they play a more independent role. Insights on the intracellular localization of Ankrd2 are crucial for the understanding of its function. Like many other proteins involved in signaling pathways it could be nuclear or it could be mainly located in the cytoplasm where it could interact with other signaling proteins in a manner similar to I-B. It is particularly interesting that Ankrd2 shows clear nuclear localization signals, both in human and mouse, but prediction programs such as PSORT indicate an uncertain localization. In this work we took particular care in determining the intracellular localization of Ankrd2. Four independent antibodies, including a monoclonal antibody against the non-ankyrin C-terminal domain, were produced and controlled for their specificity by Western blot analysis on adult skeletal muscle and on different stages of myoblast differentiation. Before the beginning of myoblast differentiation fluorescence can be seen within the nuclei of a variable percentage of cells. As the differentiation progresses, Ankrd2 seems to change localization and progressively accumulates in the cytoplasm (Fig. 5). In adult skeletal muscle the cytoplasmic localization is absolutely predominant (Fig. 6). An independent indication that the putative nuclear signal of Ankrd2 is potentially functional comes from GFP experiments (data not shown) that indicated an evident nuclear localization of a GFP construct attached to the 116 N-terminal amino-acids of Ankrd2, while the constructs with the GFP attached either in the N- or C-terminal full length Ankrd2 displayed a fluorescence pattern diffuse both in the nucleous and cytoplasm. Therefore, it is possible that this signal may be conditionally functional also in myoblasts, whereas in most adult cells it could be deactivated either by binding to specific inhibitor proteins or by a chemical/ conformational switch. This hypothesis may also explain the repeated failure in our search for Ankrd2 binding proteins, which has been extensively pursued in our laboratory by means of the yeast two-hybrid approach, without success. However, preliminary Glutathione S-transferase pull-down experiments using cell extracts from 35S-Methionine labeled myotubes and myoblasts, suggest the presence of several Ankrd2-binding proteins (data not shown). A role in controlling the activity of Ankrd2 and/or its ability to bind to other proteins could also be played by phosphorylation. This possibility is supported not only by the presence of several canonical phosphorylation sites, but also by the finding (data not shown) that
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casein kinase II is able to efficiently phosphorylate Ankrd2. An exciting hypothesis is that Ankrd2 and C-193/ CARP/MARP could be involved in the control and maintenance of an adequate muscle mass, respectively in skeletal muscle and heart. This possibility is supported by two recent reports. In rat heart, CARP is regulated by cardiac overload, including pressure overload, hypertension and heart failure, leading to a hypertrophic response (7). Similarly, in mouse skeletal muscle Ankrd2 is up regulated after muscle stretching (8), indicating a possible involvement in the signaling pathway leading to muscle hypertrophy, triggered by stretch and overload. Therefore, both proteins could play a similar role in the signaling cascade leading to muscle hypertrophy, but possibly being triggered by a different mechanism: pressure overload in heart and stretching in skeletal muscle. Interestingly, in several independent cases, the intracellular pattern of Ankrd2 that we observed in human adult skeletal muscle was not uniformly diffuse in the cytoplasm, as shown in Fig. 6a, but there was evident sarcomeric banding, overlapping the Z-disc (data not shown). It would be particularly interesting to confirm and understand this result because a direct interaction of Ankrd2 with some sarcomeric structures could give an indication of the molecular mechanism to sense muscle overload. Our immunofluorescence results also show that in normal adult skeletal muscle Ankrd2 is found in slow fibers. But not all the slow fibers, as detected by the ␣-actinin 3 antibodies, are expressing Ankrd2 at the same level (Fig. 6a). Furthermore, in some dystrophic muscles, as shown in Fig. 6b, we found that Ankrd2 was expressed together with ␣-actinin 3. In conclusion, if Ankrd2 has a role in controlling atrophy and/or hypertrophy of muscle, then it would be interesting to better investigate how slow and fast fibers respond to this control and whether Ankrd2 could be also involved in the transition between fiber types. ACKNOWLEDGMENTS We thank the following people from CRIBI, University of Padua: Elide Formentin for assistance with Northern and RT-PCR assays, Silvia Trevisan for assistance in screening the full-length libraries, Rosanna Zimbello for DNA sequencing, and Paolo Scannapieco for help with computer analysis. Thanks are also due to Mauro Sturnega and Giancarlo Lunazzi, ICGEB, Trieste, for excellent technical assistance in the immunization of animals used for antibody production and to Marta Canovas, Centro de Estudos do Genoma Humano, San Paulo, for assistance with the IF methodology. We are also grateful to Dr. Alan Beggs for supplying the ␣-actinin3 antibody, to Dr. V. Mouly (URA CNRS, Paris) for generously providing the CHQ5B cells, to Dr. S. Soddu (CRS, Rome) for supplying the C2C12
cell line and to Elisa Calabria (Biomedical Science Department, University of Padua) for providing vectors and assistance in the promoter analysis experiments. S. Kojic´ is a recipient of an ICGEB fellowship and is on leave from the Institute of Molecular Genetics and Genetic Engineering, Belgrade. The financial support of Telethon-Italy (Grant 1278 to G. Faulkner and Grant B.41 to G. Lanfranchi and G. Valle) is gratefully acknowledged. M. Vainzof and M. Zatz are supported by FAPESP-CEPID and PRONEX-CNPq.
REFERENCES 1. Mercier, J., Perez-Martin, A., Bigard, X., and Ventura, R. (1999) Mol. Aspects Med. 6, 319 –373. 2. Lanfranchi, G., Muraro, T., Caldara, F., Pacchioni, B., Pallavicini, A., Pandolfo, D., Toppo, S., Trevisan, S., Scarso, S., and Valle, G. (1996) Genome Res. 6, 35– 42. 3. Chu, W., Burns, D. K., Swerlick, R. A., and Presky, D. H. (1995) J. Biol. Chem. 270, 10236 –10245. 4. Baumeister, A., Arber, S., and Caroni, P. (1997) J. Cell Biol. 139, 1231–1242. 5. Jeyasseelan, R., Poizat, C., Baker, R. K., Abdishoo, S., Isterabadi, L. B., Lyons, G. E., and Kedest, L. (1997) J. Biol. Chem. 272, 22800 –22808. 6. Zou, Y., Evans, S., Chen, J., Kuo, H-C., Harvey, R. P., and Chien, K. R. (1997) Development 124, 793– 804. 7. Aihara, Y., Kurabayashi, M., Saito, Y., Ohyama, Y., Tanaka, T., Takeda, S., Tomaru, K., Sekiguchi, K., Arai, M., Nakamura, T., and Nagai, R. (2000) Hypertension 36, 48 –53. 8. Kemp, T. J., Sadusky, T. J., Saltisi, F., Carey, N., Moss, J., Yang, S. Y., Sasson, D. A., Goldspink, G., and Coulton, G. R. (2000) Genomics 66, 229 –241. 9. Faulkner, G., Pallavicini, A., Formentin, E., Comelli, A., Ievolella, C., Trevisan, S., Bortoletto, G., Scannapieco, P., Salamon, M., Moulay, V., Valle, G., and Lanfranchi, G. (1999) J. Cell Biol. 146, 465– 475. 10. Walter, M. A., Spillett, D. J., Thomas, P., Weissenbach, J., and Goodfellow, P. N. (1994) Nat. Genet. 7, 22–28. 11. Welle, S., Bhatt, K., and Thornton, C. A. (1999) Genome Res. 9, 506 –513. 12. Michaely, P., and Bennett, V. (1992) Trends Cell Biol. 2, 127– 129. 13. Rechsteiner, M., and Rogers, S. W. (1996) Trends Biochem. Sci. 21, 267–271. 14. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364 –368. 15. Kuo, H-C., Chen, J., Ruiz-Lozano, P., Zou, Y., Nemer, M., and Chien, K. R. (1999) Development 126, 4223– 4234. 16. Ievolella, C., Formentin, E., and Lanfranchi, G. (1998) AJ011118 skeletal muscle and cardiac protein (Mus musculus). Direct Submission to GenBank Database (30 September 1998). 17. Beggs, A. H., Byers, T. J., Knoll, J. H. M., Boyce, F. M., Bruys, G. A. P., and Kunkel, L. M. (1992) J. Biol. Chem. 267, 9281– 9288. 18. Vainzof, M., Costa, C. S., Marie, S. K., Moreira, E. S., Reed, U., Passos-Bueno, M. R., Beggs, A. H., and Zatz, M. (1997) Neuropediatrics 28, 1– 6. 19. Guttridge, D. C., Albanese, C., Reuther, J. Y., Pestell, R. G., and Baldwin, A. S., Jr. (1999) Mol. Cell Biol. 8, 5785–5799.
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Characterization of Human Skeletal Muscle Ankrd2 Alberto Pallavicini,* Snezana Kojic´,† Camilla Bean,* Mariz Vainzof,‡ Michela Salamon,* Chiara Ievolella,* Gladis Bortoletto,* Beniamima Pacchioni,* Mayana Zatz,‡ Gerolamo Lanfranchi,* Georgine Faulkner,† and Giorgio Valle* ,1 *CRIBI Biotechnology Centre, Universita` degli Studi di Padova, via Ugo Bassi 58b, I-35121 Padua, Italy; †International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy; and ‡Centro de Estudos do Genoma Humano, IB-USB, Rua do Mata˜o 106, CEP 05508-900, Sao Paulo, Brazil
Received June 4, 2001
Human Ankrd2 transcript encodes a 37-kDa protein that is similar to mouse Ankrd2 recently shown to be involved in hypertrophy of skeletal muscle. These novel ankyrin-rich proteins are related to C-193/ CARP/MARP, a cardiac protein involved in the control of cardiac hypertrophy. A human genomic region of 14,300 bp was sequenced revealing a gene organization similar to mouse Ankrd2 with nine exons, four of which encode ankyrin repeats. The intracellular localization of Ankrd2 was unknown since no protein studies had been reported. In this paper we studied the intracellular localization of the protein and its expression on differentiation using polyclonal and monoclonal antibodies produced to human Ankrd2. In adult skeletal muscle Ankrd2 is found in slow fibers; however, not all of the slow fibers express Ankrd2 at the same level. This is particularly evident in dystrophic muscles, where the expression of Ankrd2 in slow fibers seems to be severely reduced. © 2001 Academic Press Key Words: muscle proteins; muscle plasticity; hypertrophy; ankyrin; signaling pathways.
It is generally known that people can perform physical work much better after appropriate training and exercising. Both skeletal muscle and heart can increase or decrease their mass to adapt to the tasks that The cDNA sequences reported in this paper have been submitted to the GenBank/EMBL/DDJB databases with Accession Nos. AJ304805 (human Ankrd2) and AJ011118 (mouse Ankrd2). The human genomic sequence has been submitted with Accession No. AJ304804. Abbreviations used: BAC, bacterial artificial chromosome; BSA, bovine serum albumin; EST, expressed sequence tag; GFP, green fluorescent protein; FITC, fluorescein isothiocyanate; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RTPCR, reverse transcriptase PCR; SDS, sodium dodecyl sulfate. 1 To whom correspondence should be addressed. Fax: ⫹39-0498276280. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
they usually perform, but muscle plasticity is not just a matter of mass. The quality of a muscle depends to a great extent on the type of fibers of which it is composed. Muscle fibers are characterized by different subsets of specialized proteins. For instance, there are different isoforms of myosin heavy chain: isoform I is found in type I fibers which contract more slowly than type II fibers (containing type II isoforms), thus the latter are known as fast fibers and the former as slow fibers. The difference in muscle performance depends not only on the mechanics of contractile proteins, but also on the type of metabolism used, that in slow fibers is aerobic, whereas in fast fibers is anaerobic (1). Many aspects of the molecular biology of fast and slow fibers are now understood; however, very little is known about the mechanisms responsible for sensing, signaling and adjusting the mass and type of fibers of any given muscle. In this respect, we searched our database of human skeletal muscle transcripts (2) accessible at http://muscle.cribi.unipd.it, for possible transcription factors with a muscle specific pattern of expression. Transcript HSPD02860 (now called Ankrd2) revealed some very interesting features such as a nuclear sorting signal, four tandem repeated ankyrin motifs, which are typically found in several transcription factors, and a good similarity to protein C-193, a putative transcription factor found in endothelial cells (3). While the work presented in this paper was in progress some very interesting results were published on the rat and mouse orthologs of human C-193 (called respectively CARP and MARP), indicating that they are mainly expressed in heart (4 – 6) and probably involved in the control of cardiac hypertrophy (7). Recently, a paper was published by Kemp et al. (8) on mouse Ankrd2, the closest gene related to C-193/ CARP/MARP that is expressed mainly in skeletal muscle. Thus, it is possible that Ankrd2 may play a similar
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FIG. 1. Genomic organization and promoter analysis of Ankrd2. (a) Schematic representation of the genomic organization of human (AJ304804) and mouse (AJ249346) Ankrd2 genes. The lines are connecting the corresponding exons. (b) Sequence comparison of the mouse and human promoter of Ankrd2 genes. Putative binding motifs for E47, MyoD, NF-B, and the TATA box are in bold. The predicted transcription start and the first two ATG codons are also marked. The two arrows delimit the sequence region responsible for muscle expression (see Fig. 2).
role to C-193/CARP/MARP, but in skeletal muscle rather than heart. Furthermore, mouse Ankrd2 is expressed at a higher level after muscle stretching, giving a direct indication of a possible involvement of this protein in controlling muscle hypertrophy (8). In this paper we present our work on human Ankrd2 including a detailed analysis of its pattern of expression and cellular localization, both at the mRNA and protein levels. MATERIALS AND METHODS Cell culture. Cell culture conditions for primary human (CHQ5B) and mouse (C2C12) myoblast cells were as previously described (9). Differentiation medium for these cells was Dulbecco’s modified Eagle medium without serum but supplemented with 10 g/ml of insulin (Sigma I-5500, Sigma–Aldrich, St. Louis, MO) and 100 g/ml of transferrin (Sigma T-2036) or 0.4% Ultroser G (Life Technologies, Grand Island, NY). Expression studies of the Ankrd2 mRNA. The Northern blot analysis was done using mRNA filters obtained from Clontech (Clontech). Specific radiolabeled probes corresponding to the 5⬘- or 3⬘-portions of the Ankrd2 transcript were generated by PCR amplification of the cDNA clone with specific primers. Radioactive PCRs were purified and then used for hybridization utilizing the protocol suggested by
the manufacturer (NorthernMax, Ambion, Austin, TX). The signal was detected using the Cyclone Storage Phosphor System (Packard Instruments Co., Meriden, CT). Reverse transcriptase PCR (RT-PCR) assays were performed on a panel of 16 different human tissue cDNAs (Human MTC panel I and II; Clontech) using primers specific for the 3⬘ end of the Ankrd2 mRNA. Genomic mapping. Genomic mapping was performed by PCR with specific primers designed for the 3⬘ portion of the Ankrd2 transcript using the GeneBridge 4 whole-Genome Radiation Hybrid Panel (10) (Research Genetics, Huntsville, AL). The results were processed using the RHMAPPER software program (Whitehead Institute/MIT Center for Genomic Research, Cambridge, MA). Genomic sequencing. Bacterial artificial chromosome (BAC) clones containing Ankrd2 DNA (H100F12, H443C8 and H443C9) were obtained by PCR screening at the “Fondation Jean Dausset”-CEPH (Centre d’Etu`de du Polymorphisme Humaine, Paris; http://landru.cephb.fr/ services), using Ankrd2 transcript-specific primers. The H443C9 clone was used for shotgun library construction. BAC DNA was sheared using a GeneMachine Hydroshear. The fragments obtained were size selected on agarose gels and cloned into a modified pZero vector (Invitrogen). A chromosome walking approach was applied to the 768 arrayed shotgun clones and the sequences were assembled using the program SeqMan 3.61 (DNASTAR Inc., Madison, WI). The Ankrd2 genomic sequence was analyzed to determine the transcription initiation site using the program “Promoter Prediction” by Neural Network at http://www.fruitfly.org/seq_tools/promoter.html. Finally the putative
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Ankrd2 binding transcription factors were identified by the TFSearch (http://pdap1.trc.rwcp.or.jp/research/db/TFSEARCH.html) and matSearch (http://transfac.gbf.de/cgi-bin/matSearch/matsearch.pl) programs. Immunoblot analysis. Proteins from different human tissues were obtained from Clontech (Cat. Nos. 7800 to 7808, Cat. No. 7813). The protein extracts and a pre-stained molecular weight marker (Sigma C3437) were separated on 12 or 15% SDS–polyacrylamide gels and then blotted onto immobilon P membrane (Millipore, Bedford, MA). The Ankrd2 monoclonal (ascites fluid) and polyclonal antibodies were used at the same dilutions (1/200-1/400). MF-20, myosin antibody was used at a 1/10,000 dilution. This antibody (developed by Dr. D. A. Fischman) was obtained from the Developmental Studies Hybridoma Bank, University of Iowa. Goat antimouse immunoglobulin conjugated with alkaline phosphatase (Sigma A3562) was used as the second antibody in all Western blot experiments. Primary human and mouse muscle cells were harvested and resuspended in a urea buffer (8 M urea; 0.1 M NaH 2PO 4; 0.01 M Tris, pH 8.0), sonicated, centrifuged and the supernatants separated on 12 or 15% SDS–polyacrylamide gels. Immunofluorescence. Undifferentiated and differentiated primary human muscle cells grown on collagen-coated coverslips were fixed with paraformaldehyde (4%), then treated with 0.1 M glycine and permeabilized with 0.05% Tween 20 for 30 min. All wash and dilution buffers contained both bovine serum albumin (BSA) 1% and 0.05% Tween-20 to block any nonspecific binding. For these experiments the secondary antibody was fluorescein isothiocyanate (FITC)conjugated anti-mouse immunoglobulin (Sigma F4018) or goat antirabbit Texas red (Calbiochem, La Jolla, CA). All commercial immunochemicals were diluted as recommended by the suppliers. For confocal microscopy the nuclei were stained with propidium iodide (3.5 g/ml, Sigma P4170). A LSM 510 laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany) was used for confocal microscopy both at 40⫻ and 100⫻ magnification. For muscle biopsies sections of 5 m were cut using a cryostat and air-dried for 1 h and then incubated with 10% horse serum in PBS to prevent nonspecific binding. The anti-Ankrd2 antibodies monoclonal C-terminal and polyclonal N-terminal (1/40 dilution) and the anti-actinin3 antibody (1/100 dilution) obtained from Alan Beggs were used for immunocytochemistry. For double immunofluorescence experiments anti-ankrd2 and anti-actinin3 antibodies were mixed and incubated simultaneously overnight. After washing, the sections were incubated for 1 h with secondary antibodies (dilution 1/100): FITC-conjugated anti-rabbit-Ig (Sigma) and CY3-conjugated anti-mouse-Ig (Jackson Immunochemicals, West Grove, PA). An Axiophoto microscope (Carl Zeiss Microscopy), with epifluorescence, and filters for FITC and rhodamine was used for microscopy. Immunoprecipitation. Polyadenylated mRNA of adult skeletal muscle was in vitro translated using a reticulocyte lysate system (Promega, Madison, WI) and labeled with [ 35S]methionine (Amersham Pharmacia Biotech, Rainham, UK). Equal amounts of labeled proteins were mixed with the appropriate antibody and immunoprecipitated using protein A–Sepharose (Amersham Pharmacia Biotech) in a buffer containing 50 mM Hepes, pH 8.0, 250 mM NaCl, 0.1% Nonidet P40. The samples were separated by SDS–PAGE, dried, exposed using a super resolution phosphor screen and analyzed on a Packard Cyclone phosphor imager (Packard Instrument Co.). Polyclonal anti-Ankrd2 antibody, pre-immune sera and MF 20 were used at dilutions of 1/75 for these experiments. Promoter analysis. Three luciferase reporter constructs were produced to enable the identification of the minimal region involved in Ankrd2 skeletal muscle transcriptional activity. The reverse primer PROHindIII ⫹ 10 (⫹10, ⫺10) combined with PROXhoI-915 (⫺915, ⫺895), PROXhoI-616 (⫺616, ⫺596) and PROXhoI-280 (⫺280, ⫺260) were used for PCR amplification with the Ankrd2 genome as template. The PCR products were HindII/
FIG. 2. Transient expression assays to identify the minimal promoter region that confers muscle specificity. (a) Schematic diagrams of a series of luciferase constructs containing nested deletions of the Ankrd2 promoter. The construct name refers to the length of the genomic region cloned. (b) A histogram showing the luciferase activity of the different constructs in C2C12 cells at different stages of differentiation. All transfections were performed in triplicate.
XhoI digested and cloned into the pGL3-basic vector (Promega). The luciferase reporter constructs were co-transfected with the pCMV vector in mouse C2C12 cells and in mouse 3T3 cells using the lipofectamine 2000 reagent (Life Technologies). The luciferase activity, measured with the Luciferase Assay System (Promega) on the Jade luminometre (Anthos Labtec Instruments, Wals, Austria), was normalized for gal activity to account for variations in the efficiency of transfection. Since luciferase can be unstable during culture incubation the values of the luciferase activity were normalized to the pRL-SV40 vector (Promega) which was cotransfected with the pCMV vector.
RESULTS Sequencing of Human and Mouse Ankrd2 cDNA The systematic sequencing of human skeletal muscle ESTs carried out in our laboratory (2) has led to the discovery of many new genes expressed in muscle. To date, some 5300 transcripts have been identified from over 30,000 ESTs. Amongst this collection, transcript HSPD02860 (now called Ankrd2) was recognized to be particularly interesting as it shows a 43% identity at the amino acid level to C-193/CARP/MARP, a 319 amino acid nuclear protein expressed in endothelial cells (3), heart (6) and to a lesser extent in skeletal muscle (4), which is believed to play a role as a cofactor in local signaling pathways ranging from muscle morphogenesis (4) to cardiac hypertrophy (5).
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Ankyrin repeats are ubiquitous motifs involved in macromolecular interactions (12). There are at least four ankyrin motifs in detected in C-193/CARP/ MARP, mouse and human Ankrd2 proteins with possibly a fifth ankyrin repeat present only in the human protein. The Ankrd2 protein would appear to have several potential phosphorylation sites as noted
FIG. 3. Ankrd2 mRNA in human tissues detected by Northern blot and RT-PCR analysis. (a) Northern blot analysis of human tissues demonstrating patterns of expression of Ankrd2 mRNAs. A panel of mRNAs from 12 different human adult tissues: brain (br), heart (he), skeletal muscle (sk), colon (co), thymus (th), spleen (sp), kidney (ki), liver (li), small intestine (si), placenta (pl), lung (lu), and peripheral blood leukocytes (le) were probed with radioactive PCR fragments derived from either the 3⬘ untranslated region of Ankrd2 or with the 5⬘ region as indicated. The numbers on the side indicate the size (kb). (b) RT-PCR for different human tissues. The assays were performed on a panel of 16 different human tissue cDNAs (Human MTC panel I and II, Clontech) using primers specific for the 3⬘ portion of the Ankrd2 mRNA. The different tissues are indicated as in section (a), with the addition of pancreas (pa), ovary (ov), prostate (pr), testes (te), and control sample (cn). The number of cycles of PCR 26, 29, and 32 is given at the side of the figure.
The relative abundance of human Ankrd2 mRNA was predicted to be 0.055%, i.e., about 1/2000 of the polyadenylated mRNAs, as calculated from the percentage of Ankrd2 ESTs present in our muscle EST database. The strategies used for the construction of our libraries resulted in cDNA clones that reflect the concentration of each mRNA in the muscle tissue (2, 11). Our complete cDNA sequences of human and mouse Ankrd2 are available from EMBL under the Accession Nos. AJ304805 and AJ011118, respectively. The human transcript is 1159 nucleotides, whereas the mouse is slightly shorter in the 3⬘ untranslated region (1101 bp). Human and mouse sequences are very similar, the identity being 85% at the nucleotide level and 89% at the amino acid level. The nucleotide substitutions between the two species are evenly distributed along the sequences, with the only exception of a stretch of five contiguous amino acids near the N-terminal that are different between human and mouse. The ORFs of human and mouse Ankrd2 are 999 and 996 bases, respectively, encoding putative proteins of 333 and 332 amino acid residues with calculated molecular weights of 37,150 and 37,124 daltons, respectively.
FIG. 4. Expression pattern of Ankrd2 protein in human tissues and primary muscle cells. (a) Tissue distribution of Ankrd2 as demonstrated by Western blot analysis. Protein extracts from human heart and skeletal muscle (10 g) as well as from brain, placenta, testis, spleen, kidney, liver, and lung (60 g) were loaded in each lane, run on a 15% SDS–polyacrylamide gel and then blotted. The Immobilon P membrane (Millipore) was probed with mouse preimmune sera, polyclonal N- and C-terminal Ankrd2 antibodies as well as antibody to full-length Ankrd2 and a monoclonal antibody specific for an epitope from the C-terminal region of Ankrd2. All sera were used at a 1/200 dilution. (b) Immunoprecipitation of human skeletal muscle proteins obtained from in vitro translation of adult skeletal muscle mRNA using the rabbit reticulocyte lysate system (Promega). Equal amounts of [ 35S]methionine-labeled proteins were mixed with the appropriate antibody and immunoprecipitated using protein A–Sepharose and separated by SDS–PAGE. The muscle proteins were immunoprecipitated with mouse preimmune sera, anti-Ankrd2 C-terminal antibody and anti-myosin antibody (MF20 ascites fluid). Numbers on the left of the figures indicate molecular weights (kD); the rainbow [ 14C]methylated protein molecular weight marker (Amersham) was used. (c) Western blot analysis of protein extracts from mouse C2C12 cells at 0, 3, 6, and 8 days after the addition of differentiation medium. The blot was probed with mouse polyclonal antibody to N-terminal Ankrd2.
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FIG. 5. Time course of Ankrd2 expression in primary human muscle cells during differentiation as detected by indirect immunofluorescence. The mouse monoclonal C-terminal and polyclonal N-terminal Ankrd2 antibodies were used at a dilution of 1/40; myosin monoclonal antibody (MF 20) was used at 1/100 dilution. FITC-conjugated anti-mouse Ig (Sigma) was used as the second antibody at the recommended dilution. The days after the addition of differentiating medium are noted at the side of the figure. A preimmune mouse serum was used as a control giving a negative signal at all the time points (data not shown).
by computer analysis; six cAMP-dependent protein kinase phosphorylation sites, three casein kinase II sites, two calmodulin-dependent protein kinase II sites, one cGMP-dependent protein kinase site and one protein kinase C site. Ankrd2 can be phosphorylated in vitro by casein kinase II (data not shown), thus confirming the phosphorylation data, at least for casein kinase II. Another interesting motif found both in human and mouse Ankrd2, as well as C-193/CARP/MARP is the PEST sequence which serves as proteolytic signal that targets proteins for rapid destruction (13, 14). Both human and murine Ankrd2 proteins have nuclear localization motifs, however these proteins are predicted to be cytoplasmic by PSORT. Also C-193/CARP/MARP was classified as a cytoplasmic protein by PSORT despite having a nuclear localization signal and being actually localized in the nucleus both as an endogenous protein (6) and after expression of the protein in transfected cells (3–5).
Genome Organization and Promoter Analysis The Ankrd2 gene was mapped on the human chromosome region 10q23.31–23.32 using the radiation hybrid technique, as described in the experimental procedures. The closest marker is WI-3011, at an estimated distance of 4.29 centiRay with very significant lod score of 21. In order to characterize the structure of the human Ankrd2 gene, a CEPH human BAC library was screened by PCR. The positive BAC clone H443C9 was used to produce a shotgun library and a chromosome walking approach with hierarchical pooling strategies allowed the sequencing of a genomic region of 14,300 bp. The genomic DNA and cDNA comparison revealed that the gene is divided into 9 exons as shown in Fig. 1, and each of the exons 5, 6, 7 and 8 encode one ankyrin repeat, displaying a gene organization very similar to mouse Ankrd2 (8). A bioinformatic analysis of the transcription initiation site indicates an alternative ATG starting codon, twelve bases upstream from the ATG
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FIG. 6. The expression pattern of Ankrd2 in normal and dystrophic skeletal muscle as detected by immunofluorescence. (a) A normal skeletal muscle sections stained with three antibodies to Ankrd2 and an antibody to ␣-actinin3. The type II fibers are stained with ␣-actinin 3 (green) and some of the type I fibers are stained with the three anti-mouse Ankrd2 (red) antibodies. Clear sarcomeric striations can be seen with the ␣-actinin 3 antibody, whereas only weak striations can be seen with the C-terminal and full-length anti-Ankrd2 antibodies. The asterisk indicates a fiber that is negative both to ␣-actinin 3 and to Ankrd2. (b) A dystrophic muscle from a patient with an as yet unclassified form of merosin-positive congenital muscular dystrophy was labeled with the three antibodies to Ankrd2 and an antibody to ␣-actinin 3. Only the full-length Ankrd2 antibody (red) can be seen to stain some type II fibers; neither anti-Ankrd2 N- nor C-terminal antibodies (red) stained the type II fibers; ␣-actinin 3 (green) staining was used as a positive control for type II fiber staining.
codon identified in the cDNA clone, but maintaining the same reading frame. A similar analysis was carried out on the mouse genomic sequence indicating the presence of the additional twelve coding bases also in mouse, although in this case the cDNA sequences terminated at the second ATG. The hypothesis that this is the genuine start of transcription is further supported by the primer extension analysis performed on the CARP genomic sequence (15), that places the transcription initiation site in agreement with the bioinformatic analysis (8, 16). The 1157-bp 5⬘ flanking region of Ankrd2 shows several putative recognition sequences for transcription factors however interestingly the human and mouse Ankrd2 promoter sequence alignment shows only three boxes containing cis-elements specific for muscle transcription factors (see Fig. 1). In addition to the canonical muscle specific transcription factors MyoD and E47, the NF-B binding sequence was found, indicating that Ankrd2 gene expression may be regulated by the NF-B pathway as suggested for C-193 (3), the human orthologue of C-193/CARP/ MARP. To identify the minimal promoter region that confers spatial and temporal expression specificity to the Ankrd2 gene we made three luciferase reporter constructs. The longest promoter region tested contained 920 bp of the Ankrd2 5⬘-flanking sequence, while the constructs with the medium and the short region contained respectively 616 and 280 bp, as shown in Fig. 2. These Ankrd2 promoter/luciferase reporter constructs were transfected into mouse myoblasts (C2C12) and fibroblasts (3T3). The results showed that during dif-
ferentiation of myoblasts into multinucleated myotubes the level of expression of Ankrd2 is significantly increased, whereas in undifferentiated myoblasts and 3T3 cells the level of expression was barely detectable. Both cell lines were also co-transfected with a CMV promoter/lacZ reporter construct to normalize the efficiency of transfection. Interestingly, the three different constructs produced a very similar pattern of expression (Fig. 2), thus indicating that the 280 bp upstream region is sufficient to confer muscle-specific and temporal specific gene expression. However, the presence of other boxes that are specific targets for transcription factors indicates that the Ankrd2 promoter may extend beyond this 280 bp region. Expression of Ankrd2 in Different Human Tissues Northern blot analysis on 12 different human tissues using either the 3⬘ or 5⬘ untranslated regions of Ankrd2 as probes confirmed that skeletal muscle is the major site of expression of this gene and that a fainter signal is visible in hear (Figs. 3a and 3b). Since a single band is visible after hybridization with either probe this would suggest the presence of only a single Ankrd2 splicing product in muscle cells. RT-PCR of 16 different human tissue cDNAs confirmed the preferential expression of Ankrd2 mRNA in muscle tissues (Fig. 3b). Samples were taken after 26, 29 and 32 cycles of amplification and separated on agarose gels containing ethidium bromide. Lower amounts of the Ankrd2 transcript was also demonstrated in heart, kidney and prostate after 29 PCR cycles.
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Detection of Ankrd2 Protein in Muscle Cells The four different antiAnkrd2 antibodies were tested by Western blot analysis against protein extracts from different tissues, showing a very similar pattern and a high specificity for a 42 kD band found mainly in skeletal muscle (Fig. 4a). From these experiments it can be seen that Ankrd2 is present mainly in skeletal muscle and to a lesser extent in heart and kidney, confirming the results from Northern blot and PCR experiments. The antibody against the C-terminal region of Ankrd2 was assayed by immunoprecipitation (Fig. 4b) confirming the high specificity and the size of the Ankrd2 band, which is running a little faster than actin in Fig. 4b. Actin can be seen as a non-specific band (42 kDa) immunoprecipitated also with the preimmune serum control. In general, for our experiments we used the C-terminal monoclonal antibody; however, on mouse cells we used the N-terminal Ankrd2 polyclonal antibody as the monoclonal Ankrd2 antibody did not detect any signal in mouse cells (C2C12) either by Western blot or immunofluorescence, although this antibody functions normally on human cells (CHQ5B). The epitope seen by the monoclonal antibody could be absent in mouse Ankrd2, this is possible as the C-terminal region where the epitope has been mapped has some amino acid differences between the mouse and human Ankrd2 protein. During muscle cell differentiation there is an increase of Ankrd2 signal as detected by Western blot analysis of both mouse C2C12 (Fig. 4c) and human CHQ5B (data not shown) cell extracts. This pattern of up-regulation on differentiation has also been seen for MARP (4) and CARP (5). Immunofluorescence experiments were undertaken in primary human myoblasts and myotubes as well as skeletal and heart muscle tissues with the scope of pinpointing the intracellular localization of Ankrd2 protein. In general during differentiation both the number of fluorescing cells and the intensity fluorescence increases (Fig. 5). However, the pattern of fluorescence produced with the different antibodies is not identical. This is not too surprising since it is quite common that an antibody that works very well on the isolated and denatured proteins of a Western blot does not work as well on immunofluorescence where the proteins are still included in their cellular context, and vice versa. It is particularly interesting that the N-terminal antibody seems to work much better in immunofluorescence, as shown in Fig. 5, where Ankrd2 is evident before the beginning of cell differentiation and increases during this process thus reflecting the results obtained by Western blot analysis. An accurate analysis of the immunofluorescence obtained either with the monoclonal or polyclonal full-length or C-terminal
Ankrd2 antibody revealed that a limited number (5– 10%) of myoblasts show a pattern of nuclear speckles (Fig. 5). This pattern has never been detected with the N-terminal antibodies. Therefore, it is possible that the cytoplasmic Ankrd2 has the C-terminal region of the protein inaccessible, thus it can be recognized only by the anti N-terminal antibodies, while the nuclear Ankrd2 seems to have the N-terminal part inaccessible and is recognized only by the C-terminal antibodies. Localization of Ankrd2 Protein in Adult Skeletal Muscle Adult skeletal muscle from normal and dystrophic patients was studied by immunofluorescence assays, using three different anti-Ankrd2 antibodies, respectively against the N-terminal, the entire protein and the C-terminal. In normal tissue the pattern of labeling was apparently the same with the three antibodies, although the antibody against the entire protein gave a stronger reaction (Fig. 6a). An antibody against ␣-actinin 3 was used as a marker for fast fibers (17, 18), indicating that in a normal muscle Ankrd2 seems to be expressed in slow fibers (Fig. 6a). These results agree with the Ankrd2 Northern blot analyses on different types of mouse skeletal muscle, reported by Kemp et al. (8), showing that muscles rich in slow fibers, such as soleus, are expressing higher levels of Ankrd2. In our study, using anti-Ankrd2 antibodies, we can produce direct evidence to show that Ankrd2 is preferentially expressed in slow fibers of normal human skeletal muscle. However, a closer analysis of Fig. 6a reveals that some fibers, such as the one indicated with an asterisk in Fig. 6a, do not express either ␣-actinin 3 or Ankrd2. Therefore, not all slow fibers express similar levels of Ankrd2. A very different pattern of fluorescence is observed in dystrophic muscles. In the example of Fig. 6b the results from a patient with an unclassified form of merosin-positive congenital muscular dystrophy are shown. It can be seen that both N-terminal and C-terminal antibodies give a practically negative signal, whereas the antibody against the full-length protein shows a pattern “concordant” with that of ␣-actinin 3. Other altered muscles have been tested on a preliminary screen (data not shown), including Spinal Muscular Atrophy and dystrophic muscles with predominance of fast and slow fibers, with similar results. DISCUSSION Our study on Ankrd2 started a few years ago, as a part of a systematic investigation of genes expressed in human skeletal muscle. One of our newly discovered transcripts, HSPD02860 (now Ankrd2) showed some interesting features being expressed mainly in skeletal
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muscle and coding for a protein similar to the human protein C-193, sharing 43% identical amino acids. The C-193 protein was described by Chu et al. (3) as a possible regulatory factor involved in the activation of endothelial cells. Further independent studies (4 – 6) led to the discovery in mouse, rat and rabbit of CARP (Cardiac Ankyrin Repeat Protein, or Cardiac Adriamycin-Responsive Protein) and MARP (Muscle Ankyrin Repeat Protein), that are very similar to C-193, sharing about 90% identity at the amino acid level; thus, it is most likely that C-193, CARP and MARP are orthologs forms of the same protein. A 90% identity at the amino acid level is also shared between human Ankrd2 (described in this paper) and mouse Ankrd2 (8, 16). In this case, the assumption that mouse and human Ankrd2 are true orthologs is further supported by the identical genomic structure of the two genes (Fig. 1). Not only C-193/CARP/MARP is the nearest known sequence to Ankrd2, but also Ankrd2 is the nearest sequence to C-193/CARP/MARP. Given the high similarity between Ankrd2 and C-193/CARP/MARP, it is likely that they may be functionally related. This hypothesis is further supported by a strict preservation of all the recognizable structural and functional domains between the two proteins, in particular the ankyrin repeats and the PEST motifs. Since C-193/CARP/ MARP is found essentially in heart and Ankrd2 is found primarily in skeletal muscle, we could envisage that these two proteins may have parallel functions, with respective specializations for the tissue in which they are expressed. There is a strong evidence that C193/CARP/MARP is involved in transcriptional regulation, which if further supported by the recent finding (6) that in mouse C-193/CARP/MARP binds the ubiquitous transcription factor YB-1, however, the mechanism of action on specific promoters or signaling pathways remains unclear. It has been suggested that C-193 (3) like other primary response genes could be regulated by the NF-B pathway, especially since its expression was induced by IL-1, TNF-␣ and LPS, all of which activate transcription of NF-B. In this paper we show that the Ankrd2 promoter has indeed a NF-B box, both in human and mouse (Fig. 3). The possibility that Ankrd2 and C-193/CARP/MARP could play a role in the NK-B pathway is particularly interesting since in the C2C12 myoblast cell line NF-B functions as an inhibitor of myogenic differentiation, and myoblasts generated lacking NF-B activity displayed defects in cellular proliferation and cell cycle exit on differentiation (19). Interestingly, not only Ankrd2 and C-193/CARP/MARP could be regulated by the NF-B pathway, but they also share several striking similarities both with NF-B and with its inhibitor protein I-B (3). In particular, they all have multiple ankyrin repeats and phosphorylation sites. Further-
more, both Ankrd2 and C-193/CARP/MARP share with I-B putative PEST motifs, in similar positions (8). Therefore, it is possible that Ankrd2 and C-193/CARP/ MARP may function as I-B-like factors, and it would be interesting to investigate whether, at the protein level, they could interact with the canonical NF-B pathway, thus establishing a loop-back control, or whether they play a more independent role. Insights on the intracellular localization of Ankrd2 are crucial for the understanding of its function. Like many other proteins involved in signaling pathways it could be nuclear or it could be mainly located in the cytoplasm where it could interact with other signaling proteins in a manner similar to I-B. It is particularly interesting that Ankrd2 shows clear nuclear localization signals, both in human and mouse, but prediction programs such as PSORT indicate an uncertain localization. In this work we took particular care in determining the intracellular localization of Ankrd2. Four independent antibodies, including a monoclonal antibody against the non-ankyrin C-terminal domain, were produced and controlled for their specificity by Western blot analysis on adult skeletal muscle and on different stages of myoblast differentiation. Before the beginning of myoblast differentiation fluorescence can be seen within the nuclei of a variable percentage of cells. As the differentiation progresses, Ankrd2 seems to change localization and progressively accumulates in the cytoplasm (Fig. 5). In adult skeletal muscle the cytoplasmic localization is absolutely predominant (Fig. 6). An independent indication that the putative nuclear signal of Ankrd2 is potentially functional comes from GFP experiments (data not shown) that indicated an evident nuclear localization of a GFP construct attached to the 116 N-terminal amino-acids of Ankrd2, while the constructs with the GFP attached either in the N- or C-terminal full length Ankrd2 displayed a fluorescence pattern diffuse both in the nucleous and cytoplasm. Therefore, it is possible that this signal may be conditionally functional also in myoblasts, whereas in most adult cells it could be deactivated either by binding to specific inhibitor proteins or by a chemical/ conformational switch. This hypothesis may also explain the repeated failure in our search for Ankrd2 binding proteins, which has been extensively pursued in our laboratory by means of the yeast two-hybrid approach, without success. However, preliminary Glutathione S-transferase pull-down experiments using cell extracts from 35S-Methionine labeled myotubes and myoblasts, suggest the presence of several Ankrd2-binding proteins (data not shown). A role in controlling the activity of Ankrd2 and/or its ability to bind to other proteins could also be played by phosphorylation. This possibility is supported not only by the presence of several canonical phosphorylation sites, but also by the finding (data not shown) that
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casein kinase II is able to efficiently phosphorylate Ankrd2. An exciting hypothesis is that Ankrd2 and C-193/ CARP/MARP could be involved in the control and maintenance of an adequate muscle mass, respectively in skeletal muscle and heart. This possibility is supported by two recent reports. In rat heart, CARP is regulated by cardiac overload, including pressure overload, hypertension and heart failure, leading to a hypertrophic response (7). Similarly, in mouse skeletal muscle Ankrd2 is up regulated after muscle stretching (8), indicating a possible involvement in the signaling pathway leading to muscle hypertrophy, triggered by stretch and overload. Therefore, both proteins could play a similar role in the signaling cascade leading to muscle hypertrophy, but possibly being triggered by a different mechanism: pressure overload in heart and stretching in skeletal muscle. Interestingly, in several independent cases, the intracellular pattern of Ankrd2 that we observed in human adult skeletal muscle was not uniformly diffuse in the cytoplasm, as shown in Fig. 6a, but there was evident sarcomeric banding, overlapping the Z-disc (data not shown). It would be particularly interesting to confirm and understand this result because a direct interaction of Ankrd2 with some sarcomeric structures could give an indication of the molecular mechanism to sense muscle overload. Our immunofluorescence results also show that in normal adult skeletal muscle Ankrd2 is found in slow fibers. But not all the slow fibers, as detected by the ␣-actinin 3 antibodies, are expressing Ankrd2 at the same level (Fig. 6a). Furthermore, in some dystrophic muscles, as shown in Fig. 6b, we found that Ankrd2 was expressed together with ␣-actinin 3. In conclusion, if Ankrd2 has a role in controlling atrophy and/or hypertrophy of muscle, then it would be interesting to better investigate how slow and fast fibers respond to this control and whether Ankrd2 could be also involved in the transition between fiber types. ACKNOWLEDGMENTS We thank the following people from CRIBI, University of Padua: Elide Formentin for assistance with Northern and RT-PCR assays, Silvia Trevisan for assistance in screening the full-length libraries, Rosanna Zimbello for DNA sequencing, and Paolo Scannapieco for help with computer analysis. Thanks are also due to Mauro Sturnega and Giancarlo Lunazzi, ICGEB, Trieste, for excellent technical assistance in the immunization of animals used for antibody production and to Marta Canovas, Centro de Estudos do Genoma Humano, San Paulo, for assistance with the IF methodology. We are also grateful to Dr. Alan Beggs for supplying the ␣-actinin3 antibody, to Dr. V. Mouly (URA CNRS, Paris) for generously providing the CHQ5B cells, to Dr. S. Soddu (CRS, Rome) for supplying the C2C12
cell line and to Elisa Calabria (Biomedical Science Department, University of Padua) for providing vectors and assistance in the promoter analysis experiments. S. Kojic´ is a recipient of an ICGEB fellowship and is on leave from the Institute of Molecular Genetics and Genetic Engineering, Belgrade. The financial support of Telethon-Italy (Grant 1278 to G. Faulkner and Grant B.41 to G. Lanfranchi and G. Valle) is gratefully acknowledged. M. Vainzof and M. Zatz are supported by FAPESP-CEPID and PRONEX-CNPq.
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