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1.  ANKRD1, the Gene Encoding Cardiac Ankyrin Repeat Protein, Is a Novel Dilated Cardiomyopathy Gene 
Objectives
We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients.
Background
CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction.
Methods
In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription–polymerase chain reaction.
Results
Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP.
Conclusions
ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.
doi:10.1016/j.jacc.2009.02.076
PMCID: PMC2915893  PMID: 19608030
DCM; CARP; ANKRD1; mutations
2.  ZASP Interacts with the Mechanosensing Protein Ankrd2 and p53 in the Signalling Network of Striated Muscle 
PLoS ONE  2014;9(3):e92259.
ZASP is a cytoskeletal PDZ-LIM protein predominantly expressed in striated muscle. It forms multiprotein complexes and plays a pivotal role in the structural integrity of sarcomeres. Mutations in the ZASP protein are associated with myofibrillar myopathy, left ventricular non-compaction and dilated cardiomyopathy. The ablation of its murine homologue Cypher results in neonatal lethality. ZASP has several alternatively spliced isoforms, in this paper we clarify the nomenclature of its human isoforms as well as their dynamics and expression pattern in striated muscle. Interaction is demonstrated between ZASP and two new binding partners both of which have roles in signalling, regulation of gene expression and muscle differentiation; the mechanosensing protein Ankrd2 and the tumour suppressor protein p53. These proteins and ZASP form a triple complex that appears to facilitate poly-SUMOylation of p53. We also show the importance of two of its functional domains, the ZM-motif and the PDZ domain. The PDZ domain can bind directly to both Ankrd2 and p53 indicating that there is no competition between it and p53 for the same binding site on Ankrd2. However there is competition for this binding site between p53 and a region of the ZASP protein lacking the PDZ domain, but containing the ZM-motif. ZASP is negative regulator of p53 in transactivation experiments with the p53-responsive promoters, MDM2 and BAX. Mutations in the ZASP ZM-motif induce modification in protein turnover. In fact, two mutants, A165V and A171T, were not able to bind Ankrd2 and bound only poorly to alpha-actinin2. This is important since the A165V mutation is responsible for zaspopathy, a well characterized autosomal dominant distal myopathy. Although the mechanism by which this mutant causes disease is still unknown, this is the first indication of how a ZASP disease associated mutant protein differs from that of the wild type ZASP protein.
doi:10.1371/journal.pone.0092259
PMCID: PMC3960238  PMID: 24647531
3.  26S Proteasome Regulation of Ankrd1/CARP in Adult Rat Ventricular Myocytes and Human Microvascular Endothelial Cells 
Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (hrs) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.
doi:10.1016/j.bbrc.2012.07.162
PMCID: PMC3460693  PMID: 22892129
cardiomyocytes; endothelial cells; Ankrd1/CARP; 26S proteasome
4.  Ankrd2/ARPP is a novel Akt2 specific substrate and regulates myogenic differentiation upon cellular exposure to H2O2 
Molecular Biology of the Cell  2011;22(16):2946-2956.
A proteomic-based search for novel substrates of Akt was undertaken in C2C12 murine muscle cells. Our data demonstrate that Akt isoform 2 phosphorylates Ankrd2 at Serine 99 in response to H2O2 stimuli, regulating muscle differentiation rate.
Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C2C12 murine muscle cells exploiting protein characterization databases in combination with an anti–phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H2O2 triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C2C12 myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions.
doi:10.1091/mbc.E10-11-0928
PMCID: PMC3154889  PMID: 21737686
5.  Novel mutations in the sarcomeric protein myopalladin in patients with dilated cardiomyopathy 
Recently, missense mutations in titin-associated proteins have been linked to the pathogenesis of dilated cardiomyopathy (DCM). The objective of this study was to search for novel disease-associated mutations in the two human titin-binding proteins myopalladin and its amino-terminal-interacting partner cardiac ankyrin-repeat protein (CARP). In a cohort of 255 cases with familial and sporadic DCM, we analyzed the coding regions and all corresponding intron flanks located in the MYPN and CARP-encoding ANKRD1 gene. Two heterozygous missense mutations were detected in the MYPN gene (p.R955W and p.P961L), but neither of these mutations was found in 300 healthy controls. Both mutations were located in the α-actinin-binding region of myopalladin. Endomyocardial biopsies from the p.R955W carrier showed normal subcellular localization of myopalladin and α-actinin in cardiac myocytes, while their regular sarcomeric staining pattern was significantly disrupted in the p.P961L carrier, indicating that disturbed myofibrillogenesis and altered sarcomere assembly are the cause of the disease. In the ANKRD1 gene, we identified synonymous base exchanges (c.108T>C and c.-79C>T, respectively), but no non-synonymous mutations. In summary, we have identified novel missense mutations in the third immunoglobulin-like domain of myopalladin, which have either no or profound effects on the molecular composition of the sarcomere. According to our epidemiological data, the prevalence of ANKRD1 mutations seems to be lower than that of its binding partner myopalladin, indicating the clinical significance of myopalladin for the functional integrity of the sarcomeric apparatus and the protection against DCM.
doi:10.1038/ejhg.2012.173
PMCID: PMC3573205  PMID: 22892539
myopalladin; cardiac ankyrin-repeat protein; CARP; point mutation; single-nucleotide polymorphism; dilated cardiomyopathy
6.  ANKRD26 and Its Interacting Partners TRIO, GPS2, HMMR and DIPA Regulate Adipogenesis in 3T3-L1 Cells 
PLoS ONE  2012;7(5):e38130.
Partial inactivation of the Ankyrin repeat domain 26 (Ankrd26) gene causes obesity and diabetes in mice and increases spontaneous and induced adipogenesis in mouse embryonic fibroblasts. However, it is not yet known how the Ankrd26 protein carries out its biological functions. We identified by yeast two-hybrid and immunoprecipitation assays the triple functional domain protein (TRIO), the G protein pathway suppressor 2 (GPS2), the delta-interacting protein A (DIPA) and the hyaluronan-mediated motility receptor (HMMR) as ANKRD26 interacting partners. Adipogenesis of 3T3-L1 cells was increased by selective down-regulation of Ankrd26, Trio, Gps2, Hmmr and Dipa. Furthermore, GPS2 and DIPA, which are normally located in the nucleus, were translocated to the cytoplasm, when the C-terminus of ANKRD26 was introduced into these cells. These findings provide biochemical evidence that ANKRD26, TRIO, GPS2 and HMMR are novel and important regulators of adipogenisis and identify new targets for the modulation of adipogenesis.
doi:10.1371/journal.pone.0038130
PMCID: PMC3364200  PMID: 22666460
7.  Thrombocytopenia-associated mutations in the ANKRD26 regulatory region induce MAPK hyperactivation  
Point mutations in the 5′ UTR of ankyrin repeat domain 26 (ANKRD26) are associated with familial thrombocytopenia 2 (THC2) and a predisposition to leukemia. Here, we identified underlying mechanisms of ANKRD26-associated thrombocytopenia. Using megakaryocytes (MK) isolated from THC2 patients and healthy subjects, we demonstrated that THC2-associated mutations in the 5′ UTR of ANKRD26 resulted in loss of runt-related transcription factor 1 (RUNX1) and friend leukemia integration 1 transcription factor (FLI1) binding. RUNX1 and FLI1 binding at the 5′ UTR from healthy subjects led to ANKRD26 silencing during the late stages of megakaryopoiesis and blood platelet development. We showed that persistent ANKRD26 expression in isolated MKs increased signaling via the thrombopoietin/myeloproliferative leukemia virus oncogene (MPL) pathway and impaired proplatelet formation by MKs. Importantly, we demonstrated that ERK inhibition completely rescued the in vitro proplatelet formation defect. Our data identify a mechanism for development of the familial thrombocytopenia THC2 that is related to abnormal MAPK signaling.
doi:10.1172/JCI71861
PMCID: PMC3904625  PMID: 24430186
8.  Clinical significance of Ankyrin repeat domain 12 expression in colorectal cancer 
Background
Ankyrin repeat domain 12 (ANKRD12), is encoding a 224 kDa nuclear protein and most conserved at its N-terminal ankyrin repeats region and the C-terminal co-activator interacting domain. The aim of this study was to investigate the ANKRD12 mRNA expression in colorectal cancer (CRC) tumor tissues and the normal adjacent mucosa and its potential relevance to clinicopathological characteristics and prognosis.
Methods
Surgical specimens of tumor tissues (n = 68) and adjacent normal mucosa (n = 51) were obtained from CRC patients. The ANKRD12 mRNA expression was measured by quantitative real time reverse transcriptase polymerase chain reaction. The relationship between ANKRD12 mRNA expression and clinicopathological features was analyzed by appropriate statistics. Kaplan–Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between ANKRD12 expression and prognosis of CRC patients.
Results
The relative mRNA expression of ANKRD12 were significantly lower in CRC tumor tissues than in the normal adjacent mucosa (P < 0.001), and the cases with low ANKRD12 expression showed a higher frequency of liver metastasis (P = 0.015). Kaplan–Meier analysis indicated that patients (CRC without liver metastasis) with low ANKRD12 expression had poor overall survival (P = 0.041). Multivariate analysis showed that low ANKRD12 expression was an independent predictor of overall survival.
Conclusion
This study revealed that ANKRD12 mRNA were down regulated in CRC tumor tissues and low ANKRD12 expression was correlated with liver metastasis and poor survival of CRC patients.
doi:10.1186/1756-9966-32-35
PMCID: PMC3689078  PMID: 23718802
Ankyrin repeat domain 12 (ANKRD12); Colorectal cancer; Metastasis; Prognostic factor
9.  Disruption of a GATA4/Ankrd1 Signaling Axis in Cardiomyocytes Leads to Sarcomere Disarray: Implications for Anthracycline Cardiomyopathy 
PLoS ONE  2012;7(4):e35743.
Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.
doi:10.1371/journal.pone.0035743
PMCID: PMC3332030  PMID: 22532871
10.  The Ankrd 13 family of UIM-bearing proteins regulates EGF receptor endocytosis from the plasma membrane 
Molecular Biology of the Cell  2012;23(7):1343-1353.
Ankrd 13A, 13B, and 13D constitute a family of ubiquitin-interacting motif (UIM)-bearing cytoplasmic proteins. They are anchored to the plasma membrane, where they recognize the Lys63-linked polyubiquitin chains tagged to ligand-activated EGF receptor and regulate the endocytosis of EGF receptor from the cell surface in mammalian cells.
The mechanism of ubiquitin-dependent endocytosis of cell surface proteins is not completely understood. Here we examine the role of the ankyrin repeat domain (Ankrd) 13A, 13B, and 13D proteins, which constitute a functionally unknown family of ubiquitin-interacting motif (UIM)–bearing proteins, in the process. Stimulation of human HeLa cells with epidermal growth factor (EGF) rapidly induced direct binding of Ankrd 13 proteins to ubiquitinated EGF receptor (EGFR) via the UIMs. The binding was inhibited when the Ankrd 13 proteins underwent UIM-dependent monoubiquitination, suggesting that their activity is regulated by ubiquitination of themselves. Ankrd 13 proteins bound specifically to Lys-63–linked ubiquitin chains, which was consistent with a previous report that EGFR mainly undergoes Lys-63–linked polyubiquitination. Ankrd 13 proteins were anchored, via the central region and UIMs, to the plasma membrane, where they colocalized with EGFR. Finally, overexpression of wild-type as well as truncated-mutant Ankrd 13 proteins strongly inhibited rapid endocytosis of ubiquitinated EGFR from the surface in EGF-treated cells. We conclude that by binding to the Lys-63–linked polyubiquitin moiety of EGFR at the plasma membrane, Ankrd 13 proteins regulate the rapid internalization of ligand-activated EGFR.
doi:10.1091/mbc.E11-09-0817
PMCID: PMC3315809  PMID: 22298428
11.  Regulation of focal adhesion and cell migration by ANKRD28-DOCK180 interaction 
Cell Adhesion & Migration  2009;3(3):281-284.
DOCK180 is an atypical guanine nucleotide exchange factor of Rac1 identified originally as one of the two major proteins bound to the SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130Cas, and recruits the Crk-p130Cas complex to focal adhesions. Recently, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and found that ANKRD28, a protein with twenty-six ankyrin domain-repeats, interacts with the SH3 domain of DOCK180. Knockdown of ANKRD28 reduced the migration velocity and altered the distribution of focal adhesion proteins such as Crk, paxillin and p130Cas. On the other hand, the expression of ANKRD28, p130Cas, Crk and DOCK180 induced hyper-phosphorylation of p130Cas, which paralleled the induction of multiple long cellular processes. Depletion of ELMO, another protein bound to the SH3 domain of DOCK180, also retarded cell migration, but its expression together with p130Cas, Crk and DOCK180 induced extensive lamellipodial protrusion around the entire circumference without 130Cas hyperphosphorylation. These data suggest the dual modes of DOCK180-Rac regulation for cell migration.
PMCID: PMC2712811  PMID: 19458477
ankyrin; Rac; p130Cas; DOCK180; paxillin; Crk; migration; focal adhesion
12.  Titin and myosin, but not desmin, are linked during myofibrillogenesis in postmitotic mononucleated myoblasts 
The Journal of Cell Biology  1986;103(6):2185-2196.
Monoclonal antibodies specific for the muscle protein titin have been used in conjunction with muscle-specific antibodies against myofibrillar myosin heavy chains (MHCs) and desmin to study myogenesis in cultured cells. Desmin synthesis is initiated in replicating presumptive myoblasts, whereas the synthesis of titin and MHC is initiated simultaneously in their progeny, the postmitotic, mononucleated myoblasts. Both titin and MHC are briefly localized to nonstriated and thereafter to definitively striated myofibrils. At no stage during myofibrillogenesis is either protein observed as part of a sequence of mini-sarcomeres. Titin antibodies bind to the A-I junction, MHC antibodies to the A bands in nascent, maturing, and mature myofibrils. In contrast, desmin remains distributed as longitudinal filaments until well after the definitive myofibrils have aligned laterally. This tight temporal and topographical linkage between titin and myosin is also observed in postmitotic, mononucleated myoblasts and multinucleated myotubes when myofibrillogenesis is perturbed with Colcemid or taxol. Colcemid induces elongating postmitotic mononucleated myoblasts and multinucleated myotubes to round up and form Colcemid myosacs. The myofibrils that emerge in these rounded cells are deployed in convoluted circles. The time required for their nonstriated myofibrils to transform into striated myofibrils is greatly protracted. Furthermore, as Colcemid induces immense desmin intermediate filament cables, the normal spatial relationships between emerging individual myofibrils is distorted. Despite these disturbances at all stages, the characteristic temporal and spatial relationship observed in normal myofibrils between titin and MHC is observed in myofibrils assembling in Colcemid-treated cells. Newly born postmitotic mononucleated myoblasts, or maturing myotubes, reared in taxol acquire a star-shaped configuration and are induced to assemble "pseudo- striated myofibrils." Pseudo-striated myofibrils consist of laterally aggregated 1.6-micron long, thick filaments that interdigitate, not with thin filaments, but with long microtubules. These atypical myofibrils lack Z bands. Despite the absence of thin filaments and Z bands, titin localizes with its characteristics sarcomeric periodicity in pseudo-striated myofibrils. We conclude that the initiation and subsequent regulation of titin and myosin synthesis, and their spatial deployment within developing sarcomeres are tightly coupled events. These findings are discussed in terms of a model that proposes interaction between two relatively autonomous "organizing centers" in the assembly of each sarcomere.
PMCID: PMC2114608  PMID: 3536962
13.  Xtn3 is a developmentally expressed cardiac and skeletal muscle-specific novex-3 titin isoform 
Gene expression patterns : GEP  2006;6(8):913-918.
Titin proteins play an essential role in maintaining muscle function and structure. Recent work has implicated the involvement of the novex-3 titin isoform in sarcomere restructuring and disease. Unlike avian and mammalian systems, Xenopus laevis myogenesis is characterized by a wave of primary myogenesis followed by apoptosis of the primary muscles and formation of new muscles by secondary myogenesis. We show here that the Xenopus laevis novex-3 titin isoform (Xtn3) is developmentally expressed throughout the somites, heart, and primary muscles of the developing embryo. Downregulation of Xtn3 expression at tadpole stages appears to coincide with the change in myofiber composition from solely embryonic “fast” fiber types to myofibers containing both “fast” and “slow” fiber types. We demonstrate that Xtn3 is expressed early in the presomitic mesoderm and remains expressed in the somites, ventral myoblasts, and developing jaw muscles through late tailbud stage. Furthermore, we show that Xtn3 is expressed in the cardiac primordia prior to linear heart tube formation and remains expressed in the heart until tadpole stage, at which point it is downregulated in the heart except in discrete patches of cardiac cells. Finally, we demonstrate that Xtn3 transcripts are detectable in adult heart and muscle tissues.
doi:10.1016/j.modgep.2006.03.003
PMCID: PMC1635806  PMID: 16630749
Titin; TTN; Novex; Xtn3; Cardiogenesis; Cardiac; Heart; Somite; Muscle; Skeletal muscle; Pharyngeal; Myoblast; Myogenesis; Somitogenesis; Myofibril; Sarcomere; Development; Xenopus
14.  Dynamic Strength of Titin's Z-Disk End 
Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment.
doi:10.1155/2010/838530
PMCID: PMC2857871  PMID: 20414364
15.  Muscle Giants: Molecular Scaffolds in Sarcomerogenesis 
Physiological reviews  2009;89(4):1217-1267.
Myofibrillogenesis in striated muscles is a highly complex process that depends on the coordinated assembly and integration of a large number of contractile, cytoskeletal, and signaling proteins into regular arrays, the sarcomeres. It is also associated with the stereotypical assembly of the sarcoplasmic reticulum and the transverse tubules around each sarcomere. Three giant, muscle-specific proteins, titin (3–4 MDa), nebulin (600–800 kDa), and obscurin (~720–900 kDa), have been proposed to play important roles in the assembly and stabilization of sarcomeres. There is a large amount of data showing that each of these molecules interacts with several to many different protein ligands, regulating their activity and localizing them to particular sites within or surrounding sarcomeres. Consistent with this, mutations in each of these proteins have been linked to skeletal and cardiac myopathies or to muscular dystrophies. The evidence that any of them plays a role as a “molecular template,” “molecular blueprint,” or “molecular ruler” is less definitive, however. Here we review the structure and function of titin, nebulin, and obscurin, with the literature supporting a role for them as scaffolding molecules and the contradictory evidence regarding their roles as molecular guides in sarcomerogenesis.
doi:10.1152/physrev.00017.2009
PMCID: PMC3076733  PMID: 19789381
16.  Regulation of Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase (Btk) through a Novel SH3-Dependent Interaction with Ankyrin Repeat Domain 54 (ANKRD54) 
Molecular and Cellular Biology  2012;32(13):2440-2453.
Bruton's tyrosine kinase (Btk), belonging to the Tec family of tyrosine kinases (TFKs), is essential for B-lymphocyte development. Abrogation of Btk signaling causes human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). We employed affinity purification of Flag-tagged Btk, combined with tandem mass spectrometry, to capture and identify novel interacting proteins. We here characterize the interaction with ankryin repeat domain 54 protein (ANKRD54), also known as Lyn-interacting ankyrin repeat protein (Liar). While Btk is a nucleocytoplasmic protein, the Liar pool was found to shuttle at a higher rate than Btk. Importantly, our results suggest that Liar mediates nuclear export of both Btk and another TFK, Txk/Rlk. Liar-mediated Btk shuttling was enriched for activation loop, nonphosphorylated Btk and entirely dependent on Btk's SH3 domain. Liar also showed reduced binding to an aspartic acid phosphomimetic SH3 mutant. Three other investigated nucleus-located proteins, Abl, estrogen receptor β (ERβ), and transcription factor T-bet, were all unaffected by Liar. We mapped the interaction site to the C terminus of the Btk SH3 domain. A biotinylated, synthetic Btk peptide, ARDKNGQEGYIPSNYVTEAEDS, was sufficient for this interaction. Liar is the first protein identified that specifically influences the nucleocytoplasmic shuttling of Btk and Txk and belongs to a rare group of known proteins carrying out this activity in a Crm1-dependent manner.
doi:10.1128/MCB.06620-11
PMCID: PMC3434478  PMID: 22527282
17.  Expression profiles of muscle disease-associated genes and their isoforms during differentiation of cultured human skeletal muscle cells 
Background
The formation of contractile myofibrils requires the stepwise onset of expression of muscle specific proteins. It is likely that elucidation of the expression patterns of muscle-specific sarcomeric proteins is important to understand muscle disorders originating from defects in contractile sarcomeric proteins.
Methods
We investigated the expression profile of a panel of sarcomeric components with a focus on proteins associated with a group of congenital disorders. The analyses were performed in cultured human skeletal muscle cells during myoblast proliferation and myotube development.
Results
Our culture technique resulted in the development of striated myotubes and the expression of adult isoforms of the sarcomeric proteins, such as fast TnI, fast TnT, adult fast and slow MyHC isoforms and predominantly skeletal muscle rather than cardiac actin. Many proteins involved in muscle diseases, such as beta tropomyosin, slow TnI, slow MyBPC and cardiac TnI were readily detected in the initial stages of muscle cell differentiation, suggesting the possibility of an early role for these proteins as constituent of the developing contractile apparatus during myofibrillogenesis. This suggests that in disease conditions the mechanisms of pathogenesis for each of the mutated sarcomeric proteins might be reflected by altered expression patterns, and disturbed assembly of cytoskeletal, myofibrillar structures and muscle development.
Conclusions
In conclusion, we here confirm that cell cultures of human skeletal muscle are an appropriate tool to study developmental stages of myofibrillogenesis. The expression of several disease-associated proteins indicates that they might be a useful model system for studying the pathogenesis of muscle diseases caused by defects in specific sarcomeric constituents.
doi:10.1186/1471-2474-13-262
PMCID: PMC3549291  PMID: 23273262
Myogenesis; Sarcomere; Myoblast; Skeletal muscle
18.  Integrative analyses reveal novel strategies in HPV11,-16 and -45 early infection 
Scientific Reports  2012;2:515.
The interaction between human papillomavirus (HPV) and host cells is not well understood. We investigate the early stage of HPV infections by global expression profiling in a cell model, in which HaCaT cells were transfected with HPV11, HPV16 or HPV45 genomes. We report the differential expression of genes not previously implicated in HPV biology, such as the PSG family and ANKRD1, and of genes implicated in the biology of other viruses, e.g. MX1, IFI44 and DDX60. Carcinogenesis-related genes, e.g. ABL2, MGLL and CYR61, were upregulated by high-risk HPV16 and -45. The integrative analysis revealed the suppression of DNA repair by HPV11 and -16, and downregulation of cytoskeleton genes by all HPV types. Various signalling pathways were affected by the HPVs: IL-2 by HPV11; JAK-STAT by HPV16; and TGF-β, NOTCH and tyrosine kinase signalling by HPV45. This study uncovered novel strategies employed by HPV to establish infection and promote uncontrolled growth.
doi:10.1038/srep00515
PMCID: PMC3398386  PMID: 22808421
19.  Functional analysis of slow myosin heavy chain 1 and myomesin-3 in sarcomere organization in zebrafish embryonic slow muscles 
Myofibrillogenesis, the process of sarcomere formation, requires close interactions of sarcomeric proteins and various components of sarcomere structures. The myosin thick filaments and M-lines are two key components of the sarcomere. It has been suggested that myomesin proteins of M-lines interact with myosin and titin proteins and keep the thick and titin filaments in order. However, the function of myomesin in myofibrillogenesis and sarcomere organization remained largely enigmatic. No knockout or knockdown animal models have been reported to elucidate the role of myomesin in sarcomere organization in vivo. In this study, by using the gene-specific knockdown approach in zebrafish embryos, we carried out a loss-of-function analysis of myomesin-3 and slow myosin heavy chain 1 (smyhc1) expressed specifically in slow muscles. We demonstrated that knockdown of smyhc1 abolished the sarcomeric localization of myomesin-3 in slow muscles. In contrast, loss of myomesin-3 had no effect on the sarcomeric organization of thick and thin filaments as well as M- and Z-line structures. Together, these studies indicate that myosin thick filaments are required for M-line organization and M-line localization of myomesin-3. In contrast, myomesin-3 is dispensable for sarcomere organization in slow muscles.
doi:10.1016/j.jgg.2012.01.005
PMCID: PMC3971575  PMID: 22361506
Myosin; Myomesin 3; M-line; Sarcomere
20.  The Zebrafish runzel Muscular Dystrophy is Linked to the Titin Gene 
Developmental biology  2007;309(2):180-192.
Titin (also called connectin) acts as a scaffold for signaling proteins in muscle, and is responsible for establishing and maintaining the structure and elasticity of sarcomeres in striated muscle. Several human muscular dystrophies and cardiomyopathies have previously been linked to mutations in the titin gene. This study reports linkage of the runzel homozygous lethal muscular dystrophy in the zebrafish Danio rerio to a genomic interval containing the titin gene. Analysis of the genomic sequence suggests that zebrafish contain two adjacent titin loci. One titin locus lies within the genetic linkage interval and its expression is significantly reduced in runzel mutants by both immunofluorescence and protein electrophoresis. Morpholino downregulation of this same titin locus in wildtype embryos results in decreased muscle organization and mobility, phenocopying runzel mutants. Additional protein analysis demonstrates that, in wildtype zebrafish, titin isoform sizes are rapidly altered during the development of striated muscle, likely requiring a previously unrecognized need for vertebrate sarcomere remodeling to incorporate developmentally-regulated titin isoforms. Decreases of affected titin isoforms in runzel mutants during this time correlate with a progressive loss of sarcomeric organization and suggest that the unaffected titin proteins are capable of sarcomerogenesis but not sarcomere maintenance. In addition, microarray analysis of the ruz transcriptome suggests a novel mechanism of dystrophy pathogenesis, involving mild increases in calpain-3 expression and upregulation of heat shock proteins. These studies should lead to a better understanding of titin’s role in normal and diseased muscle.
doi:10.1016/j.ydbio.2007.06.015
PMCID: PMC2063437  PMID: 17678642
21.  Titin visualization in real time reveals an unexpected level of mobility within and between sarcomeres 
The Journal of Cell Biology  2011;193(4):785-798.
Contrary to prior models in which titin serves as a stable scaffold in sarcomeres, sarcomeric and soluble titin exchange dynamically in myofibers when calcium levels are low.
The giant muscle protein titin is an essential structural component of the sarcomere. It forms a continuous periodic backbone along the myofiber that provides resistance to mechanical strain. Thus, the titin filament has been regarded as a blueprint for sarcomere assembly and a prerequisite for stability. Here, a novel titin-eGFP knockin mouse provided evidence that sarcomeric titin is more dynamic than previously suggested. To study the mobility of titin in embryonic and neonatal cardiomyocytes, we used fluorescence recovery after photobleaching and investigated the contribution of protein synthesis, contractility, and calcium load to titin motility. Overall, the kinetics of lateral and longitudinal movement of titin-eGFP were similar. Whereas protein synthesis and developmental stage did not alter titin dynamics, there was a strong, inhibitory effect of calcium on titin mobility. Our results suggest a model in which the largely unrestricted movement of titin within and between sarcomeres primarily depends on calcium, suggesting that fortification of the titin filament system is activity dependent.
doi:10.1083/jcb.201010099
PMCID: PMC3166869  PMID: 21555460
22.  Signalling in sarcomeres in development and disease 
Netherlands Heart Journal  2013;21(7-8):367-371.
Sarcomeres are the smallest contractile units of heart and skeletal muscles and are essential for generation and propagation of mechanical force in these striated muscles. During the last decades it has become increasingly clear that components of sarcomeres also play a fundamental role in signal transduction in physiological and pathophysiological conditions. Mutations or misexpression of both sarcomeric contractile and non-contractile proteins have been associated with a variety of cardiac diseases. Moreover, re-expression of foetal sarcomeric proteins or isoforms during cardiac disease can be observed, emphasising the importance of understanding signalling in sarcomeres in both development and disease. The prospective of pharmacological intervention at the level of the sarcomere is now emerging and may lead to novel therapeutic strategies for the treatment of cardiac and skeletal muscle diseases. These aspects will be discussed in this brief review and recent findings, which led to novel insights into the role of the sarcomeric cytoskeleton in muscle development and disease, will be highlighted.
doi:10.1007/s12471-013-0435-6
PMCID: PMC3722383  PMID: 23864481
CHAP; Sarcomere; Cardiac; Disease; Development; Signalling
23.  Canonical Wnt signaling induces BMP-4 to specify slow myofibrogenesis of fetal myoblasts 
Skeletal Muscle  2013;3:5.
Background
The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle fiber type diversification, neuromuscular junction formation and muscle stem cell function. How Wnt proteins orchestrate such diverse activities remains poorly understood. Canonical Wnt signaling stabilizes β-catenin, which subsequently translocate to the nucleus to activate the transcription of TCF/LEF family genes.
Methods
We employed TCF-reporter mice and performed analysis of embryos and of muscle groups. We further isolated fetal myoblasts and performed cell and molecular analyses.
Results
We found that canonical Wnt signaling is strongly activated during fetal myogenesis and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific transgenic expression of a stabilized β-catenin protein led to increased oxidative myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors. Consistent with in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic differentiation of fetal myoblasts.
Conclusions
These results demonstrate a novel interaction between canonical Wnt and BMP signaling that induces myogenic differentiation towards slow muscle phenotype.
doi:10.1186/2044-5040-3-5
PMCID: PMC3602004  PMID: 23497616
Skeletal muscle; Fetal myoblasts; Canonical Wnt signaling; BMP4 signaling; Differentiation; Slow muscle specification
24.  Identification of CAP as a Costameric Protein that Interacts with Filamin C 
Molecular Biology of the Cell  2007;18(12):4731-4740.
Cbl-associated protein (CAP) is an adaptor protein that interacts with both signaling and cytoskeletal proteins. Here, we characterize the expression, localization and potential function of CAP in striated muscle. CAP is markedly induced during myoblast differentiation, and colocalizes with vinculin during costamerogenesis. In adult mice, CAP is enriched in oxidative muscle fibers, and it is found in membrane anchorage complexes, including intercalated discs, costameres, and myotendinous junctions. Using both yeast two-hybrid and proteomic approaches, we identified the sarcomeric protein filamin C (FLNc) as a binding partner for CAP. When overexpressed, CAP recruits FLNc to cell–extracellular matrix adhesions, where the two proteins cooperatively regulate actin reorganization. Moreover, overexpression of CAP inhibits FLNc-induced cell spreading on fibronectin. In dystrophin-deficient mdx mice, the expression and membrane localization of CAP is increased, concomitant with the elevated plasma membrane content of FLNc, suggesting that CAP may compensate for the reduced membrane linkage of the myofibrils due to the loss of the dystroglycan–sarcoglycan complex in these mice. Thus, through its interaction with FLNc, CAP provides another link between the myofibril cytoskeleton and the plasma membrane of muscle cells, and it may play a dynamic role in the regulation and maintenance of muscle structural integrity.
doi:10.1091/mbc.E07-06-0628
PMCID: PMC2096606  PMID: 17898075
25.  The Rho-Guanine Nucleotide Exchange Factor Domain of Obscurin Regulates Assembly of Titin at the Z-Disk through Interactions with Ran Binding Protein 9 
Molecular Biology of the Cell  2008;19(9):3782-3792.
Obscurin is an ∼800-kDa protein composed of structural and signaling domains that organizes contractile structures in striated muscle. We have studied the Rho-GEF domain of obscurin to understand its roles in morphogenesis and signaling. We used adenoviral overexpression of this domain, together with ultrastructural and immunofluorescence methods, to examine its effect on maturing myofibrils. We report that overexpression of the Rho-GEF domain specifically inhibits the incorporation of titin into developing Z-disks and disrupts the structure of the Z-disk and Z/I junction, and alters features of the A/I junction. The organization of other sarcomeric markers, including α-actinin, was not affected. We identified Ran binding protein 9 (RanBP9) as a novel ligand of the Rho-GEF domain and showed that binding is specific, with an apparent binding affinity of 1.9 μM. Overexpression of the binding region of RanBP9 also disrupted the incorporation of titin into developing Z-disks. Immunofluorescence localization during myofibrillogenesis indicated that the Rho-GEF domain assembles into sarcomeres before RanBP9, which first occurs in myonuclei and later in development translocates to the myoplasm, where it colocalizes with obscurin. Both the Rho-GEF domain and its binding region on RanBP9 bind directly to the N-terminal Ig domains of titin, which flank the Z-disk. Our results suggest that the Rho-GEF domain interacts with RanBP9 and that both can interact with the N-terminal region of titin to influence the formation of the Z-disk and A/I junction.
doi:10.1091/mbc.E08-03-0237
PMCID: PMC2526693  PMID: 18579686

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