Metalloprotease-disintegrin ADAM 12 interacts with α-actinin-1

2001 ◽  
Vol 357 (2) ◽  
pp. 353-361 ◽  
Author(s):  
Yi CAO ◽  
Qing KANG ◽  
Anna ZOLKIEWSKA
Keyword(s):  
Cell Adhesion ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Cytoplasmic Domain ◽  
C2c12 Cells ◽  
Actin Binding ◽  
Myoblast Differentiation ◽  
Adam 12

ADAM 12, a member of the ADAM family of proteins (containing ADisintegrin And Metalloprotease domain), has been implicated in differentiation and fusion of myoblasts. While the extracellular domain of ADAM 12 contains an active metalloprotease and a region involved in cell adhesion, the function of the cytoplasmic tail of ADAM 12 has been less clear. Here we show that the cytoplasmic domain of ADAM 12 interacts in vitro and in vivo with α-actinin-1, an actin-binding and cross-linking protein. Green fluorescent protein fused to ADAM 12 cytoplasmic domain co-localizes with α-actinin-1-containing actin stress fibres in C2C12 cells. The interaction between ADAM 12 and α-actinin-1 is direct and involves the 58-amino acid C-terminal fragment of ADAM 12 and the 27kDa N-terminal domain of α-actinin-1. Consistently, expression of the 27kDa fragment of α-actinin-1 in C2C12 cells using a mitochondrial targeting system results in recruitment of the co-expressed ADAM 12 cytoplasmic domain to the mitochondrial surface. Moreover, α-actinin-1 co-purifies with a transmembrane, His6-tagged form of ADAM 12 expressed in C2C12 myoblasts, indicating that the transmembrane ADAM 12 forms a complex with α-actinin-1 in vivo. These results indicate that the actin cytoskeleton may play a critical role in ADAM 12-mediated cell–cell adhesion or cell signalling during myoblast differentiation and fusion.

scholarly journals Ezrin regulated myoblast differentiation/fusion and muscle fiber specialization through PKA-NFAT-MyoD/MEF2csignalling pathway

2020 ◽  
Author(s):  
Ruo-nan Zhang ◽  
Yan Wang ◽  
Yun Liu ◽  
Xin Bao ◽  
Wei Xu ◽  
...  
Keyword(s):  
Muscle Fiber ◽  
Neuromuscular Diseases ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Type I ◽  
Over Expression ◽  
Myoblast Cells ◽  
Myofiber Type

Abstract Backgorund:Neuromuscular diseases are a kind of nervous system diseases that have a high disability rate.Ezrin’ role in skeletal muscle has not been identified. This study aims to confirm the effect and mechanism of Ezrin on myoblast differentiation and fusion, myotube size, and myofiber type.Method:By using immunoassaying and western blot analyses, Ezrin, MyHC,MEF2c, MyoG, PKAα/β/γ, PKA reg Iα, PKA reg IIβand NFATc1-c4 were detected in myoblast cells treated with Ad-Ezrin or Ad-shEzrin. Real-time PCR were used to evaluate MyoD, Myf5, MyHC-I , MyHC-IIa/b and MyHC-IIx in myoblast cells. PKA inhibitor H-89 or PKAreg I activator N6-Bz-cAMP were added into medium to confirm their relationship between Ezrin and PKA during myoblast differentiation/fusion. In vitro, Ad-NFATc1/c2 or Ad-shNFATc3/c4 were respectively transfected into C2C12 cells, myoblast differentiation/fusion, myotube size and myofiber type were assessed by using immunostaining of MyHC, MEF2c and MyoG. In vivo, transfection of Ad-Ezrin into gastrocnemius and soleus muscles for 7 days, the numbers of MyHC-1 postivemyofibers were analyzed after immunostaining of MyHC-1.Results: Ezrin expression were time-dependently increased during myoblast differentiation/fusion. Knockdown of Ezrin by shRNA delayed myoblast differentiation and fusion in a time dose-dependent pattern, as shown by immunostaining of MyHC. Conversely, over-expression of Ezrin by adenovirus time- and dosage-dependently promoted myoblastdifferentiation/fusion, and muscle fiber specialization characterized by increased MyHC I and MyHCIIa/b. Forced expression of Ezrin did not alter PKA, and PKAreg II α levels, but altered the levels of PKAreg I α/β, Myf5 and MyoD, and leading to the accumulation of MyoG+/MEF2c+ nuclei. By contrast, Ezrin knockdown significantly decreased the PKA reg I/II ratio and MyoG+/MEF2c+ nuclei. The PKA inhibitor H-89 remarkably abolished the beneficial effect of over-expressingEzrin on the numbers of MyHC+ myotubes and MyoG+/MEF2c nuclei. These opposite changes mediated by knocking down Ezrin were almost eliminated by PKAreg I activator N6-Bz-cAMP. Furthermore, over-expression of NFATc2 or knockdown of NFATc4reversed the inhibitory effect of Ezrin knockdown on myoblast differentiation/fusion, resulting in the recovery of the numbers ofMyoG+/MEF2c+ nucleiin3-nuclei+myotubes. Meanwhile, overexpression of Ezrin specifically induced type I muscle fiber specialization, which was associated with increased levels of NFATc1/c2. Furthermore, in vivo transfection ofAd-Ezrin into gastrocnemius and soleus muscles increased the numbers of MyHC-1 postivemyofibers. By contrast, knockdown of NFATc4resulted in the recovery to normal levels of MyHC-2b in Ezrin-knockdown myoblast cells, attributingtoregainingMyoDand MEF2c expression. Conclusions: Ezrin trigger myoblast differentiation and fusion, myotube size, and alters muscle fiber specialization through PKA-NFAT-MyoD/MEF2C signalling pathway.

scholarly journals Syndecan-4 regulates localization, activity and stability of protein kinase C-alpha

2004 ◽  
Vol 378 (3) ◽  
pp. 1007-1014 ◽  
Author(s):  
Eunyoung KEUM ◽  
Yeonhee KIM ◽  
Jungyean KIM ◽  
Soojin KWON ◽  
Yangmi LIM ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Heparan Sulphate ◽  
Critical Role ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Prolonged Treatment ◽  
Kinase C

During cell–matrix adhesion, syndecan-4 transmembrane heparan sulphate proteoglycan plays a critical role in the formation of focal adhesions and stress fibres. We have shown previously that the syndecan-4 cytoplasmic domain directly binds to and activates PKC-α (protein kinase C-α) in vitro [Oh, Woods and Couchman (1997) J. Biol. Chem. 272, 8133–8136]. However, whether syndecan-4 has the same activity in vivo needs to be addressed. Using mammalian two-hybrid assays, we showed that syndecan-4 interacted with PKC-α in vivo and that this interaction was mediated through syndecan-4 cytoplasmic domain. Furthermore, the activation of PKC increased the extent of interaction between syndecan-4 and PKC-α. Overexpression of syndecan-4, but not a mutant lacking its cytoplasmic domain, specifically increased the level of endogenous PKC-α and enhanced the translocation of PKC-α into both detergent-insoluble and membrane fractions. In addition, rat embryo fibroblasts overexpressing syndecan-4 exhibited a slowed down-regulation of PKC-α in response either to a prolonged treatment with PMA or to maintaining cells in suspension culture. PKC-α immunocomplex kinase assays also showed that syndecan-4 overexpression increased the activity of membrane PKC-α. Taken together, these results suggest that syndecan-4 interacts with PKC-α in vivo and regulates its localization, activity and stability.

scholarly journals Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation

1999 ◽  
Vol 10 (12) ◽  
pp. 4177-4190 ◽  
Author(s):  
Josephine C. Adams ◽  
James D. Clelland ◽  
Georgina D.M. Collett ◽  
Fumio Matsumura ◽  
Shigeko Yamashiro ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Fluorescent Protein ◽  
Cell Types ◽  
C2c12 Cells ◽  
Actin Binding ◽  
Wild Type ◽  
Inhibitory Antibody ◽  
Thrombospondin 1 ◽  
Laminin 1 ◽  
In Cells

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members α, γ, and λ, and PKCα localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCα activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin α5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.

scholarly journals Hes6 regulates myogenic differentiation

Development ◽  
2002 ◽  
Vol 129 (9) ◽  
pp. 2195-2207
Author(s):  
Judy Cossins ◽  
Ann E. Vernon ◽  
Yun Zhang ◽  
Anna Philpott ◽  
Philip H. Jones
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitor ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Xenopus Embryos ◽  
Enhancer Of Split

Hes6 is a basic helix-loop-helix transcription factor homologous to Drosophila Enhancer of Split (EoS) proteins. It is known to promote neural differentiation and to bind to Hes1, a related protein that is part of the Notch signalling pathway, affecting Hes1-regulated transcription. We show that Hes6 is expressed in the murine embryonic myotome and is induced on C2C12 myoblast differentiation in vitro. Hes6 binds DNA containing the Enhancer of Split E box (ESE) motif, the preferred binding site of Drosophila EoS proteins, and represses transcription of an ESE box reporter. When overexpressed in C2C12 cells, Hes6 impairs normal differentiation, causing a decrease in the induction of the cyclin-dependent kinase inhibitor, p21Cip1, and an increase in the number of cells that can be recruited back into the cell cycle after differentiation in culture. In Xenopus embryos, Hes6 is co-expressed with MyoD in early myogenic development. Microinjection of Hes6 RNA in vivo in Xenopus embryos results in an expansion of the myotome, but suppression of terminal muscle differentiation and disruption of somite formation at the tailbud stage. Analysis of Hes6 mutants indicates that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but that protein-protein interactions are. Thus, we demonstrate a novel role for Hes6 in multiple stages of muscle formation.

scholarly journals Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarity

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev193425
Author(s):  
Krishnanand Padmanabhan ◽  
Hanna Grobe ◽  
Jonathan Cohen ◽  
Arad Soffer ◽  
Adnan Mahly ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Polarity ◽  
Actin Polymerization ◽  
Planar Cell Polarity ◽  
Adherens Junction ◽  
Actin Binding ◽  
Thymosin Β4 ◽  
Eyelid Closure

ABSTRACTPlanar cell polarity (PCP) is essential for tissue morphogenesis and homeostasis; however, the mechanisms that orchestrate the cell shape and packing dynamics required to establish PCP are poorly understood. Here, we identified a major role for the globular (G)-actin-binding protein thymosin-β4 (TMSB4X) in PCP establishment and cell adhesion in the developing epidermis. Depletion of Tmsb4x in mouse embryos hindered eyelid closure and hair-follicle angling owing to PCP defects. Tmsb4x depletion did not preclude epidermal cell adhesion in vivo or in vitro; however, it resulted in abnormal structural organization and stability of adherens junction (AJ) due to defects in filamentous (F)-actin and G-actin distribution. In cultured keratinocytes, TMSB4X depletion increased the perijunctional G/F-actin ratio and decreased G-actin incorporation into junctional actin networks, but it did not change the overall actin expression level or cellular F-actin content. A pharmacological treatment that increased the G/F-actin ratio and decreased actin polymerization mimicked the effects of Tmsb4x depletion on both AJs and PCP. Our results provide insights into the regulation of the actin pool and its involvement in AJ function and PCP establishment.

Abstract 17273: Long Non-Coding RNA Ppp1r1b Regulates Myogenic Differentiation Through Modulating Histone 3 Methylation

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Xuedong Kang ◽  
Yan Zhao ◽  
Marlin Touma
Keyword(s):  
Histone Modification ◽  
Protein Complex ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Mouse Heart ◽  
Histone 3

Introduction: Long noncoding RNAs (lncRNAs), emerged as critical epigenetic regulators of transcriptome, play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction. In our work, we identified a novel lncRNA that regulates myogenesis. Hypothesis: LncRNA Ppp1r1b regulates myogenesis by modulating Histone 3 methylation Methods: After treated with antisense oligonucleotides (GapmeR) or siRNA against Ppp1r1b-LncRNA, real time PCR and Western blot analyses were performed to examine the expression of myogenic and sarcomere genes. Chromatin immunoprecipitation (CHIP) was used to comparatively analyze gene specific histone modification level. RNA pull-down was employed to identify the protein molecules that interact with Ppp1r1b-LncRNA. Results: By silencing Ppp1r1b-LncRNA with GapmeR, C2C12, a skeletal myoblast cell line, did not develop fully differentiated myotubes, but tend to remain in a quiescent mono-nucleated status. In vivo analysis of GapmeR injected neonatal mouse heart and in vitro siRNA silenced human skeletal myoblasts further confirmed the important role of Ppp1r1b-LncRNA on myogenesis. Members of the MyoD family of muscle-specific transcription factors (MyoD and myogenin) failed to be up-regulated during myogenic differentiation when treated with Ppp1r1b-LncRNA specific GapmeR or siRNA. Key proteins essential for establishing and maintaining normal skeletal muscle architecture, including Tcap and Dystropnin, are also suppressed in Ppp1r1b LncRNA- deficient heart. Analysis of histone modification levels at Myogenin, MyoD1 and Tcap in C2C12 cells revealed more histone tri-methylation at these myogenic and sarcomere structural genes compared to untreated cells. Additional lncRNA- protein complex isolation has further revealed insight into the biological roles of Ppp1r1b-LncRNA. Conclusions: Our results support the role of Ppp1r1b-LncRNA in promoting myogenic differentiation. Ppp1r1b-lncRNA function is mediated by inhibiting histone methylation on promoters of multiple myogenic and sarcomere genes. In particular, the identification of EZH2 in pulled Pp1r1b LncRNA: protein complex implies that Polycomb repressive complex 2 (PRC2) is involved in Ppp1r1b-LncRNA modulated myoblast differentiation.

scholarly journals Metalloprotease–disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells

2000 ◽  
Vol 352 (3) ◽  
pp. 883-892 ◽  
Author(s):  
Qing KANG ◽  
Yi CAO ◽  
Anna ZOLKIEWSKA
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Tail ◽  
Sh3 Domain ◽  
C2c12 Cells ◽  
Receptor Protein ◽  
Myoblast Differentiation ◽  
Src Tyrosine Kinase ◽  
Adam 12

ADAM 12, a member of the ADAM (protein containing a disintegrin and metalloprotease) family of metalloproteaseŐdisintegrins, has been implicated in the differentiation and fusion of skeletal myoblasts, and its expression is dramatically up-regulated in many cancer cells. While the extracellular portion of ADAM 12 contains an active metalloprotease and a cell-adhesion domain, the function of the cytoplasmic portion is much less clear. In this paper, we show that the cytoplasmic tail of ADAM 12 mediates interactions with the non-receptor protein tyrosine kinase Src. The interaction is direct, specific, and involves the N-terminal proline-rich region in the cytoplasmic tail of ADAM 12 and the Src homology 3 (SH3) domain of Src. ADAM 12 and Src co-immunoprecipitate from transfected C2C12 cells, suggesting that the two proteins form a complex in vivo. Co-expression of Src and ADAM 12, but not ADAM 9, in C2C12 cells results in activation of the recombinant Src. Moreover, endogenous ADAM 12 associates with and activates endogenous Src in differentiating C2C12 cells. These results indicate that ADAM 12 may mediate adhesion-induced signalling during myoblast differentiation.

scholarly journals A New Focal Adhesion Protein That Interacts with Integrin-Linked Kinase and Regulates Cell Adhesion and Spreading

2001 ◽  
Vol 153 (3) ◽  
pp. 585-598 ◽  
Author(s):  
Yizeng Tu ◽  
Yao Huang ◽  
Yongjun Zhang ◽  
Yun Hua ◽  
Chuanyue Wu
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Cytoskeleton Organization ◽  
Terminal Domain ◽  
Integrin Linked Kinase ◽  
Ch2 Domain ◽  
Green Fluorescent

Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH2-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti–CH-ILKBP antibodies, and green fluorescent protein–CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP–ILK–PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.

scholarly journals A Novel Muscle-Specific β1 Integrin Binding Protein (Mibp) That Modulates Myogenic Differentiation

1999 ◽  
Vol 147 (7) ◽  
pp. 1391-1398 ◽  
Author(s):  
Ji Li ◽  
Richard Mayne ◽  
Chuanyue Wu
Keyword(s):  
Cell Adhesion ◽  
Myogenic Differentiation ◽  
Binding Protein ◽  
Terminal Differentiation ◽  
C2c12 Cells ◽  
Β1 Integrin ◽  
Adhesion Receptors ◽  
C2c12 Myogenic Cells

Myogenesis is regulated by cell adhesion receptors, including integrins of the β1 family. We report the identification of a novel muscle-specific β1 integrin binding protein (MIBP). MIBP binds to the membrane-proximal cytoplasmic region shared by β1A and β1D integrins, and the binding occurs in vivo as well as in vitro. Furthermore, we show that MIBP is abundantly expressed by C2C12 myogenic cells before fusion, and the expression of MIBP is dramatically downregulated during subsequent differentiation. Finally, we show that overexpression of MIBP in C2C12 cells resulted in a suppression of fusion and terminal differentiation, suggesting that MIBP may play a key role in controlling the progression of muscle differentiation.

scholarly journals RACK1 plays a critical role in mast cell secretion and calcium mobilization by modulating F-actin dynamics

2021 ◽  
Author(s):  
Edismauro G. Freitas Filho ◽  
Elaine Z. M. da Silva ◽  
Hwei Ling Ong ◽  
William D. Swaim ◽  
Indu S. Ambudkar ◽  
...  
Keyword(s):  
Secretory Granules ◽  
Critical Role ◽  
Calcium Mobilization ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Cell Secretion ◽  
Antigen Stimulation ◽  
Cortical Regions

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and calcium mobilization. In this study RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs cells were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs there was a reduction in cortical F-actin, an increase in monomeric G-actin, and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+-secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+-stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, β-actin and the actin binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics affecting mediator secretion, and calcium signaling in MCs.

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