Lepidopteran DALP, and its mammalian ortholog HIC-5, function as negative regulators of muscle differentiation.
(17/945)
During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes, and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal-transduction molecules that determine whether myoblasts should differentiate or die are largely unknown. In this report, we describe the cloning and characterization of DALP, a small LIM-only type zinc-finger protein that is induced when the intersegmental muscles (ISMs) of the moth Manduca sexta become committed to die at the end of metamorphosis. Forced expression of death-associated LIM-only protein (DALP) in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian ortholog Hic-5, blocks differentiation and induces apoptosis in mouse C(2)C(12) myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die after removal of trophic support. Taken together, these data suggest that DALP and Hic-5 act upstream of MyoD and function as phylogenetically conserved "switches" to block muscle differentiation and induce death. (+info)
Characterization of two isoforms of the skeletal muscle LIM protein 1, SLIM1. Localization of SLIM1 at focal adhesions and the isoform slimmer in the nucleus of myoblasts and cytoplasm of myotubes suggests distinct roles in the cytoskeleton and in nuclear-cytoplasmic communication.
(18/945)
We have cloned and characterized a novel isoform of the skeletal muscle LIM protein 1 (SLIM1), designated SLIMMER. SLIM1 contains an N-terminal single zinc finger followed by four LIM domains. SLIMMER is identical to SLIM1 over the first three LIM domains but contains a novel C-terminal 96 amino acids with three potential bipartite nuclear localization signals, a putative nuclear export sequence, and 27 amino acids identical to the RBP-J binding region of KyoT2, a murine isoform of SLIM1. SLIM1 localized to the cytosol of Sol8 myoblasts and myotubes. SLIMMER was detected in the nucleus of myoblasts and, following differentiation into myotubes, was exclusively cytosolic. Recombinant green fluorescent protein-SLIM1 localized to the cytoplasm and associated with focal adhesions and actin filaments in COS-7 cells, while green fluorescent protein-SLIMMER was predominantly nuclear. SLIMMER truncation mutants revealed that the first nuclear localization signal mediates nuclear localization. The addition of the proposed nuclear export sequence decreased the level of exclusively nuclear expression and increased cytosolic SLIMMER expression in COS-7 cells. The leucine-rich nuclear export signal was required for the export of SLIMMER from the nucleus of myoblasts to the cytoplasm of myotubes. Collectively, these results suggest distinct roles for SLIM1 and SLIMMER in focal adhesions and nuclear-cytoplasmic communication. (+info)
The balance between isoforms of the prickle LIM domain protein is critical for planar polarity in Drosophila imaginal discs.
(19/945)
The tissue polarity mutants in Drosophila include a set of conserved gene products that appear to be involved in the control of cytoskeletal architecture. Here we show that the tissue polarity gene prickle (pk) encodes a protein with a triple LIM domain and a novel domain that is present in human, murine, and Caenorhabditis elegans homologs which we designate PET. Three transcripts have been identified, pk, pkM, and sple, encoding 93-, 100-, and 129-kD conceptual proteins, respectively. The three transcripts span 70 kb and share 6 exons that contain the conserved domains. The pk and sple transcripts are expressed with similar tissue-specific patterns but have qualitatively different activities. The phenotypes of pk mutants, and transgenic flies in which the different isoforms are overexpressed show that the balance between Pk and Sple is critical for the specification of planar polarity. In addition, these phenotypes suggest a tessellation model in which the alignment of wing hairs is dependent on cell shape and need not reflect fine-grained positional information. Lack of both pk and sple transcripts gives a phenotype affecting the whole body surface that is similar to those of dishevelled and frizzled (fz) suggesting a functional relationship between pk and fz signaling. (+info)
Purification and characterization of an alpha-actinin-binding PDZ-LIM protein that is up-regulated during muscle differentiation.
(20/945)
alpha-Actinin is required for the organization and function of the contractile machinery of muscle. In order to understand more precisely the molecular mechanisms by which alpha-actinin might contribute to the formation and maintenance of the contractile apparatus within muscle cells, we performed a screen to identify novel alpha-actinin binding partners present in chicken smooth muscle cells. In this paper, we report the identification, purification, and characterization of a 36-kDa smooth muscle protein (p36) that interacts with alpha-actinin. Using a variety of in vitro binding assays, we demonstrate that the association between alpha-actinin and p36 is direct, specific, and saturable and exhibits a moderate affinity. Furthermore, native co-immunoprecipitation reveals that the two proteins are complexed in vivo. p36 is expressed in cardiac muscle and tissues enriched in smooth muscle. Interestingly, in skeletal muscle, a closely related protein of 40 kDa (p40) is detected. The expression of p36 and p40 is dramatically up-regulated during smooth and skeletal muscle differentiation, respectively, and p40 colocalizes with alpha-actinin at the Z-lines of differentiated myotubes. We have established the relationship between p36 and p40 by molecular cloning of cDNAs that encode both proteins and have determined that they are the products of a single gene. Both proteins display an identical N-terminal PDZ domain and an identical C-terminal LIM domain; an internal 63-amino acid sequence present in p36 is replaced by a unique 111-amino acid sequence in p40. Analysis of the sequences of p36 and p40 suggest that they are the avian forms of the actinin-associated LIM proteins (ALPs) recently described in rat (Xia, H., Winokur, S. T., Kuo, W.-L., Altherr, M. R., and Bredt, D. S. (1997) J. Cell Biol. 139, 507-515). The expression of the human ALP gene has been postulated to be affected by mutations that cause facioscapulohumeral muscular dystrophy; thus, the characterization of ALP function may ultimately provide insight into the mechanism of this disease. (+info)
Integrin-linked kinase and PINCH: partners in regulation of cell-extracellular matrix interaction and signal transduction.
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Integrin-linked kinase (ILK) is a focal adhesion serine/threonine protein kinase that is emerging as a key signaling protein functioning at one of the early convergence points of integrin- and growth factor-signaling pathways. ILK binds to PINCH through the N-terminal ankyrin (ANK) repeat domain and the PINCH binding is crucial for focal adhesion localization of ILK. The ILK-PINCH interaction also connects ILK to Nck-2, an SH2-SH3-containing adaptor protein that interacts with components of growth factor and small GTPase signaling pathways. The kinase activity of ILK is regulated by both cell adhesion and growth factors in a phosphoinositide 3-kinase (PI3K)-dependent manner. ILK phosphorylates downstream targets such as protein kinase B (PKB, also known as Akt) and glycogen synthase kinase 3 (GSK-3) and regulates their activities. Overexpression of ILK in epithelial cells leads to striking morphological changes mimicking epithelial-mesenchymal transition, including upregulation of integrin-mediated fibronectin matrix assembly and downregulation of cell-cell adhesions. Furthermore, ILK regulates nuclear translocation of (beta)-catenin and gene expression, and promotes cell cycle progression and tumor formation. Recent genetic studies in Drosophila melanogaster and Caenorhabditis elegans have shown that lack of expression of ILK or PINCH results in phenotypes resembling those of integrin-null mutants, which demonstrates that ILK and PINCH are indispensable for integrin function during embryonic development. (+info)
Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats.
(22/945)
Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. In this study, we show that ILK is a constituent of cell-matrix focal adhesions. ILK was recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK was absent in E-cadherin-mediated cell-cell adherens junctions. In previous studies, we have identified PINCH, a protein consisting of five LIM domains, as an ILK binding protein. We demonstrate in this study that the ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeats domain, which is capable of interacting with PINCH in vitro, could also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex was markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, was unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, as an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site failed to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function. (+info)
Expression of nuclear receptor interacting proteins TIF-1, SUG-1, receptor interacting protein 140, and corepressor SMRT in tamoxifen-resistant breast cancer.
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Regulation of gene transcription as a consequence of steroid receptor-DNA interaction is mediated via nuclear receptor interacting proteins (RIPs), including coactivator or corepressor proteins, which interact with both the receptor and components of the basic transcriptional unit and vary between cell types. The aim of this study was to test the hypothesis that resistance of some breast carcinomas to tamoxifen was associated with inappropriate expression of some of these RIPs. Using Northern analysis, we observed no significant difference between the amount of either TIF-1 or SUG-1 mRNA expressed in parental MCF-7 and MCF-7 tamoxifen-resistant cell lines. However, the expression of RIP140 mRNA was lower in the resistant cell line and in the presence of estradiol, the level of RIP140 mRNA was higher in the resistant cells but not in the parental cells. In a cohort of 19 tamoxifen-resistant breast tumor samples, there was no significant difference in the level of the RIP140 and TIF-1 and corepressor SMRT mRNA compared with tamoxifen-treated tumors (n = 6) or untreated tumors (n = 21). However, SUG-1 mRNA was lower in resistant breast tumors. These data provide no support for increased expression of these RIPs or decreased expression of corepressor SMRT for being a mechanism for resistance of breast tumors to tamoxifen. (+info)
Human antigen-presenting cell/tumour cell hybrids stimulate strong allogeneic responses and present tumour-associated antigens to cytotoxic T cells in vitro.
(24/945)
Most tumours do not stimulate effective antitumour immune responses in vivo. In order to enhance the immunogenicity of human tumour cells, we fused a variety of tumour cell lines with an Epstein-Barr virus transformed B-lymphoblastoid cell line (EBV B-LCL) in vitro, to produce stable hybrid cells. Hybrid cell lines showed a marked increase in their ability to stimulate primary allogeneic T-cell responses in vitro, as compared with the parent tumour cells. The hybrid cells induced proliferation of naive (CD45RA+) as well as memory (CD45RO+) T lymphocytes, and both CD4+ and CD8+ subpopulations of T cells were directly stimulated. The stimulatory hybrids expressed human leucocyte antigen (HLA) class I and II, and a wide range of surface accessory molecules, including the T-cell co-stimulatory ligand molecules CD40, CD80 (B7.1) and CD86 (B7.2), the expression of which was required for optimal stimulation of T-cell responses. Fusion of the EBVB-LCL with a melanoma cell line (518.A2) yielded hybrid cells that expressed the melanoma-associated antigens MAGE-1 and MAGE-3, and presented these antigens to antigen-specific, HLA class I-restricted cytotoxic T-lymphocyte clones with greater efficiency than the parent melanoma cell line. These findings suggest that the generation of human antigen-presenting cell/tumour cell hybrids offers promise as an approach to cancer immunotherapy. (+info)