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1.
Cell Death Dis ; 5: e1002, 2014 Jan 16.
Article in English | MEDLINE | ID: mdl-24434510

ABSTRACT

Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-κB repressor subunit p50. In particular, we identified Gsk3ß as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.


Subject(s)
Cell Differentiation , Muscle Cells/cytology , Muscle Proteins/immunology , Muscle, Skeletal/cytology , NF-kappa B/immunology , Nuclear Proteins/immunology , Repressor Proteins/immunology , Animals , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/immunology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Cells/immunology , Muscle Proteins/genetics , Muscle, Skeletal/immunology , NF-kappa B/genetics , Nuclear Proteins/genetics , Protein Binding , Repressor Proteins/genetics
2.
Cell Death Differ ; 18(8): 1305-15, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21311568

ABSTRACT

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.


Subject(s)
Cell Nucleus/metabolism , Membrane Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/metabolism , Protein Precursors/metabolism , Anticholesteremic Agents/pharmacology , Cell Differentiation , Cells, Cultured , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lamin Type A , Lovastatin/pharmacology , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Dystrophy, Emery-Dreifuss/metabolism , Muscular Dystrophy, Emery-Dreifuss/pathology , Myoblasts/cytology , Myoblasts/metabolism , Prenylation , Stem Cells/cytology , Stem Cells/physiology
3.
Eur J Histochem ; 55(4): e36, 2011 Oct 19.
Article in English | MEDLINE | ID: mdl-22297442

ABSTRACT

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.


Subject(s)
Autophagy/drug effects , Fibroblasts/drug effects , Nuclear Proteins/metabolism , Progeria/pathology , Protein Precursors/metabolism , Sirolimus/pharmacology , Anti-Bacterial Agents/pharmacology , Blotting, Western , Cells, Cultured , Child , Chromatin/metabolism , Humans , Lamin Type A , Nuclear Envelope/drug effects , Prenylation
4.
J Med Genet ; 42(3): 214-20, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15744034

ABSTRACT

BACKGROUND: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms. OBJECTIVE: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells. METHODS: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery-Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres. RESULTS: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts. CONCLUSIONS: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.


Subject(s)
Lamin Type A/metabolism , Muscular Dystrophy, Emery-Dreifuss/metabolism , Myoblasts/metabolism , Protein Processing, Post-Translational , Animals , Cell Differentiation , Cell Line , Humans , Insulin/metabolism , Lamin Type A/genetics , Mice , Muscle Fibers, Skeletal/metabolism , Muscular Dystrophies, Limb-Girdle/genetics , Muscular Dystrophies, Limb-Girdle/metabolism , Muscular Dystrophies, Limb-Girdle/pathology , Muscular Dystrophy, Emery-Dreifuss/genetics , Phosphorylation , Signal Transduction
5.
Cell Mol Life Sci ; 60(12): 2710-20, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14685694

ABSTRACT

Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , 3-Phosphoinositide-Dependent Protein Kinases , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Platelet-Derived Growth Factor/metabolism , Protein Binding , Proto-Oncogene Proteins c-akt , cdc42 GTP-Binding Protein/metabolism
6.
Biochem Biophys Res Commun ; 277(3): 639-42, 2000 Nov 02.
Article in English | MEDLINE | ID: mdl-11062006

ABSTRACT

We recently described a novel congenital muscular dystrophy (CMD) syndrome characterized by mental retardation, microcephaly, and partial merosin deficiency on muscle biopsy. Linkage analysis excluded involvement of the known CMD loci. We now report on a study performed on the differentiation of cultured myoblasts from one patient affected by this condition to evaluate the potential to form myotubes and merosin processing in these cells. The differentiation rate was comparable to controls and myotubes were stable in culture. Biochemical analysis showed the expected 80-kDa merosin subunit in myoblasts. However, a shifted 60-kDa protein was detected in myotubes. Matrix-metalloproteinases (MMPs) zymography showed increased gelatinolytic activity, and immunoblotting identified an increased amount of membrane-type 1 matrix-metalloproteinase in pathological myotube preparations. Our results show that these CMD-derived myotubes contain a low molecular weight merosin. They further suggest that an altered regulation of MMPs can be involved in basal lamina damage.


Subject(s)
Laminin/metabolism , Muscular Dystrophies/metabolism , Cell Differentiation , Child, Preschool , Female , Humans , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinases/metabolism , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/metabolism , Muscular Dystrophies/enzymology , Muscular Dystrophies/pathology
7.
J Biol Chem ; 274(36): 25308-16, 1999 Sep 03.
Article in English | MEDLINE | ID: mdl-10464256

ABSTRACT

In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-zeta/lambda activity in plasma membranes and microsomes and (b) immunoreactive PKC-zeta and PKC-lambda in GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-zeta and PKC-lambda were also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO(4))(3) (PIP(3)) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-zeta and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-zeta pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-zeta/lambda but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-zeta. Also, insulin in situ and PIP(3) in vitro activated and stimulated autophosphorylation of a PKC-zeta mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-zeta that lacks the regulatory (presumably PIP(3)-binding) domain; this may reflect PIP(3) effects on PDK-1 or transphosphorylation by endogenous full-length PKC-zeta. Our findings suggest that insulin activates both PKC-zeta and PKC-lambda in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP(3), PDK-1-dependent phosphorylation of activation loop sites in PKC-zeta and lambda, and subsequent autophosphorylation and/or transphosphorylation.


Subject(s)
Adipocytes/metabolism , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Monosaccharide Transport Proteins/metabolism , Muscle Proteins , Protein Kinase C/metabolism , Signal Transduction/drug effects , Adipocytes/ultrastructure , Animals , Biological Transport/drug effects , Cell Line , Cell Membrane/metabolism , Cytoplasmic Granules/metabolism , Glucose Transporter Type 4 , Isoenzymes , Mice , Phosphorylation , Rats
8.
FEBS Lett ; 438(1-2): 49-54, 1998 Oct 30.
Article in English | MEDLINE | ID: mdl-9821957

ABSTRACT

Interleukin 1 (IL-1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF-kappaB. The IL-1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C-terminal portion of the type I IL-1 receptor (IL-IRI) is essential for IL-1 signalling and for IL-1 dependent activation of NF-kappaB. This portion contains a putative phosphatidylinositol 3-kinase (PI 3-kinase) binding domain (Tyr-E-X-Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3-kinase in IL-1 signalling. Here we report evidence that PI 3-kinase is recruited by the activated IL-IRI, causing rapid and transient activation of PI 3-kinase. We also show that the receptor is tyrosine phosphorylated in response to IL-1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3-kinase activation by IL-1. Our results indicate that PI 3-kinase is likely to be an important mediator of some IL-1 effects, providing docking sites for additional signalling molecules.


Subject(s)
Interleukin-1/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Interleukin-1/metabolism , Binding Sites , Consensus Sequence , Enzyme Activation , Humans , Interleukin-1/metabolism , NF-kappa B/metabolism , Osteosarcoma , Phosphorylation , Phosphotyrosine/metabolism , Precipitin Tests , Protein Binding , Receptors, Interleukin-1/chemistry , Receptors, Interleukin-1 Type I , Transcriptional Activation , Transfection , Tumor Cells, Cultured , Tyrosine/metabolism , src Homology Domains/physiology
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