Expression of functional Schistosoma mansoni Smad4: role in Erk-mediated transforming growth factor beta (TGF-beta) down-regulation.

  • Osman A
  • Niles E
  • LoVerde P
  • 15

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Abstract

Members of the transforming growth factor (TGF)-beta superfamily play pivotal roles in cell migration, differentiation, adhesion, pattern formation, and apoptosis. The family of Smad proteins acts as intracellular signal transducers of TGF-beta and related peptides. Smad4, a common mediator Smad (co-Smad), performs a central role in transmitting signals from TGF-beta, BMP, and activins. Schistosoma mansoni receptor-regulated Smad1 and SmSmad2 were previously identified and shown to act in TGF-beta signaling. Herein, we report the identification and characterization of a Smad4 homologue from S. mansoni and provide details about its role in mediation and down-regulation of TGF-beta signaling in schistosomes. In order to identify the schistosome co-Smad, we designed degenerate primers based on the sequence of the conserved MH1/MH2 domains of Smad4 proteins, which were used in PCR to amplify a 137-bp PCR product. A S. mansoni adult worm pair cDNA library was screened resulting in the isolation of a cDNA clone that encodes a 738 amino acid protein (SmSmad4). SmSmad4 was shown to interact with schistosome R-Smads (SmSmad1 and SmSmad2) in vivo and in vitro. The interaction with SmSmad2 was dependent on the receptor-mediated phosphorylation of SmSmad2. In addition, several potential phosphorylation sites for Erk1/2 kinases were identified in the SmSmad4 linker region and shown to be phosphorylated in vitro by an active mutant of mammalian Erk2. Furthermore, Erk-mediated phosphorylation of SmSmad4 decreased its interaction with the receptor-activated form of SmSmad2, in vitro. SmSmad4 was shown to complement a human Smad4 deficiency through the restoration of TGF-beta-responsiveness in MDA-MB-468 breast cancer cells.

Author-supplied keywords

  • Amino Acid
  • Amino Acid Sequence
  • Animals
  • Apoptosis
  • Blotting
  • Cell Line
  • Cloning
  • Complementary
  • Complementary: metabolism
  • DNA
  • DNA Primers
  • DNA Primers: chemistry
  • DNA-Binding Proteins
  • DNA-Binding Proteins: biosynthesis
  • Down-Regulation
  • Electrophoresis
  • Fluorescence
  • Fluorescent Antibody Technique
  • Genetic Vectors
  • Glutathione Transferase
  • Glutathione Transferase: metabolism
  • Humans
  • Immunohistochemistry
  • Indirect
  • Microscopy
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 1: metabolism
  • Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinases: metabolism
  • Molecular
  • Molecular Sequence Data
  • Peptides
  • Peptides: chemistry
  • Phosphorylation
  • Polyacrylamide Gel
  • Precipitin Tests
  • Protein Binding
  • Protein Structure
  • Reverse Transcriptase Polymerase Chain Reaction
  • Schistosoma mansoni
  • Schistosoma mansoni: metabolism
  • Sequence Homology
  • Signal Transduction
  • Smad4 Protein
  • Tertiary
  • Trans-Activators
  • Trans-Activators: biosynthesis
  • Transfection
  • Transforming Growth Factor beta
  • Transforming Growth Factor beta: metabolism
  • Tumor
  • Two-Hybrid System Techniques
  • Western

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Authors

  • Ahmed Osman

  • Edward G Niles

  • Philip T. LoVerde

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