Expression of functional Schistosoma mansoni Smad4: role in Erk-mediated transforming growth factor beta (TGF-beta) down-regulation.
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.
Osman
Ahmed
A
Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, School of Medicine, State University of New York, Buffalo, New York 14214, USA.
Niles
Edward G
EG
LoVerde
Philip T
PT
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012550:Schistosoma mansoni / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D051901:Smad4 Protein; D015534:Trans-Activators / Q000096:biosynthesis*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D010455:Peptides; C497796:SMAD4 protein, human; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases