Modulation of Smad2-mediated signaling by extracellular signal-regulated kinase.
Components of the transforming growth factor-beta and mitogen-activated protein kinase pathways interact in controlling cell growth and differentiation. We show that phosphorylation of Smad2, a mediator of the activin/transforming growth factor-beta signal, by activated extracellular signal-regulated kinase 1 (ERK1) increases the amount of Smad2 protein and leads to enhanced transcriptional activity. Epidermal growth factor increased phosphorylation of Smad2 in COS7 cells, and Smad2-dependent transcription in a mink lung epithelial cell line, L17, was enhanced by co-transfection of a constitutively active MEK1. In addition, transfection of Smad2 mutants lacking ERK sites resulted in reduced transcription, whereas mutants that mimicked ERK phosphorylation stimulated transcription. The amount of Smad2 protein was increased by transfection with a constitutively active MEK1 and reduced by co-transfection with the ERK phosphatase, HVH2. The elevation of Smad2 protein levels was because of increased half-life and resulted in increased complex formation with Smad4. A site of ERK-dependent phosphorylation on Smad2 was located to Thr(8), a site that overlaps with the calmodulin binding region. We show that calmodulin inhibits Smad2 phosphorylation by ERK1, and overexpressing calmodulin, or stimulating calmodulin activity with ionomycin, reduces Smad2 levels. These findings suggest that the ERK pathway positively regulates Smad2 signaling by phosphorylating Smad2 and that negative regulation of Smad2 signaling by calmodulin is achieved in part by inhibiting this phosphorylation.
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA. funaba@azabu-u.ac.jp
Masayuki
M
Funaba
Cole M
CM
Zimmerman
Lawrence S
LS
Mathews
D000818:Animals; D002147:Calmodulin / Q000494:pharmacology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D020935:MAP Kinase Signaling System; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D010766:Phosphorylation; D051899:Smad2 Protein; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D002147:Calmodulin; D004268:DNA-Binding Proteins; D051899:Smad2 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases