title: The tumour suppressor DiRas3 interacts with C-RAF and downregulates MEK activity to restrict cell migration.
abstract: BACKGROUND
  The mitogenic pathway, composed of RAF kinases, mitogen-activated protein kinase kinases (MEK) and extracellular signal-regulated kinases (ERK), promotes cell proliferation and migration and is upregulated in many tumours. DiRas3 (ARHI, Noey2), a mainly GTP-bound Ras-like protein with an unusual N-terminal extension, is predominantly lost in ovarian and breast cancers. Its re-expression in these tissues impairs cell proliferation, autophagy, apoptosis and cell migration. Further, loss of DiRas3 correlates with an increase in growth factor-induced ERK phosphorylation. Therefore, DIRAS3 proves to be a curious gene with remarkable tumour suppressing capabilities. However, how DiRas3 interferes with ERK phosphorylation, has remained unknown.
  
  
  RESULTS
  We demonstrate that DiRas3 associates in vivo with C-RAF and directly binds in vitro to C-RAF, which is upstream of MEK and ERK. Direct binding of DiRas3 to C-RAF is nucleotide independent, and DiRas3's N-terminal extension alone is not sufficient for binding C-RAF. DiRas3 expression inhibits the activating phosphorylations of MEK and ERK. Serum-induced recruitment of DiRas3 to the plasma membrane depends mainly on its N-terminal extension and less on its C-terminus, bound nucleotide or the presence of Ras-GTP. Correspondingly, removal of the N-terminal extension strongly decreases DiRas3's inhibition of MEK and ERK phosphorylations. Tyrosyl-phosphatases do not contribute significantly to reduction of ERK-phosphorylation byDiRas3. Consistently, downregulation of DiRas3 results in a small but significant and persistent increase in MEK and ERK phosphorylation, but does not increase phosphorylation of P38, AKT and c-Jun NH2-terminal kinase. Finally, downregulation of DiRas3 causes increased cell migration, through a mechanism that is MEK dependent.
  
  
  CONCLUSIONS
  These results support a model in which serum signals induce the recruitment of DiRas3 to the plasma membrane, where it is tethered via its N- and C-termini. At the plasma membrane, DiRas3 interacts with C-RAF to specifically suppress the activating phosphorylations on MEK and ERK, thus restricting migration of non-cancer cells. This effect is relatively small, but it is also persistent, suggesting that it contributes to the maintenance of the non-migratory phenotype of non-cancerous tissues, in which DiRas3 is expressed.
authors.affiliation: ETH Zurich, Institute of Biochemistry, Zurich 8093, Switzerland.
authors.forename: Mirko
authors.initials: M
authors.lastname: Klingauf
authors.forename: Matthias
authors.initials: M
authors.lastname: Beck
authors.forename: Ulrich
authors.initials: U
authors.lastname: Berge
authors.forename: Yagmur
authors.initials: Y
authors.lastname: Turgay
authors.forename: Stephan
authors.initials: S
authors.lastname: Heinzer
authors.forename: Peter
authors.initials: P
authors.lastname: Horvath
authors.forename: Ruth
authors.initials: R
authors.lastname: Kroschewski
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement; D015536:Down-Regulation; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C117386:DIRAS3 protein, human; D025521:Tumor Suppressor Proteins; D019908:Proto-Oncogene Proteins c-raf; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020741:rho GTP-Binding Proteins