The tumour suppressor DiRas3 interacts with C‐RAF and downregulates MEK activity to restrict cell migration

Abstract

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. 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. 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.