Araf kinase antagonizes Nodal-Smad2 activity in mesendoderm development by directly phosphorylating the Smad2 linker region

Abstract

Smad2/3-mediated transforming growth factor β signalling and the Ras-Raf-Mek-Erk cascade have important roles in stem cell and development and tissue homeostasis. However, it remains unknown whether Raf kinases directly crosstalk with Smad2/3 signalling and how this would regulate embryonic development. Here we show that Araf antagonizes mesendoderm induction and patterning activity of Nodal/Smad2 signals in vertebrate embryos by directly inhibiting Smad2 signalling. Knockdown of araf in zebrafish embryos leads to an increase of activated Smad2 with a decrease in linker phosphorylation; consequently, the embryos have excess mesendoderm precursors and are dorsalized. Mechanistically, Araf physically binds to and phosphorylates Smad2 in the linker region with S253 being indispensable in a Mek/Erk-independent manner, thereby attenuating Smad2 signalling by accelerating degradation of activated Smad2. Our findings open avenues for investigating the potential significance of Raf regulation of transforming growth factor β signalling in versatile biological and pathological processes in the future.