Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation

The Raf family of serine/threonine protein kinases couple growth factor rec eptor stimulation to mitogen activated protein kinase activation, but their own regulation is poorly understood. Using phospho-specific antisera, we s how that activated Raf-1 is phosphorylated on S338 and Y341, Expression of Raf-1 with oncogenic Ras gives predominantly S338 phosphorylation, whereas activated Src gives predominantly Y341 phosphorylation. Phosphorylation at both sites is maximal only when both oncogenic Ras and activated Src are pr esent. Raf-l that cannot interact with Ras-GTP is not phosphorylated, showi ng that phosphorylation is Ras dependent, presumably occurring at the plasm a membrane. Mutations which prevent phosphorylation at either site block Ra f-1 activation and maximal activity is seen only when both are phosphorylat ed, Mutations at S339 or Y340 do not block Raf-1 activation. While B-Raf la cks a tyrosine phosphorylation site equivalent to Y341 of Raf-1, S445 of B- Raf is equivalent to S338 of Raf-1, Phosphorylation of S445 is constitutive and is not stimulated by oncogenic Ras. However, S445 phosphorylation stil l contributes to B-Raf activation by elevating basal and consequently Ras-s timulated activity. Thus, there are considerable differences between the ac tivation of the Raf proteins; Ras-GTP mediates two phosphorylation events r equired for Raf-1 activation but does not regulate such events for B-Raf.