Isolation of temperature-sensitive mutations in the c-raf-1 catalytic domain and expression of conditionally active and dominant-defective forms of Raf-1 in cultured mammalian cells.

摘要

The c-Raf-1 kinase is converted into an oncoprotein by functional inactivation of its NH2-terminal regulatory domain and into a dominant-interfering protein by mutations that eliminate catalytic activity. This report describes a systematic charged residue-to-alanine scanning mutagenesis of the ATP-binding subdomain of the c-raf-1 gene. Two temperature-sensitive mutations were found, which were then used to construct both conditionally active and conditionally dominant-defective alleles. Stable cell lines overexpressing both types of mutants were isolated, and their phenotypes were examined. Ectopic expression of Raf-1 activity in quiescent cells was not sufficient to elicit S-phase entry, but the Raf signal could be efficiently complemented by the progression factor insulin-like growth factor I. The results point to a function of Raf-1 in the platelet-derived growth factor and epidermal growth factor pathways, leading to the establishment of competence for cell cycle entry. Ectopic expression of the dominant-defective activity in quiescent cells efficiently blocked entry into S phase. Effects of the dominant-defective protein could be detected minutes after the shift to the restrictive conditions and resulted in the rapid down-regulation of the mitogen-activated protein kinase pathway. Taken together, the phenotypes of the conditionally active and conditionally dominant-defective mutants point to a critical function of Raf-1 at very early times during exit from G0 and entry into G1.