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.
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.
Lu
K K
KK
Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
Bazarov
A V
AV
Yoon
L S
LS
Sedivy
J M
JM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002455:Cell Division; D002460:Cell Line; D048429:Cell Size / Q000235:genetics; D004789:Enzyme Activation; D006133:Growth Substances / Q000494:pharmacology; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D016196:S Phase / Q000187:drug effects; D013696:Temperature
D006133:Growth Substances; D010770:Phosphotransferases; D019908:Proto-Oncogene Proteins c-raf