Phosphorylation occurs in the amino terminus of the Raf-1 protein.
The ability of the Raf-1 protein to morphologically transform murine fibroblasts can be activated by amino-terminal deletions or substitutions. We have compared the phosphorylation states of full-length and representative transforming and non-transforming amino-terminal deletion mutants of the Raf-1 protein using phosphoamino acid analysis and tryptic phosphopeptide mapping. Several [32P]orthophosphate-labeled tryptic phosphopeptides that were present in the full-length Raf-1 protein were absent from the highly transforming 22W Raf-1 mutant (lacking 305 amino-terminal residues). Peptide-specific antisera localized Raf-1 phosphorylation sites to several amino-terminal cyanogen bromide and tryptic peptides that are deleted from the 22W protein. A major phosphorylated tryptic peptide of the Raf-1 protein was immunoprecipitated by antiserum directed against amino acid residues 257-275, a highly conserved region of the raf family. This tryptic peptide is entirely deleted from the highly transforming 22W protein. Subtractive Edman degradation and electrophoretic analysis of the immunoprecipitated tryptic peptide indicated that phosphorylation of the Raf-1 protein occurs at serine 259. Multiple phosphorylated tryptic peptide forms were immunoprecipitated by antiserum directed against Raf-1 residues 283-309. The majority of this tryptic peptide is also deleted from the highly transforming Raf-1 mutant 22W.
McGrew
B R
BR
Department of Biochemistry, University of New Hampshire, Durham 03824.
Nichols
D W
DW
Stanton
V P
VP
Cai
H
H
Whorf
R C
RC
Patel
V
V
Cooper
G M
GM
Laudano
A P
AP
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D003429:Cross Reactions; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D012694:Serine / Q000302:isolation & purification
D011518:Proto-Oncogene Proteins; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf