Role of Raf-1 conserved region 2 in regulation of Ras-dependent Raf-1 activation.
Full activation of Raf-1 requires the interaction of its CRD with Ras. The serine/threonine-rich region, CR2, of Raf-1 was implicated in Raf-1 regulation, but the underlying mechanism was unclear. Here we show that CRD loses its Ras-binding activity when expressed in connection with CR2, suggesting that CR2 masks CRD. This masking effect is abolished by substitution of Asp or Ala for Ser-259, a growth factor- and TPA-induced phosphorylation site in CR2. Treatment of COS-7 cells expressing Ha-Ras(Val-12) and Raf-1 with TPA enhances the Ha-Ras(Val-12)-dependent Raf-1 kinase activity. In contrast, the Ha-Ras(Val-12)-dependent activities of the Raf-1(S259D) and Raf-1(S259A) mutants are comparable to that of wild-type Raf-1 stimulated by both Ha-Ras(Val-12) and TPA and cannot be further stimulated by TPA treatment. These results suggest that the in vivo phosphorylation of Ser-259 may comprise a crucial step for Ras-dependent Raf-1 activation by unmasking CRD and promoting its association with Ras.
Sendoh
H
H
Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan.
Hu
C D
CD
Wu
D
D
Song
C
C
Yamawaki-Kataoka
Y
Y
Kotani
J
J
Okada
T
T
Shima
F
F
Kariya
K
K
Kataoka
T
T
D000818:Animals; D019556:COS Cells; D004789:Enzyme Activation / Q000187:drug effects; D005454:Fluorescence Polarization; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D009154:Mutation; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D010768:Phosphoserine; D019908:Proto-Oncogene Proteins c-raf; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate