TY - JOUR
T1 - Role of Raf-1 conserved region 2 in regulation of Ras-dependent Raf-1 activation
AU - Sendoh, Hiroyoshi
AU - Hu, Chang Deng
AU - Wu, Dongmei
AU - Song, Chunhua
AU - Yamawaki-Kataoka, Yuriko
AU - Kotani, Joji
AU - Okada, Tomoyo
AU - Shima, Fumi
AU - Kariya, Ken Ichi
AU - Kataoka, Tohru
N1 - Funding Information:
Abbreviations used: CRD, cysteine-rich domain; CR, conserved region; TPA, 12-O-tetradecanoylphorbol 13-acetate; RBD, Ras-binding domain; PKC, protein kinase C; PCR, polymerase chain reaction; MBP, maltose-binding protein; GAD, GAL4 transactivation domain; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PAGE, polyacrylamide gel electrophoresis; GBD, GAL4 DNA-binding domain. 1 This investigation was supported by Grants-in-Aids for Scientific Research on Priority Areas, for Scientific Research (B), and for Encouragement of Young Scientists (A) from the Ministry of Education, Science, Sports, and Culture of Japan and by a grant from the Sankyo Foundation of Life Science. 2Present address: Department of Biochemistry II, School of Medicine, University of Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan. 3To whom correspondence should be addressed. Fax: 81-78-382-5399. E-mail: [email protected].
PY - 2000/5/19
Y1 - 2000/5/19
N2 - 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. (C) 2000 Academic Press.
AB - 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. (C) 2000 Academic Press.
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U2 - 10.1006/bbrc.2000.2674
DO - 10.1006/bbrc.2000.2674
M3 - Article
C2 - 10814507
AN - SCOPUS:0034685656
SN - 0006-291X
VL - 271
SP - 596
EP - 602
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 3
ER -