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Molecular and Cellular Biology, September 1999, p. 6057-6064, Vol. 19, No. 9
Department of Physiology II, Kobe University
School of Medicine, Chuo-ku, Kobe 650-0017, Japan
Received 30 December 1998/Returned for modification 17 February
1999/Accepted 3 June 1999
To be fully activated at the plasma membrane, Raf-1 must establish
two distinct modes of interactions with Ras, one through its
Ras-binding domain and the other through its cysteine-rich domain
(CRD). The Ras homologue Rap1A is incapable of activating Raf-1 and
even antagonizes Ras-dependent activation of Raf-1. We proposed
previously that this property of Rap1A may be attributable to its
greatly enhanced interaction with Raf-1 CRD compared to Ras. On the
other hand, B-Raf, another Raf family member, is activatable by both
Ras and Rap1A. When interactions with Ras and Rap1A were measured,
B-Raf CRD did not exhibit the enhanced interaction with Rap1A,
suggesting that the strength of interaction at CRDs may account for the
differential action of Rap1A on Raf-1 and B-Raf. The importance of the
interaction at the CRD is further supported by a domain-shuffling
experiment between Raf-1 and B-Raf, which clearly indicated that the
nature of CRD determines the specificity of response to Rap1A: Raf-1,
whose CRD is replaced by B-Raf CRD, became activatable by Rap1A,
whereas B-Raf, whose CRD is replaced by Raf-1 CRD, lost its response to
Rap1A. Finally, a B-Raf CRD mutant whose interaction with Rap1A is
selectively enhanced was isolated and found to possess the double
mutation K252E/M278T. B-Raf carrying this mutation was not activated by
Rap1A but retained its response to Ras. These results indicate that the
strength of interaction with Ras and Rap1A at its CRD may be a critical determinant of regulation of the Raf kinase activity by the Ras family
small GTPases.
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Strength of Interaction at the Raf
Cysteine-Rich Domain Is a Critical Determinant of Response of Raf
to Ras Family Small GTPases
*
Corresponding author. Mailing address: Department of
Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Phone: 81-78-382-5380. Fax:
81-78-382-5399. E-mail: kataoka{at}kobe-u.ac.jp.
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