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Molecular and Cellular Biology, November 1998, p. 6698-6710, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

The RafC1 Cysteine-Rich Domain Contains Multiple Distinct Regulatory Epitopes Which Control Ras-Dependent Raf Activation

Martina Daub,1 Johannes Jöckel,1 Thomas Quack,1 Christoph K. Weber,2 Frank Schmitz,1 Ulf R. Rapp,2 Alfred Wittinghofer,1 and Christoph Block1,*

Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Dortmund,1 and Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Würzburg,2 Germany

Received 26 March 1998/Returned for modification 14 May 1998/Accepted 18 August 1998

Activation of c-Raf-1 (referred to as Raf) by Ras is a pivotal step in mitogenic signaling. Raf activation is initiated by binding of Ras to the regulatory N terminus of Raf. While Ras binding to residues 51 to 131 is well understood, the role of the RafC1 cysteine-rich domain comprising residues 139 to 184 has remained elusive. To resolve the function of the RafC1 domain, we have performed an exhaustive surface scanning mutagenesis. In our study, we defined a high-resolution map of multiple distinct functional epitopes within RafC1 that are required for both negative control of the kinase and the positive function of the protein. Activating mutations in three different epitopes enhanced Ras-dependent Raf activation, while only some of these mutations markedly increased Raf basal activity. One contiguous inhibitory epitope consisting of S177, T182, and M183 clearly contributed to Ras-Raf binding energy and represents the putative Ras binding site of the RafC1 domain. The effects of all RafC1 mutations on Ras binding and Raf activation were independent of Ras lipid modification. The inhibitory mutation L160A is localized to a position analogous to the phorbol ester binding site in the protein kinase C C1 domain, suggesting a function in cofactor binding. Complete inhibition of Ras-dependent Raf activation was achieved by combining mutations K144A and L160A, which clearly demonstrates an absolute requirement for correct RafC1 function in Ras-dependent Raf activation.

* Corresponding author. Mailing address: Abt. Strukturelle Biologie, Max-Plank-Institut für Molekulare Physiologie, Postfach 10 26 64, D-44026 Dortmund, Germany. Phone: 49 231 1206 274. Fax: 49 231 1206 230. E-mail: christoph.block{at}mpi-dortmund.mpg.de.

Molecular and Cellular Biology, November 1998, p. 6698-6710, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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