THE SOLUTION STRUCTURE OF THE RAF-1 CYSTEINE-RICH DOMAIN - A NOVEL RAS AND PHOSPHOLIPID-BINDING SITE

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals, Th e details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the i nvolvement of both Ras-binding sites in effective Raf-1-mediated trans formation provides insight into the molecular aspects and consequences of Ras-Raf interactions, The Raf-1 CRD is a member of apr emerging fa mily of domains, many of which are found within signal transducing pro teins, Several contain binding sites for diacylglycerol (or phorbol es ters) and phosphatidylserine and are believed to play a role in membra ne translocation and enzyme activation, The CRD from Raf-1 does not bi nd diacylglycerol, but interacts with Ras and phosphatidylserine. To i nvestigate the ligand-binding specificities associated with CRDs, we h ave determined the solution structure of the Raf-1 CRD using heteronuc lear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PBC). The differences are confined to regions of the CRDs in volved in binding phorbol ester in the PKC domains. Since phosphatidyl serine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar , The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.