Structure of Raf Kinase

Ser and Thr residues that serve as regulatory phosphorylation sites. Mutations are also described for these regions leading to oncogenic activation of Raf kinase. The protein kinase activity is found in the CR3 domain. 

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Fig. 9.10. Domain structure of Raf kinase. Linear representation of the functional domains of c-Rafl kinase. CR control region.

The structure of the complex indicates a specific interaction between P-sheet structures of Raf kinase and structural elements of RaplA protein belonging to the switch I region, which are thus part of the RaplA protein effector domain. Since RaplA protein has a very similar structure to Ras protein, it is assumed that Ras protein also interacts with Raf kinase via its switch I region. 

Our understanding of the mechanism of activation of Raf kinase by Ras protein is still incomplete, mainly because full-length Raf kinase cannot yet be studied biochemically and structurally. Preliminary information was obtained in experiments using a fusion protein consisting of the membrane localization signal CAAX of K-Ras protein linked to the C terminus of Raf kinase. The presence of the membrane localization sequence of the Ras protein in Raf kinase leads to its constitutive activation, and the activity of... 

Thus, lipoproteins could be injected over the surface of a lipid covered SPR sensor in a detergent free buffer solution and showed spontaneous insertion into the artificial membrane.171 Again two hydro-phobic modifications are necessary for stable insertion into the lipid layer, whereas lipoproteins with a farnesyl group only dissociate significantly faster out of the membrane. Therefore the isoprenylation of a protein is sufficient to allow interaction with membraneous structures, while trapping of the molecule at a particular location requires a second hydrophobic anchor. Interaction between the Ras protein and its effector Raf-kinase depends on complex formation of Ras with GTP (instead of the Ras GDP complex, present in the resting cell). If a synthetically modified Ras protein with a palmi-... 

Insight into the molecular interaction between Raf kinase and Ras protein was obtained from the crystal structure of the complex between the Ras binding domain of c-Rafl kinase and a Ras-like protein, RaplA protein (Nassar et al., 1995, review Wit-tinghofer and Nassar, 1996). 

Although lipid modifications are believed to be necessary for bringing soluble, cytoplasmic proteins to their membrane-bound partners, it is not obvious why transmembrane receptors with structural features favouring their anchorage in the lipid bUayer must be acylated (palmitoylated). Therefore, lipid modifications of proteins may have other roles than attachment to membranes. In some cases lipid modification may change the conformation of proteins, just like phosphorylation an example is the Raf kinase, where the membranous environment activates the kinase conformationally (see Chapter 4). 

Two structural elements of the Raf kinase have been found to interact with the Ras protein. One structural element involved in complex formation on the c-Rafl kinase side is the Ras-binding domain, encompassing amino acids 51-131 of the CR1 domain. On the Ras protein side, the switch I region is involved in this interaction, as shown by the crystal structure of the complex between the Ras-binding domain of c-Rafl kinase and the GTP form of the Ras-like protein, RaplA protein (Nassar et ah, 1995 ). These structural studies did not reveal gross conformational changes of the two proteins upon complex formation. 

Operationally, Ras effector proteins are characterized by their preferential binding to the active GTP form of Ras as compared to the inactive GDP form. In addition to Raf kinase, a number of other signal proteins have been identified to which an effector function in Ras signal conduction has been attributed (review Shields et al., 2000). These effector candidates include a very diverse collection of structurally and functionally distinct proteins, which all show preferential affinity for the active Ras form. The residues of Ras protein involved in binding these effectors lie in the region of switch I (residues 30-37) and switch II (residues 59-76) in addition, an intact Ras effector domain (residues 32-40) is required for this interaction. 

Fig. 1 Interaction network and domain structure of B-Raf. (a) Interaction network from the STRING database (http //string-db.org) for human proto-oncogene B-Raf. The lines indicate interactions between the proteins, with thickness of the lines reflecting confidence of the displayed interaction, (b) Domain structure of B-Raf and structures of individual domains, illustrating the different functional units that could be targeted by small molecule inhibitors B-Raf RBD in complex with Ras (PDB code 3kud), diacylglycerol binding Cl domain (lfaq) and the kinase domain in complex with active site inhibitor Sorafenib (luwh)...

At the time of discovery, the thienylurea lead structure 22 represented a novel structural class for kinase inhibition. Concurrently, similar structures were identified as inhibitors of P38 MAP kinase, an enzyme involved in the regulation of inflammation. Subsequently, it was shown that sorafenib inhibits Raf-1 by binding to an inactive conformation of the enzyme, a mechanism of action that has also been observed for other kinase inhibitors. ... 

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