The Ras-Raf relationship: an unfinished puzzle

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Introduction

The Raf serine/threonine kinase, originally identified as an onco-protein, is a key element in cell fate determination 1., 2.. Raf function is involved in the regulation of cell proliferation, differentiation and programmed cell death. The Raf kinase is a component of signal transduction cascades of a variety of different stimuli such as growth factors, cytokines and hormones 1., 3.. It is well established that Raf transmits its signal via the phosphorylation-dependent activation of Mek, which subsequently phosphorylates and activates the Erk MAP kinase (1). Despite 15 years of intensive research, resulting in more than 3000 publications, the mechanism of Raf activation is still an unfocused picture. Purification of the full length Raf kinase is one of the major problems. The lack of this essential tool makes biochemical studies on the protein structure very difficult. The function of the Ras GTPase is closely related to Raf and the Raf kinase was in fact the first effector element identified for Ras (4). Raf proteins have an N-terminal regulatory domain and a C-terminal kinase domain. Deletion of the N-terminal half of the proteins results in constitutively active oncogenic kinases 5., 6., 7.. The GTP-dependent interaction of the Raf N-terminal regulatory domain with Ras was found to be necessary but not sufficient for Raf activation 30., 16., 17.. Additional interaction partners and modifying enzymes are required for the induction of the enzymatic activity. Raf proteins are phosphorylated on serine, threonine and tyrosine amino acids 1., 17.. Mutational analyses have shown that the phosphorylation status of the protein determines its activity. Here we summarize the recent progress of Raf research and discuss these results in respect to the increasing knowledge of the regulation mechanisms of related GTPase/effector interactions.

Section snippets

The Ras/Raf interaction

A central question is the role of the Ras GTPase in the Raf activation process. Raf binds Ras in a GTP-dependent manner (4). The Ras/Raf interaction is mediated by two distinct contact regions 8., 9.. The first contact is made by the so-called Raf RBD (Ras binding domain) encompassing amino acids 51–131 of the human c-Raf-1 protein (10). The Raf-RBD binds to the switch I region of Ras-GTP. The switch I and switch II regions of the Ras family of small GTPases change their conformation upon

Membrane localization and activation of the RAF kinase

The Ras GTPase is membrane localized by a C-terminal farnesyl-anchor (13). Farnelysation of the Ras proteins is directed by the so-called CAAX motif. A fusion protein of the Ras-CAAX motif and the Raf kinase generates a constitutively active membrane localized kinase 14., 15.. The activity of the Raf-CAAX protein was found to be independent of Ras function in those studies. These data in combination with experiments showing that the Ras/Raf interaction by itself is insufficient in activating

Regulation of the raf related p21-activated kinases

The p21-activated kinases are induced by their interaction with the small GTPase Cdc42 (24). Similar to Raf, the Pak kinases have a N-terminal regulatory domain, mediating the GTPase interaction, and a C-terminal kinase domain. It has been shown by NMR studies, employing purified Cdc42 Q61L (1–184) and Pak (75–118) proteins, that Pak contacts the switch region of Cdc42 via its CRIB (Cdc and Rac interactive binding) motif (25). As the Ras/Raf switch/RBD interaction, the Pak CRIB/Cdc42

Summary

Raf kinases interact with GTP-loaded Ras proteins. The Ras/Raf interaction is essential for the activation of the kinase. Based on recent data we favor a model in which the Raf kinase is located to cellular membranes by its interaction with phosphatidic acid and phosphatidylserine. At the cellular membrane Raf proteins are attracted to GTP loaded Ras GTPases which might trigger profound structural changes similar to those found in Cdc42-associated Pak1 kinases (Fig. 1).

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