The Raf-1 protein kinase is the bestcharacterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-l-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins.Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine 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.Raf-1 is a serine/threonine kinase whose activation is regulated by the action of extracellular signals such as hormones and mitogens (1). A critical step in the activation of Raf-1 is its interaction with membrane-anchored Ras, an oncoprotein found to be deregulated in many human tumors (reviewed in ref.2). The activation of the Raf-1 kinase initiates the mitogenactivated protein kinase cascade, which transduces cellular growth and differentiation signals to the nucleus (3, 4). It has been shown that Ras-mediated translocation of Raf-1 to the plasma membrane is the first step of Raf activation, but Ras interaction alone is not sufficient to activate the Raf-1 kinase and other events, such as Raf-1 phosphorylation, may be required (5, 6).Raf proteins contain several conserved regions (reviewed in ref. 1). At the carboxyl (C)-terminal end is the catalytic kinase domain that shows specificity for phosphorylation of serine and threonine residues. The amino (N)-terminal region of Raf-1 is thought to be involved in negative regulation of the kinase domain, since its removal or mutation can lead to tumorigenesis (7-9). Residues 55-131, in the N terminus of Raf-1, have been shown to constitute a Ras binding site (10, 11), termed Ras-binding site I (RBS-1). The structure of this domain and the identification of the Ras-interaction surface have recently been elucidated by nuclear m...

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139 -184) may be required for Raf-1 activation. The Raf-CRD is located in the NH 2 -terminal negative regulatory domain of Raf-1 and is highly homologous to cysteinerich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH 2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.Substantial genetic, biochemical, and biological evidence supports the critical role of the Raf-1 serine/threonine kinase as a key downstream effector of Ras signaling and transformation (1, 2). Ras interaction with Raf-1 promotes the activation of Raf-1 in vivo, in part by facilitating its translocation from the cytoplasm to the plasma membrane. Activated Raf-1 phosphorylates and activates the mitogen-activated protein kinase kinases (MAPK 1 kinases; also referred to as MEKs), which in turn phosphorylate and activate the p42 and p44 MAPKs. Activated MAPKs translocate to the nucleus where they regulate the activity of transcription factors such as Elk-1 (3).Ras interaction with Raf-1 alone is not sufficient to cause full activation of Raf-1, but rather binding of Ras to Raf-1 initiates other events that lead to full activation. These additional events include tyrosine (4, 5) and serine/threonine (6 -9) phosphorylation, phospholipid binding (10, 11), and interactions with other proteins that include members of the 14-3-3 protein family and 14-3-3 associated proteins (12-17). Hence, full kinase activation involves a complex multistep process that remains to be elucidated fully.An additional complexity of Ras-mediated activation of Raf-1 is that the Ras/Raf-1 interaction is more convoluted than originally believed. We and others have shown recently that Ras interacts with two distinct Ras-binding domains in the NH 2 -terminal regulatory region of 19). The first Rasbinding domain encompasses Raf-1 residues 55-131 (20, 21) and appears to interact with Ras prior to exposure of the second binding site (19). This second binding region is contained within the Raf-1 cysteine-rich domain (residues 139 -184, designated the Raf-CRD; als...

The Pkc1-mediated cell wall integrity-signaling pathway is highly conserved in fungi and is essential for fungal growth. We thus explored the potential of targeting the Pkc1 protein kinase for developing broadspectrum fungicidal antifungal drugs through a Candida albicans Pkc1-based high-throughput screening. We discovered that cercosporamide, a broad-spectrum natural antifungal compound, but previously with an unknown mode of action, is actually a selective and highly potent fungal Pkc1 kinase inhibitor. This finding provides a molecular explanation for previous observations in which Saccharomyces cerevisiae cell wall mutants were found to be highly sensitive to cercosporamide. Indeed, S. cerevisiae mutant cells with reduced Pkc1 kinase activity become hypersensitive to cercosporamide, and this sensitivity can be suppressed under high-osmotic growth conditions. Together, the results demonstrate that cercosporamide acts selectively on Pkc1 kinase and, thus, they provide a molecular mechanism for its antifungal activity. Furthermore, cercosporamide and a ␤-1,3-glucan synthase inhibitor echinocandin analog, by targeting two different key components of the cell wall biosynthesis pathway, are highly synergistic in their antifungal activities. The synergistic antifungal activity between Pkc1 kinase and ␤-1,3-glucan synthase inhibitors points to a potential highly effective combination therapy to treat fungal infections.

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Ras proteins cycle between active, guanosine triphosphate (GTP)-bound and inactive, guanosine diphospate (GDP)-bound states to mediate signal transduction pathways that promote cell growth and differentiation. It is believed that the major physiological mechanism for Ras activation is via interaction with guanine-nucleotide exchange factors (GEFs). This interaction is highly regulated and results in elevated levels of Ras-GTP by facilitating GDP dissociation. Recently, a novel mechanism of Ras activation has been proposed, whereby nitric oxide (NO) modification of Cys-118, like GEF interaction, populates Ras in its biologically active form by stimulating GDP release. Here, we describe characterization of a variant of Ras, C118S, that is insensitive to NO modification. We have measured the GTPase activity and the GDP dissociation rate of the C118S mutant and found them to be similar to wild-type Ras. We have also analyzed the structure of this mutant using multidimensional heteronuclear NMR methods. Analysis of chemical shifts and distance restraints demonstrates that this mutation has not disrupted the structure of the protein. These results suggest that NO modification of Cys-118 may not alter Ras structure and that the basis of Ras activation by NO is destabilization of a crucial interaction between residues in the GDP-binding pocket and the nucleotide. We have also found that this mutant is a more stable form of Ras at concentrations required for NMR studies, probably due to the removal of a surface-accessible cysteine residue. This stable variant may facilitate structural and biochemical investigations of Ras and other guanine-nucleotide-binding proteins containing a cysteine at this position.