Suramin Targets the Conserved Ligand-Binding Pocket of Human Raf1 Kinase Inhibitory Protein.

Suramin was initially used to treat African sleeping sickness and has been clinically tested to treat human cancers and HIV infection in the recent years. However, the therapeutic index is low with numerous clinical side-effects, attributed to its diverse interactions with multiple biological macromolecules. Here, we report a novel binding target of suramin, human Raf1 kinase inhibitory protein (hRKIP), which is an important regulatory protein involved in the Ras/Raf1/MEK/ERK (MAPK) signal pathway. Biolayer interference technology showed that suramin had an intermediate affinity for binding hRKIP with a dissociation constant of 23.8 µM. Both nuclear magnetic resonance technology and molecular docking analysis revealed that suramin bound to the conserved ligand-binding pocket of hRKIP, and that residues K113, W173, and Y181 play crucial roles in hRKIP binding suramin. Furthermore, suramin treatment at 160 µM could profoundly increase the ERK phosphorylation level by around 3 times. Our results indicate that suramin binds to hRKIP and prevents hRKIP from binding with hRaf1, thus promoting the MAPK pathway. This work is beneficial to both mechanistically understanding the side-effects of suramin and efficiently improving the clinical applications of suramin.

Annexins and endosomal signaling.

Cell signaling and endocytosis are intimately linked in eukaryotic cells. Signaling receptors at the cell surface enter the endocytic pathway and continue to activate downstream effectors in endosomal compartments. This spatiotemporal regulation of signal transduction provides opportunity for signal diversity and a cell-specific machinery of scaffolding/targeting proteins contributes to establish compartment-specific signaling complexes. Members of the annexin (Anx) protein family, in particular AnxA1, AnxA2, and AnxA6, appear to target their interaction partners to specific membrane microdomains to contribute to the formation of compartment-specific signaling platforms along the endocytic pathway. A major challenge to understand the impact of scaffolding/targeting proteins on spatiotemporal signal transduction along endocytic pathways is the identification, isolation, and functional analysis of low-abundance signal-transducing protein complexes in endocytic compartments. Here, we describe methods to isolate endosomes and to target signaling molecules to endosomes. Applying these methodologies to suitable animal or cell models will enable the dissection of signal transduction in the endocytic compartment in the presence or absence of annexins.

Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules.

Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.

Glucocorticoid receptor interaction with 14-3-3 and Raf-1, a proposed mechanism for cross-talk of two signal transduction pathways.

The glucocorticoid receptor (GR) functions as a ligand-dependent transcription factor. In the present study we describe a specific immunoaffinity chromatography purification of GR from liver cytosol from adrenalectomized rats that may be used to identify hitherto unknown cytosolic GR interacting proteins. We have identified the ubiquitously expressed 14-3-3 as well as Raf-1, a downstream effector of Ras, as GR co-purifying proteins. In our semi-quantitative analysis liganded/activated GR showed the strongest interaction with 14-3-3 and Raf-1, but 14-3-3 was also found to co-purify with GR in a nonliganded/nonactivated state. By extensive salt washes we were also able to demonstrate that the glucocorticoid induced interaction between GR, 14-3-3, and Raf-1, respectively, is remarkably stable and withstood 2.4 m salt. The interaction between GR and 14-3-3 was also verified by 14-3-3 co-immunoprecipitation studies. Our observations that GR and Raf-1 are found within the same protein complex ("receptosome") in the cytoplasm of rat liver cells could provide a mechanistic explanation for glucocorticoid effects on the Raf-1-Ras signaling pathway.

Hepatic epidermal growth factor-regulated mitogen-activated protein kinase kinase kinase activity in the rat: lack of identity with known forms of raf and MEKK.

Mitogenic signaling involves protein kinases that phosphorylate the mitogen-activated protein kinase (MAPK) activator, MEK. In rats, basal hepatic MEK kinase activity is low in vivo in both adult rats and late gestation fetal rats, and is markedly stimulated by intraperitoneal administration of epidermal growth factor (EGF). The level of stimulated MEK phosphorylating activity is approximately 15 times higher in fetal liver than in adult liver. To identify regulated forms of the two categories of MEK kinase, Raf and MEKK, Western immunoblotting, immunoprecipitation kinase assays and immunodepletion studies were performed. Western immunoblotting confirmed that Raf-1, A-Raf, B-Raf, MEKK1 and MEKK2 were present at similar levels in E19 and adult liver. However, specific immunoprecipitation kinase assays did not detect any kinases that could account for marked EGF sensitivity or the higher level of activity in E19 fetuses. Immunodepletion studies produced a marked reduction in immunoreactive Raf/MEKK content and activity, but a minimal decrease in the ability of chromatography fractions to phosphorylate and activate recombinant MEK-1. Our results indicate that hepatic, EGF-sensitive MEK kinase activity may reside with a previously unidentified and physiologically relevant form of Raf and/or MEKK.

Localization of endogenous Grb10 to the mitochondria and its interaction with the mitochondrial-associated Raf-1 pool.

Grb10 belongs to a small family of adapter proteins that are known to interact with a number of receptor tyrosine kinases and signaling molecules. We have recently demonstrated that the Grb10 SH2 domain interacts with both the Raf-1 and MEK1 kinases. Overexpression of Grb10 genes with mutations in their SH2 domains promotes apoptosis in cultured cells, a phenotype that is reversed by concomitant overexpression of the wild type gene. Using immunofluorescence microscopy and subcellular fractionation we now show that most of the Grb10 molecules are peripherally associated with mitochondria. Following insulin-like growth factor I or serum treatment, small pools of Grb10 can also be found at the plasma membrane and in actin-rich membrane ruffles, whereas overexpression of Grb10 leads to its mislocalization to the cytosol. Two-hybrid analysis shows that the Grb10-binding site on Raf-1 co-localizes with its Ras-binding domain. Finally, we show that the endogenous Grb10 and Raf-1 proteins can be co-immunoprecipitated from a partially purified mitochondrial extract, an interaction that is enhanced following the activation of Raf-1 by ultraviolet radiation. Thus, we infer that Grb10 may regulate signaling between plasma membrane receptors and the apoptosis-inducing machinery on the mitochondrial outer membrane by modulating the anti-apoptotic activity of mitochondrial Raf-1.

Isolation of temperature-sensitive mutations in the c-raf-1 catalytic domain and expression of conditionally active and dominant-defective forms of Raf-1 in cultured mammalian cells.

The c-Raf-1 kinase is converted into an oncoprotein by functional inactivation of its NH2-terminal regulatory domain and into a dominant-interfering protein by mutations that eliminate catalytic activity. This report describes a systematic charged residue-to-alanine scanning mutagenesis of the ATP-binding subdomain of the c-raf-1 gene. Two temperature-sensitive mutations were found, which were then used to construct both conditionally active and conditionally dominant-defective alleles. Stable cell lines overexpressing both types of mutants were isolated, and their phenotypes were examined. Ectopic expression of Raf-1 activity in quiescent cells was not sufficient to elicit S-phase entry, but the Raf signal could be efficiently complemented by the progression factor insulin-like growth factor I. The results point to a function of Raf-1 in the platelet-derived growth factor and epidermal growth factor pathways, leading to the establishment of competence for cell cycle entry. Ectopic expression of the dominant-defective activity in quiescent cells efficiently blocked entry into S phase. Effects of the dominant-defective protein could be detected minutes after the shift to the restrictive conditions and resulted in the rapid down-regulation of the mitogen-activated protein kinase pathway. Taken together, the phenotypes of the conditionally active and conditionally dominant-defective mutants point to a critical function of Raf-1 at very early times during exit from G0 and entry into G1.