To identify proteins that may participate in the activation of the protein kinase Raf, proteins that interact with Raf were selected in a two-hybrid screen. Two members of the 14-3-3 protein family were isolated that interacted with both the amino terminal regulatory regions of Raf and the kinase domain of Raf, but did not compete with the guanine nucleotide-binding protein Ras for binding to Raf. 14-3-3 proteins associated with Raf in mammalian cells and accompanied Raf to the membrane in the presence of activated Ras. In yeast cells expressing Raf and MEK, mammalian 14-3-3 beta or 14-3-3 zeta activated Raf to a similar extent as did expression of Ras. Therefore, 14-3-3 proteins may participate in or be required for the regulation of Raf function. These findings suggest a role for 14-3-3 proteins in Raf-mediated signal transduction.

Induced human fibroblasts produce several mRNAs encoding interferon (IFN) activity. We previously cloned cDNA for a 1.3‐kb RNA designated IFN‐beta 2 and distinct from the 0.9‐kb IFN‐beta 1 mRNA. In vitro transcription‐‐translation mapping of the full‐length IFN‐beta 2 cDNA sequence, shows that it encodes a 23.7‐kd protein of 212 amino acids. This cDNA, fused to the SV40 early gene promoter, was transfected and amplified in Chinese hamster ovary cells and clones were obtained which constitutively produce human interferon activity. Two IFN‐beta 2 genomic clones were isolated and their expression in hamster and mouse cells also produces biologically active rIFN‐beta 2. Specific immunoassays show that IFN‐beta 2 secreted by DNA‐transformed rodent cells is a processed 21‐kd protein, whose activity is cross‐neutralized by antibodies to human IFN‐beta 1 but not to IFN‐alpha or gamma. The immunoassay also demonstrates the induction of IFN‐beta 2 secretion by fibroblasts in response to growth‐regulatory cytokines, such as interleukin‐1 and tumor necrosis factor. The function of this IFN‐beta 2 as an autoregulatory inhibitor of cell growth is discussed.

Directed cell migration is essential for a variety of important biological processes ranging from development and angiogenesis to metastasis. Ras plays a pivotal role in the signaling cascade that governs chemotaxis of fibroblasts toward platelet-derived growth factor-BB (PDGF-BB). Ras activates multiple downstream pathways, which include the extracellular signal-regulated kinase (ERK), Rac, and Ral signaling cascades. We therefore investigated the role of the Rac and ERK pathways in cell migration. We showed that migration of fibroblasts toward PDGF-BB is inhibited by expression of dominant negative Asn-17 Rac1. Blocking of the ERK pathway by either expression of dominant negative Ala-218/Ala-222-mitogen-activated protein kinase kinase (A218/A222-MEK1) or by a MEK-specific inhibitor did not inhibit migration toward PDGF-BB. In contrast, migration toward soluble fibronectin was suppressed by inhibition of the ERK pathway but not by Asn-17 Rac1 expression. These results indicate that directed cell migration mediated by different receptor classes in response to different ligands differentially utilizes the Rac and ERK pathways and suggest that Rac might play a critical role in pathological processes such as angiogenesis and metastasis.Directed cell migration or chemotaxis is a critical feature of several physiological and pathological processes, including development, wound healing, atherosclerosis, immunity, angiogenesis, and metastasis (1-4). Chemotaxis involves the sensing of a concentration gradient of chemoattractant, reorganization of the actin cytoskeleton, and subsequent movement toward the chemoattractant. Cytokine-induced reorganization of the actin cytoskeleton is mediated by members of the Rho family of GTP-binding proteins. Rho regulates stress fiber assembly, Rac controls lamellipodia formation, and Cdc42 directs the dynamics of filipodia (5-8).We have shown previously that the GTP-binding protein Ras plays a central role in the signaling cascade that governs chemotaxis of fibroblasts toward PDGF-BB 1 (9). However, the pathways downstream of Ras that control cell motility remain unknown. Ras activates several distinct effector pathways, including the ERK cascade, the pathway controlled by Rac, and the pathway initiated by RalGDS, the guanine nucleotide dissociation stimulator for RalA and -B, which are close relatives of Ras (10 -17). Since Rac has been shown to control the formation of lamellipodia induced by Ras (5, 18), it is a likely candidate for regulating Ras-mediated directed migration. A possible role for Rac in directed migration is also suggested by observations showing that Ras-and growth factor-induced activation of Rac are mediated by phosphatidylinositol 3-kinase (17, 19), which in turn has been implicated in chemotaxis (20 -22). In this study, therefore, we investigated the role of Rac in different types of cell migration: directed migration toward PDGF-BB and soluble fibronectin and random migration stimulated by lysophosphatidic acid (LPA). EXPERIMENTAL PROCEDURESCell Lines-Derivati...

Glioblastoma (GBM) is one of the most pernicious forms of cancer and currently chances of survival from this malady are extremely low. We have used the noninvasive strategy of intranasal (IN) delivery of a glioblastoma-directed adduct of curcumin (CC), CC-CD68Ab, into the brain of mouse GBM GL261-implanted mice to study the effect of CC on tumor remission and on the phenotype of the tumor-associated microglial cells (TAMs). The treatment caused tumor remission in 50% of GL261-implanted GBM mice. A similar rescue rate was also achieved through intraperitoneal infusion of a lipid-encapsulated formulation of CC, Curcumin Phytosome, into the GL261-implanted GBM mice. Most strikingly, both forms of CC elicited a dramatic change in the tumor-associated Iba1+ TAMs, suppressing the tumor-promoting Arginase1 , iNOS M2-type TAM population while inducing the Arginase1 , iNOS M1-type tumoricidal microglia. Concomitantly, we observed a marked induction and activation of microglial NF-kB and STAT1, which are known to function in coordination to cause induction of iNOS. Therefore, our novel findings indicate that appropriately delivered CC can directly kill GBM cells and also repolarize the TAMs to the tumoricidal M1 state.

We have previously demonstrated that the has a reduced intrinsic GTPase activity (4). These observations, together with its partial homology to mammalian GAP (GTPase activating protein) (12), suggest that IRAI acts to down-regulate RAS activity, and hence adenylate cyclase, by stimulating the GTPase activity of RAS proteins.In an attempt to further elucidate the components operating in the RAS-cAMP pathway, we isolated multicopy suppressor genes of the heat shock sensitive phenotype caused by the iral mutation. In this report, we describe one such suppressor gene, MSIJ, and show that MSI1 negatively regulates the cAMP level in response to glucose.In the yeast Saccharomyces cerevisiae the signal transduction for growth in response to nutrients is mediated by the RAS-cAMP pathway (1, 2). The yeast RAS proteins play an important role in this process: they activate adenylate cyclase, which induces the formation of cAMP (3, 4). The levels of this second messenger regulate, by means of cAMPdependent protein phosphorylation, the progression of the cell cycle through the early G1 phase (1).The ras proteins are highly conserved in evolution (5). The extended structural homology between the yeast and the mammalian ras proteins suggests that they may serve a fundamental function in cellular proliferation. Mutations in mammalian ras proteins have been associated with cell transformation (5); likewise, the analogous alteration in the yeast RAS2 sequence (RAS2vaSl9) causes defects in the cell cycle control exerted by nutrients (3). Although numerous indications suggest that mammalian ras proteins do not activate adenylate cyclase (6, 7), their analogy to guanine nucleotide-binding regulatory proteins (G proteins) has directed considerable attention to the study of RAS-mediated signal transduction in yeast, as this simple eukaryote is amenable to genetic approaches.Like their mammalian counterparts, yeast RAS proteins oscillate between a GDP-and a GTP-bound state, an inactive and an active form, respectively. The RAS2 protein has been shown to activate adenylate cyclase when bound to GTP, but not when bound to GDP (8). The CDC25 gene product, which is required for the regulation of adenylate cyclase, has been found to regulate the RAS proteins and may mediate nucleotide exchange of GDP for GTP (9, 10). Recently, we MATERIALS AND METHODS Yeast Strains, Media, and Plasmids. The relevant genotype of each strain-is described in the text and in the figure legends. YPD medium contains 2% Bacto peptone, 1% Bacto yeast extract, and 2% glucose. SD medium contains 0.67% yeast nitrogen base without amino acids (Difco) and 2% glucose; SC medium is SD medium supplemented with auxotrophic requirements. Sporulation medium contains 1% potassium acetate. Plasmid pd3-L was derived from pd3 (see Fig. 1) by substituting the URA3 gene with the 1.9-kilobase (kb) BamHI-Acc I LEU2 fragment from plasmid YEp13.Analysis of Heat Shock Sensitivity and Sporulation Efficiency. Heat shock experiments were performed as follows. Fresh cells were grown ...

Vemurafenib and dabrafenib selectively inhibit the v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) kinase, resulting in high response rates and increased survival in melanoma. Approximately 22% of individuals treated with vemurafenib develop cutaneous squamous cell carcinoma (cSCC) during therapy. The prevailing explanation for this is drug-induced paradoxical ERK activation, resulting in hyperproliferation. Here we show an unexpected and novel effect of vemurafenib/PLX4720 in suppressing apoptosis through the inhibition of multiple off-target kinases upstream of c-Jun N-terminal kinase (JNK), principally ZAK. JNK signaling is suppressed in multiple contexts, including in cSCC of vemurafenib-treated patients, as well as in mice. Expression of a mutant ZAK that cannot be inhibited reverses the suppression of JNK activation and apoptosis. Our results implicate suppression of JNK-dependent apoptosis as a significant, independent mechanism that cooperates with paradoxical ERK activation to induce cSCC, suggesting broad implications for understanding toxicities associated with BRAF inhibitors and for their use in combination therapies.DOI: http://dx.doi.org/10.7554/eLife.00969.001

The microtubule inhibitor vincristine is currently used to treat a variety of brain tumors, including low-grade glioma and anaplastic oligodendroglioma. Vincristine, however, does not penetrate well into brain tumor tissue, and moreover, it displays dose-limiting toxicities, including peripheral neuropathy. Mebendazole, a Food and Drug Administration-approved anthelmintic drug with a favorable safety profile, has recently been shown to display strong therapeutic efficacy in animal models of both glioma and medulloblastoma. Importantly, appropriate formulations of mebendazole yield therapeutically effective concentrations in the brain. Mebendazole has been shown to inhibit microtubule formation, but it is not known whether its potency against tumor cells is mediated by this inhibitory effect. To investigate this, we examined the effects of mebendazole on GL261 glioblastoma cell viability, microtubule polymerization and metaphase arrest, and found that the effective concentrations to inhibit these functions are very similar. In addition, using mebendazole as a seed for the National Cancer Institute (NCI) COMPARE program revealed that the top-scoring drugs were highly enriched in microtubule-targeting drugs. Taken together, these results indicate that the cell toxicity of mebendazole is indeed caused by inhibiting microtubule formation. We also compared the therapeutic efficacy of mebendazole and vincristine against GL261 orthotopic tumors. We found that mebendazole showed a significant increase in animal survival time, whereas vincristine, even at a dose close to its maximum tolerated dose, failed to show any efficacy. In conclusion, our results strongly support the clinical use of mebendazole as a replacement for vincristine for the treatment of brain tumors.

Mitogen-activated protein (MAP) kinase pathways are three-kinase modules that mediate diverse cellular processes and have been highly conserved among eukaryotes. By using a functional complementation screen in yeast, we have identified a human MAP kinase kinase kinase (MAPKKK) that shares homology with members of the mixed lineage kinase (MLK) family and therefore was called MRK (MLK-related kinase). We report the structure of the MRK gene, from which are generated two splice forms of MRK, MRK-␣ and MRK-␤, encoding for proteins of 800 and 456 amino acids, respectively. By using a combination of solid phase protein kinase assays, transient transfections in cells, and analysis of endogenous proteins in stably transfected Madin-Darby canine kidney cells, we found that MRK-␤ preferentially activates ERK6/p38␥ via MKK3/MKK6 and JNK through MKK4/MKK7. We also show that expression of wild type MRK increases the cell population in the G 2 /M phase of the cell cycle, whereas dominant negative MRK attenuates the G 2 arrest caused by ␥-radiation. In addition, exposure of cells to ␥-radiation induces MRK activity. These data suggest that MRK may mediate ␥-radiation signaling leading to cell cycle arrest and that MRK activity is necessary for the cell cycle checkpoint regulation in cells.