Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in haematopoietic cells.

Individuals with neurofibromatosis type 1 (NF1) are predisposed to certain cancers including juvenile chronic myelogenous leukaemia (JCML). The NF1 tumour-suppressor gene encodes a protein (neurofibromin) that accelerates GTP hydrolysis on Ras proteins. Here we show that primary leukaemic cells from children with NF1 show a selective decrease in NF1-like GTPase activating protein (GAP) activity for Ras but retain normal cellular GAP activity. Leukaemic cells also show an elevated percentage of Ras in the GTP-bound conformation. JCML cells are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF), and we observed a similar pattern of aberrant growth in haematopoietic cells from Nf1-/- mouse embryos. These data define a specific role for neurofibromin in negatively regulating GM-CSF signaling through Ras in haematopoietic cells and they suggest that hypersensitivity to GM-CSF may be a primary event in the development of JCML.

Modulation of nutrient sensing nuclear hormone receptors promotes weight loss through appetite suppression in mice.

AIM: Peroxisome proliferator activated receptors (PPARs) are nuclear receptors involved in glucose and lipid metabolism. Three isoforms of PPARs have been identified with different tissue distribution and biological functions. Although the pharmacology of each receptor is well studied, the physiological effect of simultaneous activation of PPARalpha, gamma and delta is only starting to emerge. We sought to determine the biological effects of a novel PPAR pan activator and elucidate the physiological mechanisms involved. METHODS: Ob/ob, diet-induced obese (DIO) or PPARalpha knockout mice were administered a novel agonist that activates all PPARs to various degrees to determine the effect on body weight, body composition, food intake and energy expenditure. In addition, serum parameters including glucose, insulin, triglycerides and ketone bodies as well as tissue acylcarnitine were evaluated. The effect of the novel agonist on liver and skeletal muscle histopathology was also studied. RESULTS: We report that simultaneous activation of all PPARs resulted in substantial weight loss in ob/ob and DIO mice. Consistent with known PPAR pharmacology, we observed that agonist treatment increased lipid oxidation, although appetite suppression was mainly responsible for the weight loss. Agonist-induced weight loss was completely absent in PPARalpha knockout mice suggesting that PPARalpha pharmacology was the major contributor to weight regulation in mice. CONCLUSIONS: Our work provides evidence that simultaneous activation of PPARalpha, gamma and delta decreases body weight by regulating appetite. These effects of the pan agonist were completely absent in PPARalpha knockout mice, suggesting that PPARalpha pharmacology was the major contributor to weight loss.

Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13.

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.

p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13.

Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.

Tau factor from Escherichia coli mediates accurate and efficient termination of transcription at the bacteriophage T3 early termination site in vitro.

The termination signal that limits transcription through the early region of bacteriophage T3 (T3Te) has been cloned and sequenced. The nucleotide sequence of T3Te is identical with that of T7Te, with the exception of a single G to U substitution in the 3' tail of the terminated transcript, and addition of an AC to the loop in the terminator stem-loop, enlarging the loop to six residues. Previous studies of the properties of T3Te have shown that this site is rho independent and is highly efficient for termination in vivo, but is used poorly in vitro during transcription with purified Escherichia coli RNA polymerase. In contrast, the equivalent site in bacteriophage T7 (T7Te) is an efficient termination signal both in vivo and in vitro. However, T3Te becomes an efficient termination site in vitro in the presence of preparations of tau factor. This factor also alters the sites of RNA chain termination found in vitro at T3Te. Transcripts formed in the presence of tau are several nucleotides shorter than those produced with RNA polymerase alone, and have 3' termini that are almost identical with transcripts found in vivo. These latter results are similar to our earlier findings with T7Te, and suggest that other rho independent terminators may act with transcription termination factors in vivo.

Discovery of a novel Raf kinase inhibitor.

We discuss the biology of Ras signal transduction and the epidemiology of ras mutations in association with disease as a background for the development of a Raf kinase inhibitor, BAY 43-9006. Knowledge of Ras effector pathways has permitted genetic validation of numerous targets involved in the Ras signaling cascade. A key Ras effector pathway involves the kinase cascade RAF/MEK/ERK (MEK: MAP/ERK kinase; ERK: extracellular signal related kinase). Indeed, we present studies of cell lines stably expressing mutant MEK constructs, which point to Raf kinase as a target for therapeutics with selective anti-tumor activity. Finally, a small molecule drug discovery program based on inhibition of Raf kinase activity is outlined and the initial pre-clinical development process of the Raf kinase inhibitor BAY 43-9006 is discussed.

Hyperactive Ras as a therapeutic target in neurofibromatosis type 1.

The NF1 gene encodes neurofibromin, a GTPase-activating protein (GAP) for members of the p21(ras) (Ras) family, which negatively regulates Ras output by accelerating the conversion of active Ras. GTP to inactive Ras.GDP. Analysis of tumors from patients with neurofibromatosis type 1 (NF1) has shown biochemical evidence of hyperactive Ras as well as frequent loss of the normal NF1 allele, consistent with its role as a tumor suppressor gene. Taken together, these data suggest that novel therapeutics directed against components of the Ras signaling cascade might provide effective treatments for certain pathological complications of NF1. Here we summarize data that support a role for hyperactive Ras in NF1 disease, including Ras processing, activation, and down-regulation. We review targets for rational drug design, provide preliminary results, and discuss implications for future studies. Am. J. Med. Genet. (Semin. Med. Genet.) 89:14-22, 1999.

Concurrent loss of the PTEN and RB1 tumor suppressors attenuates RAF dependence in melanomas harboring (V600E)BRAF.

Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring (V600E)BRAF mutations. RB1 alterations were mutually exclusive with loss of p16(INK4A), suggesting that whereas p16(INK4A) and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.

Differential regulation of rasGAP and neurofibromatosis gene product activities.

The ras-encoded p21ras proteins bind GTP very tightly, but catalyse hydrolysis to GDP very slowly. In humans, two genes encode proteins that stimulate this GTPase activity (GAP, or GTPase-activating proteins), one of relative molecular mass 120,000, referred to as p120-GAP, and another NF1-GAP, which is encoded by the neurofibromatosis type-1 gene. Both GAPs are widely expressed in mammalian tissues. Here we show that although they will both bind oncogenic mutants of p21ras, neither will stimulate their GTPase activity. NF1-GAP binds to the p21ras proteins up to 300 times more efficiently than p120-GAP. The two GAPs are inhibited to different extents by certain lipids: micromolar concentrations of arachidonate, phosphatidate and phosphatidylinositol-4,5-bisphosphate affect only NF1-GAP. This inhibition does not compete with p21ras, and lipid-inactivated NF1-GAP can still bind p21ras. We used the detergent dodecyl maltoside, which inhibits only NF1-GAP, to distinguish between the two activities in cell extracts and found both types present together in several mammalian cell lines. In contrast, GAP activity in extracts of Xenopus oocytes was not affected by dodecyl maltoside. By these criteria, the mammalian cells contain both GAP activities and the oocytes have only p120-like GAP activity. These results indicate that more than one GAP regulates p21ras in the same cell.

GTPase activating proteins.

Since Ras proteins negotiate many signalling pathways leading to cell growth or differentiation, the regulation of Ras activity is vital to cellular health. Ras activity, which derives from a collaboration between Ras and GTP, is terminated by the GTPase activating protein (GAP)-catalyzed hydrolysis of the GTP. Hence, a simple regulatory scheme emerges: extracellular signals control Ras activity via membrane receptors and GAPs. However, the signalling scenario is probably not so simple. In looking for factors which interpret Ras activity, researchers have been led to the same factors which also regulate Ras activity, namely the GAPs. Therefore, it may be that Ras proteins are actually regulators of GAPs.

Characterization of full-length neurofibromin: tubulin inhibits Ras GAP activity.

Full-length neurofibromin is a GTPase activating protein (GAP) for the Ras proto-oncogene product. Regulation of neurofibromin activity therefore has important implications for cell growth. Neurofibromin co-purifies with tubulin when expressed in insect cells. The interaction between neurofibromin and tubulin is sensitive to the microtubule depolymerizing agent colchicine. Neurofibromin GAP activity is inhibited even at low concentrations of tubulin. However, maximal inhibition of GAP activity is only approximately 70%, suggesting that the neurofibromin-tubulin complex retains residual GAP activity. This decreased activity is reflected by a 4-fold decrease in its affinity for Ras. A truncated mutant of neurofibromin with reduced sensitivity to tubulin localizes some tubulin-binding determinants to an 80 residue segment immediately N-terminal to the GAP-related domain. Since tubulin is an abundant protein in eukaryotic cells, the tubulin-neurofibromin interaction may regulate the Ras signalling pathway.

A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20.

We identified three proteins in neutrophil cytosol of molecular size 65, 62 and 68 kDa which interact in a GTP-dependent manner with rac1 and CDC42Hs, but not with rho. Purification of p65 and subsequent peptide sequencing revealed identity to rat brain PAK65 and to yeast STE20 kinase domains. Based on these sequences we screened a human placenta library and cloned the full-length cDNA. The complete amino acid sequence of the human cDNA shares approximately identity with rat brain PAK65; within the kinase domain the human protein shares > 95% and approximately 63% identity with rat PAK65 and yeast STE20 respectively. The new human (h)PAK65 mRNA is ubiquitously expressed and hPAK65 protein is distinct from either human or rat brain PAK65. Recombinant hPAK65 exhibits identical specificity to the endogenous p65; both can bind rac1 and CDC42Hs in a GTP-dependent manner. The GTP-bound forms of rac1 and CDC42Hs induce autophosphorylation of hPAK65 on serine residues only. hPAK65 activated by either rac1 or CDC42Hs is phosphorylated on the same sites. Induction of hPAK65 autophosphorylation by rac1 or CDC42Hs stimulates hPAK65 kinase activity towards myelin basic protein and once hPAK65 is activated, rac1 or CDC42Hs are no longer required to keep it active. The affinities of rac/CDC42Hs for the non-phosphorylated and phosphorylated hPAK65 were similar. hPAK65 had only a marginal effect on the intrinsic GTPase activity of CDC42Hs, but significantly affected the binding and GAP activity of p190. These data are consistent with a model in which hPAK65 functions as an effector molecule for rac1 and CDC42Hs.