Using a sensitive transfection-tumorigenicity assay, we have isolated a novel transforming gene from the DNA of two patients with chronic myelogenous leukemia. Sequence analysis indicates that the product of this gene, axl, is a receptor tyrosine kinase. Overexpression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140-kDa axl tyrosine-phosphorylated protein. Expression of axl cDNA in the baculovirus system results in the expression of the appropriate recombinant protein that is recognized by antiphosphotyrosine antibodies, confirming that the axl protein is a tyrosine kinase. (12). There are several mechanisms by which growth factor receptors can be rendered transforming. Retroviral transduction of protooncogenes can result in truncation and mutation of the normal version of the gene. This has been shown for the epidermal growth factor receptor (EGFR) (v-erbB) (18), colony-stimulating factor 1 receptor (CSF-1R) (v-fms) (68), and ros (v-ros) (52) genes. Overexpression of an otherwise normal receptor, with or without the concomitant application of ligand, can also result in neoplastic transformation, as shown for the insulinlike growth factor 1 receptor (38), EGFR (15, 77), CSF-1R (65), eph (51), and neu (16) HOS (10), has been demonstrated to be the cell surface receptor for hepatocyte growth factor, a potential growth factor for a broad spectrum of cell types as well as a mediator of liver regeneration (7). In addition, CSF-1R mediates the pleiotropic effects of its cognate ligand, CSF-1. Together, these two molecules stimulate the proliferation and differentiation of cells of the macrophage lineage (68).In an effort to determine genes involved in the progression of chronic myelogenous leukemia (CML) to acute-phase leukemia, we previously reported the identification of a transforming gene in the DNAs of two patients with CML (47). Molecular cloning and characterization indicate that this gene, which we term axl (from the Greek word anexelekto, or uncontrolled), is a receptor tyrosine kinase with a structure novel among tyrosine kinases. Our data indicate that the ax! protein has tyrosine kinase activity and is capable of transforming NIH 3T3 cells. Furthermore, axl's transforming capacity results from overexpression of axl mRNA rather than from structural mutation. MATERIALS AND METHODSIdentification of a transforming gene in CML cells. Transfections and nude mouse tumorigenicity assays were performed as described previously (47). The cell lines AF6295 and AF3642 were derived from secondary nude mouse tumors arising from transfection of DNA from blast crisis and chronic-phase CML patients, respectively. Tumors were isolated from nude mice and digested in the presence of trypsin-EDTA. A portion of these cells was then plated in plastic tissue culture flasks.Isolation of cosmid and cDNA clones. DNA fragments for cosmid cloning were generated by partial MboI digests of genomic DNA from a secondary nude mouse tumor cell line, AF6295. The restricted DNA fragment...

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here, we describe a functionally critical region of RAS located outside the effector lobe that can be targeted for inhibition. We developed a synthetic binding protein (monobody), termed NS1, that bound with high affinity to both GTP- and GDP-bound states of H- and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS disrupting RAS dimerization/nanoclustering, which in turn blocked CRAF:BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers. However, the molecular basis for the tumor- and metastasis- suppressing activity of merlin is unknown. We have now placed merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression.

Several receptor tyrosine kinases generate soluble ligand binding domains either by differential splicing resulting in a truncated RNA transcript, or by proteolytic cleavage. Although the exact role in vivo of these soluble extracellular domains is unclear, proteolysis may function to down-regulate the receptor, and soluble extracellular domains (ECD) may compete with the intact receptor binding to ligand. Axl is a member of a new class of receptor tyrosine kinases characterized by an ECD resembling cell adhesion molecules and unique sequences in the kinase domain. In addition, Axl is transforming in both fibroblast and hematopoietic cells, and appears to be involved in mesenchymal development. We now find that Axl is post-translationally processed by cleavage in a 14 amino acid region immediately NH2-terminal to the transmembrane domain resulting in a soluble ECD and a membrane bound kinase domain. The sequence of this putative cleavage site shares no homology with recognition sites of known proteases. Characterization of this proteolytic processing shows that it does not require protein synthesis or transport but is augmented by phorbol ester treatment. Since the cleavage of Axl enhances turnover of the kinase on the cell surface, we suggest that proteolytic processing down-regulates Axl kinase activity.