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.

We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrincoated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is colocalized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.The Eps15 homology (EH) 1 domain is an important proteinprotein interaction module functioning in endocytosis. The core of the EH domain-binding motif is composed of the amino acids asparagine-proline-phenylalanine (NPF) (1-3). This sequence is often found at the C terminus of EH domain-binding proteins where the free carboxylate can contribute to binding (3). The EH domain was originally identified in the epidermal growth factor receptor phosphorylation substrate Eps15 (4). Through its EH domains, Eps15 binds to epsin, a recently identified protein implicated in endocytosis (5). Eps15 is localized to the rim of clathrin-coated pits (6), likely through its interactions with AP2 (7-10) and/or with epsin (5). EH domains are also found in the yeast proteins Pan1p and End3p, which are required for endocytosis and normal organization of the actin cytoskeleton (11-15).The Src homology 3 (SH3) domain, a 50 -70-amino acid motif that binds to proline-rich ligands (16, 17) has also been implicated in endocytosis (18). For example, amphiphysins I and II are nerve terminal-enriched proteins that demonstrate SH3 domain-dependent binding to proline-rich sequences in dynamin and synaptojanin (19 -23), enzymes which function in the endocytosis of clathrin-coated vesicles (20,24,25). In fact, overexpression of the SH3 domains of amphiphysins I and II leads to a functional block in endocytosis in a number of different systems (26 -29).A link between EH and SH3 domain-mediated protein-protein interactions has been revealed with the identification and cloning of Xenopus laevis intersectin, a protein containing two N-terminal EH domains, a central helix f...

While endocytosis attenuates signals from plasma membrane receptors, recent studies suggest that endocytosis also serves as a platform for the compartmentalized activation of cellular signaling pathways. Intersectin (ITSN) is a multidomain scaffolding protein that regulates endocytosis and has the potential to regulate various biochemical pathways through its multiple, modular domains. To address the biological importance of ITSN in regulating cellular signaling pathways versus in endocytosis, we have stably silenced ITSN expression in neuronal cells by using short hairpin RNAs. Decreasing ITSN expression dramatically increased apoptosis in both neuroblastoma cells and primary cortical neurons. Surprisingly, the loss of ITSN did not lead to major defects in the endocytic pathway. Yeast two-hybrid analysis identified class II phosphoinositide 3-kinase C2␤ (PI3K-C2␤) as an ITSN binding protein, suggesting that ITSN may regulate a PI3K-C2␤-AKT survival pathway. ITSN associated with PI3K-C2␤ on a subset of endomembrane vesicles and enhanced both basal and growth factor-stimulated PI3K-C2␤ activity, resulting in AKT activation. The use of pharmacological inhibitors, dominant negatives, and rescue experiments revealed that PI3K-C2␤ and AKT were epistatic to ITSN. This study represents the first demonstration that ITSN, independent of its role in endocytosis, regulates a critical cellular signaling pathway necessary for cell survival.Intersectin (ITSN) is a modular scaffold with multiple protein interaction domains that is conserved among metazoa. At the amino terminus are two Eps15 homology (EH) domains that bind NPF motifs on proteins such as epsin (36). The EH domains are followed by a coiled-coil domain that enables ITSN to homo-and heterodimerize with proteins such as Eps15 (24). The carboxy terminus consists of five Src homology 3 (SH3) domains that interact with Pro-rich motifs on a variety of proteins, several of which are involved in regulating endocytosis. Indeed, a subset of ITSNЈs SH3 domains are potent inhibitors of clathrin-coated pit formation (26). Recent studies on the Drosophila melanogaster ortholog of ITSN, Dap160, indicate that this scaffold functions as a stabilizing or recruitment factor for components of the clathrin-coated pit (14, 17). The loss of Dap160 function results in fewer coated vesicles, as well as enlarged vesicles, indicating that ITSN functions in both the formation and maturation of endocytic vesicles. Consistent with this role in Drosophila, silencing ITSN expression in mammalian cells results in defects in epidermal growth factor receptor (EGFR) internalization (18).Although ITSN is clearly linked with endocytosis, increasing evidence suggests that ITSNЈs role within the cell is not limited to this process (1,10,18,19,(27)(28)(29)32). The cloning of mammalian ITSN revealed a longer, spliced product containing a guanine nucleotide exchange factor domain specific for Cdc42 (10, 27). Through the EH domains, ITSN activates a Jun Nterminal protein kinase-dependent pathway that cooper...

The ubiquitin-interacting motif (UIM) is a short peptide motif with the dual function of binding ubiquitin and promoting ubiquitylation. This motif is conserved throughout eukaryotes and is present in numerous proteins involved in a wide variety of cellular processes including endocytosis, protein trafficking, and signal transduction. We previously reported that the UIMs of epsin were both necessary and sufficient for its ubiquitylation. In this study, we found that many, but not all, UIM-containing proteins were ubiquitylated. When expressed as chimeric fusion proteins, most UIMs promoted ubiquitylation of the chimera. In contrast to previous studies, we found that UIMs do not exclusively promote monoubiquitylation but rather a mixture of mono-, multi-, and polyubiquitylation. However, UIMdependent polyubiquitylation does not lead to degradation of the modified protein. UIMs also bind polyubiquitin chains of varying lengths and to different degrees, and this activity is required for UIM-dependent ubiquitylation. Mutational analysis of the UIM revealed specific amino acids that are important for both polyubiquitin binding and ubiquitin conjugation. Finally we provide evidence that UIM-dependent ubiquitylation inhibits the interaction of UIM-containing proteins with other ubiquitylated cellular proteins. Our results suggest a new model for the ubiquitylation of UIM-containing proteins.The UIM 1 was first described as a peptide sequence consisting of a highly conserved ⌽-X-X-A-X-X-X-S-X-X-Ac core where ⌽ represents a hydrophobic residue and Ac is an acidic residue (1). It was identified based on the ubiquitin binding region of the RPN10 subunit of the 26 S proteasome (2, 3). The presence of UIMs in numerous proteins ranging from the MachadoJoseph disease protein (MJD1/ataxin3) to USP25, a member of the deubiquitylating enzyme family, suggests that this region is involved in regulating protein function. Indeed our previous studies and those of others have demonstrated an important role for UIMs in both ubiquitylation and in ubiquitin binding (2-11).Ubiquitylation is a post-translational modification resulting in the covalent attachment of ubiquitin through its COOHterminal Gly to the ⑀-NH 2 group of a Lys residue in a target protein. This process involves a multienzyme cascade that begins with the activation of ubiquitin in the presence of ATP and an E1 ubiquitin-activating enzyme. Subsequently the ubiquitin is transferred through a thiol-ester bond to a ubiquitin-conjugating enzyme (E2) and through the action of an E3 ubiquitin ligase is attached to the substrate by an isopeptide bond. Polyubiquitylation, the attachment of multimeric chains of ubiquitin, leads to the proteolytic destruction of proteins when Lys 48 of ubiquitin is the site of chain formation. However, ubiquitin chains formed through Lys 63 are not involved in protein degradation but rather a variety of processes including DNA repair, translation, IB kinase activation, endocytosis, and protein transport (for a review, see Ref. 12). In contrast to po...

Receptor tyrosine kinases (RTKs) are critical for normal cell growth, differentiation, and development, but they contribute to various pathological conditions when disrupted. Activation of RTKs stimulates a plethora of pathways, including the ubiquitylation and endocytosis of the receptor itself. Although endocytosis terminates RTK signaling, it has emerged as a requisite step in RTK activation of signaling pathways. We have discovered that the endocytic scaffolding protein intersectin (ITSN) cooperated with epidermal growth factor receptor (EGFR) in the regulation of cell growth and signaling. However, a biochemical link between ITSN and EGFR was not defined. In this study, we demonstrate that ITSN is a scaffold for the E3 ubiquitin ligase Cbl. ITSN forms a complex with Cbl in vivo mediated by the Src homology (SH) 3 domains binding to the Pro-rich COOH terminus of Cbl. This interaction stimulates the ubiquitylation and degradation of the activated EGFR. Furthermore, silencing ITSN by RNA interference attenuated EGFR internalization as well as activation of the extracellular signal-regulated kinasemitogen-activated protein kinase pathway, thereby demonstrating the importance of ITSN in EGFR function. Given the cooperativity between ITSN and additional RTKs, these results point to an important evolutionarily conserved, regulatory role for ITSN in RTK function that is necessary for both signaling from receptors as well as the ultimate termination of receptor signaling.