The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling.

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.

The mammalian ShcB and ShcC phosphotyrosine docking proteins function in the maturation of sensory and sympathetic neurons.

Shc proteins possess SH2 and PTB domains and serve a scaffolding function in signaling by a variety of receptor tyrosine kinases. There are three known mammalian Shc genes, of which ShcB and ShcC are primarily expressed in the nervous system. We have generated null mutations in ShcB and ShcC and have obtained mice lacking either ShcB or ShcC or both gene products. ShcB-deficient animals exhibit a loss of peptidergic and nonpeptidergic nociceptive sensory neurons, which is not enhanced by additional loss of ShcC. Mice lacking both ShcB and ShcC exhibit a significant loss of neurons within the superior cervical ganglia, which is not observed in either mutant alone. The results indicate that these Shc family members possess both unique and overlapping functions in regulating neural development and suggest physiological roles for ShcB/ShcC in TrkA signaling.

axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase.

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. The juxtaposition of fibronectin type III and immunoglobulinlike repeats in the extracellular domain, as well as distinct amino acid sequences in the kinase domain, indicate that the axl protein represents a novel subclass of receptor tyrosine kinases.

Overexpression of matrix Gla protein mRNA in malignant human breast cells: isolation by differential cDNA hybridization.

Genetic alterations are involved in the development of human breast cancer. We sought to isolate genes that are differentially expressed or suppressed in cultured human breast carcinoma cells as compared to cultured normal human breast epithelial cells by employing differential screening of selected cDNA libraries. Analysis of several clones thus isolated revealed that the matrix Gla protein (MGP) gene is overexpressed in the breast cancer cell line 600 PEI, though is transcribed at lower levels in most other mammary derived cultures. MGP requires vitamin K dependent gamma-carboxylation for its known function and thus can be inhibited by vitamin K antagonists. This raises the possibility that MGP may be among those factors that when inhibited by vitamin K antagonists reduce metastases in experimental models. Among the gene whose transcription is consistently suppressed upon mammary transformation were fibronectin and the type I keratin, K14. Differential cDNA screening therefore is an effective method of identifying genes involved in various aspects of mammary cell transformation.

Mitogenesis and endocytosis: What's at the INTERSECTIoN?

Endocytosis is a regulated physiological process by which cell surface proteins are internalized along with extracellular factors such as nutrients, pathogens, peptides, toxins, etc. The process begins with the invagination of small regions of the plasma membrane which ultimately form intracellullar vesicles. These internalized vesicles may shuttle back to the plasma membrane to recycle the membrane components or they may be targeted for degradation. One role for endocytosis is in the attenuation of receptor signaling. For example, desensitization of activated membrane bound receptors such as G-protein coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs) occurs, in part, through endocytosis of the activated receptor. However, accumulating evidence suggests that endocytosis also mediates intracellular signaling. In this review, we discuss the experimental data that implicate endocytosis as a critical component in cellular signal transduction, both in the initiation of a signal as well as in the termination of a signal. Furthermore, we focus our attention on a recently described adaptor protein, intersectin (ITSN), which provides a link to both the endocytic and the mitogenic machinery of a cell. Thus, ITSN functions at a crossroad in the biochemical regulation of cell function.

The PTB domain: a modular domain with multiple function.

The convergence of cell biology, virology, biochemistry, molecular biology and genetics, has resulted in an increased understanding of the mechanisms by which cells communicate with each other and their environment. It has become clear that signal transduction is not just a linear, stepwise activation of enzymatic cascades. Rather, signal transduction involves the specific temporal and spatial assembly of multiprotein complexes within a cell, thereby leading to specific outcomes such as growth, differentiation and apoptosis. Abrogation of these complexes can lead to diseases such as cancer. The discovery of modular protein recognition domains, eg, Src homology 2 (SH2) and SH3 domains, led to the notion that these domains promote the assembly of multiprotein signaling complexes. The phosphotyrosine binding (PTB) domain, also known as a phosphotyrosine interaction domain, was originally described as an alternative to the SH2 domain for recognition of tyrosine phosphorylated ligands. However, recent experiments suggest that this domain is more versatile than originally described and is capable of binding a broad range of ligands including phospholipids and nonphosphorylated proteins. Thus, PTB domains possess a high degree of flexibility in ligand binding, thereby allowing for a wider range of interactions with cellular targets.

Intersectin 1 is required for neuroblastoma tumorigenesis.

Intersectin 1 (ITSN1) is a scaffold protein that regulates diverse cellular pathways, including endocytosis and several signal transduction pathways, including phosphatidylinositol 3-kinase, Class IIß (PI3K-C2ß). ITSN1's transforming potential in vitro suggests that this scaffold protein may be involved in human tumorigenesis. Herein, we demonstrate that ITSN1 is expressed in primary human neuroblastoma tumors and tumor cell lines and is necessary for their in vitro and in vivo tumorigenic properties. Silencing ITSN1 significantly inhibits the anchorage independent growth of tumor cells in vitro and tumor formation in xenograft assays independent of MYCN status. Overexpression of the ITSN1 target, PI3K-C2ß, rescues the soft agar growth of ITSN1-silenced cells demonstrating the importance of the ITSN1-PI3K-C2ß pathway in neuroblastoma tumorigenesis. These findings represent the first demonstration that the ITSN1-PI3K-C2ß pathway has a requisite role in human cancer, specifically neuroblastomas.

The transforming receptor tyrosine kinase, Axl, is post-translationally regulated by proteolytic cleavage.

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.

Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways.

Intersectin is a member of a growing family of adaptor proteins that possess conserved Eps15 homology (EH) domains as well as additional protein recognition motifs. In general, EH domain-containing proteins play an integral role in clathrin-mediated endocytosis. Indeed, intersectin functions in the intermediate stages of clathrin-coated vesicle assembly. However, recent evidence suggests that components of the endocytic machinery also regulate mitogenic signaling pathways. In this report, we provide several lines of evidence that intersectin has the capacity to activate mitogenic signaling pathways. First, intersectin overexpression activated the Elk-1 transcription factor in an MAPK-independent manner. This ability resides within the EH domains, as expression of the tandem EH domains was sufficient to activate Elk-1. Second, intersectin cooperated with epidermal growth factor to potentiate Elk-1 activation; however, a similar level of Elk-1 activation was obtained by expression of the tandem EH domains suggesting that the coiled-coil region and SH3 domains act to regulate the EH domains. Third, intersectin expression was sufficient to induce oncogenic transformation of rodent fibroblasts. And finally, intersectin cooperated with progesterone to accelerate maturation of Xenopus laevis oocytes. Together, these data suggest that intersectin links endocytosis with regulation of pathways important for cell growth and differentiation.

Involvement of NH(2)-terminal sequences in the negative regulation of Vav signaling and transforming activity.

Deletion of the NH(2)-terminal 65 amino acids of proto-Vav (to form onco-Vav) activates its transforming activity, suggesting that these sequences serve a negative regulatory role in Vav function. However, the precise role of these NH(2)-terminal sequences and whether additional NH(2)-terminal sequences are also involved in negative regulation have not been determined. Therefore, we generated additional NH(2)-terminal deletion mutants of proto-Vav that lack the NH(2)-terminal 127, 168, or 186 amino acids, and assessed their abilities to cause focus formation in NIH 3T3 cells and to activate different signaling pathways. Since Vav mutants lacking 168 or 186 NH(2)-terminal residues showed a several 100-fold greater focus forming activity than that seen with deletion of 65 residues, residues spanning 66 to 187 also contribute significantly to negative regulation of Vav transforming activity. The increase in Vav transforming activity correlated with the activation of the c-Jun, Elk-1, and NF-kappaB transcription factors, as well as increased transcription from the cyclin D1 promoter. Tyrosine 174 is a key site of phosphorylation by Lck in vitro and Lck-mediated phosphorylation has been shown to be essential for proto-Vav GEF function in vitro. However, we found that an NH(2)-terminal Vav deletion mutant lacking this tyrosine residue (DeltaN-186 Vav) retained the ability to be phosphorylated by Lck in vivo and Lck still caused enhancement of DeltaN-186 Vav signaling and transforming activity. Thus, Lck can stimulate Vav via a mechanism that does not involve Tyr(174) or removal of NH(2)-terminal regulatory activity. Finally, we found that NH(2)-terminal deletion enhanced the degree of Vav association with the membrane-containing particulate fraction and that an isolated NH(2)-terminal fragment (residues 1-186) could impair DeltaN-186 Vav signaling. Taken together, these observations suggest that the NH(2) terminus may serve as a negative regulator of Vav by intramolecular interaction with COOH-terminal sequences to modulate efficient membrane association.

The src homology 2 and phosphotyrosine binding domains of the ShcC adaptor protein function as inhibitors of mitogenic signaling by the epidermal growth factor receptor.

Upon ligand activation, the epidermal growth factor receptor (EGFR) becomes tyrosine-phosphorylated, thereby recruiting intracellular signaling proteins such as Shc. EGFR binding of Shc proteins results in their tyrosine phosphorylation and subsequent activation of the Ras and Erk pathways. Shc interaction with activated receptor tyrosine kinases is mediated by two distinct phosphotyrosine interaction domains, an NH2-terminal phosphotyrosine binding (PTB) domain and a COOH-terminal Src homology 2 (SH2) domain. The relative importance of these two domains for EGFR binding was examined by determining if expression of the isolated SH2 or PTB domain of ShcC would inhibit EGFR signaling. The SH2 domain potently inhibited numerous aspects of EGFR signaling including activation of Erk2 and the Elk-1 transcription factor as well as EGFR-dependent transformation. Furthermore, the SH2 domain inhibited focus formation by the Neu oncoprotein, another EGFR family member. Surprisingly, inhibition of the EGFR by the SH2 domain did not involve stable association with the receptor. In contrast, the PTB domain associated quite well with the receptor yet had little effect on EGFR signaling. Although the EGFR cytoplasmic tail contains consensus binding sites for the PTB and SH2 domains of ShcC, and both domains of ShcC interact with the receptor in vitro, the SH2 domain is more potent for inhibiting receptor function in vivo. However, inhibition is not due to stable association with the receptor, suggesting that the SH2 domain is binding to a heretofore unknown protein(s) necessary for proper EGFR function.

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells.

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cgamma1 (PLCgamma1). We used recombinant glutathione-S-transferase (GST)-PLCgamma1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCgamma1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCgamma1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.

A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain.

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.

Intersectin can regulate the Ras/MAP kinase pathway independent of its role in endocytosis.

We previously identified intersectin, a multiple EH and SH3 domain-containing protein, as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. More recently, we identified mammalian Sos, a guanine-nucleotide exchange factor for Ras, as an intersectin SH3 domain-binding partner. We now demonstrate that overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4 degrees C or with phenylarsine oxide, treatments previously established to inhibit EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation.

Binding specificity and mutational analysis of the phosphotyrosine binding domain of the brain-specific adaptor protein ShcC.

Shc proteins (hereafter referred to as ShcA) represent major substrates of tyrosine phosphorylation by a wide variety of growth factors and cytokines. We have recently described a novel ShcA-like protein, ShcC, which like ShcA contains an NH2-terminal phosphotyrosine binding domain (PTB), a central effector region (CH1) and a COOH-terminal Src homology 2 domain (SH2). Both the SH2 and PTB domains of ShcC bind a similar profile of proteins as the comparable regions of ShcA. In an effort to define the functional differences or similarities between ShcA and ShcC, we have further characterized the PTB domain of ShcC. Using a degenerate phosphopeptide library screen, we show that the PTB domain of ShcC preferentially binds the sequence His-hydrophobic-Asn/hydrophobic-Asn-Pro-Ser/Thr-Tyr(P). This sequence is similar to the binding site for the ShcA PTB domain, suggesting that these two proteins may have overlapping specificities. In addition, random mutagenesis of the ShcC PTB domain has identified several amino acids important for PTB function (Gly32, Glu63, Ala136, Gly139, and Asp140). Mutation of these amino acids dramatically reduces the affinity of the ShcC PTB domain for the activated epidermal growth factor receptor in vitro.

Expression of axl, a transforming receptor tyrosine kinase, in normal and malignant hematopoiesis.

We previously reported the cloning, and characterization of a receptor tyrosine kinase, axl, from two patients with chronic myelogenous leukemia. Herein, we describe the expression pattern of axl in normal and malignant hematopoietic tissue axl message is detected in normal human bone marrow but not significantly in normal blood leukocytes. Cell separation experiments showed that axl is expressed in hematopoietic CD34+ progenitor and marrow stromal cells, at low levels in peripheral monocytes, but not in lymphocytes or granulocytes. Consistent with the normal pattern of axl expression, axl RNA was found predominantly in diseases of the myeloid lineage: 39 of 66 (59%) patients with myeloproliferative disorders (acute myeloid leukemia, chronic myeloid leukemia (CML) in chronic phase, CML in myeloid blast crisis, and myelodysplasia) showed significant axl transcription, as compared with 1 of 45 (2%) lymphoid leukemias (chronic lymphocytic leukemia, acute lymphocytic leukemia, and CML in lymphoid blast crisis). Treatment of K562 cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), administration of interferon alpha (IFN alpha) to normal monocytes, and treatment of U937 cells with TPA and IFN tau significantly induced axl expression, supporting a role for this kinase in the intracellular signaling of myeloid cells through a variety of biochemical pathways. These results suggest that the axl kinase may be operative in normal and malignant myeloid biology.

Rek, a gene expressed in retina and brain, encodes a receptor tyrosine kinase of the Axl/Tyro3 family.

Rek (retina-expressed kinase) has been identified as a putative novel receptor-type tyrosine kinase of the Axl/Tyro3 family with a potential role in neural cell development. rek clones were isolated from a chick embryonic brain cDNA library with a DNA probe obtained by reverse transcriptase-polymerase chain reaction of mRNA from Müller glia-like cells cultured from chick embryonic retina. Sequence analysis indicated that Rek is a protein of 873 amino acids with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains with eight predicted N-glycosylation sites. Two consensus src homology 2 domain binding sites are present in the cytoplasmic domain, suggesting that Rek activates several signal transduction pathways. Northern analysis of rek mRNA revealed a 5.5-kilobase transcript in chick brain, retina, and kidney and in primary cultures of retinal Müller glia-like cells. Rek protein was identified by immunoprecipitation and immunoblotting as a 140-kDa protein expressed in the chick retina at embryonic days 6-13, which corresponded to the major period of neuronal and glial differentiation. Transfection of rek cDNA into COS cells resulted in transient expression of a putative precursor of 106 kDa that autophosphorylated in immune complex protein kinase assays. Overexpression of rek cDNA in mouse NIH3T3 fibroblasts resulted in activation of the 140-kDa rek kinase and induction of morphologically transformed foci. These properties indicated that Rek has oncogenic potential when overexpressed, but its normal function is likely to be related to cell-cell recognition events governing the differentiation or proliferation of neural cells.

Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

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 clathrin-coated 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 co-localized 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.