MET(PRC) mutations in the Ron receptor result in upregulation of tyrosine kinase activity and acquisition of oncogenic potential.

Ron and Met are structurally related receptor tyrosine kinases that elicit a complex biological response leading to invasive growth. Naturally occurring point mutations activate the Met kinase in papillary renal carcinomas (MET(PRC) mutations). By site-directed mutagenesis, we generated homologous amino acid substitutions in the Ron kinase domain and analyzed the biochemical and biological properties of the mutant receptors. Among the mutations studied, D(1232)H and M(1254)T displayed transforming activity in NIH3T3 cells, inducing focus formation and anchorage-independent growth. The D(1232)H and M(1254)T substitutions resulted in increased Ron autophosphorylation both in vivo and in vitro and constitutive binding to intracellular signal transducers. Both mutations yielded a dramatic increase in catalytic efficiency, indicating a direct correlation between kinase activity and oncogenic potential. Molecular modeling of the Ron D(1232)H mutation suggests that this single amino acid substitution favors the transition of the kinase from the inactive to the active state. These data demonstrate that point mutations can confer transforming activity to the Ron receptor and show that RON is a potential oncogene.     

Activation of the JNK pathway is essential for transformation by the Met oncogene.

The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.     

Dimerization mediated through a leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase.

Oncogenic activation of the met (hepatocyte growth factor/scatter factor) receptor tyrosine kinase involves a genomic rearrangement that generates a hybrid protein containing tpr-encoded sequences at its amino terminus fused directly to the met-encoded receptor kinase domain. Deletion of Tpr sequences abolishes the transforming ability of this protein, implicating this region in oncogenic activation. We demonstrate, by site-directed mutagenesis and coimmunoprecipitation experiments, that a leucine zipper motif within Tpr mediates dimerization of the tpr-met product and is essential for the transforming activity of the met oncogene. By analogy with ligand-stimulated activation of receptor tyrosine kinases, we propose that constitutive dimerization mediated by a leucine zipper motif within Tpr is responsible for oncogenic activation of the Met kinase. The possibility that this mechanism of activation represents a paradigm for a class of receptor tyrosine kinase oncogenes activated by DNA rearrangement is discussed.     

An oncogenic point mutation confers high affinity ligand binding to the neu receptor. Implications for the generation of site heterogeneity.

The neu protooncogene encodes a receptor tyrosine kinase homologous to the receptor for the epidermal growth factor. The oncogenic potential of neu is released upon chemical carcinogenesis, which replaces a glutamic acid for a valine residue, within the single transmembrane domain. This results in constitutive receptor dimerization and activation of the intrinsic catalytic function. To study the implications of the oncogenic mutation and the consequent receptor dimerization on the interaction with the yet incompletely characterized ligand of p185neu, we constructed chimeric proteins between the ligand binding domain of the epidermal growth factor receptor and the transmembrane and cytoplasmic domains of the normal or the transforming Neu proteins. The chimeric receptors displayed cellular and biochemical differences characteristic of the normal and the transforming Neu proteins and therefore may reliably represent the ligand binding functions of the two receptor forms. Analyses of ligand binding revealed qualitative and quantitative differences that were a result of the single mutation; whereas the normal chimera (valine version) displayed two populations of binding sites with approximately 90% of the receptors in the low affinity state, the transforming receptor (glutamic acid version) showed a single population of binding sites with relatively high affinity. Kinetics measurements indicated that the difference in affinities was because of slower rates of both ligand association and ligand dissociation from the constitutively dimerized mutant receptor. It therefore appears that the oncogenic mutation, by permanently dimerizing the receptor, establishes a high affinity ligand binding state which is functionally equivalent to the ligand-occupied normal receptor. Our conclusion is further supported by the rates of endocytosis of the wild-type and the mutant receptor. Hence, these results provide the first experimental evidence from living cells which supports a model that attributes the heterogeneity of ligand binding sites to the state of oligomerization of receptor tyrosine kinases.     

The RON and MET oncogenes are co-expressed in human ovarian carcinomas and cooperate in activating invasiveness

RON is a member of the receptor tyrosine kinase gene family that includes the MET oncogene, whose germline mutations have been causally related to human tumorigenesis. In vitro, RON and MET receptors cross-talk, synergize in intracellular signaling, and cooperate in inducing morphogenic responses. Here we show that the RON and MET oncogenes were expressed in 55% and 56% of human ovarian carcinomas, respectively, and were significantly coexpressed in 42% (P < 0.001). In ovarian carcinoma samples and cell lines we did not find mutations in RON and MET gene kinase domain, nor coexpression of RON and MET receptor ligands (MSP and HGF, respectively). We show that motility and invasiveness of ovarian cancer cells coexpressing MET and RON receptors were elicited by HGF and, to a lesser extent, by MSP. More interestingly, invasion of both reconstituted basement membrane and collagen gel was greatly enhanced by the simultaneous addition of the two ligands. These data suggest that coexpression of the MET and RON receptors confer a selective advantage to ovarian cancer cells and might promote ovarian cancer progression.     

Cross-talk between RON receptor tyrosine kinase and other transmembrane receptors

RON is a transmembrane receptor tyrosine kinase that mediates biological activities of Macrophage Stimulating Protein (MSP). MSP is a multifunctional factor regulating cell adhesion, motility, growth and survival. MSP binding to RON causes receptor tyrosine phosphorylation leading to up-regulation of RON catalytic activity and subsequent activation of downstream signaling molecules. Recent studies show that RON is spatially and functionally associated with other transmembrane molecules including adhesion receptors integrins and cadherins, and cytokine and growth factor receptors IL-3 betac, EPOR and MET. For example, MSP-induced cell shape change is mediated via RON-activated IL-3 betac receptor. Activation of integrins causes MSP-independent RON phosphorylation, and the integrin/RON collaboration regulates cell survival. Thus, RON can be activated without MSP by ligand stimulation of RON-associated receptors, and MSP-activated RON can cause ligand-independent activation of RON-associated receptors. As a result of the receptor cross-activation RON-specific pathways become a part of a signal transduction network of other receptors, and conversely signaling pathways activated by other receptors can be used by RON. This receptor collaboration extends the spectrum of cellular responses generated by MSP and by putative ligands of RON-associated receptors. However signaling pathways involved in the receptor cross-talk and underlying activation mechanisms remain to be investigated. The purpose of this review is to summarize data and to discuss a role of cross-talk between RON and other transmembrane receptors.     

Met Activation and Receptor Dimerization in Cancer: A Role for the Sema Domain

Ligand dependent activity of receptor tyrosine kinases is critical for modulatingdownstream signaling and cell proliferation. In normal cellular context, hepatocytegrowth factor (HGF) regulates MET kinase activation and mediates cell proliferation,migration and motility. Recent elucidation of the MET extracellular domain suggests thatthe Sema domain, which bears structural similarity to other Semaphorins and Plexinfamily members, plays a critical role in ligand mediated receptor activation.Overexpression of MET which is observed in many cancers leads to ligand independentreceptor dimerization and activation. Evidence to support a role for the Sema domain incancer and therapeutic implications of targeting the Met Sema domain are discussed inthis review.     

Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met(p+1loop)-->Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr(p+1loop) residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr(p+1loop)-->Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK(Metp+1loop) with 2B mutation and wild-type EPH members the Thr(p+1loop) residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET(M918T), nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr(p+1loop) residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.     

The soluble sema domain of the RON receptor inhibits macrophage-stimulating protein-induced receptor activation

RON is a receptor tyrosine kinase of the MET family that is involved in cell proliferation, cell survival, and cell motility in both normal and disease states. Macrophage-stimulating protein (MSP) is the RON ligand whose binding to RON causes receptor activation. RON is a trans-membrane heterodimer comprised of one alpha- and one beta-chain originating from a single-chain precursor and held together by several disulfide bonds. The intracellular part of RON contains the kinase domain and regulatory elements. The extracellular region is characterized by the presence of a sema domain (a stretch of approximately 500 amino acids with several highly conserved cysteine residues), a PSI (plexin, semaphorins, integrins) domain, and four immunoglobulin-like folds. Here we show that a soluble, secreted molecule representing the sema domain of RON (referred to as ron-sema) has a dominant negative effect on the ligand-induced receptor activation and is capable of inhibiting RON-dependent signaling pathways and cellular responses. Results suggest that the sema domain of RON participates in ligand binding by the full-length receptor. The ability of ron-sema to suppress growth of MSP-responsive cells in culture, including cancer cells, points to a potential therapeutic use of this molecule, and forced expression of it could potentially be used as a gene therapy tool for treating MSP-dependent types of cancer.     

Characterization of some molecular mechanisms governing autoactivation of the catalytic domain of the anaplastic lymphoma kinase

NPM/ALK is an oncogenic fusion protein expressed in approximately 50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr --> Phe) three potential autophosphorylation sites contained in the "YXXXYY" motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.     

Geldanamycins trigger a novel Ron degradative pathway, hampering oncogenic signaling

Ron, the tyrosine kinase receptor for macrophage-stimulating protein is responsible for proliferation and migration of cells from different tissues. Ron can acquire oncogenic potential by single point mutations in the kinase domain, and dysregulated Ron signaling has been involved in the development of different human cancers. We have previously shown that ligand-activated Ron recruits the negative regulator c-Cbl, which mediates its ubiquitylation and degradation. Here we report that Ron is ubiquitylated also by the U-box E3 ligase C-terminal Hsc70-interacting protein (CHIP), recruited via chaperone intermediates Hsp90 and Hsc70. Gene silencing shows that CHIP activity is necessary to mediate Ron degradation upon cell treatment with Hsp90 inhibitors geldanamycins. The oncogenic Ron(M1254T) receptor escapes from c-Cbl negative regulation but retains a strong association with CHIP. This constitutively active mutant of Ron displays increased sensitivity to geldanamycins, enhanced physical interaction with Hsp90, and more rapid degradation rate. Cell growth and migration, as well as the transforming potential evoked by Ron(M1254T), are abrogated upon Hsp90 inhibition. These data highlight a novel mechanism for Ron degradation and propose Hsp90 antagonists like geldanamycins as suitable pharmacological agents for therapy of cancers where altered Ron signaling is involved.     

Oncogenic forms of the neu/HER2 tyrosine kinase are permanently coupled to phospholipase C gamma.

The neu/HER2 proto-oncogene encodes a transmembrane tyrosine kinase homologous to receptors for polypeptide growth factors. The oncogenic potential for the presumed receptor is released through multiple genetic mechanisms including a specific point mutation, truncation at the extracellular domain and overexpression of the protooncogene. Here we show that all these modes of oncogenic activation result in a constitutively phosphorylated neu protein and an increase in tyrosine phosphorylation of a phosphatidylinositol-specific phospholipase (PLC gamma). The examined transforming neu/HER2 proteins, unlike the normal gene product, also co-immunoprecipitated with PLC gamma molecules. A kinase-defective mutant of a transforming neu failed to mediate both tyrosine phosphorylation and association with PLC gamma, suggesting direct interaction of the neu kinase with PLC gamma. This possibility was examined by employing a chimeric protein composed of the extracellular ligand-binding domain of the epidermal growth factor receptor and the neu cytoplasmic portion. The chimeric receptor mediated rapid ligand-dependent modification of PLC gamma on tyrosine residues. It also physically associated, in a ligand-dependent manner, with the phosphoinositidase. Based on the presented results we suggest that the mechanism of cellular transformation by the neu/HER2 receptor involves tyrosine phosphorylation and activation of PLC gamma.     

Activation of Ras/Erk pathway by a novel MET-interacting protein RanBPM

MET is a receptor protein-tyrosine kinase (RPTK) for hepatocyte growth factor (HGF), which is a multifunctional cytokine controlling cell growth, morphogenesis, and motility. MET overexpression has been identified in a variety of human cancers. Oncogenic missense mutations of the tyrosine kinase domain of the MET gene have been identified in human papillary renal cell carcinomas. In this study, RanBPM, also known as RanBP9, is identified as a novel interacting protein of MET through yeast two-hybrid screen. RanBPM contains a conserved SPRY (repeats in splA and RyR) domain. We demonstrate that RanBPM can interact with MET in vitro and in vivo, and the interaction can be strengthened by HGF stimulation. RanBPM interacts with the tyrosine kinase domain of MET through its SPRY domain. We show that RanBPM can induce GTP-Ras association and Erk phosphorylation and elevate serum response element-luciferase (SRE-LUC) expression, indicating that RanBPM can activate the Ras-Erk-SRE pathway. We demonstrate that RanBPM, which itself is not a guanine exchange protein, stimulates Ras activation by recruiting Sos. On the cellular level, A704 cells, a human renal carcinoma cell line, transfected with RanBPM exhibit increased migration ability. Our data suggest that RanBPM, functioning as an adaptor protein for the MET tyrosine kinase domain, can augment the HGF-MET signaling pathway and that RanBPM overexpression may cause constitutive activation of the Ras signaling pathway.     

Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes

The role of Raf and MAPK (mitogen-activated protein kinase) during the maturation of Xenopus oocytes was investigated. Treatment of oocytes with progesterone resulted in a shift in the electrophoretic mobility of Raf at the onset of germinal vesicle breakdown (GVBD), which was coincident with the activation of MAPK. Expression of a kinase- defective mutant of the human Raf-1 protein (KD-RAF) inhibited progesterone-mediated MAPK activation. MAPK activation was also inhibited by KD-Raf in oocytes expressing signal transducers of the receptor tyrosine kinase (RTK) pathway, including an activated tyrosine kinase (Tpr-Met), a receptor tyrosine kinase (EGFr), and Ha-RasV12. KD- RAF completely inhibited GVBD induced by the RTK pathway. In contrast, KD-RAF did not inhibit GVBD and the progression to Meiosis II in progesterone-treated oocytes. Injection of Mos-specific antisense oligodeoxyribonucleotides inhibited MAPK activation in response to progesterone and Tpr-Met, but failed to inhibit these events in oocytes expressing an oncogenic deletion mutant of Raf-1 (delta N'Raf). Injection of antisense oligodeoxyribonucleotides to Mos also reduced the progesterone- and Tpr-Met-induced electrophoretic mobility shift of Xenopus Raf. These results demonstrate that RTKs and progesterone participate in distinct yet overlapping signaling pathways resulting in the activation of maturation or M-phase promoting factor (MPF). Maturation induced by the RTK pathway requires activation of Raf and MAPK, while progesterone-induced maturation does not. Furthermore, the activation of MAPK in oocytes appears to require the expression of Mos.     

Src redox regulation: there is more than meets the eye

Src-family kinases are critically involved in the control of cytoskeleton organization and in the generation of integrin-dependent signaling responses, inducing tyrosine phosphorylation of many signaling and cytoskeletal proteins. Activity of the Src family of tyrosine kinases is tightly controlled by inhibitory phosphorylation of a carboxy-terminal tyrosine residue, inducing an inactive conformation through binding with its SH2 domain. Dephosphorylation of C-ter tyrosine, as well as its deletion of substitution with phenylalanine in oncogenic Src kinases, leads to autophosphorylation at a tyrosine in the activation loop, thereby leading to enhanced Src activity. Beside this phophorylation/dephosphorylation circuitry, cysteine oxidation has been recently reported as a further mechanism of enzyme activation. Mounting evidence describes Src activation via its redox regulation as a key outcome in several circumstances, including growth factor and cytokines signaling, integrin-mediated cell adhesion and motility, membrane receptor cross-talk as well in cell transformation and tumor progression. Among the plethora of data involving Src kinase in physiological and pathophysiological processes, this review will give emphasis to the redox component of the regulation of this master kinase.