Academic literature on the topic 'Tpr-MET'

MET, RON, and SEA are members of a gene family encoding tyrosine kinase receptors with distinctive properties. Besides mediating growth, they control cell dissociation, motility ("scattering"), and formation of branching tubules. While there are transforming counterparts of MET and SEA, no oncogenic forms of RON have yet been identified. A chimeric Tpr-Ron, mimicking the oncogenic form of Met (Tpr-Met) was generated to investigate its transforming potential. For comparison, a chimeric Tpr-Sea was also constructed. Fusion with Tpr induced constitutive activation of the Ron and Sea kinases. While Tpr-Sea was more efficient than Tpr-Met in transformation, Tpr-Ron did not transform NIH 3T3 cells. The differences in the transforming abilities of Tpr-Met and Tpr-Ron were linked to the functional features of the respective tyrosine kinases using the approach of swapping subdomains. Kinetic analysis showed that the catalytic efficiency of Tpr-Ron is five times lower than that of Tpr-Met. Moreover, constitutive activation of Ron resulted in activation of the MAP kinase signaling cascade approximately three times lower than that attained by Tpr-Met. However, constitutive activation of Ron did induce a mitogenic-invasive response, causing cell dissociation, motility, and invasion of extracellular matrices. Tpr-Ron also induced formation of long, unbranched tubules in tridimensional collagen gels. These data show that RON has the potential to elicit a motile-invasive rather than a transformed phenotype.

We determined the nucleotide sequence of the rearranged trp-met genomic locus and the corresponding portions of the unrearranged tpr and met genomic fragments. The breakpoints occur at one end of a stretch of 21 A residues that follow an Alu repetitive sequence in the tpr locus and within a group of 3 A residues in the met proto-oncogene locus. We conclude that the fusion between the tpr locus on chromosome 1 and the met locus on chromosome 7 resulted from a recombination event.

We determined the nucleotide sequence of the rearranged trp-met genomic locus and the corresponding portions of the unrearranged tpr and met genomic fragments. The breakpoints occur at one end of a stretch of 21 A residues that follow an Alu repetitive sequence in the tpr locus and within a group of 3 A residues in the met proto-oncogene locus. We conclude that the fusion between the tpr locus on chromosome 1 and the met locus on chromosome 7 resulted from a recombination event.

tpr-met, a tyrosine kinase oncogene, is the activated form of the met proto-oncogene that encodes the receptor for hepatocyte growth factor/scatter factor. The tpr-met product (p65tpr-met) was tested for its ability to induce meiotic maturation in Xenopus oocytes. While src and abl tyrosine kinase oncogene products have previously been shown to be inactive in this assay, p65tpr-met efficiently induced maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD) together with the associated increase in ribosomal S6 subunit phosphorylation. tpr-met-mediated MPF activation and GVBD was dependent on the endogenous c-mosxe, while the increase in S6 protein phosphorylation was not significantly affected by the loss of mos function. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine inhibits tpr-met-mediated GVBD at concentrations that prevent insulin- but not progesterone-induced oocyte maturation. Moreover, maturation triggered by tpr-met is also inhibited by cyclic AMP-dependent protein kinase. This is the first demonstration that a tyrosine kinase oncogene product, p65tpr-met, can induce meiotic maturation in Xenopus oocytes and activate MPF through a mos-dependent pathway, possibly the insulin or insulinlike growth factor 1 pathway.

tpr-met, a tyrosine kinase oncogene, is the activated form of the met proto-oncogene that encodes the receptor for hepatocyte growth factor/scatter factor. The tpr-met product (p65tpr-met) was tested for its ability to induce meiotic maturation in Xenopus oocytes. While src and abl tyrosine kinase oncogene products have previously been shown to be inactive in this assay, p65tpr-met efficiently induced maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD) together with the associated increase in ribosomal S6 subunit phosphorylation. tpr-met-mediated MPF activation and GVBD was dependent on the endogenous c-mosxe, while the increase in S6 protein phosphorylation was not significantly affected by the loss of mos function. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine inhibits tpr-met-mediated GVBD at concentrations that prevent insulin- but not progesterone-induced oocyte maturation. Moreover, maturation triggered by tpr-met is also inhibited by cyclic AMP-dependent protein kinase. This is the first demonstration that a tyrosine kinase oncogene product, p65tpr-met, can induce meiotic maturation in Xenopus oocytes and activate MPF through a mos-dependent pathway, possibly the insulin or insulinlike growth factor 1 pathway.

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.

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.

ABSTRACT A Met-hepatocyte growth factor receptor oncoprotein, Tpr-Met, generated by chromosomal rearrangement, fuses a protein dimerization motif with the cytoplasmic domain of the Met receptor, producing a cytosolic, constitutively activated tyrosine kinase. Although both the Met receptor and the Tpr-Met oncoprotein associate with the same substrates, activating mutations of the Met receptor in hereditary papillary renal carcinomas have different signaling requirements for transformation than Tpr-Met. This suggests differential activation of membrane-localized pathways by oncogenic forms of the membrane-bound Met receptor but not by the cytoplasmic Tpr-Met oncoprotein. To establish which pathways might be differentially regulated, we have localized the constitutively activated Tpr-Met oncoprotein to the membrane using the c-src myristoylation signal. Membrane localization enhances cellular transformation, focus formation, and anchorage-independent growth and induces tumors with a distinct myxoid phenotype. This correlates with the induction of hyaluronic acid (HA) and the presence of a distinct form of its receptor, CD44. A pharmacological inhibitor of phosphoinositide 3′ kinase (PI3′K), inhibits the production of HA, and conversely, an activated, plasma membrane-targeted form of PI3′K is sufficient to enhance HA production. Furthermore, the multisubstrate adapter protein Gab-1, which couples the Met receptor with PI3′K, enhances Met receptor-dependent HA synthesis in a PI3′K-dependent manner. These results provide a positive link to a role for HA and CD44 in Met receptor-mediated oncogenesis and implicate PI3′K in these events.

The Met protooncogene encodes a receptor tyrosine kinase that is deregulated by point mutation, and overexpression/amplification in a number of human tumours. The Met receptor is also oncogenically activated following genomic rearrangement which generates a cytoplasmic, constitutively activated fusion protein, Tpr-Met. In addition to autophosphorylation sites within the catalytic domain, the carboxy terminus of Tpr-Met/Met contains a single major site of autophosphorylation, tyrosine 489. This tyrosine residue represents a unique multisubstrate binding site, capable of binding numerous intracellular proteins, and is critical for the biological activities of both the Met receptor and Tpr-Met oncoprotein. Addition of the c-src myristoylation sequence to the amino terminus of the normally cytoplasmic Tpr-Met, localizes Tpr-Met to plasma membranes and enhances cellular transformation, in vitro invasion, and tumourigenicity. Furthermore, a membrane targetted Tpr-Met is localized to a similar subcellular compartment as the Met receptor, and alters the complement of signalling proteins required for efficient transformation. In this respect, a membrane localized Tpr-Met resembles oncogenic forms of the transmembrane Met receptor, and provides a model with which to study transformation by Met receptor oncoproteins. Significantly, membrane localization of Tpr-Met induces a phosphoinositide 3' kinase (PI3' K) dependent autocrine loop, involving the production of hyaluronic acid (HA), and post-translational modification of the cell surface receptor for HA, CD44. PI3'K activity and the HA/CD44 autocrine loop, are dependent on the multisubstrate binding site, tyrosine 489, and tyrosine residue 498, a residue with no previously described biochemical function. Although the exact mechanisms by which PI3'K regulates HA production are unclear, the induction of a HA/CD44 autocrine loop may represent a novel mechanism by which deregulated receptor tyrosine kinases increase their onco

Activation of the Met receptor tyrosine kinase by its ligand, Hepatocyte Growth Factor (HGF), leads to mitogenesis, cell motility, morphogenesis, and angiogenesis. Mutational analysis has demonstrated the requirement of a single tyrosine within the carboxy-terminus (Y1356) of the Met receptor for the recruitment and activation of all Met-dependent signalling pathways and for the transformation of fibroblasts by the Tpr-Met oncogene. The selective abolishment of Grb2 from the Tpr-Met oncoprotein, by generating an asparagine to histidine mutation two amino acids downstream from Y1356 (N1358H), led to a reduction in Tpr-Met-mediated transformation of fibroblasts. Moreover, Met receptor studies demonstrate that while a Grb2 binding site is not required for epithelial cell motility, it is critical for the formation of branching tubules when cells are suspended in a collagen matrix. This suggests that Grb2-dependent pathways are involved in the organization and polarization of epithelial cells following Met receptor stimulation.
Grb2 associated molecules, Gab1 and Cbl, are highly phosphorylated following stimulation of the Met receptor. Moreover, signaling pathways associated with Gab1 are critical for branching tubulogenesis in epithelial cells. Expression of a constitutively active version of Cbl, 70z-Cbl, results in an epithelial-mesenchymal transition, leading to the breakdown of cellular junctions and reorganization of the actin cytoskeleton. The amino-terminal SH2 domain is the minimal region required to induce morphological changes, which may be mediated through its interaction with the Met receptor, and/or an unidentified protein of 150 kDa.

Certaines protéines comme l'onconase ou le facteur TAT du virus du SIDA ont l'étonnante capacité de pouvoir pénétrer à l'intérieur des cellules malgré leur forte taille. Ces protéines, peuvent être internalisées spontanément par les cellules, car elles possèdent dans leur séquence, un domaine particulier appelé domaine de transduction. Dans la première partie de cette thèse, nous avons utilisé le domaine de transduction de TAT (VIH) pour vectoriser Tpr-Met, une protéine kinase qui agit sur la prolifération, la survie, la migration des cellules. Nous avons construit, exprimé et purifié une protéine chimérique TAT-Tpr-Met et avons mis au point sa renaturation in vitro. TAT-Tpr-Met pénètre dans plusieurs types cellulaires en culture et augmente la prolifération, la survie et la mobilité des cellules. Nous avons ensuite montré que le prétraitement de progéniteurs hépatiques avec cette molécule améliorait leur prise de greffe, après transplantation dans le foie de souris. Ce travail montre qu'il est possible d'utiliser un domaine de transduction pour modifier transitoirement les capacités d'intégration des cellules, sans altérer le génome. Dans la deuxième partie de cette thèse, nous avons construit une protéine fusion entre le domaine de transduction de TAT et la sous-unité catalytique de la télomèrase (hTERT). Au cours de ce travail, nous avons identifié un domaine de transduction naturel dans hTERT. Nous avons fait synthétisé ce domaine sous forme d'un peptide lié à FITC, et avons pu montrer qu'il avait la capacité d'être internalisé par les cellules humaines in vitro. Nous avons produit la sous unité catalytique de la télomèrase à partir de cellules d'insectes infectées par des baculovirus recombinants, et avons également mis au point un protocole pour purifier plusieurs milligrammes de cette protéine, ainsi que son variant dominant négatif, en l'exprimant dans la levure. Les études menées avec la protéine recombinante ont démontré que la protéine hTERT avait une capacité d'internalisation naturelle. Cette propriété suggère que la protéine hTERT, et son variant dominant négatif pourraient respectivement être utilisés comme activateur et inhibiteur de la télomèrase
Some proteins such as onconase or TAT from the HIV have an amazing ability to penetrate inside the cells, despite their heavy size. These proteins can be internalized by the cells spontaneously, as they have in their sequence, a field called transduction domain. In the first part of this thesis, we used the domain of transduction from TAT (HIV) to vectorize TPR-Met, a protein kinase that acts on the proliferation, survival, cell migration. We built, expressed and purified chimeric protein TAT-TPR-Met and have developed its renaturation in vitro. TAT-TPR-Met enter s several cell types in culture and increases the proliferation, survival and mobility of cells. We then showed that pretreatment with liver progenitors of this molecule improved their presence into the liver after transplantation in the mouse. This work shows that it is possible to use a domain of transduction to change temporarily the integration capacity of cells, without altering the genome. In the second part of this thesis, we built a protein fusion between the protein transduction domain of TAT and the catalytic subunit of telomerase (hTERT) gene. During this work, we have identified a natural transduction domain in hTERT. We have made this area synthesized form of a peptide linked to FITC, and were able to show he had the ability to be internalized by human cells in vitro. We produced the catalytic subunit of telomerase from insect cells infected with recombinant baculovirus, and have also developed a protocol to purify several milligrams of this protein and its variant dominant negative, expressing in yeast. Studies with the recombinant protein showed that the protein hTERT had a natural ability to internalize. This property suggests that the protein hTERT and its dominant negative variant respectively could be used as activator and inhibitor of telomerase

碩士
國立中興大學
生物醫學研究所
93
The c-met proto-oncogene encodes the hepatocyte growth factor receptor. Tpr-met is a hybrid gene resulting from a chromosomal rearrangement between chromosome 1 and chromosome 7 with its upstream region derived from the TPR locus (Translocated Promoter Region) fused to downstream sequences encoding the Met kinase. Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase, participates in the control of cell migration, cell cycle progression, and cell survival. In this study, I found that Tpr-Met interacted with FAK both in vivo and in vitro. Mutations at the Tyr-482 or Tyr-489 of Tpr-Met abolished this interaction. Removal of the phosphorylation of Tpr-Met by alkaline phosphatase in vitro decreased its interaction with FAK. Moreover, a synthetic phosphopeptide corresponding to the sequence surrounding the Tyr-482 and Tyr-489 was capable of blocking the interaction. Finally, the NH2-terminal domain of FAK was found to be sufficient for binding to Tpr-Met. Taken together, I found a novel interaction between Tpr-Met and FAK, which is mediated by the phosphorylated Tyr-482 and Tyr-489 of Tpr-Met and the NH2-terminal domain of FAK.

碩士
國立中興大學
生命科學系
92
Abstract Keywords：FAK, Tpr-Met, oncogene, phosphorylation, tumor, transformation Focal adhesion kinase (FAK), a 125 kDa cytoplasmic protein tyrosine kinase localized in focal contacts, plays a crucial role in the control of integrin- mediated cellular functions. We have previously demonstrated that increased expression of FAK renders epithelial cells susceptible to transformation by hepatocyte growth factor (HGF) stimulation (Chan et al., 2002, J. Biol. Chem. 277, 50373-50379). The HGF receptor is encoded by the proto-oncogene c-met, which can be uncongenially activated through a chromosomal rearrangement that creates a hybrid gene tpr-met. Tpr-Met, a 65 kDa protein tyrosine kinase which is constitutively active and phosphorylated on tyrosine residues. In this study, I attempted to examine the potential interaction between FAK and Tpr-Met. My results showed that Tpr-Met stimulates the tyrosine phosphorylation of FAK in intact cells and is capable of directly phosphorylating recombinant FAK in vitro. Moreover, I demonstrated that Tpr-Met is associated with FAK both in vivo and in vitro. Mutations at the Tyr-482 and Tyr-489 of Tpr-Met impair the ability of Tpr-Met to bind and phosphorylate FAK. The NH2- and COOH-terminal domains of FAK are sufficient for Tpr-Met binding in vivo and in vitro. My results showed that the expression of FAK enhances Tpr-Met induced anchorage-independent cell growth and cell invasiveness. The ability of these cells to invade Matrigel correlated with activation and increased expression of matrix metalloprotease-2. Biochemical analysis revealed that FAK is important for Tpr-Met-induced activation of ERK, JNK, c-Jun, and AKT, but not STAT-3. Together, FAK may serve as a platform for Tpr-Met to amplify the signals to the downstream and play an important role in Tpr-Met-induced cell transformation.