Loss of function effect of RET mutations causing Hirschsprung disease.

We have introduced three Hirschsprung (HSCR) mutations localized in the tyrosine kinase domain of RET into the RET/PTC2 chimaeric oncogene which is capable of transforming NIH3T3 mouse fibroblasts and of differentiating pC12 rat pheochromocytoma cells. The three HSCR mutations abolished the biological activity of RET/PTC2 in both cell types and significantly decreased its tyrosine phosphorylation. By contrast, a rare polymorphism in exon 18 does not alter the transforming capability of RET/PTC2 or its tyrosine phosphorylation. These data suggest a loss of function effect of HSCR mutations which might act through a dominant negative mechanism. Our model system is therefore capable of discriminating between causative HSCR mutations and rare polymorphisms in the tyrosine kinase domain of RET.

The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma.

RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins.

Bmx tyrosine kinase has a redundant function downstream of angiopoietin and vascular endothelial growth factor receptors in arterial endothelium.

The Bmx gene, a member of the Tec tyrosine kinase gene family, is known to be expressed in subsets of hematopoietic and endothelial cells. In this study, mice were generated in which the first coding exon of the Bmx gene was replaced with the lacZ reporter gene by a knock-in strategy. The homozygous mice lacking Bmx activity were fertile and had a normal life span without an obvious phenotype. Staining of their tissues using beta-galactosidase substrate to assess the sites of Bmx expression revealed strong signals in the endothelial cells of large arteries and in the endocardium starting between days 10.5 and 12.5 of embryogenesis and continuing in adult mice, while the venular endothelium showed a weak signal only in the superior and inferior venae cavae. Of the five known endothelial receptor tyrosine kinases tested, activated Tie-2 induced tyrosyl phosphorylation of the Bmx protein and both Tie-2 and vascular endothelial growth factor receptor 1 (VEGFR-1) stimulated Bmx tyrosine kinase activity. Thus, the Bmx tyrosine kinase has a redundant role in arterial endothelial signal transduction downstream of the Tie-2 and VEGFR-1 growth factor receptors.

Molecular characterization of a thyroid tumor-specific transforming sequence formed by the fusion of ret tyrosine kinase and the regulatory subunit RI alpha of cyclic AMP-dependent protein kinase A.

The ret oncogene frequently has been found activated in papillary thyroid carcinomas. A previous characterization of ret activation revealed recombination of its tyrosine kinase domain and sequences derived from an uncharacterized locus (D10S170). The mechanism leading to this recombination was identified as a paracentric inversion of the long arm of chromosome 10, inv(10)(q11.2q21), with the breakpoints occurring where ret and D10S170 were mapped. To further characterize the activation of ret in papillary thyroid carcinomas, we have now isolated and sequenced a second type of ret oncogenic rearrangement not involving the D10S170 locus. The nucleotide sequence indicated that the transforming activity was created by the fusion of the ret tyrosine kinase domain with part of the RI alpha regulatory subunit of protein kinase A (PKA). This is the first example of an oncogenic activity involving a PKA gene. PKA is the main intracellular cyclic AMP receptor, and its RI alpha subunit gene is located on chromosome 17q. RI alpha-ret transcripts encode two isoforms of the chimeric protein (p76 and p81), which display constitutive tyrosine phosphorylation as well as a tyrosine kinase enzymatic activity. Under nonreducing conditions, both isoforms are found in a dimeric configuration because of both homo- and heterodimer formation. Thus, the in vivo activation of ret in human papillary thyroid carcinomas is provided by the fusion of its tyrosine kinase domain with different genes and can be mediated by different mechanisms of gene rearrangement.

The Bmx tyrosine kinase is activated by IL-3 and G-CSF in a PI-3K dependent manner.

Cytoplasmic protein tyrosine kinases play crucial roles in signaling via a variety of cell surface receptors. The Bmx tyrosine kinase, a member of the Tec family, is expressed in hematopoietic cells of the granulocytic and monocytic lineages. Here we show that Bmx is catalytically activated by interleukin-3 (IL-3) and granulocyte-colony stimulating factor (G-CSF) receptors. Activation of Bmx required phosphatidylinositol 3-kinase (PI-3K) as demonstrated by the ability of PI-3K inhibitors to block the activation signal. A green fluorescent protein (GFP) tagged Bmx was translocated to cellular membranes upon co-expression of a constitutively active form of PI-3K, further indicating a role for PI-3K in signaling upstream of Bmx. The expression of wild type Bmx in 32D myeloid progenitor cells resulted in apoptosis in the presence of G-CSF, while cells expressing a kinase dead mutant of Bmx differentiated into mature granulocytes. However, Bmx did not modulate IL-3-dependent proliferation of the cells. These results demonstrate distinct effects of Bmx in cytokine induced proliferation and differentiation of myeloid cells, and suggest that the stage specific expression of Bmx is critical for the differentiation of myeloid cells. Oncogene (2000) 19, 4151 - 4158

RET activation by germline MEN2A and MEN2B mutations.

The RET proto-oncogene encodes a receptor tyrosine kinase (TK). It has been shown that distinct germline mutations in the RET proto-oncogene are associated with the dominantly inherited cancer syndromes multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC) as well as Hirschsprung disease (HSCR), a congenital disorder characterised by absent enteric innervation. In this study, we have transfected NIH3T3 and PC12 phaeochromocytoma cells with MEN2A (Cys634-> Arg) and MEN2B (Met918-> Thr) RET constructs. Both caused transformation of the NIH3T3 cells and differentiation of PC12 cells. The Ret (MEN2A) and Ret (MEN2B) proteins were constitutively phosphorylated on tyrosine, and their in vitro kinase activity was significantly higher than that of the wild type protein. The MTC cell line TT carries a CYs634-> Trp MEN2A mutation, and we have shown by immunoelectronmicroscopy that Ret is clustered on the cell surface in a manner reminiscent of ligand-induced aggregation of cell surface receptors. RET is activated, as RET/PTC oncogene, by somatic rearrangements which link the TK domain to a constitutive dimerization interface in papillary thyroid carcinomas. We have compared the biological and biochemical activity of the TK domains of the wild type and MEN 2B Ret in the context of the RET/PTC. The results show that the MEN 2B mutation significantly increases the TK domain enzymatic activity suggesting that dimerization may be still necessary for MEN 2B Ret to express its full activity.

The oncogenic versions of the Ret and Trk tyrosine kinases bind Shc and Grb2 adaptor proteins.

Proto-TRK and proto-RET genes encode receptor type tyrosine kinases. Oncogenic rearrangements of both proto-oncogenes have been detected with a significant frequency in human papillary thyroid carcinomas. Chimeric Ret and Trk oncoproteins, encoded by different rearrangements of proto-TRK and proto-RET genes, display a constitutive phosphorylation on tyrosine. Moreover, it has been shown that phosphorylated tyrosine receptors, activated by their ligands, form multiprotein complexes responsible for transducing mitogenic or differentiation signals. We have therefore begun to analyse in this study the signal transduction pathways triggered by different Ret and Trk oncoproteins. We have shown that the SH2 domain of the adaptor protein Shc coimmunoprecipitates with all the Ret and Trk oncoproteins as well as with NGF-activated proto-Trk receptor. Tyrosine phosphorylation of Trk proteins both normal and oncogenic is necessary for their binding to Shc. In addition, in cells containing either Ret or Trk oncoproteins, Shc proteins are constitutively phosphorylated on tyrosine and bound to Grb2. Only in in vitro experiments were Ret and Trk oncoproteins shown to bind the SH2 region of Grb2. Finally, when proto-Trk product is stimulated by NGF, Shc phosphorylation and association with Grb2 are induced. In conclusion, we have shown that Ret and Trk oncoproteins can form multiprotein complexes, however, the functional meaning of the described interactions has to be elucidated.

Key role of Shc signaling in the transforming pathway triggered by Ret/ptc2 oncoprotein.

The RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of protoRET, a gene encoding two protein isoforms of a transmembrane tyrosine kinase receptor. By using Ret/ptc2 short isoform (iso9), we have previously demonstrated that Tyr586 (Tyr1062 of protoRet) is the docking site for both the PTB and the SH2 domains of Shc. To determine the relevance of this interaction for the transforming activity of Ret/ptc oncogenes, we have generated and characterized novel Ret/ptc mutants unable to activate Shc: Ret/ptc2 long isoform (iso51)-Y586F and both isoforms of Ret/ptc2-N583A. These mutants neither activate Shc nor transform NIH3T3 cells. Since Tyr1062 shows features of a multifunctional docking site, we have used a Shc mutant (Shc Y317F) to directly assess Shc role. We have demonstrated that in our cell system Shc Y317F behaves like a dominant interfering mutant on the activation of the Grb2-Sos pathway by endogenous Shc triggered by Ret/ptc2. A strong reduction of the transforming activity of Ret/ptc2 in presence of this mutant was also demonstrated. Our data suggest that Shc activation play a key role in the transforming pathways triggered by Ret/ptc oncoproteins. Moreover, we have shown that coexpression of the Shc-Y317F mutant with Ret/ptc2 specifically causes apoptosis, and that the surviving cells lose the long-term expression of one of the two genes.

Identification of Shc docking site on Ret tyrosine kinase.

The RET proto-oncogene encodes two isoforms of a receptor type tyrosine kinase which plays a role in neural crest and kidney development. Distinct germ-line mutations of RET have been associated with the inherited cancer syndromes MEN2A, MEN2B and FMTC as well as with the congenital disorder Hirschsprung disease (HSCR), whereas somatic rearrangements (RET/PTCs) have been frequently detected in the papillary thyroid carcinoma. Despite these findings, suggesting a relevant role for RET product in development and neoplastic processes, little is known about the signalling triggered by this receptor. In this study, we have demonstrated that the transducing adaptor molecule Shc is recruited and activated by both Ret isoforms and by the rearranged cytoplasmatic Ret/ptc2 oncoproteins as well as by the membrane bound receptor activated by MEN2A or by MEN2B associated mutations. Moreover, our analysis has identified the Ret tyrosine residue and the Shc domains involved in the interaction. In fact, here we show that both the phosphotyrosine binding domains of Shc, PTB and SH2, interact with Ret/ptc2 in vitro. However, PTB domain binds 20 folds higher amount of Ret/ptc2 than SH2. The putative binding site for either SH2 and PTB domains has been identified as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this finding, by using RET/PTC2-Y586F mutant, we have demonstrated that this tyrosine residue, the last amino acid but one before the divergence of the two Ret isoforms, is the docking site for Shc.

A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities.

The vascular endothelial growth factor (VEGF) and the VEGF-C promote growth of blood vessels and lymphatic vessels, respectively. VEGF activates the endothelial VEGF receptors (VEGFR) 1 and 2, and VEGF-C activates VEGFR-3 and VEGFR-2. Both VEGF and VEGF-C are also potent vascular permeability factors. Here we have analyzed the receptor binding and activating properties of several cysteine mutants of VEGF-C including those (Cys156 and Cys165), which in other platelet-derived growth factor/VEGF family members mediate interchain disulfide bonding. Surprisingly, we found that the recombinant mature VEGF-C in which Cys156 was replaced by a Ser residue is a selective agonist of VEGFR-3. This mutant, designated DeltaNDeltaC156S, binds and activates VEGFR-3 but neither binds VEGFR-2 nor activates its autophosphorylation or downstream signaling to the ERK/MAPK pathway. Unlike VEGF-C, DeltaNDeltaC156S neither induces vascular permeability in vivo nor stimulates migration of bovine capillary endothelial cells in culture. These data point out the critical role of VEGFR-2-mediated signal transduction for the vascular permeability activity of VEGF-C and strongly suggest that the redundant biological effects of VEGF and VEGF-C depend on binding and activation of VEGFR-2. The DeltaNDeltaC156S mutant may provide a valuable tool for the analysis of VEGF-C effects mediated selectively via VEGFR-3. The ability of DeltaNDeltaC156S to form homodimers also emphasizes differences in the structural requirements for VEGF and VEGF-C dimerization.

Induction of TNF-sensitive cellular phenotype by c-Myc involves p53 and impaired NF-kappaB activation.

Normal fibroblasts are resistant to the cytotoxic action of tumor necrosis factor (TNF), but are rendered TNF-sensitive upon deregulation of c-Myc. To assess if oncoproteins induce the cytotoxic TNF activity by modulating TNF signaling, we investigated the TNF-elicited signaling responses in fibroblasts containing a conditionally active c-Myc protein. In association with cell death, c-Myc impaired TNF-induced activation of phospholipase A2, JNK protein kinase and cell survival-signaling-associated NF-kappaB transcription factor complex. The TNF-induced death of mouse primary fibroblasts expressing deregulated c-Myc was inhibited by transient overexpression of the p65 subunit of NF-kappaB, which increased NF-kappaB activity in the cells. Unlike other TNF-induced signals, TNF-induced accumulation of the wild-type p53 mRNA and protein was not inhibited by c-Myc. TNF, with c-Myc, induced apoptosis in mouse primary fibroblasts but only weakly in p53-deficient primary fibroblasts. The C-terminal domain of p53, which is a transacting dominant inhibitor of wild-type p53, failed to inhibit apoptosis by c-Myc and TNF, suggesting that the cell death was not dependent on the transcription-activating function of p53. Taken together, the present findings show that the cytotoxic activity of TNF towards oncoprotein-expressing cells involves p53 and an impaired signaling for survival in such cells.