The DNA Rearrangement That Generates the TRK-T3 Oncogene Involves a Novel Gene on Chromosome 3 Whose Product Has a Potential Coiled-Coil Domain

Abstract

Oncogenic rearrangements of the NTRK1 gene (also designated TRKA), encoding one of the receptors for the nerve growth factor, are frequently detected in thyroid carcinomas. Such rearrangements fuse the NTRK1 tyrosine kinase domain to 5-end sequences belonging to different genes. In previously reported studies we have demonstrated that NTRK1 oncogenic activation involves two genes, TPM3 and TPR, both localized similarly to the receptor tyrosine kinase, on the q arm of chromosome 1. Here we report the characterization of a novel NTRK1-derived thyroid oncogene, named TRK-T3. A cDNA clone, capable of transforming activity, was isolated from a transformant cell line. Sequence analysis revealed that TRK-T3 contains 1,412 nucleotides of NTRK1 preceded by 598 nucleotides belonging to a novel gene that we have named TFG (TRK-fused gene). The TRK-T3 amino acid sequence displays, within the TFG region, a coiled-coil motif that could endow the oncoprotein with the capability to form complexes. The TRK-T3 oncogene encodes a 68-kDa cytoplasmic protein reacting with NTRK1-specific antibodies. By sedimentation gradient experiments the TRK-T3 oncoprotein was shown to form, in vivo, multimeric complexes, most likely trimers or tetramers. The TFG gene is ubiquitously expressed and is located on chromosome 3. The breakpoint producing the TRK-T3 oncogene occurs within exons of both the TFG gene and the NTRK1 gene and produces a chimeric exon that undergoes alternative splicing. Molecular analysis of the NTRK1 rearranged fragments indicated that the chromosomal rearrangement is reciprocal and balanced and involves loss of a few nucleotides of germ line sequences.Chromosomal rearrangements producing chimeric oncogenes are frequently associated with human cancer, and several lines of evidence suggest that they are involved in the pathogenesis of their respective tumors (31).Among solid tumors, papillary thyroid carcinoma provides a unique model of a frequent generation of chimeric oncogenes. In this type of neoplasia, in fact, two proto-oncogenes, RET and NTRK1 (also named TRKA), have been found rearranged in about 50% of the samples assayed (5). RET and NTRK1 both encode transmembrane receptor tyrosine kinases (RTKs), whose expression is highly tissue specific, being restricted to specific components of the peripheral nervous system (1, 38, 40). The common mechanism of activation is represented by somatic rearrangements juxtaposing their tyrosine kinase (TK) domain to 5Ј-end sequences derived from unrelated loci and producing chimeric oncogenes whose products display a constitutive and ectopic TK enzymatic activity (29).The NTRK1 gene encodes one of the receptors for the nerve growth factor (NGF) (15, 16) and is located on the q arm of chromosome 1 (24, 26). NTRK1 was originally detected as an oncogene in a human colon carcinoma; this activated version of the gene was generated by a chromosomal rearrangement fusing the NTRK1 TK domain with sequences of a tropomyosin gene, TPM3, and was designated the ''TRK oncogene'' (21).Afterwards, ...

Detection of novel mRNA splice variants of human ING4 tumor suppressor gene

“…Immunofluorescence experiments with anti-myc 9E10 antibodies (Figure 6b, top) showed that all the variant ING4 proteins are localized in the nucleus, similarly to the wild-type. As control, the lack of reactivity of mock-transfected cells, and the cytoplasmic localization of TFG-myc protein (Greco et al, 1995) are shown. Moreover, Western blot analysis of nuclear and cytoplasmic extracts from ING4-myc-transfected HEK293T cells showed that all the ING4 variants retain the capability to enter the nucleus (Figure 6b, bottom).…”

Role of the TFG N-terminus and coiled-coil domain in the transforming activity of the thyroid TRK-T3 oncogene

“…TRK-T3 encodes a 68 kDa cytoplasmic protein displaying a constitutive tyrosine phosphorylation and capable to transform NIH3T3 mouse ®broblasts (Greco et al, 1995).…”

Biological activity of the thyroid TRK-T3 oncogene requires signalling through Shc

“…The T3/WT plasmid contains the TRK-T3 cDNA inserted into the pRC/CMV expression vector (Greco et al, 1995), which carries the neomycin resistance gene. The T3/Y291F mutant carries the mutation of Tyr291 to phenylalanine (F291) and was constructed by means of site-directed mutagenesis using an in vitro oligonucleotide mutagenesis system (Altered Sites in vitro Mutagenesis System, Promega).…”

“…In the TRK-T3 oncogene, produced by a t(1;3) translocation, the NTRK1 TK domain is juxtaposed to sequences of the TFG gene (Greco et al, 1995), which encodes a protein of unknown function containing a coiled-coil domain, putative phosphorylation sites for PKC and CK2 and glycosylation sites (Mencinger et al, 1997;Mencinger and Aman, 1999). The TFG coiled-coil domain plays an important role in TRK-T3 activation and its consequent biological activity insofar as it mediates the dimerization leading to the ligand-independent tyrosine phosphorylation of the TK domain.…”