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

Abstract

The thyroid TRK-T3 oncogene results from the fusion of the tyrosine kinase (TK) domain of NTRK1 (one of the receptors for the Nerve Growth Factor) on chromosome 1 to sequences of a novel gene, TFG, on chromosome 3. The 68 kDa TRK-T3 fusion oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform mouse NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain most likely responsible for the constitutive, ligand-independent activation of the receptor tyrosine kinase activity. We have previously shown that TRK-T3 oncoprotein forms, in vivo, complexes of three or four molecules. By mean of di erent experimental approaches, we show here that TRK-T3 activity depends on oligomers formation. In addition, the analysis of di erent TRK-T3 mutants indicates that the TFG coiled-coil domain and its Nterminal region are both required for the activation and the fully transforming activity of the TRK-T3 oncoprotein, although, most likely, they play a role in di erent steps of the transforming process. The deletion of the coiled-coil domain abrogates the oligomers formation leading to a constitutive activation; the deletion of the Nterminal region, although not a ecting phosphorylation and complexes formation, abrogates transformation, thus suggesting a role in cellular localization and/or interaction with substrata.

Pathobiology of NPM-ALK and variant fusion genes in anaplastic large cell lymphoma and other lymphomas

“…The fusion of TFG with NTRK1 Leukemia forms the oncogene TRK-T3 that has transforming activity in various models. [196][197][198] The ALK breakpoint in these translocations was the same as in the classical t(2;5). Wild-type TFG is constitutively expressed in many different tissues.…”

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

“…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). The T3/ABN kinase-defective mutant carries the mutation of the ATP binding site Lys339 to Ala (Greco et al, 1998). In ShcY317F mutant the Tyr317 is mutated to Phe.…”

“…Role of Shc in TRK-T3 signalling E Roccato et al of TRK-T3, as it was undetectable when the T3/ABN kinase-dead mutant (Greco et al, 1998) was used ( Figure 3A). We next investigated the ability of ShcY317F to recruit Grb2 upon TRK-T3 activation.…”

“…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. Moreover, also the regions outside the coiled-coil domain are required for TRK-T3 transforming activity (Greco et al, 1998) in ways that are currently under investigation.…”

An in vivo functional genetic screen reveals a role for the TRK-T3 oncogene in tumor progression

“…The 68 kDa TRK-T3 fusion oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform mouse NIH 3T3 cells (Greco et al, 1995(Greco et al, , 1998. We describe here an unexpected and new role for TRK-T3 in tumor progression.…”