Role of TFG sequences outside the coiled-coil domain in TRK-T3 oncogenic activation

Abstract

The TRK-T3 oncoprotein, isolated from a human papillary thyroid tumor, arises from the fusion between the N-terminal domain of the TFG gene and the tyrosine kinase domain of the NTRK1 receptor. The 68 kDa TRK-T3 oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain, which mediates protein oligomerization essential for the oncogene constitutive activation, and several consensus sites for protein interaction. In this study, we investigate the role of TFG sequences outside the coiledcoil domain on TRK-T3 activation, We constructed four mutants carrying different deletions of TFG sequences and expressed them in mammalian cells. By performing biochemical and biological assays we demonstrated that all the deleted regions are required for TRK-T3 activation, as they are involved in different mechanisms such as protein processing, formation of stable and/or functional complexes, and possible interaction with other proteins. By constructing site-specific mutants, we demonstrated a crucial role for a PB1 domain and a considerable contribution of an SH2-binding motif in TRK-T3 oncogenic activation. This work establishes an important role for TFG sequences outside the coiled-coil domain in the activation of the thyroid TRK-T3 oncogene.

Analysis of SHP-1-mediated Down-regulation of the TRK-T3 Oncoprotein Identifies Trk-fused Gene (TFG) as a Novel SHP-1-interacting Protein

“…Altogether these data demonstrate that the TRK-T3 oncoprotein displays a dual interaction with the SHP-1 phosphatase; the NTRK1 portion binds the SHP-1 catalytic domain whereas the TFG portion interacts with SHP-1 SH2 domains. The latter interaction was further confirmed by GST pull-down experiments performed with the T3/L2 mutant, in which all the TFG sequences except the coiled-coil domain had been deleted (18). As shown in Fig.…”

“…The TFG sequences outside the coiled-coil domain play an important role in TRK-T3 oncogenic activation. Their deletion interferes with different mechanisms involving protein processing, formation of stable and/or functional complexes, and possible interactions with other proteins (18). In particular, the PB1 domain appears to have a crucial role since a single point mutation at a conserved Lys residue completely abrogates TRK-T3 activity, thus demonstrating the significance of this region in oncogenic activation.…”

“…All the mutants were inserted into the pRC/CMV expression vector. The T3/L2 mutant, inserted into the pIRESneo expression vector, is deleted for all the TFG sequences except the coiled-coil domain (18). The T3/ABN kinase-defective mutant, inserted into the pRC/CMV expression vector, carries the Lys 339 to Ala mutation of the ATP-binding site (17).…”

“…In addition to the coiled-coil domain, the TFG protein also contains several consensus sites, which suggest possible interactions with other proteins. These include a PB1 domain (18), putative phosphorylation sites for PKC and CK2, glycosylation sites, as well as SH2-and SH3-binding sites (19). Several of these sites are identical in TFG proteins from different species, indicating that the protein might be involved in basic cellular processes (16).…”

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

“…Further mapping of the TFG sequence of TRK-T3 revealed binding motifs for PKC, CK2 and SH3 (Mencinger et al, 1997), which could activate cell survival pathways. It has been demonstrated that TRK-T3 can activate various signal transduction pathways in transformed cells including PLC, JNK and ERK1/2 MAP kinases (Roccato et al, 2003). Activation of such signaling pathways may well explain why expression of TRK-T3 allows tumor cells to proliferate in growth factor-deprived conditions.…”