NPC associating domain

NPC associating domain

Figure 1. Schematic representation of NTRK1, TRKs and TPR proteins. In the oncoproteins the portions contributed by NTRK1 and activating sequences are indicated. The gray portions represent coiled-coil domains. On TPR the break sites of Met, TRK-T1 and TRK-T2 oncoproteins are indicated by arrows. TM: transmembrane domain, TK: tyrosine kinase domain, NPC: nuclear pore complex.

(Greco, A. et al., 1997; Greco, A. et al., 1992). TRK-T3 is activated by TFG, a novel gene on chromosome 3q11-12 (Greco, A. et al, 1995). All TRK oncogenes but TRK-T1 retain the NTRK1 transmembrane domain. TRK oncogenes display constitutive, ligand-independent tyrosine kinase activity.

In experimental models TRK oncogenes recapitulate the biological effects of the NTRK1 receptor upon NGF stimulation. In fact, they induce morphological transformation of NIH3T3 mouse fibroblasts, and neuronal-like differentiation of PC12 cells (Greco, A. et al., 1993b). The mechanisms by which TRK oncogenes mediate their effects have been in part elucidated (Figure 2). TRK oncoproteins interact to and activate PLCy She, FRS2, FRS3, IRS1 and IRS2. All these molecules, except PLCy, are recruited by the same tyrosine residue, corresponding to Tyr490 of NTRK1, most likely in a competitive fashion. Such interaction site is crucial for oncogenic activity, in so far its mutation to phenylalanine completely abrogate TRK oncogene biological activity. Moreover, by using a Shc dominant-negative mutant unable to recruit GRB2, we showed a crucial role of Shc adaptor in TRK-T3 biological activity. Conversely, mutation of the interaction site did not affect the oncogenic activity. It is worth noting that our studies on TRK oncogenes allowed the identification of novel proteins interacting with NTRK1 kinase, such as IRS1, IRS2 and FRS3 (Miranda, C. et al., 2001; Roccato, E. et al., 2002; Ranzi, V. et al., 2003).

The capability of TPM3, TPR and TFG to activate chimeric tyrosine kinase onco-genes is not restricted to NTRK1; in fact, they have been found fused to other kinase genes. TPM3 and TFG were reported to fuse to ALK in anaplastic large cell lymphoma

Figure 2. TRK oncogenes signaling pathways. Tyrosine residues are indicated with the number of the corresponding aminoacids of NTRK1.

(Hernandez, L. et al., 2002; Lamant, L. et al., 1999). TPR was first identified as part of the MET oncogene in HOS cells, fused to the TK domain of the hepatocyte growth factor receptor (Park, M. et al., 1986); subsequently it was detected fused to the raf oncogene during the transfection of a rat hepatocarcinoma (Ishikawa, F. et al., 1987). Interestingly, TPR and TFG were first identified in rearranged, oncogenic versions. TPM3 gene encodes a non-muscle tropomyosin isoform. TPR gene encodes a large protein of the nuclear pore complex; recent studies have shown that TPR is a phos-phorylated protein involved in mRNA export, through the formation of complexes with different interacting proteins (Shibata, S. et al., 2002; Green, D. M. et al., 2003). TFG encodes a protein of still unknown function.

Role of activating sequences in TRK oncogenic activation

Despite the diversity in structure and function, all the NTRK1 activating proteins contain coiled-coil domains that promote protein dimerization/multimerization. Coiled-coil domains are characterized by heptad repeats with the occurrence of apolar residues

Figure 3. Prediction of coiled-coil domains in TPM3, TPR and TFG with the use of Paircoil program (Berger B., PNAS vol 92, 1995. pag. 8259-8263). The vertical scale represents relative coiled-coil probability; the horizontal scale represents amino acids number.

preferentially in the first (a) and fourth (d) positions (Lupas, A., 1996; Lupas, A. et al., 1991). This confers to the proteins the capability to fold into a-helices that are wound into a superhelix. In Figure 3 the coiled-coil domains detected in TRK activating sequences by sequence analysis with the COIL program are shown.

TPM3 contains numerous, overlapping coiled-coil domains. Several coiled-coil domains are present in TPR, and two of them fall in the region contained in MET and TRK-T1 oncogenes. It has been reported that mutations within the first coiled-coil domain drastically reduces MET transforming activity (Rodrigues, G. A. et al., 1993). TFG contains a single coiled-coil domain, of approximately three heptads, shorter than typical coiled-coil domains (Figures 3 and 4). However, the presence of a hydrophobic residue in position a, would increase the strength of association, despite the short length (Greco, A. et al., 1995). The contribution of TFG coiled-coil domain to TRK-T3 oncogenic activation has been elucidated by studies employing mutants where the domain was either deleted or mutated at leucine residues in position d of each hep-tad. We have demonstrated that coiled-coil domain plays a crucial role in TRK-T3 oncogenic activation by mediating oncoprotein complexes formation, an essential step for tyrosine kinase activation. Our studies support the model by which coiled-coil domains mediate protein oligomerization of RTK oncogenes leading to constitutive, ligand-independent tyrosine kinase activity (Greco, A. et al., 1998). The TFG coiled-coil domain is predicted to fold into trimers. By size-exclusion chromatography we have demonstrated that the wild type TRK-T3 protein is part ofhigh molecular weight complexes, compatible with the assembly of six oncoproteins molecules, or including other proteins. However, the precise composition of TRK-T3 complexes remains to be determined (Roccato, E. et al., 2003).

Studies on receptor and non receptor tyrosine kinase chimeric oncogenes have demonstrated that the importance of activating genes is related to the coiled-coil domains which mediate the activation above reported. However, an attractive hypothesis is the possibility that activating sequences may contribute with other functions, apart from dimerization. In addition, since the oncogenic rearrangements disabled one allele of the activating gene, the reduced activity of the corresponding protein could play a role in thyroid carcinogenesis. In this respect the thyroid TRK-T3 oncogene represents an attractive model for at least three reasons: 1) the activating portion is provided by TFG, a gene coding for a novel protein whose function remains to be unveiled; 2) the TFG portion contained in TRK-T3 display a single, short coiled-coil domain, corresponding to 14% of the aminoacid residues; 3) TFG might interact with other proteins (see later).

After our initial isolation as part of the TRK-T3 oncogene, the normal TFG counterpart was cloned and characterized (Figure 4).

Figure 4. Schematic representation of the TRK-T3 oncogene. The portions contributed by TFG and NTRK1 are indicated. In the box the TFG aminoacidic sequence is reported; specific domains and consensus sites are indicated. CC: coiled-coil domain; TM: transmembrane domain; CK2: putative phosphorylation site for CK2; PKC: putative phosphorylation site for PKC.

Figure 4. Schematic representation of the TRK-T3 oncogene. The portions contributed by TFG and NTRK1 are indicated. In the box the TFG aminoacidic sequence is reported; specific domains and consensus sites are indicated. CC: coiled-coil domain; TM: transmembrane domain; CK2: putative phosphorylation site for CK2; PKC: putative phosphorylation site for PKC.

TFG gene is ubiquitously expressed in human adult tissues and it is conserved among several species, including C. elegans. In addition to the coiled-coil domain, the TFG protein also contains putative phosphorylation sites for PKC and CK2, glycosilation sites, as well SH2- and SH3-binding sites (Mencinger, M. et al., 1997). Several of these sites are identical in TFG proteins from different species, indicating that the protein might be involved in basic cell processes (Mencinger, M. et al., 1999). We have recently identified a PB1 domain, which encompasses almost entirely the TFG N-terminal portion (Roccato, E. et al., 2003). PB1 is a novel protein module mediating protein oligomerization: in fact it is capable of binding to target proteins containing PC motifs and, as recently discovered, other PB1 domains (Terasawa, H. et al., 2001; Nakamura, K. et al., 2003). Based on the peculiarity ofTFG, we have recently focused our interest on the role of sequences outside the coiled-coil domain in TRK-T3 onco-genic activation. On the whole our studies demonstrate that the regions outside the coiled-coil domain give a great contribution to TRK-T3 activation. When deleted, complexes formation is unaffected; however transforming activity is reduced to different extent. More detailed information was provided by studies employing point mutants: transforming activity was significantly reduced by mutating a putative SH2-binding motif, whereas it was abrogated by the mutation of the conserved Lys residue within the PB1 domain. These evidences strongly support the notion that proteins interacting with TFG might play a role in TRK-T3 oncogenic activity (Roccato, E. et al., 2003). The identification of such proteins will give an important contribution to the definition of the modalities by which TRK-T3 triggers thyroid carcinogenesis, as well as to the discovery of TFG normal function.

Genomic features of NTRK1 oncogenic rearrangements

The NTRK1 genomic rearrangements present in the tumor DNA have been cloned and characterized (Figure 5) (Greco, A. et al., 1997; Greco, A. et al., 1995, Butti, M.G. et al, 1995). All the breakpoints fall within a NTRK1 region of 2.9 Kb, showing a GC content of 58.8% (Greco, A. et al., 1993a). The NTRK1 rearrangements are balanced; in fact, in addition to the oncogenic rearrangement containing the moiety of the receptor, the reciprocal product of the rearrangement, containing the portion of NTRK1 fused to the 3' portion of the activating genes, was present in tumor DNA.

Figure 5. Genomic structure of the NTRK1 gene. The break sites of the different TRK oncogenes are indicated.

Sequencing of the breakpoint regions showed no homologies between the joined extremities. This suggests that the Non Homologous End Joining (NHEJ) mechanism, which requires little or not sequence homology, could be involved in the generation of TRK oncogenes. The NHEJ pathway is activated by ionizing radiations, and this is consistent with the association of PTC with therapeutic or accidental radiation exposure. Analysis of the breakpoint regions in different tumors demonstrated that all the rearrangements are conservative, involving deletion, insertion or duplication ofonly few nucleotides. In a tumor carrying the TRK-T2 oncogene a peculiar rearrangement was found, with the end of NTRK1 joined to sequences from chromosome 17; however, such additional rearrangement does not contribute to oncogenic activation (Greco, A. et al., 1997).

No cytogenetic studies are available for tumors carrying NTRK1 rearrangements; therefore the type of chromosomal rearrangement generating TRK oncogenes is not documented. A t(1 ;3) translocation is most likely responsible for the generation of TRK-T3 oncogene (TFG/NTRK1 rearrangement). For TRK, TRK-T1 and TRK-T2, produced by rearrangements with genes located on the q arm of chromosome 1, similarly to NTRK1, three mechanisms of rearrangement can be postulated: deletion, inversion, and reciprocal translocation between the two chromosome 1 homologues. The presence in the tumor DNA of the reciprocal products of the rearrangement allowed us to exclude the deletion. Sequence data recently available in public databases unveiled that NTRK1 has transcriptional orientation opposite to that of TPM3 and TPR. Therefore, intrachromosomal inversion is the only mechanism capable to produce TRK, TRK-T1 and TRK-T2 oncogenes (Figure 6).

The thyroid epithelium is very prone to chromosomal rearrangements. These include the RET and NTRK1 oncogenes in PTC, and the PAX8/PPARy fusion gene associated with follicular thyroid tumors. This predisposition to gene rearrangements is a peculiarity of thyroid epithelium, at variance with other epithelia, and the understanding of the molecular basis underlying such predisposition represents a fascinating topic. Recently, Nikiforova et al (2000) have shown that, in thyroid interphase nuclei, RET and H4 loci display a distance reduced with respect to other cell type, and suggested that this spatial contiguity may provide the structural basis for the generation of the thyroid RET/H4 (PTC1) oncogene. It is very important to assess whether or not this attractive model also apply to NTRK1 and its partners, rearranging genes. Another possibility is that the high frequency of chromosomal rearrangements in thyroid tumors might reflect the thyrocyte intrinsic capability to repair DNA DSBs, either spontaneous or induced. Yang et al (1999) showed that human thyrocytes exposed in vitro to ionizing radiations failed to induce apoptosis; instead, they showed a significant increase of DNA end-joining activity. In this respect the analysis of the DNA repair kinetics and the status of the enzymes involved in DNA repair in human thyrocytes deserve to be investigated.

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