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.

Abstract

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 (DlOS170). The mechanism leading to this recombination was identified as a paracentric inversion of the long arm of chromosome 10, inv(10)(qll.2q21), with the breakpoints occurring where ret and DlOS170 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 DlOS170 locus. The nucleotide sequence indicated that the transforming activity was created by the fusion of the ret tyrosine kinase domain with part of the Rle 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 RIax subunit gene is located on chromosome 17q. Rlo-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.

Grb2 binding to the different isoforms of Ret tyrosine kinase

“…In fact, we had previously demonstrated that heterodimerization between the two isoforms can occur (Bongarzone et al, 1993) and, as a consequence, here we have observed that pY620 inhibits the binding of Grb2 with both isoforms.…”

“…The experiments were performed with constitutively activated oncogenic Ret versions, encoded by RET/PTC2 (Bongarzone et al, 1993), a thyroid carcinoma-speci®c oncogene generated by a somatic rearrangement of RET and by RET-C634R , a RET point mutant associated to the inherited cancer syndrome MEN2A. The two oncogenic proteins represent cytosolic and membranebound forms of Ret kinase, respectively.…”

Papillary thyroid carcinoma: 6 cases from 2 families with associated lymphocytic thyroiditis harbouring RET/PTC rearrangements

“…In regard to the second question, we are unable to verify it because we do not have enough biological material to study by XL-PCR the genomic sequences where the break-points are generally located in the different RET/PTC rearrangements known until present (Grieco et al, 1990;Bongarzone et al, 1993;Santoro et al, 1994;Fugazzola et al, 1996;Bounacer et al, 1997;Suárez, 1998b). Finally, concerning the third question, there was no evidence in the clinical history of our patients of a possible low-dose irradiation and/or a chemical agent aggression.…”

“…Indeed, contrasting with the high and very similar frequency (about 60%) of activation by rearrangement observed in the therapeutic or accidental radiation-associated tumours (Bounacer et al, 1997), the frequency of ret activation by this mechanism in 'spontaneous' thyroid tumours varies widely between different studies: from 2.5% to 34% (Bongarzone et al, 1989(Bongarzone et al, , 1993Grieco et al, 1990;Santoro et al, 1994;Delvincourt et al, 1996;Bounacer et al, 1997). It has been postulated that this variation observed in 'spontaneous' tumours, could be the result of the different geographical origins of populations studied, the age at tumour occurrence, or the sensitivity of the experimental methods used to detect the rearrangement.…”

Expression of Ret/PTC1, -2, -3, -Δ3 and -4 in German papillary thyroid carcinoma

“…In thyrocytes, another alteration of ret, chimerical proteins named ret/PTC generated by chromosomal translocation, may cause papillary thyroid carcinoma. The 3' tyrosine kinase domain of ret is fused to the 5' part of the H4 (PTC1), Rla (PTC2) or elel (PTC3) gene, resulting in constitutional expression of ret and phosphorylation of ret and other target proteins (Grieco et al, 1990;Bongarzone et al, 1993;Santoro et al, 1994). This event was specific for papillary thyroid carcinoma (Santoro et al, 1993), but the reported frequencies varied widely from 2.5% to 30% of these tumours Zou et al, 1994).…”