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Tyrosines outside the kinase core and dimerization are required for the mitogenic activity of RET/ptc2
K Durick1, V J Yao, M G Borrello
1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0654, USA.
Abstract:
Defects in the c-ret proto-oncogene, a member of the protein tyrosine kinase receptor family, have recently been linked to two types of genetic syndromes, Hirschsprung's disease and the multiple endocrine neoplasia family of inherited cancers. RET/ptc2 is the product of a papillary thyroid carcinoma translocation event between the genes coding for c-ret and the type I alpha regulatory subunit of protein kinase A (RI alpha) (Lanzi, C., Borrello, M., Bongarzone, I., Migliazza, A., Fusco, A., Grieco, M., Santoro, M., Gambetta, R., Zunino, F., Della Porta, G., and Pierotti, M. (1992) Oncogene 7, 2189-2194). The resulting 596-residue protein contains the first two-thirds of RI alpha and the entire tyrosine kinase domain of c-ret (RETtk). An in vivo assay of growth stimulatory effects was developed, which consisted of microinjecting a RET/ptc2 expression plasmid into the nuclei of 10T1/2 mouse fibroblasts and observing the incorporation of 5-bromodeoxyuridine. This assay was used to determine that only the dimerization domain of RI alpha fused to RETtk is required for RET/ptc2's mitogenic activity. In addition, all of the reported Hirschsprung's disease point mutations in the RETtk (S289P, R421Q, and R496G) inactivate RET/ptc2 in our assay, confirming that these are loss of function mutations. Two tyrosines outside the conserved kinase core were also identified that are essential for full mitogenic activity of RET/ptc2. These two tyrosines, Tyr-350 and Tyr-586, are potential sites for Src homology 2 and phosphotyrosine binding domain interactions.
Insights
Defects in the c-ret proto-oncogene are linked to genetic syndromes. The RET/ptc2 protein
Area of Science:
- Oncogene research
- Molecular biology
- Cancer genetics
Background:
- The c-ret proto-oncogene, a protein tyrosine kinase receptor, is implicated in genetic syndromes like Hirschsprung's disease and inherited cancers.
- RET/ptc2 is a fusion protein resulting from a papillary thyroid carcinoma translocation, combining parts of c-ret and protein kinase A's RI alpha subunit.
Purpose of the Study:
- To investigate the functional domains of the RET/ptc2 oncoprotein responsible for its mitogenic activity.
- To validate the role of specific RET tyrosine kinase mutations in Hirschsprung's disease as loss-of-function mutations.
Main Methods:
- Development of an in vivo growth stimulatory assay using microinjection of RET/ptc2 expression plasmids into 10T1/2 mouse fibroblasts.
- Assessment of 5-bromodeoxyuridine incorporation to measure mitogenic effects.
- Analysis of specific RET/ptc2 mutations, including Hirschsprung's disease-associated point mutations and tyrosine substitutions.
Main Results:
- The dimerization domain of RI alpha fused to the RET tyrosine kinase (RETtk) is sufficient for RET/ptc2's mitogenic activity.
- Hirschsprung's disease mutations (S289P, R421Q, R496G) inactivate RET/ptc2, confirming their loss-of-function nature.
- Two specific tyrosines (Tyr-350 and Tyr-586) outside the kinase core are crucial for full mitogenic activity, suggesting roles in protein interactions.
Conclusions:
- The dimerization domain of RI alpha and specific tyrosine residues are critical for RET/ptc2's oncogenic function.
- Loss-of-function mutations in RETtk are associated with Hirschsprung's disease.
- Tyr-350 and Tyr-586 represent potential interaction sites for signaling proteins involved in RET-mediated mitogenesis.