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Identification and biochemical characterization of p70TRK, product of the human TRK oncogene
G Mitra1, D Martin-Zanca, M Barbacid
1Developmental Oncology Section, Frederick Cancer Research Facility, MD 21701.
Abstract:
TRK is a human transforming gene generated in a colon carcinoma by a somatic rearrangement that fused a nonmuscle tropomyosin gene to sequences that shared extensive homology with members of the tyrosine-protein kinase supergene family. These sequences are likely to be derived from a transmembrane receptor gene whose putative ligand binding domain has been replaced by tropomyosin. In the present studies, we have expressed the entire coding sequences of the TRK oncogene as well as its protein kinase-related carboxyl-terminal domain in Escherichia coli. Antisera raised against these bacteria-synthesized TRK polypeptides has allowed us to identify the gene product of the TRK oncogene as a 70-kDa protein. Immunoprecipitates containing p70TRK have an associated protein kinase activity specific for tyrosine residues. Moreover, p70TRK is phosphorylated in vivo in serine (75%), threonine (20%), and tyrosine (5%) residues. Finally, immunofluorescence and cellular fractionation studies indicate that p70TRK is preferentially located in the cytoplasmic fraction.
Insights
The TRK oncogene, a fusion of tropomyosin and tyrosine-protein kinase sequences, produces a 70-kDa protein (p70TRK). This protein exhibits tyrosine kinase activity and is primarily located in the cytoplasm.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- The TRK oncogene arises from a somatic rearrangement in colon carcinoma.
- It results from the fusion of a nonmuscle tropomyosin gene with tyrosine-protein kinase gene sequences.
- The TRK oncogene likely originates from a transmembrane receptor gene with a tropomyosin-substituted ligand-binding domain.
Purpose of the Study:
- To express the TRK oncogene and its protein kinase domain in Escherichia coli.
- To characterize the gene product of the TRK oncogene.
- To investigate the enzymatic activity and cellular localization of the TRK gene product.
Main Methods:
- Expression of TRK oncogene sequences in Escherichia coli.
- Generation of antisera against synthesized TRK polypeptides.
- Immunoprecipitation and in vivo phosphorylation studies.
- Immunofluorescence and cellular fractionation.
Main Results:
- Identified the TRK oncogene product as a 70-kDa protein (p70TRK).
- Demonstrated associated protein kinase activity specific for tyrosine residues in p70TRK immunoprecipitates.
- Showed in vivo phosphorylation of p70TRK on serine (75%), threonine (20%), and tyrosine (5%).
- Found p70TRK preferentially located in the cytoplasmic fraction.
Conclusions:
- The TRK oncogene encodes a 70-kDa protein with tyrosine kinase activity.
- p70TRK undergoes phosphorylation on multiple residues, including tyrosine.
- The cytoplasmic localization of p70TRK suggests its role in intracellular signaling pathways.