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Tyrosine kinase activity may be necessary but is not sufficient for c-erbB1-mediated tissue-specific tumorigenicity
D C Connolly1, S L Toutenhoofd, N J Maihle
1Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
Abstract:
Expression of mutant avian c-erbB1 genes results in tissue-specific transformation in chickens. Site-directed mutagenesis was used to generate kinase-defective mutants of several tissue-specific v-erbB transforming mutants by replacement of the ATP-binding lysine residue in the kinase domain with an arginine residue. These kinase-defective v-erbB mutants were analyzed for their in vitro and in vivo transforming potentials. Specifically, kinase-defective mutants of erythroleukemogenic, hemangioma-inducing, and sarcomagenic v-erbB genes were assessed for their oncogenic potential. In vitro transformation potential was assessed by soft-agar colony formation in primary cultures of chick embryo fibroblasts (CEF). In vivo transformation potential was determined by infection of 1-day-old line 0 chicks with concentrated recombinant retrovirus and then monitoring of birds for tumor formation. These transformation assays demonstrate that kinase activity is absolutely essential for transformation by tissue-specific transforming mutants of the avian c-erbB1 gene. Since all of the tissue-specific v-erbB mutants characterized to date exhibit tyrosine kinase activity in vitro but do not transform all tissues in which they are expressed, we conclude that v-erbB-associated tyrosine kinase activity may be necessary but is not sufficient to induce tumor formation.
Insights
Kinase activity is essential for avian c-erbB1 gene transformation in chickens. However, v-erbB tyrosine kinase activity alone is not sufficient to cause tumor formation in all tissues.
Area of Science:
- Oncogenesis
- Molecular Biology
- Avian Viral Oncogenes
Background:
- Mutant avian c-erbB1 genes induce tissue-specific transformation in chickens.
- The v-erbB oncoprotein is a constitutively active receptor tyrosine kinase.
Purpose of the Study:
- To investigate the role of kinase activity in tissue-specific transformation mediated by avian c-erbB1.
- To determine if v-erbB tyrosine kinase activity is sufficient for oncogenesis.
Main Methods:
- Site-directed mutagenesis to create kinase-defective v-erbB mutants.
- In vitro transformation assays using soft-agar colony formation in chick embryo fibroblasts (CEF).
- In vivo tumor formation assays in 1-day-old chicks infected with recombinant retroviruses.
Main Results:
- Kinase activity is absolutely essential for transformation by tissue-specific v-erbB mutants.
- Kinase-defective v-erbB mutants lost their transforming potential.
- All tested v-erbB mutants exhibited tyrosine kinase activity but did not transform all tissues.
Conclusions:
- Tyrosine kinase activity is a necessary but not sufficient factor for v-erbB-induced tumor formation.
- Additional cellular factors likely regulate the tissue specificity of v-erbB oncogenesis.