Molecular Cloning of Avian Sarcoma Virus CT10 and Characterization of its Protein Product

Author

Bruce J. Mayer

Date of Award

1989

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

Four previously uncharacterized avian sarcoma viruses were screened and two of these, RPL30 and CTIO, were found to encode apparently novel oncogenes. Biologically active CTIO DNA was molecularly cloned and the nucleotide sequence was determined. The CTIO genome encodes a gag-fusion polypeptide of 47 kilodaltons, termed p47^gag-crk. This protein contains blocks of sequence similarity to a noncatalytic, potentially regulatory region found in the nonreceptor tyrosine kinases, a phosphoinositide-specific phospholipase C, and the ras GTPase activator protein; no homology was found to any known catalytic domain. Potential roles for the homologous domains, termed SH2 and SH3, in normal signal transduction and in the biological activity of p47^gag-crk are discussed. Biochemical data demonstrated that phosphotyrosine levels on at least three cellular proteins were greatly elevated in CTI0-infected cells, and that a tyrosine kinase activity was immunoprecipitated in association with p47^gag-crk. A specific antiserum that recognizes the c-crk-derived portion of the oncogene product was generated, and a number of proteins were identified that were phosphorylated in in vitro kinase reactions of v-crk immunoprecipitates or were coimmunoprecipitated with the v-crk protein. All of the phosphoproteins that specifically coprecipitate with p47^gag-crk contained high levels of phosphotyrosine; these proteins include the three major phosphotyrosine-containing proteins of CTI0-infected cells. Preliminary experiments demonstrated that p47^gag-crk, the crlc-associated tyrosine kinase activity, and the major phosphotyrosine containing proteins of infected cells were localized on membranes in the nonionic detergent-insoluble cytoskeletal matrix. A series of 10 v-crk mutants were constructed and their biological activity was assessed. Lesions within the SH2 or SH3 domains decreased or abolished biological activity, and viral gag sequences were also required for full transforming activity. Mutations in the nonhomologous regions between the SH2 and SH3 domains had no effect. Biochemical analysis of mutant-infected cells demonstrated that in all cases increased phosphotyrosine levels correlated with transformation.

Comments

A thesis submitted to the faculty of the Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Recommended Citation

Mayer, Bruce J., "Molecular Cloning of Avian Sarcoma Virus CT10 and Characterization of its Protein Product" (1989). Student Theses and Dissertations. 366.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/366