Title

Biological function of p58(gag)

Date of Award

1997

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Coralie A. C. Carraway, Committee Chair

Abstract

P58$\rm\sp{gag}$ is a truncated retroviral Gag protein located at the microfilament-membrane interface of the MAT-C1 ascites subline of the 13762 rat mammary adenocarcinoma. It contains proline-rich regions that are similar to the consensus sequences for Src homology 3 (SH3) ligands. Direct binding of the c-Src SH3 and c-Abl SH3 domain GST-fusion proteins to p58$\rm\sp{gag}$ was demonstrated by overlay analyses and affinity chromatography. The association of c-Src with p58$\rm\sp{gag}$ was demonstrated by the co-immunoprecipitation of in vitro-translated p58$\rm\sp{gag}$ with platelet c-Src by anti-c-Src antibody. Further, either platelet or baculovirus-expressed c-Src could bind to a GST-p58$\rm\sp{gag}$ fusion protein. Finally, the GST-p58$\rm\sp{gag}$ fusion protein, but not GST, was phosphorylated by platelet or baculovirus-expressed c-Src, but not by a kinase-negative c-Src variant expressed in insect cells. The binding of p58$\rm\sp{gag}$ to intact c-Abl was demonstrated by showing in vitro-translated c-Abl binding to purified GST-p58$\rm\sp{gag}$. Binding of p58$\rm\sp{gag}$ to c-Abl resulted in the phosphorylation of GST-p58$\rm\sp{gag}$ in the binding complex. The phosphorylation of GST-p58$\rm\sp{gag}$ by c-Abl was further confirmed by using purified baculorvirus-expressed GST-c-Abl to phosphorylate the purified p58$\rm\sp{gag}$. The association of c-Abl SH3 with p58$\rm\sp{gag}$ was confirmed by failure of a baculovirus-expressed, SH3-deleted GST-c-Abl mutant to bind to in vitro translated-p58$\rm\sp{gag}.$ The binding site of p58$\rm\sp{gag}$ to the SH3 domain of c-Abl was located to a 14 amino acid region in p58$\rm\sp{gag}$ by mapping C-terminal deletion mutants of GST-p58$\rm\sp{gag}$ and by peptide competition experiments. Our results suggest that p58$\rm\sp{gag}$ may play a important role in recruitment signal components such as c-Src and c-Abl to the microfilament-membrane interface, and that the association of retroviral Gag protein with cellular tyrosine kinases may be important for virus maturation.In vitro studies had shown that p58$\rm\sp{gag}$ bound to F-actin and acted as a filament capping protein. Transient transfection and stable transfection of p58$\rm\sp{gag}$ in Cos-7 cells or MDCK cells respectively resulted in depletion of stress fibers near the ventral cell surface. The F-actin binding domains in p58$\rm\sp{gag}$ were mapped by using deletion mutants. The F-actin binding regions in p58$\rm\sp{gag}$ were delineated as regions 427-369 and 304-209 of the C-terminus of p58$\rm\sp{gag}.$

Keywords

Biology, Molecular; Health Sciences, Oncology

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9824549