Biological function of p58(gag)

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

Coralie A. C. Carraway, Committee Chair


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$\rm\sp{gag}.$


Biology, Molecular; Health Sciences, Oncology

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