Purification and Activity of the PP60SRC Protein Kinase

Author

Michael E. Greenberg

Date of Award

1982

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Gerald Edelman

Related Theses

View more theses from the Gerald Edelman Laboratory

Keywords

pp60 src, tyrosine kinase, cell transformation, Rous Sarcoma virus, vinculin, 34 kD phosphoprotein

Abstract

Transformation of cells by Rous Sarcoma virus results from the action of a single virally coded phosphoprotein with a molecular weight of 60,000 daltons (pp60^src). The expression of pp60^src results in many significant changes in the cell's phenotype which include disruption of the cytoskeleton and the deregulation of cell growth. During the last few years significant progress has been made in many laboratories towards understanding the structure and function of pp60^src (reviewed by Erikson et al., 1980). This thesis work describes procedures that were developed for purifying and characterizing pp60^src and some of its target proteins. The src protein associated kinase activity was purified several thousand fold, in detergent-free solution. Analysis of the purified protein by immunoprecipitation indicated that the pp60^src had degraded during the purification to a 52,000 dalton species. Detergents were found to be necessary to extract pp60^src from cell membranes in an undegraded form. A second reproducible procedure was developed to purify pp60^src as an intact protein in the presence of detergents. The purified protein migrated as a monomer with an approximate molecular weight of 60,000 daltons when analyzed on glycerol gradients or by gel filtration. The purification schemes successfully removed phosphatases and inhibitors so that it was possible to study the kinase activity of pp60^src in solution under controlled conditions. Both casein and TBR IgG were phosphorylated at tyrosine residues by partially purified pp60^src. The src protein did not phosphorylate histones or phosvitin and the phosphorylation of casein was not enhanced in the presence of cAMP. The interaction of pp60^src with more physiological substrates was also examined. No direct interaction of pp60^src with purified cytoskeletal components was detected but several of these proteins were phosphorylated at tyrosine residues by pp60^src in vitro including tubulin, actin, vinculin, myosin, and filamin. Other proteins including the 34 kD phosphoprotein identified by Radke and Martin (1979) and cytosolic malate dehydrogenase were also phosphorylated by pp60^src Further biochemical characterization of the partially purified pp52^src and pp60^src preparations resulted in the identification of a 50 kD protein that appears to interact with the src protein. The 50 kD protein was found to be phosphorylated in a Ca²⁺-calmodulin dependent manner and this phosphorylation was inhibited by the calmodulin inhibitor chlorpromazine. The Ca²⁺-calmodulin activated phosphorylation of the 50 kD protein is not pp60^src- induced since it occurs to a significant extent in samples prepared from non-transformed chick embryo cells. Several experiments suggested that pp52^src binds to the 50 kD protein. Ca²⁺-calmodulin regulation was further implicated in the in vivo action of pp60^src. The calmodulin inhibitor chlorpromazine was observed to inhibit the pp60^src-induced deregulation of DNA synthesis. The drug had no detectable in vivo effect on the pp60^src-associated kinase activity. While a variety of in vitro and in vivo experiments from different laboratories (Sefton et al., 1981a; Radke and Martin, 1979; Brugge et al., 1981) have identified several pp60^src substrates, the role of these proteins in cell transformation is still not understood. To begin characterization of the src targets, monoclonal antibodies were prepared against vinculin and the 34 kD protein. Four different monoclonal antibodies were raised which immunoprecipitated a 130,000 dalton phosphoprotein from both normal and transformed cells. By immunofluorescence the antibodies were found to stain chick embryo fibroblasts at sites of cell contact with the substratum. The immunoprecipitation and immunofluorescence data demonstrated that the four antibodies recognized vinculin. One monoclonal antibody was identified which immunoprecipitated a 34 kD phosphoprotein from normal and transformed cells. A second antibody was isolated which recognized a non-phosphorylated 34 kD protein. It was determined that the 34 kD phosphoprotein was phosphorylated at a tyrosine residue in a transformation specific manner and it is probably the same protein originally identified by Radke and Martin (1979). The tissue distribution and subcellular localization of the two 34 kD proteins was examined using the monoclonal antibodies. The 34 kD phosphoprotein was localized, by indirect immunofluorescent staining, to the plasma membrane. The antibody to the non-phosphorylated protein specifically stained the nucleoli. The monoclonal antibodies to vinculin and the 34 kD phosphoprotein allowed the rapid purification and characterization of these substrates from normal and transformed cells. The purified target proteins and the specific antibodies should make it possible to further probe the biochemical mechanisms involved in cellular transformation by pp60^src.

Comments

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

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Recommended Citation

Greenberg, Michael E., "Purification and Activity of the PP60SRC Protein Kinase" (1982). Student Theses and Dissertations. 587.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/587