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|Title:||Tyrosine-Specific Protein Phosphorylation in Cells Transformed by Rous Sarcoma Virus or Stimulated by Mitogens|
|Department / Program:||Microbiology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The transforming proteins of several RNA tumor viruses as well as a number of membrane receptors for polypeptide growth factors possess a tyrosine-specific protein kinase activity. Genetic and biochemical evidence indicates that this enzymatic activity is involved at the early stages of oncogenic transformation or mitogenic stimulation. In order to identify the cellular substrates for these kinases, radio-labeled lysates from cells transformed by an RNA tumor virus, Rous sarcoma virus (RSV), or stimulated by a variety of polypeptide growth factors were fractionated on SDS-PAGE gels. The relative phosphoamino acid composition of the gel-fractionated phosphoproteins was determined by partial acid hydrolysis followed by two-dimensional paper electrophoresis to separate the phosphoamino acids. A minimum of 30 proteins was shown to undergo phosphorylation on tyrosine during oncogenic transformation by RSV. In contrast, mitogenic stimulation with polypeptide growth factors triggered the phosphorylation of only two proteins with M(,r)'s of 40 and 42 kilodaltons, the latter being the predominant phosphorylation. The phosphorylation of these proteins was also stimulated by tumor promoters, agents that induce many of the phenotypic alterations seen in RSV-transformed cells. The mitogen-stimulated phosphorylations occurred rapidly and transiently. These observations suggest that the phosphorylation of the 40 and 42K proteins is important for mitogenesis. Other investigators showed that the 42K protein is constitutively phosphorylated in RSV-transformed cells, suggesting a possible involvement for this protein in the process of viral oncogenesis. Studies with a cell variant that is mitogenically unresponsive to TPA indicate that the phosphorylation of the 42K protein is not sufficient to stimulate DNA synthesis, although it may be a necessary condition. The phosphorylation of the 40 and 42K proteins occurs via a calmodulin or phospholipid-dependent reaction since it is inhibited by phenothiazines. A partial purification procedure for the 42K protein was devised. This procedure is currently being used to obtain sufficient antigen for the purpose of raising specific antisera, reagents that will be invaluable to further characterize the biological role of this phosphoprotein.|
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1985.
|Date Available in IDEALS:||2014-12-16|