Immunochemical and immunohistochemical techniques were used to determine the role of post-translational modifications in the regulation of DNA polymerase α in Rat-1(tsLA24/RSV) cells. Immunoaffinity purification following sucrose gradient fractionation showed two immunospecific polypeptides of Mᵣ ≃ 185,000 and 220,000 only in those fractions exhibiting DNA polymerase α activity. The Mᵣ ≃ 220,000 polypeptide was shown to be phosphorylated, primarily at serine residues. Incubation of cell lysates with immobilized alkaline phosphatase reduced enzyme activity and subsequent readdition of ATP, but not ATP-γ-S, restored activity suggesting the involvement of an endogenous serine protein kinase. This kinase may be a cAMP dependent protein kinase because prior incubation of the catalytic subunit stimulated DNA polymerase α activity 3-4 fold. In the absence of serum growth factors or pp60ˢʳᶜ, DNA polymerase α activity and semi-conservative DNA replication rates in growth arrested cells were severely depressed. However, both polymerase activity and DNA synthetic rates were subsequently restored by either activation of pp60ˢʳᶜ by temperature shift or by serum addition. DNA polymerase α protein was found primarily in the nucleus of all cells in log phase, growth arrested or subsequently stimulated cultures, independent of whether the cells were replicating DNA. Stimulation by either pp60ˢʳᶜ or serum did not alter DNA polymerase α localization within the cell nor lead to a preferential synthesis of Mᵣ ≃ 220,000 peptide or proteolytic conversion of the Mᵣ ≃ 220,000 peptide to smaller peptides, but did result in phosphorylation of the Mᵣ ≃ 220,000 polypeptide. This phosphorylation was not apparent in serum deprived, growth arrested cells. It is suggested that pp60ˢʳᶜ acts to initiate DNA synthesis through the temporal activation of DNA polymerase α through a mechanism similar to that used by serum growth factors and that phosphorylation by a serine protein kinase serves an important function.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/184054 |
| Date | January 1987 |
| Creators | DONALDSON, ROBERT WILLIAM. |
| Contributors | Gerner, Eugene, Bowden, G. T., Meyskens, F. L., Tischler, M. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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