A Protein Kinase Induced by Pseudorabies Virus in Infected Cells

Katan, Matilda (1985) A Protein Kinase Induced by Pseudorabies Virus in Infected Cells. PhD thesis, University of Glasgow.

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This thesis describes studies of the phosphorylation of proteins during the infection of baby hamster kidney fibroblasts (BHK cells) with the swine herpes virus, pseudorabies virus, with particular emphasis on the protein kinase activites in infected cells. Protein kinases present in the cytosol of BHK cells infected with pseudorabies virus were compared with those present in the cytosol of uninfected cells. The cytosol was first subjected to chromatography on DEAE-cellulose and fractions analysed for protein kinase activities. In the fractions from uninfected cells the activities were found to correspond to the following previously characterized protein kinases: i) cyclic AMP-dependent protein kinase type I; ii) cyclic AMP-dependent protein kinase type II; iii) protein kinase C; iv) casein kinase I; v) casein kinase II; vi) a less clearly defined protamine kinase which is probably the proteolytic fragment of protein kinase C. In BHK cells infected with pseudorabies virus the protein kinases listed above were present in amounts comparable to those in uninfected cells. However, a new protein kinase activity was detected in infected cells, appearing about 4 h after infection and increasing during the following 6 h, at least. This protein kinase designated virus-induced protein kinase (ViPK), was the subject of subsequent studies. The new protein kinase was further purified either by Blue A dye-ligand chromatography or by high-performance size-exclusion and ion- exchange chromatography. The latter procedure resulted in 100-fold purification of the enzyme. The partially-purified preparations were used to characterize the new protein kinase. It had an apparent molecular weight of 68,000 on the basis of size-exclusion chromatography, and had a sedimentation coefficient of 4.3S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km=60 muM), but not GTP, as phosphate donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A striking characteristic of the protein kinase was a high KC1 concentration optimum with the persistence of activity up to 800 mM KC1, at least. These characteristics distinguish it from other protein kinases found in BHK cells or reported to be present in other eukaryotic cells. Further studies attempted to relate the new protein kinase activity to proteins which become phosphorylated during viral infection. The results obtained show that the major virion phosphoproteins were not phosphorylated by the new protein kinase in vitro, even though that activity was also present in the virion. An enzyme activity, present in BHK cells and a similar activity from virus particles, both with characteristics consistent with those of casein kinase II, phosphorylated these proteins in vitro. Thus, it is possible that these viral proteins synthesized de novo in infected cells can be recognized as substrates by cellular casein kinase II. Analysis of viral non-structural proteins showed that one of the proteins tested, viral DNase, served as a substrate iin vitro for a partially-purified preparation of the new protein kinase. Studies of the phosphorylation of ribosomal protein S6 in vitro by protein kinase activities from infected cells showed that the new protein kinase and also some other cellular protein kinases (cyclic AMP-dependent protein kinases and protein kinase C) can catalyse this reaction. Specific phosphorylation of S6 by the new protein kinase was only observed in certain conditions, e. g. 600 mM KC1 or 7-10 mM spermine. Phosphopeptide maps of S6 following phosphorylation in vivo and in vitro by the new kinase were similar. However, the enzyme catalysed the incorporation of only two phosphate groups per molecule of S6 while the stoichiometry of phosphorylation in vivo was up to five phosphate groups per S6 molecule. This finding does not support the possibility that the new protein kinase is responsible for the complete phosphorylation of ribosomal protein S6 observed in vivo. One of the cellular kinases, protein kinase C appears to be a better candidate, on the basis of the results of studies of stoichiometry and phosphopeptides.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry, Virology
Date of Award: 1985
Depositing User: Enlighten Team
Unique ID: glathesis:1985-76559
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:09
Last Modified: 19 Nov 2019 14:09
URI: https://theses.gla.ac.uk/id/eprint/76559

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