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Catalytic properties of a human cytomegalovirus-induced protein kinase
Abstract:
Human cytomegalovirus, a DNA virus whose genome contains a fragment of transforming DNA, induces a threonine-serine protein kinase having a molecular mass of 68 kDa (p68). p68 was extracted from cells 96-144 h after infection, and immunoprecipitated with a monoclonal antibody (F6b). Antibody-enzyme complexes were immobilized on heat/formaldehyde-inactivated Staphylococcus aureus. The best substrates for p68 were acidic proteins, phosvitin and casein. Glycogen synthase, phosphorylase alpha and histones were phosphorylated at rates not higher than 1-4% that obtained with phosvitin as substrate. ATP and GTP were equally good substrates of p68. p68 is able to autophosphorylate at the same residues (i.e. threonine and serine) as the protein substrates. Autophosphorylation does not seem to represent an intermediate in substrate phosphorylation. The protein kinase activity of p68 was not enhanced by cAMP, calcium ions, or polyamines like spermine or spermidine. Only at low Mg2+ concentration spermine enhanced by 68% the rate of casein phosphorylation. Heparin, a potent inhibitor of casein kinase II, inhibits p68 activity too, but ten-times higher concentrations were required for the same degree of inhibition. Quercetin, a bioflavonoid, acts as a strong inhibitor of p68 protein kinase activity. The inhibitory effect of quercetin was competitive towards the nucleotide substrate (Ki = 2.8 microM), and non-competitive towards the protein substrate (Ki = 15 microM).
Insights
Human cytomegalovirus induces a novel protein kinase (p68) that phosphorylates acidic proteins. Quercetin strongly inhibits this kinase activity, offering potential therapeutic insights.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Human cytomegalovirus (HCMV), a DNA virus, possesses transforming DNA fragments.
- HCMV infection induces a specific threonine-serine protein kinase, designated p68.
Purpose of the Study:
- To characterize the enzymatic activity and substrate specificity of the HCMV-induced p68 protein kinase.
- To investigate potential inhibitors of p68 kinase activity.
Main Methods:
- Extraction and immunoprecipitation of p68 from infected cells using a monoclonal antibody (F6b).
- Immobilization of antibody-enzyme complexes on Staphylococcus aureus.
- Assay of kinase activity using various protein substrates (phosvitin, casein, glycogen synthase, phosphorylase alpha, histones) and nucleotide substrates (ATP, GTP).
- Investigation of the effects of various modulators (cAMP, Ca2+, polyamines, heparin, quercetin) on kinase activity.
Main Results:
- p68 preferentially phosphorylated acidic proteins like phosvitin and casein.
- ATP and GTP were equally effective as phosphate donors.
- p68 exhibited autophosphorylation on threonine and serine residues.
- Quercetin demonstrated potent inhibition, acting competitively towards nucleotide substrates and non-competitively towards protein substrates.
- Heparin showed inhibitory effects, but required higher concentrations than for casein kinase II.
Conclusions:
- HCMV-induced p68 is a distinct threonine-serine protein kinase with specific substrate preferences.
- Quercetin is a strong inhibitor of p68 kinase activity, with defined inhibition kinetics.
- Understanding p68's function and inhibition may offer targets for antiviral therapies.