Acta NaturaeActa NaturaeActa Naturae2075-8251Acta Naturae Ltd1078510.32607/20758251-2010-2-3-110-121ArticlesСтатьиResearch ArticleNew Test System for Serine/Threonine Protein Kinase Inhibitors Screening: E. coli APHVIII/ Pk25 designNew Test System for Serine/Threonine Protein Kinase Inhibitors Screening: E. coli APHVIII/ Pk25 designBekkerO BAlekseevaM GOsolodkinD IPalyulinV AElizarovS MZefirovN SDanilenkoV Nvalerid@rutenia.ruVavilov Institute of General Genetics, Russian Academy of SciencesLomonosov Moscow State UniversityBach Institute of Biochemistry, Russian Academy of Sciences1509201023VOL 2, NO3 (2010)ТОМ 2, №3 (2010)11012117012020Copyright ©; 2010, Bekker O.B., Alekseeva M.G., Osolodkin D.I., Palyulin V.A., Elizarov S.M., Zefirov N.S., Danilenko V.N.Copyright ©; 2010, Bekker O.B., Alekseeva M.G., Osolodkin D.I., Palyulin V.A., Elizarov S.M., Zefirov N.S., Danilenko V.N.2010Bekker O.B., Alekseeva M.G., Osolodkin D.I., Palyulin V.A., Elizarov S.M., Zefirov N.S., Danilenko V.N.Bekker O.B., Alekseeva M.G., Osolodkin D.I., Palyulin V.A., Elizarov S.M., Zefirov N.S., Danilenko V.N.https://creativecommons.org/licenses/by/4.0https://actanaturae.ru/2075-8251/article/view/10785

An efficient test system for serine/threonine protein kinase inhibitors screening has been developed based on the E. coli protein system APHVIII/Pk25. Phosphorylation of aminoglycoside phosphotransferase VIII (APHVIII) by protein kinases enhances resistance of the bacterial cell to aminoglycoside antibiotics, e.g. kanamycin. Addition of protein kinase inhibitors prevents phosphorylation and increases cell sensitivity to kanamycin. We have obtained modifications of APHVIII in which phosphorylatable Ser146 was encompassed into the canonical autophosphorylation sequence of Streptomyces coelicolor Pk25 protein kinase. Mutant and wild-type aphVIII were cloned into E. coli with the catalytic domain of pk25. As a result of the expression of these genes, accumulation of corresponding proteins was clearly observed. Extracted from bacterial lysates, Pk25 demonstrated its ability to autophosphorylate. It was shown that variants of E. coli containing both aphVIII and pk25 were more resistant to kanamycin than those carrying only aphVIII. Protein kinase inhibitors of the indolylmaleimide class actively inhibited Pk25 and reduced cell resistance to kanamycin. Modeling of APHVIII and Pk25 3D structures showed that pSer146 is an analog of phosphoserine in the ribose pocket of protein kinase A. Pk25 conformation was similar to that of PknB of Mycobacterium tuberculosis. Potential indolylmaleimide inhibitors were docked into the ATP-binding pocket of Pk25. The designed test system can be used for the primary selection of ATP-competitive small molecule protein kinase inhibitors.

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