• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Oct 15, 2000; 351(Pt 2): 393–402.
PMCID: PMC1221375

DNA repair protein O6-alkylguanine-DNA alkyltransferase is phosphorylated by two distinct and novel protein kinases in human brain tumour cells.


We showed recently that human O(6)-alkylguanine-DNA alkyltransferase (AGT), an important target for improving cancer chemotherapy, is a phosphoprotein and that phosphorylation inhibits its activity [Srivenugopal, Mullapudi, Shou, Hazra and Ali-Osman (2000) Cancer Res. 60, 282-287]. In the present study we characterized the cellular kinases that phosphorylate AGT in the human medulloblastoma cell line HBT228. Crude cell extracts used Mg(2+) more efficiently than Mn(2+) for phosphorylating human recombinant AGT (rAGT) protein. Both [gamma-(32)P]ATP and [gamma-(32)P]GTP served as phosphate donors, with the former being twice as efficient. Specific components known to activate protein kinase A, protein kinase C and calmodulin-dependent kinases did not stimulate the phosphorylation of rAGT. Phosphoaminoacid analysis after reaction in vitro with ATP or GTP showed that AGT was modified at the same amino acids (serine, threonine and tyrosine) as in intact HBT228 cells. Although some of these properties pointed to casein kinase II as a candidate enzyme, known inhibitors and activators of casein kinase II did not affect rAGT phosphorylation. Fractionation of the cell extracts on poly(Glu/Tyr)-Sepharose resulted in the adsorption of an AGT kinase that modified the tyrosine residues and the exclusion of a fraction that phosphorylated AGT on serine and threonine residues. In-gel kinase assays after SDS/PAGE and non-denaturing PAGE revealed the presence of two AGT kinases of 75 and 130 kDa in HBT228 cells. The partly purified tyrosine kinase, identified as the 130 kDa enzyme by the same assays, was strongly inhibited by tyrphostin 25 but not by genestein. The tyrosine kinase used ATP or GTP to phosphorylate the AGT protein; this reaction inhibited the DNA repair activity of AGT. Evidence that the kinases might physically associate with AGT in cells was also provided. These results demonstrate that two novel cellular protein kinases, a tyrosine kinase and a serine/threonine kinase, both capable of using GTP as a donor, phosphorylate the AGT protein and affect its function. The new kinases might serve as potential targets for strengthening the biochemical modulation of AGT in human tumours.

Full Text

The Full Text of this article is available as a PDF (293K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Pegg AE, Dolan ME, Moschel RC. Structure, function, and inhibition of O6-alkylguanine-DNA alkyltransferase. Prog Nucleic Acid Res Mol Biol. 1995;51:167–223. [PubMed]
  • Mitra S, Kaina B. Regulation of repair of alkylation damage in mammalian genomes. Prog Nucleic Acid Res Mol Biol. 1993;44:109–142. [PubMed]
  • Srivenugopal KS, Yuan XH, Friedman HS, Ali-Osman F. Ubiquitination-dependent proteolysis of O6-methylguanine-DNA methyltransferase in human and murine tumor cells following inactivation with O6-benzylguanine or 1,3-bis(2-chloroethyl)-1-nitrosourea. Biochemistry. 1996 Jan 30;35(4):1328–1334. [PubMed]
  • Gerson SL, Willson JK. O6-alkylguanine-DNA alkyltransferase. A target for the modulation of drug resistance. Hematol Oncol Clin North Am. 1995 Apr;9(2):431–450. [PubMed]
  • Dolan ME, Pegg AE. O6-benzylguanine and its role in chemotherapy. Clin Cancer Res. 1997 Jun;3(6):837–847. [PubMed]
  • Spiro TP, Gerson SL, Liu L, Majka S, Haaga J, Hoppel CL, Ingalls ST, Pluda JM, Willson JK. O6-benzylguanine: a clinical trial establishing the biochemical modulatory dose in tumor tissue for alkyltransferase-directed DNA repair. Cancer Res. 1999 May 15;59(10):2402–2410. [PubMed]
  • Davis BM, Roth JC, Liu L, Xu-Welliver M, Pegg AE, Gerson SL. Characterization of the P140K, PVP(138-140)MLK, and G156A O6-methylguanine-DNA methyltransferase mutants: implications for drug resistance gene therapy. Hum Gene Ther. 1999 Nov 20;10(17):2769–2778. [PubMed]
  • Srivenugopal KS, Mullapudi SR, Shou J, Hazra TK, Ali-Osman F. Protein phosphorylation is a regulatory mechanism for O6-alkylguanine-DNA alkyltransferase in human brain tumor cells. Cancer Res. 2000 Jan 15;60(2):282–287. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Edara S, Kanugula S, Goodtzova K, Pegg AE. Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine. Cancer Res. 1996 Dec 15;56(24):5571–5575. [PubMed]
  • Blackwell JR, Horgan R. A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett. 1991 Dec 16;295(1-3):10–12. [PubMed]
  • Xu-Welliver M, Leitão J, Kanugula S, Pegg AE. Alteration of the conserved residue tyrosine-158 to histidine renders human O6-alkylguanine-DNA alkyltransferase insensitive to the inhibitor O6-benzylguanine. Cancer Res. 1999 Apr 1;59(7):1514–1519. [PubMed]
  • Boyle WJ, van der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. [PubMed]
  • Braun S, Abdel Ghany M, Lettieri JA, Racker E. Partial purification and characterization of protein tyrosine kinases from normal tissues. Arch Biochem Biophys. 1986 Jun;247(2):424–432. [PubMed]
  • Adamíková L, Resnick RJ, Tomáska L. Enrichment of yeast protein tyrosine kinase activity by substrate affinity chromatography. Yeast. 1996 Jul;12(9):833–838. [PubMed]
  • Martensen TM. Chemical properties, isolation, and analysis of O-phosphates in proteins. Methods Enzymol. 1984;107:3–23. [PubMed]
  • Kameshita I, Fujisawa H. A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem. 1989 Nov 15;183(1):139–143. [PubMed]
  • Ostrowski LE, Pegram CN, Von Wronski MA, Humphrey PA, He XM, Shiota S, Mitra S, Brent TP, Bigner DD. Production and characterization of antipeptide antibodies against human O6-methylguanine-DNA methyltransferase. Cancer Res. 1991 Jul 1;51(13):3339–3344. [PubMed]
  • Brent TP, von Wronski M, Pegram CN, Bigner DD. Immunoaffinity purification of human O6-alkylguanine-DNA alkyltransferase using newly developed monoclonal antibodies. Cancer Res. 1990 Jan 1;50(1):58–61. [PubMed]
  • Cui T, Sugrue RJ, Xu Q, Lee AK, Chan YC, Fu J. Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. Virology. 1998 Jul 5;246(2):409–417. [PubMed]
  • Lindberg RA, Quinn AM, Hunter T. Dual-specificity protein kinases: will any hydroxyl do? Trends Biochem Sci. 1992 Mar;17(3):114–119. [PubMed]
  • Becker W, Joost HG. Structural and functional characteristics of Dyrk, a novel subfamily of protein kinases with dual specificity. Prog Nucleic Acid Res Mol Biol. 1999;62:1–17. [PubMed]
  • Guerra B, Boldyreff B, Sarno S, Cesaro L, Issinger OG, Pinna LA. CK2: a protein kinase in need of control. Pharmacol Ther. 1999 May-Jun;82(2-3):303–313. [PubMed]
  • Gatica M, Hinrichs MV, Jedlicki A, Allende CC, Allende JE. Effect of metal ions on the activity of casein kinase II from Xenopus laevis. FEBS Lett. 1993 Jan 4;315(2):173–177. [PubMed]
  • Leroy D, Heriché JK, Filhol O, Chambaz EM, Cochet C. Binding of polyamines to an autonomous domain of the regulatory subunit of protein kinase CK2 induces a conformational change in the holoenzyme. A proposed role for the kinase stimulation. J Biol Chem. 1997 Aug 15;272(33):20820–20827. [PubMed]
  • Hathaway GM, Lubben TH, Traugh JA. Inhibition of casein kinase II by heparin. J Biol Chem. 1980 Sep 10;255(17):8038–8041. [PubMed]
  • Critchfield JW, Coligan JE, Folks TM, Butera ST. Casein kinase II is a selective target of HIV-1 transcriptional inhibitors. Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6110–6115. [PMC free article] [PubMed]
  • al-Obeidi FA, Wu JJ, Lam KS. Protein tyrosine kinases: structure, substrate specificity, and drug discovery. Biopolymers. 1998;47(3):197–223. [PubMed]
  • Chen Z, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D, Maniatis T. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes Dev. 1995 Jul 1;9(13):1586–1597. [PubMed]
  • Montagnoli A, Fiore F, Eytan E, Carrano AC, Draetta GF, Hershko A, Pagano M. Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation. Genes Dev. 1999 May 1;13(9):1181–1189. [PMC free article] [PubMed]
  • Hannan RD, Hempel WM, Cavanaugh A, Arino T, Dimitrov SI, Moss T, Rothblum L. Affinity purification of mammalian RNA polymerase I. Identification of an associated kinase. J Biol Chem. 1998 Jan 9;273(2):1257–1267. [PubMed]
  • Cardenas ME, Walter R, Hanna D, Gasser SM. Casein kinase II copurifies with yeast DNA topoisomerase II and re-activates the dephosphorylated enzyme. J Cell Sci. 1993 Feb;104(Pt 2):533–543. [PubMed]
  • Goldenring JR, Casanova JE, DeLorenzo RJ. Tubulin-associated calmodulin-dependent kinase: evidence for an endogenous complex of tubulin with a calcium-calmodulin-dependent kinase. J Neurochem. 1984 Dec;43(6):1669–1679. [PubMed]
  • Hofmann K, Bucher P, Falquet L, Bairoch A. The PROSITE database, its status in 1999. Nucleic Acids Res. 1999 Jan 1;27(1):215–219. [PMC free article] [PubMed]
  • Vora RA, Pegg AE, Ealick SE. A new model for how O6-methylguanine-DNA methyltransferase binds DNA. Proteins. 1998 Jul 1;32(1):3–6. [PubMed]
  • Wibley JE, Pegg AE, Moody PC. Crystal structure of the human O(6)-alkylguanine-DNA alkyltransferase. Nucleic Acids Res. 2000 Jan 15;28(2):393–401. [PMC free article] [PubMed]
  • Fainstein E, Einat M, Gokkel E, Marcelle C, Croce CM, Gale RP, Canaani E. Nucleotide sequence analysis of human abl and bcr-abl cDNAs. Oncogene. 1989 Dec;4(12):1477–1481. [PubMed]
  • Firmbach-Kraft I, Byers M, Shows T, Dalla-Favera R, Krolewski JJ. tyk2, prototype of a novel class of non-receptor tyrosine kinase genes. Oncogene. 1990 Sep;5(9):1329–1336. [PubMed]
  • Winston JT, Koepp DM, Zhu C, Elledge SJ, Harper JW. A family of mammalian F-box proteins. Curr Biol. 1999 Oct 21;9(20):1180–1182. [PubMed]
  • Jans DA, Hübner S. Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol Rev. 1996 Jul;76(3):651–685. [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem Compound links
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...