DNA-PK assay

Anderson, Carl W.; Connelly, Margery A.

Title: DNA-PK assay

Patent · Tue Oct 12 00:00:00 EDT 2004

OSTI ID:1175070

Anderson, Carl W.; Connelly, Margery A.

The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

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Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Organization:: USDOE

DOE Contract Number:: AC02-98CH10886

Assignee:: Brookhaven Science Associates (Upton, NY)

Patent Number(s):: 6,803,203

Application Number:: 09/695,437

OSTI ID:: 1175070

Country of Publication:: United States

Language:: English

References (16)

Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Lees-Miller, S. P.; Chen, Y. R.; Anderson, C. W. Molecular and Cellular Biology, Vol. 10, Issue 12 https://doi.org/10.1128/MCB.10.12.6472	journal	December 1990
Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography Andersson, Lennart; Porath, Jerker Analytical Biochemistry, Vol. 154, Issue 1 https://doi.org/10.1016/0003-2697(86)90523-3	journal	April 1986
Selective adsorption of phosphoproteins on gel-immobilized ferric chelate Muszynska, Grazyna; Andersson, Lennart; Porath, Jerker Biochemistry, Vol. 25, Issue 22 https://doi.org/10.1021/bi00370a018	journal	November 1986
[3] Protein kinase phosphorylation site sequences and consensus specificity motifs: Tabulations Pearson, Richard B.; Kemp, Bruce E. Methods in Enzymology https://doi.org/10.1016/0076-6879(91)00127-I	book	January 1991
[2] Measurement of chemical phosphate in proteins Buss, Janice E.; Stull, James T. Methods in Enzymology https://doi.org/10.1016/0076-6879(83)99035-3	book	January 1983
Hsp70 binds specifically to a peptide derived from the highly conserved domain (I) region of P53 Lam, Kelvin T.; Calderwood, Stuart K. Biochemical and Biophysical Research Communications, Vol. 184, Issue 1 https://doi.org/10.1016/0006-291X(92)91174-O	journal	April 1992
[4] Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA Jackson, Richard J.; Hunt, Tim Methods in Enzymology https://doi.org/10.1016/S0076-6879(83)96008-1	book	January 1983
Studies of the kinetics and ionic requirements for the phosphorylation of ribosomal protein S6 after fertilization of Arbacia punctulata eggs Ballinger, Dennis G.; Bray, Sarah J.; Hunt, Tim Developmental Biology, Vol. 101, Issue 1 https://doi.org/10.1016/0012-1606(84)90129-5	journal	January 1984
Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Lees-Miller, S. P.; Sakaguchi, K.; Ullrich, S. J. Molecular and Cellular Biology, Vol. 12, Issue 11 https://doi.org/10.1128/MCB.12.11.5041	journal	November 1992
A synthetic peptide substrate specific for casein kinase-1 Agostinis, Patrizia; Pinna, Lorenzo A.; Meggio, Flavio FEBS Letters, Vol. 259, Issue 1 https://doi.org/10.1016/0014-5793(89)81498-X	journal	December 1989
Formation of stable p53 homotetramers and multiples of tetramers Stenger, Judith E.; Mayr, Gregory A.; Mann, Kristine Molecular Carcinogenesis, Vol. 5, Issue 2 https://doi.org/10.1002/mc.2940050204	journal	January 1992
[10] Design and use of peptide substrates for protein kinases Kemp, Bruce E.; Pearson, Richard B. Methods in Enzymology https://doi.org/10.1016/0076-6879(91)00134-I	book	January 1991
The human DNA-activated protein kinase phosphorylates simian virus 40 T antigen at amino- and carboxy-terminal sites. Chen, Y. R.; Lees-Miller, S. P.; Tegtmeyer, P. Journal of Virology, Vol. 65, Issue 10 https://doi.org/10.1128/JVI.65.10.5131-5140.1991	journal	January 1991
An immunochemical analysis of the human nuclear phosphoprotein p53 Vojtěšek, B.; Bártek, J.; Midgley, C. A. Journal of Immunological Methods, Vol. 151, Issue 1-2 https://doi.org/10.1016/0022-1759(92)90122-A	journal	July 1992
The Human DNA-Activated Protein Kinase, DNA-PK: Substrate Specificity Anderson, Carl W.; Connelly, Margery A.; Lees-Miller, Susan P. Methods in Protein Structure Analysis https://doi.org/10.1007/978-1-4899-1031-8_35	book	January 1995
Isolation of phosphorylated peptides and proteins on ion exchange papers Glass, D. B.; Masaracchia, R. A.; Feramisco, J. R. Analytical Biochemistry, Vol. 87, Issue 2 https://doi.org/10.1016/0003-2697(78)90707-8	journal	July 1978

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