Cell Signaling Technology

Product Pathways - Cell Cycle / Checkpoint

Phospho-DNA-PK (Ser2056) Antibody #4215

Applications Reactivity Sensitivity MW (kDa) Source
W H (Mk) Endogenous 450 Rabbit

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

Phospho-DNA-PK (Ser2056) Antibody recognizes endogenous levels of DNA-PK protein only when phosphorylated at Ser2056.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ser2056 of human DNA-PK protein. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from HCT 116 cells, untreated (-) or treated (+) with neocarzinostatin (NCS, 1 hr, 10 μM), using Phospho-DNA-PK (Ser2056) Antibody (upper) and DNA-PK Antibody #4602 (lower).

Background

DNA-dependent protein kinase (DNA-PK) is an important factor in the repair of double-stranded breaks in DNA. Cells lacking DNA-PK or in which DNA-PK is inhibited fail to show proper nonhomologous end-joining (NHEJ) (1-7). DNA-PK is composed of two DNA-binding subunits (Ku70 and Ku86) and one 450 kDa catalytic subunit (DNA-PKcs) (8). It is thought that a heterodimer of Ku70 and Ku86 binds to double-stranded DNA broken ends before DNA-PKcs binds and is activated (1,9). Activated DNA-PKcs is a serine/threonine kinase that has been shown to phosphorylate a number of proteins in vitro, including p53, transcription factors, RNA polymerase, and Ku70/Ku86 (10,11). DNA-PKcs autophosphorylation at multiple sites, including Thr2609 and Ser2056, results in an inactivation of DNA-PK kinase activity and NHEJ ability (12,13). It has been demonstrated, however, that DNA-PK preferentially phosphorylates substrates before it autophosphorylates, suggesting that DNA-PK autophosphorylation may play a role in disassembly of the DNA repair machinery (14,15). Autophosphorylation at Thr2609 has also been shown to be required for DNA-PK-mediated double strand break repair, and phosphorylated DNA-PK co-localizes with H2A.X and 53BP1 at sites of DNA damage (16). Phosphorylation at Ser2056 occurs in response to double-stranded DNA breaks and ATM activation (17).

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  2. Hartley, K. O. et al. (1995) Cell 82, 840-856.
  3. Rosenzweig, K. E. et al. (1997) Clin. Cancer Res. 3, 1149-1156.
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  7. Chen, S. et al. (2001) J. Biol. Chem. 276, 24323-24330.
  8. Jeggo, P.A. (1997) Mutat. Res. 384, 1-14.
  9. Suwa, A. et al. (1994) Proc. Natl. Acad. Sci. USA 91, 6904-6908.
  10. Anderson, C.W. and Lees-Miller, S.P. (1992) Crit. Rev. Eukaryot. Gene Expr. 2, 283-314.
  11. Kuhn, A. et al. (1995) Genes Dev. 9, 193-203.
  12. Chan, D.W. and Lees-Miller, S.P. (1996) J. Biol. Chem. 271, 8936-8941.
  13. Douglas, P. et al. (2002) Biochem. J. 368, 243-251.
  14. Lees-Miller, S. P. et al. (1992) Mol. Cell. Biol. 12, 5041-5049.
  15. Jackson, S. P. et al. (1990) Cell 63, 155-165.
  16. Chan, D. W. et al. (2002) Genes Dev. 16, 2333-2338.
  17. Yajima, H. et al. (2009) J Mol Biol 385, 800-10.

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