DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination.
Nonhomologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. Several proteins, including the DNA-dependent protein kinase (DNA-PK), XRCC4 and DNA ligase IV, are required for nonhomologous end joining both in vitro and in vivo. Since XRCC4 is recruited to the DNA double-strand break with DNA-PK, and because the protein kinase activity of DNA-PK is required for its in vivo function, we reasoned that XRCC4 could be a potential physiological substrate of DNA-PK. Here, we have used mass spectrometry to map the DNA-PK phosphorylation sites in XRCC4. Two major phosphorylation sites (serines 260 and 318), as well as several minor sites were identified. All of the identified sites lie within the carboxy-terminal 100 amino acids of XRCC4. Substitution of each of these sites to alanine (in combination) reduced the ability of DNA-PK to phosphorylate XRCC4 in vitro by at least two orders of magnitude. However, XRCC4-deficient cells that were complemented with XRCC4 lacking DNA-PK phosphorylation sites were analogous to wild type XRCC4 with respect to survival after ionizing radiation and ability to repair DSBs introduced during V(D)J recombination.
Department of Biochemistry & Molecular Biology, University of Calgary, 3330 Hospital Drive, NW, Calgary, Alta, Canada T2N 1N4.
Yaping
Y
Yu
Wei
W
Wang
Qi
Q
Ding
Ruiqiong
R
Ye
Dawn
D
Chen
Dennis
D
Merkle
David
D
Schriemer
Katheryn
K
Meek
Susan P
SP
Lees-Miller
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002470:Cell Survival / Q000528:radiation effects*; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004307:Dose-Response Relationship, Radiation; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011839:Radiation, Ionizing; D011995:Recombination, Genetic; D045567:VDJ Recombinases / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D045567:VDJ Recombinases; D000409:Alanine