The DNA-dependent protein kinase catalytic subunit is phosphorylated in vivo on threonine 3950, a highly conserved amino acid in the protein kinase domain.
The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.
Douglas
Pauline
P
Department of Biochemistry, University of Calgary, Calgary, Alberta, Canada.
Cui
Xiaoping
X
Block
Wesley D
WD
Yu
Yaping
Y
Gupta
Shikha
S
Ding
Qi
Q
Ye
Ruiqiong
R
Morrice
Nick
N
Lees-Miller
Susan P
SP
Meek
Katheryn
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002460:Cell Line; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011839:Radiation, Ionizing; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019319:Okadaic Acid; D013912:Threonine; D011494:Protein Kinases; D051747:DNA-Activated Protein Kinase