The DNA-dependent protein kinase catalytic subunit phosphorylation sites in human Artemis.
Artemis protein has irreplaceable functions in V(D)J recombination and nonhomologous end joining (NHEJ) as a hairpin and 5' and 3' overhang endonuclease. The kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is necessary in activating Artemis as an endonuclease. Here we report that three basal phosphorylation sites and 11 DNA-PKcs phosphorylation sites within the mammalian Artemis are all located in the C-terminal domain. All but one of these phosphorylation sites deviate from the SQ or TQ motif of DNA-PKcs that was predicted previously from in vitro phosphorylation studies. Phosphatase-treated mammalian Artemis and Artemis that is mutated at the three basal phosphorylation sites still retain DNA-PKcs-dependent endonucleolytic activities, indicating that basal phosphorylation is not required for the activation. In vivo studies of Artemis lacking the C-terminal domain have been reported to be sufficient to complement V(D)J recombination in Artemis null cells. Therefore, the C-terminal domain may have a negative regulatory effect on the Artemis endonucleolytic activities, and phosphorylation by DNA-PKcs in the C-terminal domain may relieve this inhibition.
Ma
Yunmei
Y
Department of Pathology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90089-9176, USA.
Pannicke
Ulrich
U
Lu
Haihui
H
Niewolik
Doris
D
Schwarz
Klaus
K
Lieber
Michael R
MR
D016367:Baculoviridae / Q000235:genetics; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004720:Endonucleases; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C426344:DCLRE1C protein, human; D004720:Endonucleases