Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr²⁶⁰⁹, Ser²⁶¹², Thr²⁶²⁰, Ser²⁶²⁴, Thr²⁶³⁸ and Thr²⁶⁴⁷) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr²⁶⁰⁹, Ser²⁶¹², Thr²⁶³⁸, Thr²⁶⁴⁷ and Ser³²⁰⁵) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser²⁶¹², Thr²⁶³⁸ and Thr²⁶⁴⁷ in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.