c-Jun is phosphorylated by the DNA-dependent protein kinasein vitro; definition of the minimal kinase recognition motif

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Abstract
The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here, we show that the DNA-PK modifies c-Jun in vitro and that serlne residue 249 (Ser-249) is required for phosphorylatlon to occur. This residue corresponds to one of three sites of c-Jun that are phosphorylated in vivo and which negatively regulate c-Jun DNA binding in vitro. However, we find that phosphorylation of c-Jun by the DNA-PK does not interfere with DNA binding, Indicating that phosphorylation at other sites is required for this effect. Mutagenesls of the phosphorylated region of c-Jun reveals that the primary amino acid sequence recognised by the DNA-PK consists of the sequence Ser–Gin, and that adjacent addle residues potentiate kinase activity. Furthermore, when this site is placed within the context of a second protein, It confers DNA-PK directed phosphorylation upon that protein. Our findings will facilitate identification of DNA-PK phosphorylation sites in other transcription factors.