Identification of phosphorylation sites on transcription factor Sp1 in response to DNA damage and its accumulation at damaged sites.
DNA damage induces hyper-phosphorylation of the Sp1 transcriptional factor. We have demonstrated that ionizing radiation-associated DNA double-strand breaks (DSBs) induce phosphorylation of at least Ser-56 and Ser-101 residues on Sp1 in an ATM-dependent manner. UV irradiation- or hydroxyurea (HU)-induced replicative stress results in phosphorylation of only the Ser-101 residue. Furthermore, silencing of the ATM- and Rad3-related protein (ATR) in ATM-deficient cells treated with HU abrogated the Ser-101 phosphorylation. Thus, phosphorylation of Ser-101 on Sp1 appears to be a general response to DNA damage dependent on both ATM and ATR. Although silencing of Sp1 expression by siRNA targeting resulted in an increase in sensitivity to ionizing radiation (IR), the Ser-101 phosphorylation did not affect transcriptional activity from the Sp1 responsive promoter. Confocal laser microscopy analysis revealed co-localization of phosphorylated Sp1 at Ser-101 with phosphorylated ATM at Ser-1981, the affected sites representing DSBs. These observations suggest that phosphorylated Sp1 might play a role in DNA repair at damage sites rather than functioning in transcriptional regulation.
Iwahori
Satoko
S
Division of Virology, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan.
Yasui
Yoshihiro
Y
Kudoh
Ayumi
A
Sato
Yoshitaka
Y
Nakayama
Sanae
S
Murata
Takayuki
T
Isomura
Hiroki
H
Tsurumi
Tatsuya
T
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004249:DNA Damage; D004261:DNA Replication / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila; D004789:Enzyme Activation / Q000528:radiation effects; D006801:Humans; D010766:Phosphorylation / Q000528:radiation effects; D010768:Phosphoserine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011839:Radiation, Ionizing; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000528:radiation effects; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; D025521:Tumor Suppressor Proteins; D010768:Phosphoserine; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases