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p53蛋白質のDNA結合と傷害DNA認識におよぼすJNKによるリン酸化の影響：源　利成¹, Victor ADLER^3,, 磨伊正義², Ze'ev RONAI^3, (金沢大・がん研・¹遺伝子診断, ²腫瘍外科, ^3,Derald H. Ruttenberg Cancer Ctr., Mount Sinai Sch. of Med.)

JNK phosphorylation of p53 decreases DNA binding and increases DNA damage recognition：Toshinari MINAMOTO¹, Victor ADLER^3,, Masayoshi MAI², Ze'ev RONAI^3, (Div. of ¹Diag. Mol. Pathol. and ²Surg. Oncol., Cancer Res. Inst., Kanazawa Univ., ^3,Derald H. Ruttenberg Cancer Ctr., Mount Sinai Sch. of Med.)

We have examined the effect of p53 phosphorylation on sequence-specific DNA binding, transcriptional activities and recognition of damaged DNA. Using baculovirus and bacterially expressed human wild-type p53 proteins (p53^wt) we showed that while phosphorylation by either protein kinase A (PKA) or casein kinase II (CKII) led to modest (10-15%) increase, phosphorylation by DNA-dependent protein kinase (DNA-PK) led to a marked increase (120%) in p53wt binding to the p21^CIP/WAF1 promoter sequence. Conversely phosphorylation by Jun N-terminal kinase (JNK) decreased (30-50%) p53^wt binding to p21 target sequence. p53 phosphomutants at codon 9 (p53⁹) or 15 (p53¹⁵) gained DNA binding activity by phosphorylation with PKA, CKII or JNK (25-70%). Comparing with PKA and CKII, phosphorylation by JNK led to the highest increase in p53 affinity to damaged DNA. As in vivo changes in p53 phosphorylation are also expected to occur after exposure to DNA damage, we have monitored transcriptional activities mediated by p21^CIP/WAF1 promoter- driven CAT construct in p53 null cells transfected with wild-type or mutant p53 cDNAs. While transcriptional activities of p53^wt increased in UV-treated cells, p53⁹ or p53¹⁵ exhibit high transcriptional activities that were inhibited upon UV treatment. In all our data showed that JNK phosphorylation of p53 increases its affinity for association with damaged DNA, while decreasing its DNA binding activities. Increased binding and possibly transcriptional activities of p53 after UV irradiation could be attributed to subsequent phosphorylation of p53 by kinase(s) other than JNK, including DNA-PK. Our study points to the delicate balance of p53 phosphorylation by multiple kinases, each of which exert different biological activities.