Jun NH₂-Terminal Kinase Phosphorylation of p53 on Thr-81 Is Important for p53 Stabilization and Transcriptional Activities in Response to Stress

Abstract

The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH₂-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.

ACKNOWLEDGMENTS

We thank Victor Adler, Karen Quadrini, and Ekaterina Matusevich for technical assistance, Roger Davis, Michael Karin, and Audrey Minden for stress kinase constructs, Xei Wu and Toru Ouchi for p53 constructs, and Hidetoshi Tahara for NHF. We also thank James Manfredi for advice on conformation analysis.

This study was supported by grant CA78419 from the National Cancer Institute (to Z.R.) and by an NIH shared instrumentation grant (1 S10 RR0 9145-01) and NSF Major Research Instrumentation grant (DBI-9 724504).