MEKK1/JNK signaling stabilizes and activates p53

Activation of the tumor suppressor p53 by stress and damage stimuli often correlates with induction of stress kinases, Jun-NH(2) kinase (JNK). As JNK association with p53 plays an important role in p53 stability, in the present study we have elucidated the relationship between the JNK-signaling pathway and p53 stability and activity. Expression of a constitutively active form of JNKK upstream kinase, mitogen-activated protein kinase kinase kinase (Delta MEKK1), increased the level of the exogenously transfected form of p53 in p53 null (10.1) cells as well as of endogenous p53 in MCF7 breast cancer cells. Increased p53 level by forced expression of Delta MEKK1 coincided with a decrease in p53 ubiquitination in vivo and with prolonged p53 half-life. Computerized modeling of the JNK-binding site (amino acids 97-116; p7 region) enabled us to design mutations of exposed residues within this region. Respective mutations (p53(101-5-8)) and deletion (p53(Delta p7)) forms of p53 did not exhibit the same increase in p53 levels upon Delta MEKK1 expression. In vitro phosphorylation of p53 by JNK abolished Mdm2 binding and targeting of p53 ubiquitination. Similarly, Delta MEKK1 expression increased p53 phosphorylation by immunopurified JNK and dissociated p53-Mdm2 complexes. Transcriptional activity of p53, as measured via mdm2 promoter-driven luciferase, exhibited a substantial increase in Delta MEKK1-expressing cells. Cotransfection of p53 and Delta MEKK1 into p53 null cells potentiated p53-dependent apoptosis, suggesting that MEKK1 effecters contribute to the ability of p53 to mediate programmed cell death. Our results point to the role of MEKK1-JNK signaling in p53 stability, transcriptional activities, and apoptotic capacity as part of the cellular response to stress.