MEKK 1 / JNK signaling stabilizes and activates p 53

Activation of the tumor suppressor p53 by stress and damage stimuli often correlates with induction of stress kinases, Jun-NH2 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 (DMEKK1), 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 DMEKK1 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 (p53101-5-8) and deletion (p53Dp7) forms of p53 did not exhibit the same increase in p53 levels upon DMEKK1 expression. In vitro phosphorylation of p53 by JNK abolished Mdm2 binding and targeting of p53 ubiquitination. Similarly, DMEKK1 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 DMEKK1-expressing cells. Cotransfection of p53 and DMEKK1 into p53 null cells potentiated p53-dependent apoptosis, suggesting that MEKK1 effectors 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. Being a key target for inactivation in human cancer, the p53 tumor suppressor gene has been implicated in cell cycle control, DNA repair, replicative senescence, and programmed cell death (reviewed in refs. 1 and 2). The ability of p53 to elicit diverse regulatory functions is likely to depend on its phosphorylation pattern which is conformation dependent (3). p53 phosphorylation is mediated by several cellular kinases including casein kinase I, casein kinase II, protein kinase A, CDK7, DNA-activated protein kinase, and Jun-NH2 kinase (JNK) (3–8). Although kinases that efficiently phosphorylate p53 both in vitro and in vivo were successfully identified, the direct relationship between p53 phosphorylation by a specific signaling cascade and its ability to elicit its biologic activities in response to DNA damage is not fully understood. Among protein kinases that are expected to produce stress-activated phosphorylation of p53 are JNK and DNAactivated protein kinase, which reportedly phosphorylate residues within the amino terminal domain of p53 (4–6). DNA-activated protein kinase mediated phosphorylation of Ser-15, and Ser-37 has been shown to contribute to p53 accumulation (9). Ser-15 was identified as one of the major sites on p53 that is phosphorylated by cellular stress (10). Because the p53 response to stress and damage is preserved in cells from severe combined immunodeficient mice (10–12), we examined the role of JNK in this response. JNKs are a family of stress kinases induced by change in redox potential, heat shock, osmotic shock, UV irradiation, and inflammatory cytokines (13–16). JNK activity requires mitogenactivated protein kinases kinase (MEKK) 1–4 which phosphorylates MKK4/7. MKK4/7, in turn, phosphorylates JNK on residues 183 and 185 (17–20). Activated JNK phosphorylates its substrates, c-Jun, ATF2, ELK1, and p53 (3, 13–14, 21). Although JNK activities in response to stress and damage have been implicated in cell growth control as well as in apoptosis (22–23), little is known about JNK effectors that elicit apoptosis. JNK exerts an important regulatory function through its ability to target the ubiquitination of its unphosphorylated associated proteins (e.g., c-Jun, ATF2) in normal-growing cells. Upon its activation in response to stress, JNK mediates phosphorylation, which protects its substrates from ubiquitination and degradation (24–26). Similar to c-Jun, wild-type p53 has a short half-life, which is prolonged in response to stress or DNA damage (27). Key contributors to p53 half-life are its associated proteins Mdm2 and JNK (28, 29). Common to the ability of both Mdm2 and JNK to associate with p53 is their requirement for a specific conformation of the p53 protein (3, 9), which depends on its phosphorylation status and association with other cellular proteins. Changes in p53 conformation in response to stress-mediated phosphorylation of p53 have been documented (9, 30). JNK association with p53 requires residues 97–155 of p53 as concluded from the observation that p53 deleted of the first 97 amino acids binds well to JNK, whereas p53 that lacks the first 155 amino acids fails to associate with JNK (3). Further analysis identified amino acids 97–116 as the primary p53 sequence domain required for JNK association (29). p53 deleted of amino acids 97–116 no longer associates with JNK (29). Identifying p53 as a JNK substrate led us to examine the biologic significance of p53 phosphorylation by JNK. Herein, we provide evidence that MEKK1/JNK signaling increases p53 stability and transcriptional activation and that MEKK1/JNK potentiates the ability of p53 to initiate programmed cell death. The mechanisms underlying the effect of MEKK1/JNK signaling on p53 stability and functions are discussed. MATERIALS AND METHODS Cells. 10.1 are p53 null mouse fibroblast cells (31), 293T are adenovirus-transformed human kidney cells that express the simian virus 40 large T antigen (32). Both 10.1 and 293T cells were maintained in DMEM (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) and antibiotics in 5% CO2. MCF7 is a human breast cancer cell line maintained in RPMI 1640 supplemented with 10% fetal bovine serum and antibiotics. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9510541-6$2.00y0 PNAS is available online at www.pnas. org. Abbreviations: MEKK1, mitogen-activated protein kinases kinase 1 (also known as JNKKK); DMEKK1, a constitutively active form of MEKK1 which lacks the first 350 amino acids; JNK, Jun-NH2-terminal kinase; HA, hemagglutinin tag. ‡To whom reprint requests should be addressed at: Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1130, New York, NY 10029-6574. e-mail: zeevoronai@smtplink. mssm.edu.