14-3-3gamma binds to MDMX that is phosphorylated by UV-activated Chk1, resulting in p53 activation.
It has been shown that MDMX inhibits the activity of the tumor suppressor p53 by primarily cooperating with the p53 feedback regulator MDM2. Here, our study shows that this inhibition can be overcome by 14-3-3gamma and Chk1. 14-3-3gamma was identified as an MDMX-associated protein via an immuno-affinity purification-coupled mass spectrometry. Consistently, 14-3-3gamma directly interacted with MDMX in vitro, and this interaction was stimulated by MDMX phosphorylation in vitro and in cells. Interestingly, in response to UV irradiation, the wild-type, but not the kinase-dead mutant, Chk1 phosphorylated MDMX at serine 367, enhanced the 14-3-3gamma-MDMX binding and the cytoplasmic retaining of MDMX. The Chk1 specific inhibitor UCN-01 repressed all of these effects. Moreover, overexpression of 14-3-3gamma, but not its mutant K50E, which did not bind to MDMX, suppressed MDMX-enhanced p53 ubiquitination, leading to p53 stabilization and activation. Finally, ablation of 14-3-3gamma by siRNA reduced UV-induced p53 level and G1 arrest. Thus, these results demonstrate 14-3-3gamma and Chk1 as two novel regulators of MDMX in response to UV irradiation.
Jin
Yetao
Y
Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA.
Dai
Mu-Shui
MS
Lu
Steven Z
SZ
Xu
Yingda
Y
Luo
Zhijun
Z
Zhao
Yingming
Y
Lu
Hua
H
D048948:14-3-3 Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D016193:G1 Phase / Q000528:radiation effects; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014466:Ultraviolet Rays
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1