Phosphorylation of Thr18 and Ser20 of p53 in Ad-p53-induced apoptosis.
The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53's ability to induce cell cycle arrest versus apoptosis, particularly because wild-type p53 cells are resistant to Ad-p53-induced apoptosis. Here we use Ad-p53 constructs to test the hypothesis that simultaneous phosphorylation of p53 at threonine 18 (Thr18) and serine 20 (Ser20) is causally associated with p53-mediated apoptosis. Studies using phosphorylation-specific antibodies demonstrated that p53-induced apoptosis correlates with phosphorylation of p53 at Thr18 and Ser20 but not with carboxy-terminal phosphorylation (Ser392). To prove a causal relationship between apoptosis and Thr18 and Ser20 phosphorylation of p53, the effects of an adenoviral p53 construct that was not phosphorylated (Ad-p53) was compared with a Thr18/Ser20 phosphomimetic construct (Ad-p53-18D20D) in wild-type p53 gliomas. Whereas treatment with Ad-p53 resulted only in cell cycle arrest, treatment with Ad-p53-18D20D induced dramatic apoptosis. Microarray and Western blot analyses showed that only Ad-p53-18D20D was capable of inducing expression of apoptosis-inducing proteins. Chromatin immunoprecipitation assays indicated that the protein product of Ad-p53-18D20D, but not Ad-p53, was capable of binding to apoptosis-related genes. We thus conclude that phosphorylation of Thr18 and Ser20 is sufficient for inducing p53-mediated apoptosis in glioma cells. These results have implications for p53 gene therapy and inform other strategies that aim to restore p53 function.
Nakamizo
Akira
A
Department of Neurosurgery, Brain Tumor Center, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit 442, Houston, TX 77030-4009, USA.
Amano
Toshiyuko
T
Zhang
Wei
W
Zhang
Xin-Qiao
XQ
Ramdas
Latha
L
Liu
Ta-Jen
TJ
Bekele
B Nebiyou
BN
Shono
Tadahisa
T
Sasaki
Tomio
T
Benedict
William F
WF
Sawaya
Raymond
R
Lang
Frederick F
FF
D000256:Adenoviridae; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D047468:Immunoprecipitation; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D013912:Threonine; D012694:Serine