Model-driven experimental approach reveals the complex regulatory distribution of p53 by the circadian factor Period 2.
The circadian clock and cell cycle networks are interlocked on the molecular level, with the core clock loop exerting a multilevel regulatory role over cell cycle components. This is particularly relevant to the circadian factor Period 2 (Per2), which modulates the stability of the tumor suppressor p53 in unstressed cells and transcriptional activity in response to genotoxic stress. Per2 binding prevents Mdm2-mediated ubiquitination of p53 and, therefore, its degradation, and oscillations in the peaks of Per2 and p53 were expected to correspond. However, our findings showed that Per2 and p53 rhythms were significantly out-of-phase relative to each other in cell lysates and in purified cytoplasmic fractions. These seemingly conflicting experimental data motivated the use of a combined theoretical and experimental approach focusing on the role played by Per2 in dictating the phase of p53 oscillations. Systematic modeling of all possible regulatory scenarios predicted that the observed phase relationship between Per2 and p53 could be simulated if (i) p53 was more stable in the nucleus than in the cytoplasm, (ii) Per2 associates to various ubiquitinated forms of p53, and (iii) Per2 mediated p53 nuclear import. These predictions were supported by a sevenfold increase in p53's half-life in the nucleus and by in vitro binding of Per2 to the various ubiquitinated forms of p53. Last, p53's nuclear shuttling was significantly favored by ectopic expression of Per2 and reduced because of Per2 down-regulation. Our combined theoretical/mathematical approach reveals how clock regulatory nodes can be inferred from oscillating time course data.
Integrated Cellular Responses Laboratory, Department of Biological Sciences, Biocomplexity Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
Tetsuya
T
Gotoh
Department of Mathematical Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34131, Korea; finkielc@vt.edu jaekkim@kaist.ac.kr.
Jae Kyoung
0000-0001-7842-2172
JK
Kim
Integrated Cellular Responses Laboratory, Department of Biological Sciences, Biocomplexity Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
Jingjing
J
Liu
Integrated Cellular Responses Laboratory, Department of Biological Sciences, Biocomplexity Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
Marian
M
Vila-Caballer
Integrated Cellular Responses Laboratory, Department of Biological Sciences, Biocomplexity Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
Philip E
PE
Stauffer
Computational Cell Biology Laboratory, Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
John J
JJ
Tyson
Integrated Cellular Responses Laboratory, Department of Biological Sciences, Biocomplexity Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; finkielc@vt.edu jaekkim@kaist.ac.kr.
Carla V
CV
Finkielstein
D002467:Cell Nucleus / Q000378:metabolism; D057906:Circadian Clocks / Q000235:genetics; D003198:Computer Simulation; D005786:Gene Expression Regulation; D045325:HCT116 Cells; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D056950:Period Circadian Proteins / Q000378:metabolism*; D021381:Protein Transport; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000378:metabolism; D054875:Ubiquitination
C421902:PER2 protein, human; D056950:Period Circadian Proteins; D016159:Tumor Suppressor Protein p53; D057149:Ubiquitinated Proteins; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2
Period 2; circadian rhythms; mathematical modeling; p53; tumor suppressor