title: Controlled access of p53 to the nucleus regulates its proteasomal degradation by MDM2.
abstract: The tumor suppressor p53 can be sent to the proteasome for degradation by placing its nucleo-cytoplasmic shuttling under ligand control. Endogenous p53 is ubiquitinated by MDM2 in the nucleus, and controlling the access of p53 to the nuclear compartment regulates its ubiquitination and proteasomal degradation. This was accomplished by the use of a protein switch that places nuclear translocation under the control of externally applied dexamethasone. Fluorescence microscopy revealed that sending protein switch p53 (PS-p53) to the nucleus produces a distinct punctate distribution in both the cytoplasm and nucleus. The nuclear role in accessing the proteasome was investigated by inhibiting classical nuclear export with leptomycin B. Trapping PS-p53 in the nucleus only allows this punctate staining in that compartment, suggesting that PS-p53 must translocate first to the nuclear compartment for cytoplasmic punctate staining to occur. The role of MDM2 binding was explored by inhibiting MDM2/p53 binding with nutlin-3. Inhibition of this interaction blocked both nuclear export and cytoplasmic and nuclear punctate staining, providing evidence that any change in localization after nuclear translocation is due to MDM2 binding. Further, blocking the proteolytic activity of the proteasome maintained the nuclear localization of the construct. Truncations of p53 were made to determine smaller constructs still capable of interacting with MDM2, and their subcellular localization and degradation potential was observed. PS-p53 and a smaller construct containing the two MDM2 binding regions of p53 (Box I + V) were indeed degraded by the proteasome as measured by loss of enhanced green fluorescent protein that was also fused to the construct. The influence of these constructs on p53 gene transactivation function was assessed and revealed that PS-p53 decreased gene transactivation, while PS-p53(Box I + V) did not significantly change baseline gene transactivation.
authors: Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
authors: James R
authors: JR
authors: Davis
authors: Mohanad
authors: M
authors: Mossalam
authors: Carol S
authors: CS
authors: Lim
mesh_terms: D021581:Active Transport, Cell Nucleus; D000970:Antineoplastic Agents / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism*; D002626:Chemistry, Pharmaceutical; D003593:Cytoplasm / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D008024:Ligands; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence; D009154:Mutation; D010879:Piperazines / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D005231:Fatty Acids, Unsaturated; D007093:Imidazoles; D008024:Ligands; D010879:Piperazines; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; C482205:nutlin 3; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D046988:Proteasome Endopeptidase Complex; C038753:leptomycin B
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