Evidence that the human cytomegalovirus IE2-86 protein binds mdm2 and facilitates mdm2 degradation.
Levels of the p53 tumor suppressor protein are increased in human cytomegalovirus (HCMV)-infected cells and may be important for HCMV pathogenesis. In normal cells p53 levels are kept low due to an autoregulatory feedback loop where p53 activates the transcription of mdm2 and mdm2 binds and ubiquitinates p53, targeting p53 for proteasomal degradation. Here we report that, in contrast to uninfected cells, mdm2 was undetectable upon treatment of infected fibroblasts with the proteasome inhibitor MG132. Cellular depletion of mdm2 was reproducible in p53-null cells transfected with the HCMV IE2-86 protein, but not with IE172, independently of the endogenous mdm2 promoter. IE2-86 also prevented the emergence of presumably ubiquitinated species of p53. The regions of IE2-86 important for mdm2 depletion were those containing the sequences corresponding to the putative zinc finger and C-terminal acidic motifs. mdm2 and IE2-86 coimmunoprecipitated in transfected and infected cell lysates and in a cell-free system. IE2-86 blocked mdm2's p53-independent transactivation of the cyclin A promoter in transient-transfection experiments. Pulse-chase experiments revealed that IE2-86 but not IE1-72 or several loss-of-function IE2-86 mutants increased the half-life of p53 and reduced the half-life of mdm2. Short interfering RNA-mediated depletion of IE2-86 restored the ability of HCMV-infected cells to accumulate mdm2 in response to proteasome inhibition. Taken together, the data suggest that specific interactions between IE2-86 and mdm2 cause proteasome-independent degradation of mdm2 and that this may be important for the accumulation of p53 in HCMV-infected cells.
CB #7295, Lineberger Comprehensive Cancer Center, Rm. 32-026, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295, USA.
Zhigang
Z
Zhang
David L
DL
Evers
Joseph F
JF
McCarville
Jean-Christophe
JC
Dantonel
Shu-Mei
SM
Huong
Eng-Shang
ES
Huang
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D019925:Cyclin A / Q000235:genetics; D006207:Half-Life; D006801:Humans; D017874:Immediate-Early Proteins / Q000502:physiology*; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D015534:Trans-Activators / Q000502:physiology*; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
D019925:Cyclin A; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D007976:Leupeptins; D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D051736:Proto-Oncogene Proteins c-mdm2; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde