Cytomegalovirus Promoter Up-regulation Is the Major Cause of Increased Protein Levels of Unstable Reporter Proteins after Treatment of Living Cells with Proteasome Inhibitors

Beatriz Alvarez-Castelao,Idoia Martín-Guerrero,África García-Orad,José G Castaño

doi:10.1074/jbc.m109.004101

Abstract

Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.

Highlights

We show here that this accumulation is mainly due to Second, active ubiquitin is transferred to one of the several transcriptional up-regulatiTohniosfatrhteiccleythoamsebgealeonviwruitshpdrroamwontebry thUeBaCu/Eth2orusb.iIqnuFitiign.-3co, nthjuegsaatimnge iemnzaygmesesw. eTrehen ubiquitin is by proteasome inhibitors aunsdemdetdoiarteepdr,eastelenatstthine prearstu,lbtsy oAfPd1iffeurseunatlleyxtpraenrsimferernetdatlocaomndeimtiobnersoffotrhEeGEF3Pudb2iqmuiRtiNnAligase family transactivation
We have presented experimental evidence showing that transcriptional up-regulation of the CMV promoter by proteasome inhibitors is mainly responsible of the increased levels of unstable GFP in treated cells rather than inhibition of the degradation of the reporter protein by the UPS
In the context of the CMV promoter it that end and based on the results shown above, it is clear that an would be expected that proteasome inhibitors could prevent increase in the amounts of unstable GFP proteins is needed to NF␬B/Rel activation by inhibition of I␬␤ degradation (45), actiaccurately measure its degradation because of the low steady- vate AP1 by stabilization of both Jun and Fos transcription facstate levels of the unstable proteins

Summary

EXPERIMENTAL PROCEDURES

Untreated cells showed little fluorescence, and after addition of proteasome inhibitors a dose-dependent increase in the amount of the reporter fusion protein was invariably obtained in parallel with increase levels of the reported protein as demonstrated by Western blot analysis (Fig. 1). Fluorescence microscopy analysis of EYFP-C-Cot transiently transfected PC12 and NIH3T3 cells treated with different doses of proteasome inhibitors. Dation of similar amplification efficiencies for target and refer- Transcriptional Regulation of CMV-driven Fluorescence ence genes (100 Ϯ 5%), the relative expression levels were Reporter Constructs—The above results could be a particular analyzed by the ⌬⌬CT comparative method (20) from three property of our reporter EYFP-C-Cot construct, so we decided different experiments. Results are expressed as -fold C-terminal amino acids 422– 461 from ornithine decarboxylchanges, taking the control (no treatment) as a reference value ase), a direct substrate of the 26 S proteasome

RESULTS

Findings

DISCUSSION