Methods for measuring PERK activation

Similar to IRE1α, PERK also undergoes transautophosphorylation upon ER stress. Phosophorylation levels of PERK can be detected by a phospho-specific PERK antibody (Cell Signaling Technologies, Danvers, MA). Upon activation, PERK phosphorylates eIF2α to reduce global mRNA translation. Measuring eIF2α phosphorylation levels by immunoblot using anti-phopho-eIF2α specific antibody (Cell Signaling, Danvers, MA) indirectly reflects PERK activation. However it must be noted that other eIF2α kinases exist and therefore proper controls should be included to confirm PERK dependent eIF2α phosphorylation.(1)

Methods for measuring ATF6α activation

As mentioned previously, in response to ER stress, ATF6α (90 kDa) transits to the Golgi apparatus and cleaved by SP1 and S2P producing a 50 kDa form which translocates to the nucleus to activate transcription of UPR genes [2]. By transfecting cells with a GFP-ATF6α fusion protein, ATF6 translocation events upon ER stress can be monitored by fluorescence microscropy.(3)

Studying UPR downstream markers and responses

Immunostaining and immunofluorescence for downstream markers of the UPR

Immunostaining can also be used to measure UPR activation. [CO4]The advantage of immunostaining is that we can study tissues from patients or mouse models with ER stress-related diseases. Many of the antibodies discussed previously could be used for immunocytochemistry. In addition, CHOP, BiP and PDI antibodies can be used as indicators of cells undergoing ER stress conditions. CHOP is regulated under the PERK-eIF2α-ATF4 pathway and has been shown to have a role in ER stress mediated apoptosis. However, it must be cautioned that many commercially available antibodies for detection of CHOP expression fail specificity evaluation [4]. BiP is a central regulator of the UPR stress sensors as well as an ER chaperone to assist protein folding. BiP is highly expressed in the ER and can be used as an ER marker. PDI is involved in oxidative protein folding in the ER lumen and its expression is induced by ER stress.

Measuring transcriptional activation of the UPR

Upon ER stress conditions, activated master regulators of the UPR communicate to the nucleus to regulate the transcription of genes involved in protein folding and processing to increase the ER protein folding capacity, ERAD and autophagy components to reduce the ER workload, and cell survival and death factors to determine the fate of the cell depending on the ER stress condition.(5)

Many of the genes regulated by the UPR contain unique cis-acting response elements within their promoters. These include ERSE (ER stress response element, 5'-CCAAT-N9-CCACG-3'), ERSE-II (ER stress response element II, 5’-ATTGG-N1-CCACG-3’) and the UPRE (Unfolded Protein Response element, 5’-TGACGTGG/A-3’). (6)

 

Footnotes

1.Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D. Mol Cell. 2000;5:897–904. [PubMed]

2. Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D. Mol Cell. 2000;6:1099–1108. [PubMed]

3. Yoshida H, Haze K, Yanagi H, Yura T, Mori K. The Journal of biological chemistry. 1998;273:33741–33749. [PubMed]

4. Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R, Brown MS, Goldstein JL. Mol Cell. 2000;6:1355–1364. [PubMed]

5. Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M, Mori K. Mol Cell Biol. 2000;20:6755–6767. [PMC free article] [PubMed]

6. Haze K, Yoshida H, Yanagi H, Yura T, Mori K. Mol Biol Cell. 1999;10:3787–3799. [PMC free article] [PubMed]