ADP Ribosylation in the ER Stress Response

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Sci. Signal.  13 Nov 2012:
Vol. 5, Issue 250, pp. ec293
DOI: 10.1126/scisignal.2003769

The unfolded protein response (UPR) allows cells to respond to the increasing demand of the endoplasmic reticulum (ER) and involves the activation of three transmembrane ER-localized stress sensors: the kinases PERK and IRE-1α and the transcription factor ATF6. Jwa and Chang identified poly(ADP-ribose) polymerase (PARP)16 as an ER-localized transmembrane protein with the N-terminal and catalytic domains in the cytoplasm and the C-terminal domain in the lumen of the ER. Analysis of glutathione S-transferase (GST) fusion proteins expressed in and purified from bacteria showed that GST-PARP16 was catalytically active and ADP-ribosylated itself in a manner dependent on the conserved residues His152 and Tyr182.Using an ER microsomal preparation, ADP-ribosylation activity toward PARP16 was detected in microsomes from cells expressing green fluorescent protein (GFP) fused to PARP16 but not from cells expressing GFP-PARP16H152Q Y182A. Overexpression of GFP-PARP16 resulted in ER morphological changes typical of cells undergoing the ER stress response, and knockdown of PARP16 increased cell death in response to three different activators of the ER stress response. GFP-PARP16 ADP-ribosylation activity was increased in ER microsomes prepared from cells exposed to ER stress stimuli, and ADP-ribosylation of GFP-PERK and GFP-IRE-1α was enhanced in the ER microsomes from cells exposed to ER stress and was inhibited in ER microsomes from cells in which PARP16 was knocked down. Overexpression of GFP-PARP16, but not GFP-PARP16H152Q Y182A, stimulated PERK and IRE-1α activity, and knockdown of PARP16 compromised downstream responses mediated by PERK and IRE-1α in response to ER stress. In vitro assays suggested that GST-PARP–mediated ADP ribosylation stimulated the kinase activities of GFP-PERK and GFP–IRE-1α and the endonuclease activity of GFP–IRE-1α and also suggested that PARP16 is a substrate of each of these kinases. Activation of PERK and IRE-1α requires dissociation from the ER chaperone BiP, and knockdown of PARP16 prevented the dissociation of BiP from these two enzymes, suggesting that PARP16 not only ADP-ribosylates these two kinases on the cytosolic side of the ER but may also facilitate their release from BiP on the lumenal side of the ER.

M. Jwa, P. Chang, PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1α-mediated unfolded protein response. Nat. Cell Biol. 14, 1223–1230 (2012). [PubMed]

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