Signaling in the general control response (the yeast counterpart of the mammalian integrated stress response) is dependent on phosphorylation of eIF2α, as substitution of the residue corresponding to serine 51 in yeast eIF2α prevents activation of genes by amino acid starvation (Williams et al. 1989
; Dever et al. 1992
). We have found previously that the integrated stress response requires the activity of the eIF2α kinases PERK and GCN2, and is mediated by translational regulation of ATF4 (Harding et al. 2000a
). Here, we extend these observations to show that a GSE that promotes the dephosphorylation of eIF2α is able to block the integrated stress response. Activation of the stress-induced eIF2α kinases is unaffected by this GSE, indicating that the latter does not interfere with the development of the proximal stress signal. Our findings further support the notion that the activation of gene expression by the integrated stress response is mediated by eIF2α phosphorylation in mammalian cells.
The GSE we identified is a product of the GADD34
gene. Its ability to interfere with signaling in the integrated stress response correlates with its ability to bind the catalytic subunit of PP1 and to dephosphorylate eIF2α. Binding to PP1c is shared by the structurally related HSV protein γ1
34.5, and lysates of cells infected with γ1
34.5-expressing virus also have markedly increased activity of an eIF2α phosphatase (He et al. 1997
, He et al. 1998
). Together, these findings suggest that GADD34 exerts its activity on the integrated stress response by promoting the dephosphorylation of eIF2α. It seems likely that HSV has coopted this cellular mechanism to prevent host protein synthesis shutdown when the eIF2α kinase PKR is activated by viral infection, as suggested originally by Roizman and colleagues (He et al. 1998
). Although γ1
34.5's essential role in viral infectivity is well established, it is not known if inhibition of host gene activation is part of this essential role. In this regard, it is interesting to note that a dominant negative mutation in the yeast PP1c homologue, GLC7, is able to derepress a transcriptional program that is dependent on signaling by the eIF2α kinase, Gcn2p (Wek et al. 1992
Suppression of the integrated stress response by the COOH-terminal fragment of GADD34 is significantly greater than that by the full-length protein. This difference does not appear to be due to the higher level of expression of the smaller fragment, or to significantly reduced ability to associate with the catalytic subunit of the phosphatase ( C). Furthermore, the full-length GADD34 was able to impart high levels of phosphatase activity to lysates of transfected cells or to immune complexes obtained from transfected cells ( B and D). This suggests that its ability to promote the dephosphorylation of eIF2α was unmasked by cell lysis and detergent extraction. We noted that the COOH-terminal active fragment of GADD34 is distributed throughout the cytoplasm, whereas the full-length protein is localized to a reticular structure costained with antiserum to ER markers ( C and data not shown). It is tempting to speculate that the reduced activity of the full-length protein may be due to its sequestration at an inactive site. If correct, this suggests a means to regulate GADD34 activity posttranslationally as well as at the level of the gene's expression.
What might be the physiological significance of GADD34's potential activity as an inhibitor of the integrated stress response? It had previously been noted that eIF2α phosphorylation and protein synthesis inhibition are transient in stressed cells (Prostko et al. 1992
, Prostko et al. 1993
). GADD34 is activated under physiologically stressful conditions (Fornace et al. 1989
; Doutheil et al. 1999
), and its profile of induction is very similar to other targets of the integrated stress response such as CHOP
. Indeed, we find that GADD34 induction by ER stress is dependent on the activity of the eIF2α kinases PERK and GCN2 ( and ). Therefore, it seems likely that transcriptional induction of GADD34 is part of a negative feedback loop that attenuates signaling in the integrated stress response. Other stress-responsive signaling pathways have elaborated components that serve similar negative feedback functions. For example, the mitogen-activated protein kinase pathway is inhibited by dual-specificity phosphatases that are transcriptionally induced by mitogen-activated protein kinase signaling (English et al. 1999
; Camps et al. 2000
), and cytokine signaling is inhibited by products of suppressor of cytokine signaling (SOCS) genes that are themselves positively regulated at the transcriptional level by binding of cytokines to their receptors (Krebs and Hilton 2000
). The possible application of this principle to the integrated stress response is depicted in cartoon form in .
Figure 8 Model depicting GADD34's role in inhibiting signaling through the integrated stress response pathway. GADD34 is induced transcriptionally by the eIF2α kinases PERK and GCN2. It associates with PP1c to reduce levels of eIF2α phosphorylation (more ...)
There are several theoretical reasons why the phosphorylation of eIF2α may have evolved as a transient signal in stressed cells. Sustained eIF2α phosphorylation is lethal to cells in culture (Srivastava et al. 1998
), and in ischemic neurons, sustained eIF2α phosphorylation correlates with death in vivo (DeGracia et al. 1997
; Paschen and Doutheil 1999
). Early in the UPR, high levels of eIF2α phosphorylation inhibit translation of most proteins, including some like BiP that are induced later in the course of the response (Harding et al. 2000a
, Figure 5C therein). We speculate that in addition to inhibiting further signaling, the activation of GADD34 and the attendant delayed dephosphorylation of eIF2α provide a means for cells to translate mRNAs like BiP, whose induction occurred earlier, during the active phase of the integrated stress response.