Abstract
Stress imposed on the endoplasmic reticulum (ER) induces the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2) on Ser51. This results in transient inhibition of general translation initiation while concomitantly activating a signaling pathway that promotes the expression of genes whose products improve ER function. Conversely, dephosphorylation of eIF2alphaSer51 is accomplished by protein phosphatase 1 (PP1c) complexes containing either the protein CReP or GADD34, which target PP1c to eIF2. Here, we demonstrate that the Src homology (SH) domain-containing adaptor Nck is a key component of a molecular complex that controls eIF2alpha phosphorylation and signaling in response to ER stress. We show that overexpression of Nck decreases basal and ER stress-induced eIF2alpha phosphorylation and the attendant induction of ATF4 and CHOP. In contrast, we demonstrate that the mouse embryonic fibroblasts lacking both isoforms of Nck (Nck1-/-Nck2-/-) show higher levels of eIF2alpha phosphorylation and premature induction of ATF4, CHOP, and GADD34 in response to ER stress and finally, are more resistant to cell death induced by prolonged ER stress conditions. We establish that a significant amount of Nck protein localizes at the ER and is in a complex with eIF2 subunits. Further analysis of this complex revealed that it also contains the Ser/Thr phosphatase PP1c, its regulatory subunit CReP, and dephosphorylates eIF2alpha on Ser51 in vitro. Overall, we demonstrate that Nck as a component of the CReP/PP1c holophosphatase complex contributes to maintain eIF2alpha in a hypophosphorylated state. In this manner, Nck modulates translation and eIF2alpha signaling in response to ER stress.
Highlights
In a previous study, we uncovered a novel function for Nck in modulating mRNA translation at the level of initiation through its direct interaction with the -subunit of the eukaryotic initiation factor 2 [9]. eIF2 is a heterotrimeric complex (␣, , and ␥-subunit) that in part drives the initiation of mRNA translation by carrying out the delivery of the methionyl-initiator tRNA to the 40 S ribosomal subunit [10]
Four eIF2␣ kinases have been identified: 1) HRI, which couples mRNA translation with heme availability in erythroid cells [13], 2) GCN2, which is activated in response to amino acid deprivation [14], 3) PKR, a component of the antiviral response activated by double-strand RNA [15], and 4) PERK (PKR-like endoplasmic reticulum kinase), a type 1 transmembrane protein resident of the endoplasmic reticulum (ER) and activated upon accumulation of improperly folded secretory proteins [16, 17]
Among other genes regulated by ATF4 is GADD34, which coordinates the recovery of protein synthesis by interacting with the catalytic subunit of protein phosphatase 1 (PP1c) and targeting PP1c toward eIF2 to promote eIF2␣ dephosphorylation [23]
Summary
We uncovered a novel function for Nck in modulating mRNA translation at the level of initiation through its direct interaction with the -subunit of the eukaryotic initiation factor 2 (eIF2) [9]. eIF2 is a heterotrimeric complex (␣-, -, and ␥-subunit) that in part drives the initiation of mRNA translation by carrying out the delivery of the methionyl-initiator tRNA to the 40 S ribosomal subunit [10]. The effects of overexpressing Nck on eIF2␣ phosphorylation resemble those observed after CReP and GADD34 overexpression (Fig. 1A, bottom panels), two related PP1c regulatory subunits found in holophosphatase complexes that dephosphorylate eIF2␣Ser51 in non-stressed [38] and ER-stressed cells
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