As activation of ATF6, PERK and IRE1 acts as ER-proximal UPR signal transducers, we first monitored the liberation of the cleaved ATF6 cytosolic fragment, ATF6f, and accumulation of phosphorylated PERK and XBP-1s (the form of XBP-1 derived from its spliced mRNA).
The results in Figure 6a show that, inactivation of PERK by PERK-DN attenuated eIF2 α phosphorylation and had little effect on cell proliferation, whereas Mar-induced eIF2 α phosphorylation was blunted by PERK-DN, leading to the blocking of LC3BII accumulation and partial restoration of viable cells as well as decreased cell death.
We also accounted for subcellular localization of pERK by considering its accumulation into the nucleus (detailed equations in Materials and methods Section 2).
Through live imaging and biochemical studies, we find that loss of Kri1l causes the accumulation of misfolded proteins and excessive PERK activation-dependent autophagy in HSPCs.
Four distinct eIF2α kinases can catalyze phosphorylation at this single residue, each acting in response to different cellular stress conditions: PERK senses accumulation of unfolded polypeptides in the lumen of the endoplasmic reticulum (ER), GCN2 responds to amino acid starvation and UV-light, PKR responds to viral infection, and HRI responds to heme deficiency.
Loss of Kri1l, a critical component of SSU complex, causes ribosomal biogenesis defects, accumulation of misfolded proteins and activation of PERK-eif2a signaling.
Accumulation of misfolded/unfolded proteins is known to trigger activation of PERK, which subsequently phosphorylates eif2a to inhibit protein synthesis.
The expression profile of PERK, a major ER stress sensor upregulated upon accumulation of ubiquitinylated and misfolded proteins (Walter and Ron 2011), was studied in retrogradely labeled CSMN (Supplementary Fig. 4 C–I ).
When cells encounter ER stress or misfolded protein accumulation, an ER-resident chaperone, GRP78, dissociates from PERK, IRE1 and ATF6, leading to autophosphorylation of PERK, IRE1 and mobilization of ATF6 for activation.
In mammals, three ER transmembrane proteins, IRE1, ATF6, and PERK, respond to the accumulation of unfolded proteins in the ER lumen [ 5].
