Calcineurin interacts with PERK and dephosphorylates calnexin to relieve ER stress in mammals and frogs.
The accumulation of misfolded proteins within the endoplasmic reticulum (ER) triggers a cellular process known as the Unfolded Protein Response (UPR). One of the earliest responses is the attenuation of protein translation. Little is known about the role that Ca2+ mobilization plays in the early UPR. Work from our group has shown that cytosolic phosphorylation of calnexin (CLNX) controls Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) 2b.Here, we demonstrate that calcineurin (CN), a Ca2+ dependent phosphatase, associates with the (PKR)-like ER kinase (PERK), and promotes PERK auto-phosphorylation. This association, in turn, increases the phosphorylation level of eukaryotic initiation factor-2 alpha (eIF2-alpha) and attenuates protein translation. Data supporting these conclusions were obtained from co-immunoprecipitations, pull-down assays, in-vitro kinase assays, siRNA treatments and [35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+ concentrations and involved the cytosolic domain of PERK. CN levels were rapidly increased by ER stressors, which could be blocked by siRNA treatments for CN-Aalpha in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-alpha. CN knockdown in Xenopus oocytes predisposed them to induction of apoptosis. We also found that CLNX was dephosphorylated by CN when Ca2+ increased. These data were obtained from [gamma32P]-CLNX immunoprecipitations and Ca2+ imaging measurements. CLNX was dephosphorylated when Xenopus oocytes were treated with ER stressors. Dephosphorylation was pharmacologically blocked by treatment with CN inhibitors. Finally, evidence is presented that PERK phosphorylates CN-A at low resting levels of Ca2+. We further show that phosphorylated CN-A exhibits decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is re-established.Our data suggest two new complementary roles for CN in the regulation of the early UPR. First, CN binding to PERK enhances inhibition of protein translation to allow the cell time to recover. The induction of the early UPR, as indicated by increased P-elF2alpha, is critically dependent on a translational increase in CN-Aalpha. Second, CN dephosphorylates CLNX and likely removes inhibition of SERCA2b activity, which would aid the rapid restoration of ER Ca2+ homeostasis.
PubMed ID: 20700529
PMC ID: PMC2916823
Article link: PLoS One.
Grant support: P01 AG19316-06 NIA NIH HHS , R01 GM55372 NIGMS NIH HHS
Genes referenced: canx cntrl eif2ak2 elf2 fgfr1 hspa5 hspa9 isyna1 ppp3ca sult2a1
Morpholinos referenced: ppp3ca MO2 ppp3ca MO3
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|Figure 1. CLNX is dephosphorylated during ER stress by CN.(A) IPs of [γ32P]ATP-labeled CLNX from oocytes in the absence (0 minutes) or presence (15, 30 and 60 minutes) of Tg (1 µM) were performed. The samples were resolved through 12% SDS-PAGE, transferred to nitrocellulose and P-CLNX visualized by autoradiography (top panel). For loading control, a Western blot of CLNX was performed in oocyte microsomal extracts before the IPs (bottom panel). Histogram depicts the relative intensity of each band relative to the corresponding density of the CLNX Western blot. Notice that exogenous CLNX is expressed at higher levels than endogenous CLNX and that its autoradiographic signal is significantly higher than the signal from endogenous levels of phosphorylatioed CLNX (Figure S1). (B) Immunodetection by Western blotting of control oocytes and CLNX overexpressing oocytes. Top panel shows endogenous and exogenous CLNX. Middle panels show phosphorylated eIF2α (P-elF2α) and BiP (Assay Designs cat# SPA-826) in each corresponding cytosolic fraction. Lower panel shows α-actin loading controls. (C) Samples from Tg-treated oocytes that were pre-incubated CsA (200 nM) and FK506 (20 nM) for 16 hours are presented in lane 3. Immunodetection of CLNX by Western blotting was used as a loading control (lower panels). Histogram depicts the mean intensity of each band relative to the corresponding density in the Western blots of overexpressed CLNX. DMSO (0.05% v/v) is used as the vehicle control. Notice that control oocytes injected only with CsA/FK506 do not exhibit increased stress as indicated by Western blot analysis of eIF2α -P or BiP (Figure S2). (D) Samples from Tm-treated oocytes (lanes 2 and 4) that were pre-incubated or not with inhibitors CsA and FK506 as indicated above are shown in lanes 3 and 4. The middle panels show Western blots of CLNX of the oocyte microsomal extracts before IPs. Histogram shows the relative intensities of P-CLNX compared to overexpressed CLNX. Methanol (0.05%v/v) is used as the vehicle control. Data represents 3 independent experiments with 10 oocytes per group.|
|Figure 2. Tm treatment increases cytosolic Ca2+.(A) Images of Fura-2 loaded oocytes before (0 minutes) and after (15 minutes) Tm treatment. Ca2+ levels are presented as fura-2 fluorescence ratios of 340 to 380 nm excitation. The intensity scale bar for these images is presented in B and C. (B) Time course of Fura-2 ratio (Ratio340/380) changes in response to Tm treatment (2.5 µg/ml, added at arrow). (C) Histogram of the average Ratio340/380 (n = 9 oocytes, pooled from 3 independent experiments) at rest (0 minutes) and after Tm treatment (15 minutes).|
|Figure 4. PERK auto-phosphorylation and kinase activity increases with the interaction of CN-A and PERK.(A) GST-cPERK and CN-Aα/B were incubated with [γ32P]ATP, resolved through 12% SDS-PAGE and visualized by autoradiography as described in Materials and Methods. Phosphorylation levels are shown for GST-cPERK in the presence (lanes 1 and 2) and absence (lanes 3 and 4) of CaM for high (H, 1.2 µM) and low (L, 30 nM) Ca2+, for GST-alone (lane 5), for GST-cPERK K/A in high Ca2+ (lane 6), for GST-cPERK without CN-A (lane 7) and for CN-A α/B without GST-cPERK (lane 8). Histogram corresponding to densitometric analysis of cPERK auto-phosphorylation (B) or CN-A phosphorylation (C) from the average of three independent experiments (n = 3), using as 100% control value lane 1 in B and lane 2 in C, respectively. See the Commassie blue gel for the loading control of the autoradiogram (Figure S6). (D) Kinase assay was performed as described above in the presence of 2 µM Ca2+, but adding increasing amounts of CN-Aα/B and in the presence of eIF2α (50 nM). Histogram corresponding to densitometric analysis of cPERK auto-phosphorylation (E) or CN-A phosphorylation (F) from three independent experiments (n = 3). One asterisk corresponds to a statistical significant difference (p<0.05, ANOVA test) and two asterisks denote a statistical significant difference of (p<0.001; ANOVA test).|
|Figure 8. Role of CN in the early phases of ER stress.(1) Resting conditions of the ER: CLNX is phosphorylated, interacting with SERCA 2b and inhibiting its activity. CLNX is also interacting with the ribosome, increasing the capacity of protein folding. PERK is associated with BiP, which prevents its autophosphorylation. Protein processing and folding is optimal (depicted by spirals). (2) ER stress: unfolded proteins accumulate in the ER lumen, BiP dissociates from PERK, permitting its dimerization and autophosphorylation, which leads to attenuation of protein synthesis. At the same time, Ca2+ is released, activating CN, inducing dephosphorylation of CLNX, thereby removing pump inhibition. (3) CN levels are increased, leading to the association of CN with pre-activated PERK, which induces further PERK auto-phosphorylation, increasing the phosphorylation level of eIF2α. This emphasizes the protein translation inhibition. If cell Ca2+ levels are restored (1), CN becomes phosphorylated by PERK, decreasing its activity. CN expression also returns to resting levels further reducing its signaling. These steps, in combination with a full Ca2+ store and BiP re-association with PERK, restore normal protein translation and ER homeostasis.|