Calcineurin Interacts with PERK and Dephosphorylates Calnexin to Relieve ER Stress in Mammals and Frogs

Abstract

BackgroundThe 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.Methodology/Principal FindingsHere, 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 α (eIF2-α) 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-Aα in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-α. 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 [γ32P]-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.Conclusions/SignificanceOur 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-elF2α, is critically dependent on a translational increase in CN-Aα. Second, CN dephosphorylates CLNX and likely removes inhibition of SERCA2b activity, which would aid the rapid restoration of ER Ca2+ homeostasis.

Insulin Secretion and Ca2+ Dynamics in β-Cells Are Regulated by PERK (EIF2AK3) in Concert with Calcineurin

“…Hence we turned toward examining the possibility that PERK may mediate Ca 2ϩ regulation through interaction with calcineurin (14), a Ca 2ϩ /calmodulin-dependent protein phosphatase. In addition to a multiplicity of functions in modulating Ca 2ϩ /calmodulindependent processes in the cell, CN was more recently shown to bind to PERK and increase its activation (53). The CN-dependent activation of PERK was also shown to be enhanced by increased cytoplasmic Ca 2ϩ levels.…”

“…Interaction of SERCA and Calnexin Is Negatively Regulated by PERK-SERCA pump activity is negatively regulated through interaction with calnexin, an ER chaperone protein 26,38). To determine whether PERK affects the interaction between SERCA and calnexin, a co-immunoprecipitation experiment with SERCA antibody was performed.…”

“…␤-Cells-Previous studies in other mammalian and amphibian cell types showed that calcineurin, a Ca 2ϩ -dependent protein phosphatase (14), interacts with PERK, and dephosphorylates calnexin (26). This raises the possibility that PERK regulates SERCA activity and SOCE in ␤-cells through a CN-dependent pathway.…”

Gene Expression Patterns in Functionally Different Cochlear Compartments of the Newborn Rat

“…The high expression levels of the calcium-binding proteins Calm3 and of the purinergic ion channel P2rx2 in the OC and the SV made us assume that the corresponding proteins have a role to play in cell survival or cell repair. Calcineurin plays an important role in refilling Ca 2+ stores of the endoplasmatic reticulum and maintaining optimum conditions for protein processing and folding (Bollo et al, 2010).…”

Viral Replication Strategies: Manipulation of ER Stress Response Pathways and Promotion of IRES-Dependent Translation

“…When excess proteins accumulate in the ER lumen, BiP dissociates from its three transmembrane sensors, resulting in the functional activation of the 3 major arms of the UPR. PERK and IRE1 are activated and undergo homodimerization and auto-phosphorylation (Bollo, et al, 2010, Liu, et al, 2000, Oikawa & Kimata, 2010, triggering their downstream genes. The activation of the IRE1 pathway leads to the splicing of X box binding protein 1 (XBP-1) (Lee, et al, 2002).…”