INIMEC - CONICET   05467
INSTITUTO DE INVESTIGACION MEDICA MERCEDES Y MARTIN FERREYRA
Unidad Ejecutora - UE
artículos
Título:
Calcineurin interacts with PERK and dephosphorylates Calnexin to relieve ER stress in mammals and frogs
Autor/es:
BOLLO M, PAREDES R M, ZHELEZNOVA N, HOLSTEIN D CAMACHO P AND. LECHLEITER JD
Revista:
PLOS ONE
Editorial:
PUBLIC LIBRARY SCIENCE
Referencias:
Lugar: San Francisco, CA 94111, USA; Año: 2010 vol. 5 p. 1 - 14
ISSN:
1932-6203
Resumen:
Background: 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.
(SERCA) 2b.
phosphorylation of calnexin (CLNX) controls Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase
(SERCA) 2b.
(SERCA) 2b.
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.
(SERCA) 2b.
phosphorylation of calnexin (CLNX) controls Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase
(SERCA) 2b.
(SERCA) 2b.
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.
(SERCA) 2b.
phosphorylation of calnexin (CLNX) controls Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase
(SERCA) 2b.
(SERCA) 2b.
2+ 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.
(SERCA) 2b.
2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+-ATPase
(SERCA) 2b.
Methodology/Principal Findings: 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 a (eIF2-a) 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+
[35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+
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+
[35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+
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 a (eIF2-a) 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+
[35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+
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+
[35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+
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 a (eIF2-a) 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+
[35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+
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+
[35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+
a (eIF2-a) 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+
[35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+
in-vitro kinase assays, siRNA treatments and
[35S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca2+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-Aa in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced
increases in phosphorylated elF2-a. 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 [c32P]-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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
found that CLNX was dephosphorylated by CN when Ca2+ increased. These data were obtained from [c32P]-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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
increases in phosphorylated elF2-a. 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 [c32P]-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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
found that CLNX was dephosphorylated by CN when Ca2+ increased. These data were obtained from [c32P]-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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
a in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced
increases in phosphorylated elF2-a. 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 [c32P]-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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
found that CLNX was dephosphorylated by CN when Ca2+ increased. These data were obtained from [c32P]-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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
a. 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 [c32P]-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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
2+ increased. These data were obtained from [c32P]-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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
2+ 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.
decreased phosphatase activity, consistent with this regulatory mechanism being shut down as ER homeostasis is
re-established.
2+. 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/Significance: 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-elF2a, is critically dependent on a translational increase in CN-Aa. Second, CN
dephosphorylates CLNX and likely removes inhibition of SERCA2b activity, which would aid the rapid restoration of ER Ca2+
dephosphorylates CLNX and likely removes inhibition of SERCA2b activity, which would aid the rapid restoration of ER Ca2+
CN binding to PERK enhances inhibition of pro