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Requirement for kinase-induced conformational change in eukaryotic initiation factor 2alpha (eIF2alpha) restricts phosphorylation of Ser51.

ABSTRACT: As phosphorylation of eukaryotic translation initiation factor 2? (eIF2?) on Ser51 inhibits protein synthesis, cells restrict this phosphorylation to the antiviral protein kinase PKR and related eIF2? kinases. In the crystal structure of the PKR-eIF2? complex, the C-terminal lobe of the kinase contacts eIF2? on a face remote from Ser51, leaving Ser51 ? 20 ? from the kinase active site. PKR mutations that cripple the eIF2?-binding site impair phosphorylation; here, we identify mutations in eIF2? that restore Ser51 phosphorylation by PKR with a crippled substrate-binding site. These eIF2? mutations either disrupt a hydrophobic network that restricts the position of Ser51 or alter a linkage between the PKR-docking region and the Ser51 loop. We propose that the protected state of Ser51 in free eIF2? prevents promiscuous phosphorylation and the attendant translational regulation by heterologous kinases, whereas docking of eIF2? on PKR induces a conformational change that regulates the degree of Ser51 exposure and thus restricts phosphorylation to the proper kinases.

SUBMITTER: Dey M

PROVIDER: S-EPMC3060252 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Requirement for kinase-induced conformational change in eukaryotic initiation factor 2alpha (eIF2alpha) restricts phosphorylation of Ser51.

Dey Madhusudan M Velyvis Algirdas A Li John J JJ Chiu Elaine E Chiovitti David D Kay Lewis E LE Sicheri Frank F Dever Thomas E TE

Proceedings of the National Academy of Sciences of the United States of America 20110228 11

As phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) on Ser51 inhibits protein synthesis, cells restrict this phosphorylation to the antiviral protein kinase PKR and related eIF2α kinases. In the crystal structure of the PKR-eIF2α complex, the C-terminal lobe of the kinase contacts eIF2α on a face remote from Ser51, leaving Ser51 ∼ 20 Å from the kinase active site. PKR mutations that cripple the eIF2α-binding site impair phosphorylation; here, we identify mutations in eIF2α ...[more]

PMID: 21368187

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Project description:Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)(2) (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-kappaB activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0-300 microM) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 muM with marked suppression at >100 microM. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2alpha (eIF2alpha) kinase. Indeed, we found a marked (approximately 4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2alpha-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2alpha kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2alpha kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes.

Dataset Information

Requirement for kinase-induced conformational change in eukaryotic initiation factor 2alpha (eIF2alpha) restricts phosphorylation of Ser51.

Publications

Requirement for kinase-induced conformational change in eukaryotic initiation factor 2alpha (eIF2alpha) restricts phosphorylation of Ser51.

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