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The double-stranded RNA-dependent protein kinase differentially regulates insulin receptor substrates 1 and 2 in HepG2 cells.

ABSTRACT: Initially identified to be activated upon virus infection, the double-stranded RNA-dependent protein kinase (PKR) is best known for triggering cell defense responses by phosphorylating eIF-2?, thus suppressing RNA translation. We as well as others showed that the phosphorylation of PKR is down-regulated by insulin. In the present study, we further uncovered a novel function of PKR in regulating the IRS proteins. We found that PKR up-regulates the inhibitory phosphorylation of IRS1 at Ser312, which suppresses the tyrosine phosphorylation of IRS1. This effect of PKR on the phosphorylation of IRS1 is mediated by two other protein kinases, JNK and IKK. In contrast, PKR regulates IRS2, another major IRS family protein in the liver, at the transcriptional rather than the posttranslational level, and this effect is mediated by the transcription factor, FoxO1, which has been previously shown to be regulated by insulin and plays a significant role in glucose homeostasis and energy metabolism. In summary, we found for the first time that initially known as a virus infection response gene, PKR regulates the upstream central transmitters of insulin signaling, IRS1 and IRS2, through different mechanisms.

SUBMITTER: Yang X

PROVIDER: S-EPMC2947480 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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The double-stranded RNA-dependent protein kinase differentially regulates insulin receptor substrates 1 and 2 in HepG2 cells.

Yang Xuerui X Nath Aritro A Opperman Michael J MJ Chan Christina C

Molecular biology of the cell 20100804 19

Initially identified to be activated upon virus infection, the double-stranded RNA-dependent protein kinase (PKR) is best known for triggering cell defense responses by phosphorylating eIF-2α, thus suppressing RNA translation. We as well as others showed that the phosphorylation of PKR is down-regulated by insulin. In the present study, we further uncovered a novel function of PKR in regulating the IRS proteins. We found that PKR up-regulates the inhibitory phosphorylation of IRS1 at Ser312, whi ...[more]

PMID: 20685959

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Project description:Double-stranded RNA-dependent protein kinase (PKR) is a eukaryotic initiation factor 2? kinase that inhibits mRNA translation under stress conditions. PKR also mediates inflammatory and apoptotic signaling independently of translational regulation. Congestive heart failure is associated with cardiomyocyte hypertrophy, inflammation, and apoptosis, but the role of PKR in left ventricular hypertrophy and the development of congestive heart failure has not been examined.We observed increased myocardial PKR expression and translocation of PKR into the nucleus in humans and mice with congestive heart failure. To determine the impact of PKR on the development of congestive heart failure, PKR knockout and wild-type mice were exposed to pressure overload produced by transverse aortic constriction. Although heart size increased similarly in wild-type and PKR knockout mice after transverse aortic constriction, PKR knockout mice exhibited very little pulmonary congestion, well-preserved left ventricular ejection fraction and contractility, and significantly less myocardial fibrosis compared with wild-type mice. Bone marrow-derived cells from wild-type mice did not abolish the cardiac protective effect observed in PKR knockout mice, whereas bone marrow-derived cells from PKR knockout mice had no cardiac protective effect in wild-type mice. Mechanistically, PKR knockout attenuated transverse aortic constriction-induced tumor necrosis factor-? expression and leukocyte infiltration and lowered cardiac expression of proapoptotic factors (Bax and caspase-3), so that PKR knockout hearts were more resistant to transverse aortic constriction-induced cardiomyocyte apoptosis. PKR depletion in isolated cardiomyocytes also conferred protection against tumor necrosis factor-?- or lipopolysaccharide-induced apoptosis.PKR is a maladaptive factor upregulated in hemodynamic overload that contributes to myocardial inflammation, cardiomyocyte apoptosis, and the development of congestive heart failure.

Project description:Vaccinia virus has recently been used as an expression vector for gene delivery and an oncolytic agent for cancer therapy. Although it has been established that interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) and RNase L interfere with viral replication, little else is known about the other host factors that might affect viral replication and virus-mediated host cell killing. In this study, we evaluated the roles of c-Jun NH2-terminal kinase (JNK) in oncolytic vaccinia virus replication and vaccinia virus-mediated host cell killing. We found that JNK knockout mouse embryonic fibroblasts (MEFs) were more susceptible to oncolytic vaccinia virus infection than wild-type MEFs. Moreover, viral replication and the production of infectious viral progeny were up to 100-fold greater in JNK-deficient MEFs than in wild-type MEFs. A similar result was observed for wild-type vaccinia virus. The increased killing of infected cells and the production of viral progeny was also observed in wild-type MEFs that had been treated with JNK inhibitors and in human colon cancer cells that had been transfected with dominant-negative JNK constructs. Moreover, testing on several human lung cancer cell lines and HeLa cells showed an inverse correlation between levels of JNK expression and susceptibility to oncolytic vaccinia virus. Our study also revealed that oncolytic virus infection-mediated PKR activation was blocked or diminished in JNK-deficient MEFs. The adenovirus-mediated ectopic expression of human PKR in JNK-deficient MEFs reduced vaccinia virus replication to the levels observed in wild-type MEFs, indicating that JNK is required for vaccinia virus to efficiently activate PKR. Our results demonstrated that the cellular status of JNK function can dramatically affect oncolytic vaccinia virus replication and vaccinia virus-mediated host cell killing. This finding may enable further improvements in oncolytic virotherapy using vaccinia virus.

Project description:UnlabelledStress granules (SGs) are dynamic cytoplasmic repositories containing translationally silenced mRNAs that assemble upon cellular stress. We recently reported that the SG nucleating protein G3BP1 promotes antiviral activity and is essential in double-stranded RNA-dependent protein kinase (PKR) recruitment to stress granules, thereby driving phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we delineate the mechanism for SG-dependent PKR activation. We show that G3BP1 and inactive PKR directly interact with each other, dependent on both the NTF2-like and PXXP domains of G3BP1. The G3BP1-interacting protein Caprin1 also directly interacts with PKR, regulates efficient PKR activation at the stress granule, and is also integral for the release of active PKR into the cytoplasm to engage in substrate recognition. The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. Our data links stress responses and their resultant SGs with innate immune activation through PKR without a requirement for foreign double-stranded RNA (dsRNA) pattern recognition.ImportanceOur previous work indicates that stress granules have antiviral activity and mediate innate immunity through functions of G3BP1; however, the mechanistic details of these functions were not resolved. We show that much of the antiviral activity of stress granules is contingent on the function of PKR in a complex with G3BP1 and Caprin1. The PKR-G3BP1-Caprin1 complex undergoes dynamic transitioning within and outside stress granules to accomplish PKR activation and translational repression. This mechanism appears to function distinctly from canonical pattern recognition of double-stranded RNA by PKR. Therefore, this mechanism bridges the stress response with innate immunity, allowing the cell to respond to many cellular stressors and amplify the pathogen pattern recognition systems of innate immunity.

Dataset Information

The double-stranded RNA-dependent protein kinase differentially regulates insulin receptor substrates 1 and 2 in HepG2 cells.

Publications

The double-stranded RNA-dependent protein kinase differentially regulates insulin receptor substrates 1 and 2 in HepG2 cells.

Similar Datasets

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