Identification of the PKR Nuclear Interactome Reveals Roles in Ribosome Biogenesis, mRNA Processing and Cell Division

Blalock, William L.; Piazzi, Manuela; Bavelloni, Alberto; Raffini, Mirco; Faenza, Irene; D’Angelo, Antonio; Cocco, Lucio

doi:10.1002/jcp.24529

Cited by 27 publications

(51 citation statements)

References 51 publications

(82 reference statements)

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“…For example, substrates of pPKR might be enriched near the chromosome region and recruit PKR. A recent mass spectrometry analysis on PKR interactome revealed that pPKR interacts with proteins involved in chromatin modification and cell division, such as BUB3 and NPM1 (Blalock et al 2014). Alternatively, it is possible that there may be different classes of dsRNAs that are localized to the chromosome region during mitosis and tether PKR.…”

Section: Discussionmentioning

confidence: 99%

PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

Kim

Lee

Park³

et al. 2014

Genes Dev.

117

126

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dsRNA-dependent protein kinase R (PKR) is a ubiquitously expressed enzyme well known for its roles in immune response. Upon binding to viral dsRNA, PKR undergoes autophosphorylation, and the phosphorylated PKR (pPKR) regulates translation and multiple signaling pathways in infected cells. Here, we found that PKR is activated in uninfected cells, specifically during mitosis, by binding to dsRNAs formed by inverted Alu repeats (IRAlus). While PKR and IRAlu-containing RNAs are segregated in the cytosol and nucleus of interphase cells, respectively, they interact during mitosis when nuclear structure is disrupted. Once phosphorylated, PKR suppresses global translation by phosphorylating the α subunit of eukaryotic initiation factor 2 (eIF2α). In addition, pPKR acts as an upstream kinase for c-Jun N-terminal kinase and regulates the levels of multiple mitotic factors such as CYCLINS A and B and POLO-LIKE KINASE 1 and phosphorylation of HISTONE H3. Disruption of PKR activation via RNAi or expression of a transdominant-negative mutant leads to misregulation of the mitotic factors, delay in mitotic progression, and defects in cytokinesis. Our study unveils a novel function of PKR and endogenous dsRNAs as signaling molecules during the mitosis of uninfected cells.

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Section: Discussionmentioning

confidence: 99%

PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

Kim

Lee

Park³

et al. 2014

Genes Dev.

117

126

View full text Add to dashboard Cite

show abstract

“…The analysis was performed as described elsewhere (41)(42)(43). Briefly, gel slices were washed in 100 mM ammonium bicarbonate (pH 8) and 50% acetonitrile (ACN), until completely destained, and then were digested with sequencing-grade trypsin (Promega, Madison, WI, USA) at 37°C.…”

Section: Ms Analysismentioning

confidence: 99%

Prohibitin 2 represents a novel nuclear AKT substrate during all‐ trans retinoic acid–induced differentiation of acute promyelocytic leukemia cells

et al. 2014

Self Cite

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The AKT/PKB kinase is essential for cell survival, proliferation, and differentiation; however, aberrant AKT activation leads to the aggressiveness and drug resistance of many human neoplasias. In the human acute promyelocytic leukemia cell line NB4, nuclear AKT activity increases during all-trans retinoic acid (ATRA)-mediated differentiation. As nuclear AKT activity is associated with differentiation, we sought to identify the nuclear substrates of AKT that were phosphorylated after ATRA treatment. A proteomics-based search for nuclear substrates of AKT in ATRA-treated NB4 cells was undertaken by using 2D-electrophoresis/mass spectrometry (MS) in combination with an anti-AKT phospho-substrate antibody. Western blot analysis, an in vitro kinase assay, and/or site-directed mutagenesis were performed to further characterize the MS findings. MS analysis revealed prohibitin (PHB)-2, a multifunctional protein involved in cell cycle progression and the suppression of oxidative stress, to be a putative nuclear substrate of AKT. Follow-up studies confirmed that AKT phosphorylates PHB2 on Ser-91 and that forced expression of the PHB2(S91A) mutant results in a rapid loss of viability and apoptotic cell death. Activation of nuclear AKT during ATRA-mediated differentiation results in the phosphorylation of several proteins, including PHB2, which may serve to coordinate nuclear-mitochondrial events during differentiation.

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“…12 Although PKR is primarily found in the cytoplasm where it regulates translation, about 20% of PKR is nuclear where it has been shown to regulate RNA splicing, mRNA stability, and ribosome biogenesis and affect diseases including acute myeloid leukemia AML. 16–18 …”

Section: Introductionmentioning

confidence: 99%

Discriminating Self and Non-Self by RNA: Roles for RNA Structure, Misfolding, and Modification in Regulating the Innate Immune Sensor PKR

Hull

Bevilacqua

2016

Acc. Chem. Res.

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CONSPECTUS Pathogens are recognized by the innate immune system in part via their unique and complex RNA signatures. A key sensor in human innate immunity is the RNA-activated protein kinase PKR, which has two double-stranded RNA (dsRNA) binding motifs (dsRBMs) at its N-terminus. Early studies described PKR as being activated potently by long stretches of perfect dsRNA, a signature typical of viruses. More recently, we and others have found that PKR is also activated by RNAs having structural defects such as bulges and internal loops. This article describes advances in our understanding of the ability of PKR to detect diverse foreign RNAs and how that recognition plays significant roles in discriminating self from non-self. The experiments discussed employ a wide range of techniques including activation assays, native polyacrylamide gel electrophoresis (PAGE), protein footprinting, and small angle X-ray scattering (SAXS). We discuss how misfolding and dimerization of RNA lead to activation of PKR. We also present recent findings on the activation of PKR by varied bacterial functional RNAs including ribozymes and riboswitches, which are among the few structured RNAs known to interact with PKR in a site-specific manner. Molecular models for how these structured RNAs activate PKR are provided. Studies by SAXS revealed that PKR straightens bent RNAs. Most external and internal RNA cellular modifications introduced in vitro and found naturally, such as the m7G cap and m6A group, abrogate activation of PKR, but other modifications, such as 5’-ppp and 2’-fluoro groups, are immunostimulatory and potential anticancer agents. Genome-wide studies of RNA folding in vitro and in vivo have provided fresh insights into general differences in RNA structure amongst bacteria, viruses, and human. These studies suggest that in vivo, cellular human RNAs are less folded than once thought, unwound by helicases, destabilized by m6A modifications, and often bound up with proteins—conditions known to abrogate activation of PKR. It thus appears that non-self RNAs are detected as unmodified, naked RNAs with appreciable secondary and tertiary structure. Observation that PKR is activated by structured but otherwise diverse RNAs is consistent both with the broad-spectrum nature of innate immunity and with the non-specific recognition of RNA by the dsRBM family. These findings provide a possible explanation for the apparent absence of protein-free structured human RNAs, such as ribozymes and riboswitches.

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Identification of the PKR Nuclear Interactome Reveals Roles in Ribosome Biogenesis, mRNA Processing and Cell Division

Cited by 27 publications

References 51 publications

PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

Prohibitin 2 represents a novel nuclear AKT substrate during all‐ trans retinoic acid–induced differentiation of acute promyelocytic leukemia cells

Discriminating Self and Non-Self by RNA: Roles for RNA Structure, Misfolding, and Modification in Regulating the Innate Immune Sensor PKR

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