scholarly journals Stress Granules Regulate Double-Stranded RNA-Dependent Protein Kinase Activation through a Complex Containing G3BP1 and Caprin1

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Lucas C. Reineke ◽  
Nancy Kedersha ◽  
Martijn A. Langereis ◽  
Frank J. M. van Kuppeveld ◽  
Richard E. Lloyd
Keyword(s):  
Pattern Recognition ◽  
Innate Immunity ◽  
Protein Kinase ◽  
Antiviral Activity ◽  
Stress Granules ◽  
Initiation Factor ◽  
Dependent Protein Kinase ◽  
Α Subunit ◽  
Double Stranded Rna ◽  
Dependent Protein

ABSTRACT Stress 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. IMPORTANCE Our 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.

scholarly journals The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR)

2002 ◽  
Vol 366 (1) ◽  
pp. 175-186 ◽  
Author(s):  
Xu HUANG ◽  
Brian HUTCHINS ◽  
Rekha C. PATEL
Keyword(s):  
Protein Kinase ◽  
Essential Role ◽  
Initiation Factor ◽  
Serum Starvation ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
The Third ◽  
Protein Activator ◽  
Dependent Protein ◽  
Domain 3

One of the key mediators of the antiviral and antiproliferative actions of interferon is double-stranded-RNA-dependent protein kinase (PKR). PKR activity is also involved in the regulation of cell proliferation, apoptosis and signal transduction. We have recently identified PACT, a novel protein activator of PKR, as an important modulator of PKR activity in cells in the absence of viral infection. PACT heterodimerizes with PKR and activates it by direct protein—protein interactions. Endogenous PACT acts as an activator of PKR in response to diverse stress signals, such as serum starvation and peroxide or arsenite treatment, and is therefore a novel, stress-modulated physiological activator of PKR. In this study, we have characterized the functional domains of PACT that are required for PKR activation. Our results have shown that, unlike the N-terminal conserved domains 1 and 2, the third conserved domain of PACT is dispensable for its binding of double-stranded RNA and inter action with PKR. However, a deletion of domain 3 results in a loss of PKR activation ability, in spite of a normal interaction with PKR, thereby indicating that domain 3 plays an essential role in PKR activation. Purified recombinant domain 3 could also activate PKR efficiently in vitro. Our results indicate that, although dispensable for PACT's high-affinity interaction with PKR, the third motif is essential for PKR activation. In addition, domain 3 and eukaryotic initiation factor 2α both interact with PKR through the same region within PKR, which we have mapped to lie between amino acid residues 318 and 551.

scholarly journals Genetic Involvement of a cAMP-Dependent Protein Kinase in a G Protein Signaling Pathway Regulating Morphological and Chemical Transitions in Aspergillus nidulans

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 591-600
Author(s):  
Kiminori Shimizu ◽  
Nancy P Keller
Keyword(s):  
Protein Kinase ◽  
Aspergillus Nidulans ◽  
Signaling Pathway ◽  
G Protein ◽  
Radial Growth ◽  
Morphological Development ◽  
Dependent Protein Kinase ◽  
Α Subunit ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Abstract In the filamentous fungus Aspergillus nidulans, a heterotrimeric G protein α-subunit and an RGS domain protein, encoded by fadA and flbA, respectively, regulate production of the carcinogenic metabolite sterigmatocystin (ST) and asexual spores (i.e., conidia). We investigated the genetic involvement of the cAMP-dependent protein kinase catalytic subunit (PkaA), a potential downstream target of FadA activity, in ST production and conidiation. Relative to wild type, sporulation was decreased in the pkaA overexpression strain but was not totally absent, as occurs in ΔflbA or fadAG42R (fadA-dominant active) strains. Deletion of pkaA resulted in a hyper-conidiating strain with limited radial growth. This phenotype was epistatic to mutation in flbA or fadA; the double mutants ΔpkaA; ΔflbA and ΔpkaA; fadAG42R recovered sporulation and their radial growth was severely restricted. PkaA overexpression also negatively regulated AflR, the ST biosynthesis-specific transcription factor, both transcriptionally and post-transcriptionally. Deletion of pkaA restored ST production in the ΔflbA background but not in the fadAG42R background. These data provide genetic evidence that the FlbA/FadA signaling pathway regulating ST production and morphological development is partially mediated through PkaA.

scholarly journals Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase.

1995 ◽  
Vol 14 (24) ◽  
pp. 6095-6106 ◽  
Author(s):  
Y. L. Yang ◽  
L. F. Reis ◽  
J. Pavlovic ◽  
A. Aguzzi ◽  
R. Schäfer ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dependent Protein Kinase ◽  
Double Stranded Rna ◽  
Dependent Protein
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