The binding of double‐stranded RNA and adenovirus VAI RNA to the interferon‐induced protein kinase

Galabru, Julien; Katze, Michael G.; Robert, N.; Hovanessian, Ara G.

doi:10.1111/j.1432-1033.1989.tb14485.x

Cited by 133 publications

(86 citation statements)

References 41 publications

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“…This form can be dephosphorylated by a PP1-like phosphatase to regenerate the form of PKR with high affinity for RNA and low eIF2α kinase activity. This model for PKR activation is consistent with the finding that autophosphorylation of PKR, but not phosphorylation of eIF2α by autophosphorylated PKR, is inhibited by the adenovirus RNA inhibitor VA I RNA (41).…”

Section: Discussionsupporting

confidence: 89%

“…The discovery of autophosphorylation sites in the dsRBD of PKR and the observation that one of those sites is near the protein/ RNA interface in a complex between the dsRBD and an RNA activator led us to speculate that phosphorylation could influence the RNA-binding properties of the enzyme (23,24,26). This is consistent with previous reports indicating that once autophosphorylated, PKR no longer responds to RNA regulators, including kinase inhibiting RNAs (41). Given the number of autophosphorylation sites found in PKR to date (10 sites mapped, possibly 14 total), we concluded that identification of sites involved in controlling RNA binding by site-directed mutagenesis may be problematical, particularly given the possibility that the phosphorylation effect on RNA affinity may be cumulative, with no particularly important single site (21)(22)(23)(24).…”

Section: Discussionsupporting

confidence: 87%

“…Viral RNA inhibitors cannot inhibit PKR once it is autophosphorylated and active (41). A virus that uses an RNA inhibitor of PKR apparently needs to intercept the enzyme in its dephospho state.…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Jammi

Beal

2001

Nucleic Acids Research

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The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2α kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2α kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA·Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho-and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2α.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 87%

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Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Jammi

Beal

2001

Nucleic Acids Research

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“…The antibody-bound DAI was washed four times with buffer I and three times with buffer II (10 mM Tris-HCl [pH 7.5], 100 mM KCl, 0.1 mM EDTA, 100 U of aprotinin per ml, 20% glycerol). DAI was eluted by using guanidinium hydrochloride and dialyzed as previously described (9). For production of polyclonal antibody, New Zealand White rabbits were subcutaneously injected with 100 g of the purified DAI expressed in E. coli along with an equal volume of Freund's complete adjuvant.…”

Section: Methodsmentioning

confidence: 99%

“…It is generally thought that dsRNA binding stimulates the autokinase activity of DAI and that autophosphorylation locks the kinase domain into a configuration that can bind and phosphorylate eIF-2␣ in the absence of dsRNA activators (8,9,20). The kinetics of activation are second order for enzyme concentration (20), and partially phosphorylated DAI purifies as a dimer, whereas unphosphorylated DAI exists as a monomer (21).…”

mentioning

confidence: 99%

Structural Requirements for Double-Stranded RNA Binding, Dimerization, and Activation of the Human eIF-2α Kinase DAI in Saccharomyces cerevisiae

Romano

Green

Barber

et al. 1995

Molecular and Cellular Biology

120

192

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The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the ␣ subunit of translation initiation factor 2 (eIF-2␣). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2␣; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2␣ phosphorylation.A response to starvation and stress common to mammalian and yeast cells is the inhibition of protein synthesis by phosphorylation of the ␣ subunit of translation initiation factor 2 (eIF-2␣). The first step in translation initiation is the formation of a ternary complex composed of eIF-2 (made up of three nonidentical subunits), GTP, and charged initiator tRNA Met (Met-tRNA i Met ). This ternary complex interacts with the small ribosomal subunit, forming a 43S preinitiation complex, which then binds to mRNA and assembles an 80S initiation complex at the AUG start codon. The eIF-2 is released from the ribosome in an eIF-2 ⅐ GDP binary complex. To initiate another round of translation, the GDP bound to eIF-2 must be replaced by GTP, which requires the guanine nucleotide exchange factor, eIF-2B (reviewed in reference 29). Phosphorylation of the ␣ subunit of eIF-2 on the serine residue at position 51 impairs guanine nucleotide exchange on eIF-2 by inhibiting eIF-2B activ...

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Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.

et al. 1992

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The human p68 kinase is an interferon‐regulated enzyme that inhibits protein synthesis when activated by double‐stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N‐terminus, shown to bind double‐stranded RNA in vitro and expression of a mutant form of the alpha‐subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha‐subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.

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The binding of double‐stranded RNA and adenovirus VAI RNA to the interferon‐induced protein kinase

Cited by 133 publications

References 41 publications

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

Structural Requirements for Double-Stranded RNA Binding, Dimerization, and Activation of the Human eIF-2α Kinase DAI in Saccharomyces cerevisiae

Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.

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