Glen N. Barber

Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.

Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain II. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors.

Mutants of the RNA-dependent protein kinase (PKR) lacking double-stranded RNA binding domain I can act as transdominant inhibitors and induce malignant transformation

Recently we reported that introduction of catalytically inactive PKR molecules into NIH 3T3 cells causes malignant transformation and the development of tumors in nude mice. We have proposed that PKR may be a tumor suppressor gene possibly because of its translational inhibitory properties. We have now designed and characterized a number of PKR mutants encoding proteins that retain their catalytic competence but are mutated in their regulatory double-stranded RNA (dsRNA) binding domains (RBDs). RNA binding analysis revealed that PKR proteins either lacking or with point mutations in the first RBD (RBD-1) bound negligible amounts of dsRNA activator or adenovirus VAI RNA inhibitor. Despite the lack of binding, such variants remained functionally competent but were much less active than wild-type PKR. PKR variants completely lacking RBD-1 were largely unresponsive to dsRNA in activation assays but could be activated by heparin. To complement these studies, we evaluated the effects of point mutations in RBD-1 or the removal of either RBD-1 or RBD-2 on the proliferation rate of mouse 3T3 cells. We were unsuccessful at isolating stably transformed cells expressing RBD-1 point mutants or RBD-2-minus mutants. In contrast, NIH 3T3 cells, which constitutively expressed PKR proteins that lacked RBD-1, were selected. These cells displayed a transformed phenotype and caused tumors after inoculation in nude mice. Further, levels of endogenous eIF-2 alpha phosphorylation in RBD-1-minus cell lines were reduced, suggesting that such mutants act in a dominant negative manner to inhibit the function of endogenous PKR. These results emphasize the importance of RBD-1 in PKR control of cell growth and provide additional evidence for the critical role played by PKR in the regulation of malignant transformation.

Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae.

The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha). 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 alpha; 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 alpha phosphorylation.

The P58 cellular inhibitor complexes with the interferon-induced, double-stranded RNA-dependent protein kinase, PKR, to regulate its autophosphorylation and activity.

The 58-kDa protein, referred to as P58, is a cellular inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. The P58 protein inhibits both the autophosphorylation of PKR and the phosphorylation of the PKR natural substrate, the α subunit of eukaryotic initiation factor eIF-2. Sequence analysis revealed that P58 is a member of the tetratricopeptide family of proteins. Utilizing experimental approaches, which included coprecipitation or coselection of native and recombinant wild-type and mutant proteins, we found that P58 can efficiently complex with the PKR protein kinase. Attempts to map the P58 interactive sites revealed a correlation between the ability of P58 to inhibit PKR in vitro and bind to PKR. The DnaJ sequences, present at the carboxyl terminus of P58, were dispensable for binding in vitro, while sequences containing the eIF-2 α similarity region were essential for efficient complex formation. Furthermore, not all tetratricopeptide motifs were necessary for PKR-P58 interactions. Initial experiments to map the binding domains present in PKR showed that P58 complexed with PKR molecules that lacked the first RNA binding domain but did not bind to a PKR mutant containing only the amino terminus. These data, taken together, demonstrate that P58 inhibits PKR through a direct interaction, which is likely independent of the binding of double-stranded RNA to the protein kinase.

Detection of protein kinase homologues and viral RNA-binding domains utilizing polyclonal antiserum prepared against a baculovirus-expressed ds RNA-activated 68,000-Da protein kinase

The P68 protein kinase (referred to as P68 based on its M(r) of 68,000 in human cells) is a serine/threonine kinase induced by interferon treatment and activated by dsRNAs. The kinase is under tight controls in virus-infected cells since once activated, it phosphorylates its natural substrate eukaryotic initiation factor 2 (elF-2), leading to potential limitations in functional elF-2 and decreases in protein synthesis initiation. To further delineate the molecular mechanisms underlying kinase regulation, we attempted to express the P68 protein kinase in insect cells using a baculovirus vector. Repeated efforts to isolate recombinant baculoviruses containing a wild-type kinase failed, whereas recombinants expressing a nonfunctional kinase with a catalytic domain II mutation were readily isolated. When used to infect Spodoptera frugiperda cells, the recombinant virus expressed the exogenous mutant protein at almost 5-10% of the total proteins synthesized. We then purified the kinase by immunoaffinity chromatography to raise monospecific antiserum which recognized not only the human native wild-type P68, but also kinase homologues in murine, bovine, and monkey cells as determined by immunoblot and immunoprecipitation analysis. Fortunately, kinase function also could be assayed using this antibody since the human and nonhuman kinase homologues, present in immunoprecipitates, were autophosphorylated and phosphorylated the natural substrate, elF-2 alpha. Further, this antiserum recognized epitopes throughout the molecule including the amino and carboxyl termini in contrast to the available monoclonal antibody. In vitro assays using the polyclonal antibody revealed the importance of the amino terminus, especially amino acids 1-97, in the binding of the kinase to viral RNA activators and inhibitors. Finally, we determined that the P68 amino terminus was both necessary and sufficient for binding dsRNA as we were able to transfer dsRNA-binding properties to a reporter gene product previously unable to bind RNA.