Site-specific modification and RNA crosslinking of the RNA-binding domain of PKR

Abstract

RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates and inhibits the function of the translation initiation factor eIF-2. PKR is activated in vitro by binding RNA molecules with extensive duplex structure. To further define the nature of the RNA regulation of PKR, we have prepared and characterized site-specifically modified proteins consisting of the PKR 20 kDa RNA-binding domain (RBD). Here we show that the two cysteines found naturally in this domain can be altered by site-directed mutagenesis without loss of RNA binding affinity or the RNA-regulated kinase activity. Introduction of cysteine residues at other sites in the PKR RBD allows for site-specific modification with thiol-selective reagents. PKR RBD mutants reacted selectively with a maleimide to introduce a photoactivatable cross-linking aryl azide at three different positions in the protein. RNA crosslinking efficiency was found to be dependent on the amino acid modified, suggesting differences in access to the RNA from these positions in the protein. One of the amino acid modifications that led to crosslinking of the RNA is located at a residue known to be an autophosphorylation site, suggesting that autophosphorylation at this site could influence the RNA binding properties of PKR. The PKR RBD conjugates described here and other similar reagents prepared via these methods are applicable to future studies of PKR-RNA complexes using techniques such as photocrosslinking, fluorescence resonance energy transfer and affinity cleaving.

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

“…Our technique generates ∼100 µg purified dephosphoPKR per liter of culture whose in vitro The advent of a technique for generating wild-type PKR in a dephosphorylated state allowed us to directly assess the effect of autophosphorylation on RNA binding affinity. 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).…”

“…1; 23,24). Furthermore, one of these residues (S33) has been shown to be in proximity to the RNA in the bound complex (26). These results suggested that autophosphorylation may have an effect on the RNAbinding properties of the enzyme (26).…”

“…PKR RNA ligands were obtained by transcription using T7 RNA polymerase as previously described (26,31). The dissociation constants for PP1-treated FLAG-PKR binding to a 92 nt RNA activator and an RNA inhibitor derived from adenovirus (VA I RNA) were obtained by native gel shift assays as previously described (32).…”

“…This RNA was chosen for these studies because it binds to the dsRBD of PKR with high affinity to form a saturatable complex well-suited for gel mobility shift experiments (32). Furthermore, photocrosslinking experiments involving this RNA and a PKR isolated dsRBD indicated that a previously identified autophosphorylation site (Ser33) was located near the protein/RNA interface in the complex, suggesting that autophosphorylation may play a role in regulating RNA binding (26).…”

“…Furthermore, one of these residues (S33) has been shown to be in proximity to the RNA in the bound complex (26). These results suggested that autophosphorylation may have an effect on the RNAbinding properties of the enzyme (26). Interestingly, although wild-type PKR and several site-directed mutants have been overexpressed using a variety of different expression systems in various organisms, the extent to which kinase active recombinant enzyme can be stimulated by RNA in vitro has been disputed (6,27).…”