Activation of protein kinase PKR requires dimerization-induced cis-phosphorylation within the activation loop

Abstract

Protein kinase R (PKR) functions in a plethora of cellular processes, including viral and cellular stress responses, by phosphorylating the translation initiation factor eIF2α. The minimum requirements for PKR function are homodimerization of its kinase and RNA-binding domains, and autophosphorylation at the residue Thr-446 in a flexible loop called the activation loop. We investigated the interdependence between dimerization and Thr-446 autophosphorylation using the yeast Saccharomyces cerevisiae model system. We showed that an engineered PKR that bypassed the need for Thr-446 autophosphorylation (PKR(T446∼P)-bypass mutant) could function without a key residue (Asp-266 or Tyr-323) that is essential for PKR dimerization, suggesting that dimerization precedes and stimulates activation loop autophosphorylation. We also showed that the PKR(T446∼P)-bypass mutant was able to phosphorylate eIF2α even without its RNA-binding domains. These two significant findings reveal that PKR dimerization and activation loop autophosphorylation are mutually exclusive yet interdependent processes. Also, we provide evidence that Thr-446 autophosphorylation during PKR activation occurs in a cis mechanism following dimerization.

Keywords: Activation Loop; Phosphorylation; Protein Kinase RNA (PKR); Protein Kinases; Protein Phosphorylation; Stress Response; eIF2α.

FIGURE 1.

Functional coupling between the phosphorylated activation loop and the helix-αC. A, analyses of PKR KD structures. The structural coordinates of PKR-KD-K296R (PDB ID: 3UIU) and PKR- KD bound to AMP-PNP and eIF2α (PDB ID: 2A19) were aligned in a single PyMol file by pairwise alignment. Proteins of the PKR-KD (gray) and the PKR-KD-K296R (green) are represented as ribbons. For clarity, the entire structure of eIF2α and several structural elements in PKR are omitted. The conserved residues at the helix-αC (E308, R307, and K304), β-strand 3 (β-3, K296), hinge (E367), activation loop (T446), and catalytic loop (R413 and D414) are shown. The activation loop (region between the DFG and SPE motifs) is colored in purple. The phosphate moiety (red) of the phosphorylated Thr-446 salt-bridges (thin dotted line in gray) with the residue Arg-413 from the catalytic loop and residues Lys-304 and Arg-307 from the helix-αC. The thick purple dotted line in the PKR-KD-K296R structure represents the unresolved portion of the activation loop. The K296R mutation is shaded yellow. B, in vivo analysis of PKR mutants by growth in yeast. The yeast strain H17 (eIF2B-desensitive) expressing WT PKR or indicated mutants were serially diluted and spotted on SD and SGal media. The Slg⁻ phenotype on SGal medium indicates the functionality of PKR alleles. C, in vivo analysis of PKR and eIF2α phosphorylation. Whole cell extracts were prepared from yeast cells indicated in B and subjected to Western blot analysis using antibodies of phosphorylated eIF2α (eIF2α∼P), eIF2α, and phosphorylated PKR (PKR∼P) and PKR.

FIGURE 2.

Thr-446 phosphorylation occurs in a cis mechanism following dimerization. A, heterologous dimerization domains activate the PKR KD. The LIM-PKR^KD fusion gene in the plasmid pC901 with a URA3 selectable marker and the Ldb-PKR^KD fusion gene in the plasmid pC903 with a TRP selectable marker were introduced into WT yeast strain H2557 as well as S51A strain. The sign (+) means the presence, whereas the sign (−) means the absence of pC901/pC903 plasmids but contains the respective vector plasmid. The indicated D414A mutation was introduced in to the LIM-PKR^KD fusion construct. Transformants were tested for growth on SD and SGal media. The PKR was inactive when the yeast grew on SGal medium, but active if the yeast did not grow. B, analysis of Thr-446 autophosphorylation and eIF2α phosphorylation. Yeast strains expressing indicated LIM- PKR^KD (WT or D414A) and Ldb-PKR^KD fusion protein were grown in the SGal medium and whole cell extracts were subjected to Western blot analysis using either a Thr-446-phosphospecific (T446∼P) or Ser-51 phosphospecific (eIF2α∼P) antibody followed by a polyclonal antibody of PKR or eIF2α. C, models represent mono- or dimeric form of LIM- and Ldb-PKR^KD fusion proteins. The bi-lobal PKR-KD is shown in gray whereas the activation loops in purple solid lines with a phosphorylated residue (red circle). The inactive form of the kinase domain is colored orange.

FIGURE 3.

The Thr-446 phosphorylation does not occur in trans mechanism. A, in vitro analysis of PKR autophosphorylation. Purified PKR-K296R and GST-PKR^KD fusion proteins were incubated in a kinase reaction buffer. The reaction products were then separated by SDS-PAGE and subjected to Western blot analyses using phosphospecific antibody of Thr-446 followed by a polyclonal antibody of PKR. B, analysis of Thr-446 phosphorylation. GST-PKR^KD co-expressed with WT PKR or PKR-K296R mutant in yeast. Whole cell extracts were prepared from these cells and subjected to Western blot analysis using a Thr-446 phosphospecific antibody of PKR (T446∼P). The membrane was stripped and re-probed with a polyclonal antibody of PKR.

FIGURE 4.

Functional substitution of activation loop residues of PKR by residues from the activation loop of phosphorylase kinase 1 (Phk1). A, in vivo analysis of PKR mutants by growth in yeast. The yeast strain H17 (eIF2B desensitive) or MY71 (eIF2α-S51A) harboring indicated WT PKR and its derivatives were serially diluted and spotted on SD and SGal media. B, in vivo analysis of eIF2α phosphorylation by PKR mutants. Whole cell extracts were prepared from yeast cells indicated in A and subjected to Western blot analysis using phosphospecific antibodies against Ser-51 (eIF2α∼P). The membrane was stripped and re-probed with a polyclonal antibody of eIF2α and PKR. C, in vitro analysis of eIF2α phosphorylation by PKR mutants. Purified PKR protein (WT, K296R, or PKR^phk1) was mixed with the recombinant eIF2α and [γ-³³P]ATP in a reaction buffer for 10 min. The reaction products were then separated using SDS-PAGE. The gel was stained, dried, and subjected to autoradiography to monitor the incorporation of ³³P in eIF2α proteins. D, catalytic function of PKR^phk1 chimera requires an active interaction between the activation loop and the helix-αC. Left panels, eIF2B desensitive strain H17 expressing indicated PKR mutants were tested for growth on SD and SGal media. Right panels, yeast cells were grown in the presence of galactose (10%) and harvested after 2 h. Whole cell extracts were then prepared and subjected to Western blot analyses using antibodies of phosphorylated eIF2α (eIF2α∼P), eIF2α, and PKR.

FIGURE 5.

Kinase domains of PKR and Phk1 are structurally similar. A, residue E182 in Phk1 corresponds to the residue Thr-446 in PKR. The activation loop residues (boxed) of PKR, Phk1, and Chk1 were compared by pairwise alignment. The conserved residues are colored in green. A red arrowhead shows Thr-446 of PKR. B, kinase domains of PKR and Phk1 superimpose well on each other. Structural coordinates of the kinase domains of PKR (PDB ID: 2A1A) and Phk1 (PDB ID: 2PHK) were superimposed, using computer software PyMol. Proteins are shown as a ribbon presentation. For clarity, several structural elements are omitted. Only the helix-αC (colored orange in PKR whereas gray in Phk1) and the activation loop (colored purple both in PKR and Phk1) in the active site regions are shown. The conserved Arg-413 of the RD motif (R413 colored green) contacts with phosphorylated Thr-446 in PKR, whereas the corresponding Arg-148 in Phk1 (R148 colored green) contacts with phospho-mimetic E182. The catalytic base aspartate (D414 in PKR or D149 in Phk1) is colored in blue.

FIGURE 6.

Salt-bridge interactions at the dimer interface are partially important for PKR^phk1 function. A, intra- and inter-molecular salt-bridge interactions at the PKR-KD dimer interface. A dimer of the PKR kinase domains (PDB ID: 2A19) is represented as a ribbon diagram. For clarity, only the interacting N-terminal lobes are shown. Several intra- and inter-molecular hydrophobic and salt-bridge interactions stabilize the dimer interface between protomer 1 (gray) and protomer 2 (green). Only two prominent salt-bridge interactions (R262·D266 and D289·Y293·Y323) are zoomed in a separate box. B, in vivo analysis of PKR mutants by growth in yeast and by eIF2α phosphorylation. The gcn2Δ yeast strains harboring indicated PKR mutants were tested for growth on SD and SGal media (upper panels). Whole cell extracts from these cells were subjected to Western blot analyses using antibodies of phosphorylated eIF2α (eIF2α∼P) and eIF2α.

FIGURE 7.

The kinase domain of PKR^phk1 chimera is partially active. A, in vivo analysis of PKR mutants by growth in yeast. The WT (eIF2α-WT) and S51A (eIF2α-S51A) yeast strains harboring indicated PKR mutants were serially diluted and tested for growth on SGal medium. B, PKR^KD-Phk1 phosphorylates eIF2α. Whole cell extracts from cells expressing indicated PKR derivatives were subjected to Western blot analyses using antibodies of phosphorylated eIF2α (eIF2α∼P) and eIF2α. C, PKR^KD-Phk1 can activate the GCN4 translational control. Whole cell extracts of yeast cells carrying a GCN4 LacZ reporter plasmid p180 were prepared and β-gal activities were monitored as described previously (36).

FIGURE 8.

Maturation and activation of PKR by autophosphorylation reactions. A conceptual model of the inactive PKR in which the RBD2 is bound to the kinase domain (40) and the activation loop (solid purple line) is un-phosphorylated. Binding of dsRNA or PACT (light blue star) to the RBD releases the autoinhibition (40). Consequently, the kinase domains of PKR adopt a favorable dimeric configuration (even with an inactive KD partner shown in gray) that facilitates autophosphorylation on the residue Thr-446 in cis. As shown by dotted lines, the phosphorylated Thr-446 couples with the helix-αC (orange cylinder) and an active closed conformation is achieved. Then PKR phosphorylates other residues in trans (shown in red circles) and becomes fully active.