Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition.
The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.
Dey
Madhusudan
M
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Cao
Chune
C
Dar
Arvin C
AC
Tamura
Tomohiko
T
Ozato
Keiko
K
Sicheri
Frank
F
Dever
Thomas E
TE
D002384:Catalysis; D019281:Dimerization; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D019892:eIF-2 Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D012330:RNA, Double-Stranded; D019892:eIF-2 Kinase