Biomedical subjects

Chune Cao

Publications and source records attributed to Chune Cao.

5 recordsLinked to original sources

PubMed2007

Conserved intermolecular salt bridge required for activation of protein kinases PKR, GCN2, and PERK.

The protein kinases PKR, GCN2, and PERK phosphorylate translation initiation factor eIF2alpha to regulate general and genespecific protein synthesis under various cellular stress conditions. Recent x-ray crystallographic structures of PKR and GCN2 revealed distinct dimeric configurations of the kinase domains. Whereas PKR kinase domains dimerized in a back-to-back and parallel orientation, the GCN2 kinase domains displayed an antiparallel orientation. The dimerization interfaces on PKR and GCN2 were localized to overlapping surfaces on the N-terminal lobes of the kinase domains but utilized different intermolecular contacts. A key feature of the PKR dimerization interface is a salt bridge interaction between Arg(262) from one protomer and Asp(266) from the second protomer. Interestingly, these two residues are conserved in all eIF2alpha kinases, although in the GCN2 structure, the two residues are too remote to interact. To test the importance of this potential salt bridge interaction in PKR, GCN2, and PERK, the residues constituting the salt bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases, the double mutation designed to restore the salt bridge interaction with opposite polarity resulted in a functional kinase. Thus, the salt bridge interaction and dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. These results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain, whereas the GCN2 structure may represent an inactive state of the kinase.

Animals↗

PubMed2005

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.

Catalysis↗

PubMed2005

PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha.

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.

Amino Acid Sequence↗

PubMed2003

X-ray structure of translation initiation factor eIF2gamma: implications for tRNA and eIF2alpha binding.

The x-ray structure of the gamma-subunit of the heterotrimeric translation initiation factor eIF2 has been determined to 2.4-A resolution. eIF2 is a GTPase that delivers the initiator Met-tRNA to the P site on the small ribosomal subunit during a rate-limiting initiation step in translation. The structure of eIF2gamma closely resembles that of EF1A.GTP, consisting of an N-terminal G domain followed by two beta-barrels arranged in a closed configuration with domain II packed against the G domain in the vicinity of the Switch regions. The G domain of eIF2gamma has an unusual zinc ribbon motif, not previously found in other GTPases. Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of eIF2gamma that bind the alpha-subunit and Met-tRNA(i)(Met), respectively. These structural, biochemical, and genetic results provide new insights into eIF2 ternary complex assembly.

Amino Acid Motifs↗

PubMed2002

Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation.

Initiation factors IF2 in bacteria and eIF2 in eukaryotes are GTPases that bind Met-tRNA(i)(Met) to the small ribosomal subunit. eIF5B, the eukaryotic ortholog of IF2, is a GTPase that promotes ribosomal subunit joining. Here we show that eIF5B GTPase activity is required for protein synthesis. Mutation of the conserved Asp-759 in human eIF5B GTP-binding domain to Asn converts eIF5B to an XTPase and introduces an XTP requirement for subunit joining and translation initiation. Thus, in contrast to bacteria where the single GTPase IF2 is sufficient to catalyze translation initiation, eukaryotic cells require hydrolysis of GTP by both eIF2 and eIF5B to complete translation initiation.

Catalysis↗