Fan Zhang

Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2α Kinases PKR and GCN2

ABSTRACT The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit α (eIF2α) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2α kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

Tight binding of the phosphorylated alpha subunit of initiation factor 2 (eIF2alpha) to the regulatory subunits of guanine nucleotide exchange factor eIF2B is required for inhibition of translation initiation.

ABSTRACT Translation initiation factor 2 (eIF2) is a heterotrimeric protein that transfers methionyl-initiator tRNAMet to the small ribosomal subunit in a ternary complex with GTP. The eIF2 phosphorylated on serine 51 of its α subunit [eIF2(αP)] acts as competitive inhibitor of its guanine nucleotide exchange factor, eIF2B, impairing formation of the ternary complex and thereby inhibiting translation initiation. eIF2B is comprised of catalytic and regulatory subcomplexes harboring independent eIF2 binding sites; however, it was unknown whether the α subunit of eIF2 directly contacts any eIF2B subunits or whether this interaction is modulated by phosphorylation. We found that recombinant eIF2α (glutathioneS-transferase [GST]–SUI2) bound to the eIF2B regulatory subcomplex in vitro, in a manner stimulated by Ser-51 phosphorylation. Genetic data suggest that this direct interaction also occurred in vivo, allowing overexpressed SUI2 to compete with eIF2(αP) holoprotein for binding to the eIF2B regulatory subcomplex. Mutations in SUI2 and in the eIF2B regulatory subunit GCD7 that eliminated inhibition of eIF2B by eIF2(αP) also impaired binding of phosphorylated GST-SUI2 to the eIF2B regulatory subunits. These findings provide strong evidence that tight binding of phosphorylated SUI2 to the eIF2B regulatory subcomplex is crucial for the inhibition of eIF2B and attendant downregulation of protein synthesis exerted by eIF2(αP). We propose that this regulatory interaction prevents association of the eIF2B catalytic subcomplex with the β and γ subunits of eIF2 in the manner required for GDP-GTP exchange.

Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop.

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2α to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA bindingin vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2α kinase function of PKR.

Interactions of Eukaryotic Translation Initiation Factor 3 (eIF3) Subunit NIP1/c with eIF1 and eIF5 Promote Preinitiation Complex Assembly and Regulate Start Codon Selection

ABSTRACT The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the eIF2-GTP-Met- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{i}}^{\mathit{Met}}\) \end{document} ternary complex (TC) to form the multifactor complex (MFC). We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui− phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui− phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui− phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo.

A triad of subunits from the Gal11/tail domain of Srb mediator is an in vivo target of transcriptional activator Gcn4p

ABSTRACT The Srb mediator is an important transcriptional coactivator for Gcn4p in the yeast Saccharomyces cerevisiae. We show that three subunits of the Gal11/tail domain of mediator, Gal11p, Pgd1p, and Med2p, and the head domain subunit Srb2p make overlapping contributions to the interaction of mediator with recombinant Gcn4p in vitro. Each of these proteins, along with the tail subunit Sin4p, also contributes to the recruitment of mediator by Gcn4p to target promoters in vivo. We found that Gal11p, Med2p, and Pgd1p reside in a stable subcomplex in sin4Δ cells that interacts with Gcn4p in vitro and that is recruited independently of the rest of mediator by Gcn4p in vivo. Thus, the Gal11p/Med2p/Pgd1p triad is both necessary for recruitment of intact mediator and appears to be sufficient for recruitment by Gcn4p as a free subcomplex. The med2Δ mutation impairs the recruitment of TATA binding protein (TBP) and RNA polymerase II to the promoter and the induction of transcription at ARG1, demonstrating the importance of the tail domain for activation by Gcn4p in vivo. Even though the Gal11p/Med2p/Pgd1p triad is the only portion of Srb mediator recruited efficiently to the promoter in the sin4Δ strain, this mutant shows high-level TBP recruitment and wild-type transcriptional induction at ARG1. Hence, the Gal11p/Med2p/Pgd1p triad may contribute to TBP recruitment independently of the rest of mediator.

Domains of eIF1A that mediate binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in vivo

Translation initiation factor 1A (eIF1A) is predicted to bind in the decoding site of the 40S ribosome and has been implicated in recruitment of the eIF2–GTP–Met‐tRNAiMet ternary complex (TC) and ribosomal scanning. We show that the unstructured C‐terminus of eIF1A interacts with the C‐terminus of eIF5B, a factor that stimulates 40S–60S subunit joining, and removal of this domain of eIF1A diminishes translation initiation in vivo. These findings support the idea that eIF1A–eIF5B association is instrumental in releasing eIF1A from the ribosome after subunit joining. A larger C‐terminal truncation that removes a 310 helix in eIF1A deregulates GCN4 translation in a manner suppressed by overexpressing TC, implicating eIF1A in TC binding to 40S ribosomes in vivo. The unstructured N‐terminus of eIF1A interacts with eIF2 and eIF3 and is required at low temperatures for a step following TC recruitment. We propose a modular organization for eIF1A wherein a core ribosome‐binding domain is flanked by flexible segments that mediate interactions with other factors involved in recruitment of TC and release of eIF1A at subunit joining.

Interdependent Recruitment of SAGA and Srb Mediator by Transcriptional Activator Gcn4p

ABSTRACT Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

The C-terminal region of eukaryotic translation initiation factor 3a (eIF3a) promotes mRNA recruitment, scanning, and, together with eIF3j and the eIF3b RNA recognition motif, selection of AUG start codons.

ABSTRACT The C-terminal domain (CTD) of the a/Tif32 subunit of budding yeast eukaryotic translation initiation factor 3 (eIF3) interacts with eIF3 subunits j/Hcr1 and b/Prt1 and can bind helices 16 to 18 of 18S rRNA, suggesting proximity to the mRNA entry channel of the 40S subunit. We have identified substitutions in the conserved Lys-Glu-Arg-Arg (KERR) motif and in residues of the nearby box6 element of the a/Tif32 CTD that impair mRNA recruitment by 43S preinitiation complexes (PICs) and confer phenotypes indicating defects in scanning and start codon recognition. The normally dispensable CTD of j/Hcr1 is required for its binding to a/Tif32 and to mitigate the growth defects of these a/Tif32 mutants, indicating physical and functional interactions between these two domains. The a/Tif32 CTD and the j/Hcr1 N-terminal domain (NTD) also interact with the RNA recognition motif (RRM) in b/Prt1, and mutations in both subunits that disrupt their interactions with the RRM increase leaky scanning of an AUG codon. These results, and our demonstration that the extreme CTD of a/Tif32 binds to Rps2 and Rps3, lead us to propose that the a/Tif32 CTD directly stabilizes 43S subunit-mRNA interaction and that the b/Prt1-RRM-j/Hcr1-a/Tif32-CTD module binds near the mRNA entry channel and regulates the transition between scanning-conducive and initiation-competent conformations of the PIC.

Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Δ cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

Evidence that complex formation by Bas1p and Bas2p (Pho2p) unmasks the activation function of Bas1p in an adenine-repressible step of ADE gene transcription.

Bas1p and Bas2p (Pho2p) are Myb-related and homeodomain DNA binding proteins, respectively, required for transcription of adenine biosynthetic genes in Saccharomyces cerevisiae. The repression of ADE genes in adenine-replete cells involves down-regulation of the functions of one or both of these activator proteins. A LexA-Bas2p fusion protein was found to activate transcription from a lexAop-lacZ reporter independently of both BAS1 function and the adenine levels in the medium. In contrast, a LexA-Bas1p fusion activated the lexAop reporter in a BAS2-dependent and adenine-regulated fashion. The DNA binding activity of Bas2p was not needed for its ability to support activation of the lexAop reporter by LexA-Bas1p, indicating that LexA-Bas1p recruits Bas2p to this promoter. The activation functions of both authentic Bas1p and LexA-Bas1p were stimulated under adenine-repressing conditions by overexpression of Bas2p, suggesting that complex formation by these proteins is inhibited in adenine-replete cells. Replacement of Asp-617 with Asn in Bas1p or LexA-Bas1p allowed either protein to activate transcription under repressing conditions in a manner fully dependent on Bas2p, suggesting that this mutation reduces the negative effect of adenine on complex formation by Bas1p and Bas2p. Deletions of N-terminal and C-terminal segments from the Bas1p moiety of LexA-Bas1p allowed high-level activation by the truncated proteins independently of Bas2p and adenine levels in the medium. From these results we propose that complex formation between Bas1p and Bas2p unmasks a latent activation function in Bas1p as a critical adenine-regulated step in transcription of the ADE genes.