JBC Oz Biosciences

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, J.
Right arrow Articles by Dever, T. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, J.
Right arrow Articles by Dever, T. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 274, Issue 45, 32198-32203, November 5, 1999


The Interferon-induced Double-stranded RNA-activated Protein Kinase PKR Will Phosphorylate Serine, Threonine, or Tyrosine at Residue 51 in Eukaryotic Initiation Factor 2alpha *

Jingfang Lu, Eileen B. O'Hara, Bruce A. Trieselmann, Patrick R. Romano, and Thomas E. DeverDagger

From the Laboratory of Eukaryotic Gene Regulation, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2716

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The family of eukaryotic initiation factor 2alpha (eIF2alpha ) protein kinases plays an important role in regulating cellular protein synthesis under stress conditions. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate Ser-51 on the alpha  subunit of the translation initiation factor eIF2. By using an in vivo assay in yeast, the substrate specificity of these three eIF2alpha kinases was examined by substituting Ser-51 in eIF2alpha with Thr or Tyr. In yeast, phosphorylation of eIF2 inhibits general translation but derepresses translation of the GCN4 mRNA. All three kinases phosphorylated Thr in place of Ser-51 and were able to regulate general and GCN4-specific translation. In addition, both PKR and HRI were found to phosphorylate eIF2alpha -S51Y and stimulate GCN4 expression. Isoelectric focusing analysis of eIF2alpha followed by detection using anti-eIF2alpha and anti-phosphotyrosine-specific antibodies demonstrated that PKR and HRI phosphorylated eIF2alpha -S51Y on Tyr in vivo. These results provide new insights into the substrate recognition properties of the eIF2alpha kinases, and they are intriguing considering the potential for alternate substrates for PKR in cellular signaling and growth control pathways.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The human interferon-induced double-stranded RNA-activated protein kinase PKR, which functions in the cellular antiviral defense mechanism, is a member of a family of structurally related Ser/Thr kinases that specifically phosphorylate Ser-51 on the alpha  subunit of the translation initiation factor eIF21 (1, 2). The binding of double-stranded RNA, thought to be generated during viral infections, is proposed to alter the conformation of PKR and activate the kinase to autophosphorylate (1, 2). The active, phosphorylated form of PKR can then phosphorylate eIF2alpha on Ser-51 and convert eIF2 into an inhibitor of its guanine nucleotide exchange factor eIF2B, resulting in the inhibition of translation initiation (1, 2). The other members of the eIF2alpha kinase family are the mammalian heme-regulated inhibitor of translation (HRI) that is activated by heme deprivation, the apparently ubiquitous kinase GCN2, first identified in yeast but also found in flies and mammals, which is activated under conditions of amino acid or purine nucleotide deprivation (1-4), and the newly identified mammalian kinase PERK or PEK, a transmembrane kinase located in the endoplasmic reticulum that is activated under conditions of endoplasmic reticulum stress (5, 6). In the yeast Saccharomyces cerevisiae, low level phosphorylation of eIF2alpha by GCN2 alters the pattern of translation reinitiation on the GCN4 mRNA and induces GCN4 expression (2). Increased synthesis of GCN4, a transcriptional activator of amino acid biosynthetic genes, enables cells to withstand amino acid starvation conditions. The mammalian eIF2alpha kinases PKR and HRI can substitute for GCN2 in yeast to phosphorylate eIF2alpha and stimulate GCN4 translation (7). In addition, high level phosphorylation of eIF2alpha in yeast by mutationally hyperactivated alleles of GCN2 or by overexpression of PKR or HRI severely inhibits general translation initiation and impairs cell growth (7-10).

In addition to regulating translation by phosphorylating eIF2alpha , PKR has been proposed to play roles in cell signaling (11) and growth control (12). In addition, several reports have proposed additional substrates for PKR (13-16). In biochemical analyses, PKR has been shown to phosphorylate intact eIF2alpha or a 12-residue peptide containing the Ser-51 phosphorylation site (17). As part of our studies aimed to define the in vivo substrate recognition properties of PKR, we chose to examine the ability of PKR, HRI, and GCN2 to phosphorylate Thr or Tyr in place of Ser at residue 51 in eIF2alpha . In general, protein kinases phosphorylate either Ser/Thr or Tyr residues, and these two classes of protein kinases are mutually exclusive. In fact, when Ser or Thr was substituted for a Tyr autophosphorylation site in the p130gag-fps Tyr kinase of fujinami sarcoma virus, neither hydroxyamino acid was phosphorylated, and the mutant kinases had reduced enzymatic and oncogenic activities (18). Similarly, structural analyses of Ser/Thr and Tyr kinases support the view that the active sites of these enzymes are designed to ensure substrate selectivity (19, 20). Our analyses have revealed that the three eIF2alpha kinases can phosphorylate Thr in place of Ser-51 in eIF2alpha and, in addition, that PKR and HRI can phosphorylate Tyr in place of eIF2alpha -Ser-51. These results suggest that PKR and HRI may have a rather flexible active site that can accommodate Tyr as well as Ser/Thr, and these results have interesting implications regarding the substrate recognition properties of PKR.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- The SUI2-S51T and SUI2-S51Y alleles encoding, respectively, the yeast eIF2alpha -S51T and eIF2alpha -S51Y proteins were constructed using the polymerase chain reaction. The 5'-TCC-3' (Ser-51) codon of SUI2 was changed to either 5'-ACC-3' (Thr-51) or 5'-TAC-3' (Tyr-51). The SUI2 alleles were transferred to a low copy number LEU2/CEN4 vector to create pC105 (Ser-51), pC107 (S51T), and pC110 (S51Y) or to the high copy number LEU2 vector pRS425 (21) to create pC136 (S51Y). The wild-type SUI2 (Ser-51) and SUI2-S51A (22) alleles were subcloned into pRS425 to create the high copy number LEU2 SUI2 (pC133) and SUI2-S51A (pC135) plasmids. The low copy number URA3 plasmids carrying wild-type GCN2 (p722), GCNc-516 (GCN2c-E532K-E1522K, p1056), or GCN2c-513 (GCN2c-M719V-E1537G, p1052) have been described (9). The high copy number pEMBLyex4 plasmids that express human PKR (p1420) or PKR-K296R (p1421) and rabbit HRI (p1246) or HRI-K199R (p1247) under the control of a yeast GAL-CYC1 chimeric promoter were described previously (7).

Strains-- Standard methods were used for culturing and transformation of yeast strains (23). The strains H1925 (Mata ura3-52 leu2-3 leu2-112 trp1-Delta 63 sui2Delta gcn2Delta , p919[SUI2 URA3], p1108[GCN4-lacZ TRP1] integrated at trp1-Delta 63); H2507 (Mata ura3-52 leu2-3 leu2-112 trp1-Delta 63 sui2Delta gcn2Delta , p919[SUI2 URA3]); and J101 (Mata ura3-52 leu2-3 leu2-112 trp1-Delta 63 sui2Delta gcn2Delta gcn3Delta , p919[SUI2 URA3]) were transformed with low or high copy number LEU2 plasmids containing the SUI2 mutant alleles, and then the transformants were transferred to medium containing 5-fluoroorotic acid to evict the URA3 plasmid carrying wild-type SUI2. The various eIF2alpha kinase plasmids were introduced into the resultant strains by transformation selecting for Ura prototrophy.

Assays of GCN4-lacZ Expression and Isoelectric Focusing (IEF) Gel Electrophoresis-- Identical cell growth conditions were used for GCN4-lacZ expression and IEF gel electrophoresis assays. For strains expressing HRI or HRI-K199R, pre-cultures were incubated 2 days in SR medium (2% raffinose in place of glucose in SD medium) and then inoculated 1:50 into fresh SR medium and harvested after overnight growth. For strains expressing PKR or PKR-K296R, pre-cultures were grown 2 days in SD medium, and then cells (~0.1 A600 units) were inoculated into SGR medium (10% galactose plus 2% raffinose in place of glucose in SD medium) and harvested after overnight growth. The methods for growing strains expressing GCN2 or GCN2c kinases, cell harvesting and breaking, beta -galactosidase assays, and IEF gel electrophoresis have been described previously (22).

Immunodetection and Immunoprecipitation Methods-- To detect phosphotyrosine on immunoblots, the membrane was probed with affinity purified rabbit anti-phosphotyrosine polyclonal antibodies (2.3 µg/ml) in 1× TBS-T (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.25% Tween 20) containing 3% bovine serum albumin. The anti-phosphotyrosine antibodies were a kind gift of Dr. R. Friesel, Holland Laboratory, American Red Cross, Rockville, MD. In addition, anti-phosphotyrosine antibodies were obtained from Zymed Laboratories Inc. (South San Francisco, CA). To detect PKR, the membrane was probed with monoclonal anti-PKR antibodies (71/10) in 1× TBS-T containing 5% nonfat dry milk, as described previously (7, 24).

PKR expression from the plasmid pET-PKR was induced in the Escherichia coli strain BL21(DE3)pLysS as described previously (24). Cells were pelleted, suspended in lysis buffer (20 mM Tris-HCl (pH 7.5), 50 mM KCl, 150 mM NaCl, 20% glycerol, 1% Triton X-100, 0.5 mM EDTA) containing inhibitors (1 mM phenylmethylsulfonyl fluoride, 7 µg/ml pepstatin, 50 mM NaF, 35 mM beta -glycerol phosphate), and then disrupted by sonication. PKR expression from the plasmid p1420 was induced in a derivative of the gcn2Delta yeast strain H2507 expressing eIF2alpha -S51A as described above for the IEF analysis. Cells were pelleted, suspended in lysis buffer, and broken with glass beads in a Braun homogenizer as described previously (22). To induce PKR expression, HeLa cells were treated overnight with interferon and then harvested, washed with cold phosphate-buffered saline, suspended in 0.5× RIPA buffer (25 mM Tris-HCl (pH 7.5), 75 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.05% SDS) containing inhibitors (see above), and broken by subjecting them to freeze-thaw cycles three times. Following breakage, the lysates from all three cell types were cleared by centrifugation. Whole cell extracts were incubated with anti-phosphotyrosine antibodies prebound to protein A-Sepharose beads or with beads alone in 0.2 ml of lysis buffer at 4 °C for 1 h with rocking. The beads were washed three times each with 0.5 ml of lysis buffer and then boiled 5 min in SDS loading buffer.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of Translation in Yeast Cells Expressing eIF2alpha -S51T or eIF2alpha -S51Y in Place of Wild-type eIF2alpha -- To initiate an analysis of substrate recognition by the eIF2alpha kinases, we examined translational regulation in yeast strains in which Thr or Tyr was substituted for Ser-51 in eIF2alpha . Plasmids that express wild-type or inactive forms of PKR or HRI or containing wild-type GCN2 or hyperactivated GCN2c alleles were introduced into gcn2Delta yeast strains expressing either eIF2alpha -S51T (Thr-51), eIF2alpha -S51Y (Tyr-51), eIF2alpha -S51A (Ala-51), or wild-type eIF2alpha (Ser-51). Expression of the GCN2c-513 kinase or PKR was toxic in strains expressing either the Ser-51 or Thr-51 forms of eIF2alpha ; however, the toxicity of the GCN2c kinase was slightly reduced in the Thr-51 strain (Fig. 1, A and B, GCN3 sectors). In contrast, the eIF2alpha -S51A and eIF2alpha -S51Y mutations completely suppressed the toxic effects of the GCN2c and PKR kinases. Translational regulation by the eIF2alpha kinases is dependent on both phosphorylation of eIF2alpha and the ability of phosphorylated eIF2 to inhibit its guanine nucleotide exchange factor eIF2B. The fact that expression of PKR and the other kinases showed no toxicity in the eIF2alpha -S51Y strain suggests two possibilities as follows: 1) these kinases fail to phosphorylate eIF2alpha -S51Y, or 2) the phosphorylated form of eIF2alpha -S51Y is a weaker inhibitor of eIF2B than phosphorylated wild-type eIF2alpha . This inhibition of eIF2B by phosphorylated eIF2 is dependent on the alpha  subunit of eIF2B, encoded by GCN3 in yeast (2, 7). Deletion of GCN3 suppressed the toxicity resulting from expression of GCN2c-513 or PKR in yeast strains expressing either the Ser-51 or Thr-51 forms of eIF2alpha (Fig. 1, A and B, gcn3Delta sectors). These results suggest that the GCN2c and PKR kinases efficiently phosphorylate eIF2alpha on Ser or Thr at residue 51 and that the phosphorylated forms of wild-type eIF2alpha and eIF2alpha -S51T inhibit translation via the same mechanism.


View larger version (83K):
[in this window]
[in a new window]
  		Fig. 1.   Growth rate analysis and amino acid analog sensitivity of yeast strains expressing different eIF2alpha alleles and various eIF2alpha kinases. The indicated eIF2alpha proteins (S, wild-type eIF2alpha ; T, eIF2alpha -S51T; Y, eIF2alpha -S51Y; or A, eIF2alpha -S51A) were expressed in the isogenic strains H2507 (gcn2Delta GCN3) and J101 (gcn2Delta gcn3Delta ) as described under "Experimental Procedures." A, the resulting strains were then transformed with the plasmid p1052 carrying the GCN2c-513 allele. The indicated transformants were streaked on SD plates and incubated 2 days at 30 °C. B, the strains expressing the various eIF2alpha proteins were transformed with the plasmid p1420 that expresses the human PKR kinase under the control of a galactose-inducible promoter. The indicated transformants were streaked on an SGal plate (10% galactose) and incubated 6 days at 30 °C. C, the strains described above, expressing the various eIF2alpha proteins, were transformed with plasmids encoding the indicated eIF2alpha kinases as follows: GCN2 (p722), PKR (p1420), PKR-K296R (p1421), HRI (p1246), and HRI-K199R (p1247). Expression of GCN2 was directed by the authentic GCN2 promoter, whereas the mammalian kinases were expressed under the control of a yeast galactose-inducible promoter. Patches of transformants were grown to confluence on SD medium and replica-plated to SGal medium and SGal plus 3-AT (10 mM) medium. Plates were incubated at 30 °C for 3 days.

A more sensitive assay of translational regulation in yeast is to monitor GCN4 expression. In wild-type strains phosphorylation of eIF2alpha by GCN2 stimulates GCN4 expression and enables cells to grow on medium containing 3-aminotriazole (3-AT), an inhibitor of histidine biosynthesis. Expression of GCN2 or HRI, but not the catalytically inactive HRI-K199R, conferred resistance to 3-AT in gcn2Delta strains expressing either the Ser-51 or Thr-51 forms of eIF2alpha but not in strains expressing eIF2alpha -S51A or eIF2alpha -S51Y (Fig. 1C). High level expression of PKR was lethal in strains expressing wild-type eIF2alpha or eIF2alpha -S51T, independent of 3-AT (Fig. 1C, left and right panels). In addition, expression of PKR, but not the catalytically inactive PKR-K296R, in eIF2alpha -S51Y strains conferred resistance to 3-AT, suggesting that PKR can phosphorylate eIF2alpha on Tyr at residue 51 and that phosphorylated eIF2alpha -S51Y can inhibit eIF2B and regulate GCN4 translation. Whereas the eIF2alpha -S51Y strain expressing HRI failed to grow on SGal (10% galactose) medium containing 3-AT (Fig. 1C), it was partially resistant to 3-AT when grown on SR (2% raffinose) medium (data not shown).

To provide a quantitative measure of the regulation of GCN4 expression in the strains expressing the various eIF2alpha proteins and kinases, expression of a GCN4-lacZ reporter was assayed. For the GCN2 and GCN2c-513 kinases the induction of GCN4-lacZ expression was similar in strains expressing either the Ser-51 or Thr-51 forms of eIF2alpha (Fig. 2). However, GCN4 expression in the eIF2alpha -S51Y strain was almost identical to that observed in a strain expressing the non-phosphorylatable Ala-51 form of eIF2alpha . In strains expressing wild-type eIF2alpha , eIF2alpha -S51T, or eIF2alpha -S51Y, PKR generated 2-5-fold higher GCN4-lacZ expression than inactive PKR-K296R (Fig. 2). In addition, GCN4-lacZ expression was 2-fold higher in eIF2alpha -S51Y versus eIF2alpha -S51A strains expressing PKR. These results are consistent with the 3-AT-resistant phenotype noted for eIF2alpha -S51Y strains expressing PKR (Fig. 1C) and support the idea that PKR can phosphorylate eIF2alpha on Tyr at residue 51. 


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2.   Regulation of GCN4 expression by GCN2 and PKR in yeast strains expressing various eIF2alpha mutant proteins. The indicated kinases were expressed in derivatives of the gcn2Delta strain H1925 containing the various eIF2alpha proteins. beta -Galactosidase activities expressed from an integrated wild-type GCN4-lacZ fusion were measured in whole cell extracts and are the averages of 2-3 independent transformants; S.Es. were 32% or less. For strains expressing GCN2 or GCN2c-513, cells were grown under either non-starvation conditions where GCN4 expression is repressed (R) or under amino acid starvation conditions imposed by the addition of 10 mM 3-AT, where GCN4 expression is derepressed (DR). The PKR and PKR-K296R proteins were expressed from a yeast GAL-CYC1 hybrid promoter. For assays, cells were grown exponentially in SD medium, where kinase expression is low, and then shifted to SGR-inducing medium. Cells were harvested after overnight growth in inducing medium. The GCN4-lacZ expression values obtained for GCN2 and PKR cannot be directly compared because the different media used for these cultures resulted in altered basal levels of GCN4-lacZ expression, as is apparent in the eIF2alpha -S51A strain.

IEF Analysis Reveals Phosphorylation of eIF2alpha in Vivo on Ser, Thr, or Tyr at Residue 51-- When analyzed by IEF-PAGE, eIF2alpha resolves as a doublet. The lower species of this doublet co-migrates with eIF2alpha from strains expressing mutant kinases or eIF2alpha -S51A and represents basal eIF2alpha , whereas the upper species of eIF2alpha is phosphorylated on residue 51 (7, 22). In IEF-PAGE analyses GCN2, GCN2c-516, and more active GCN2c-513 kinases were found to phosphorylate eIF2alpha on Ser or Thr but not Tyr or Ala at residue 51 (Fig. 3, A and B), consistent with the results of the genetic tests. In addition, phosphorylation of eIF2alpha was readily detected in strains expressing wild-type eIF2alpha , eIF2alpha -S51T, or eIF2alpha -S51Y and either PKR or HRI but not PKR-K296R or HRI-K199R (Fig. 3C, upper panel, and data not shown).


View larger version (39K):
[in this window]
[in a new window]
  		Fig. 3.   IEF gel electrophoresis of eIF2alpha from yeast strains expressing various eIF2alpha proteins and the GCN2, GCN2c, or PKR kinase. Plasmids that express the indicated eIF2alpha kinases were introduced into gcn2Delta yeast strains expressing the indicated eIF2alpha proteins from low copy number plasmids. A and B, strains expressing GCN2 and GCN2c kinases. Yeast strains expressing the indicated eIF2alpha proteins were transformed with plasmids that express wild-type GCN2 (p722), or the constitutively activated GCN2c-516 (p1056), or GCN2c-513 (p1052) kinases under the control of the natural GCN2 promoter. Cells were grown under nonstarvation conditions (R) or amino acid starvation conditions invoked by the addition of 10 mM 3-AT (DR), as indicated. C, strains expressing PKR. Plasmids expressing wild-type PKR (p1420) or the inactive mutant PKR-K296R (p1421) under the control of a GAL-CYC1 hybrid promoter were introduced into gcn2Delta yeast strains expressing the indicated eIF2alpha proteins. Extracts were prepared from cells grown exponentially in SD medium and then shifted to SGR medium to induce PKR expression. Immunoblot analysis using PKR monoclonal antibodies on 50-µg aliquots of the same extracts used for IEF-PAGE are aligned below the IEF data.

To confirm that PKR and HRI were phosphorylating eIF2alpha -S51Y on Tyr, a second IEF-PAGE analysis was performed. In yeast strains lacking the endogenous eIF2alpha kinase GCN2 and expressing either wild-type eIF2alpha or eIF2alpha -S51Y, the expression of wild-type PKR or HRI resulted in eIF2alpha focusing as a doublet on IEF gels (Fig. 4, upper panel, lanes 2, 4, 5, and 7). When the same blot from Fig. 2 (upper panel) was probed with anti-phosphotyrosine antibodies (Fig. 4, lower panel), cross-reactive bands were only detected in samples from strains expressing eIF2alpha -S51Y and a wild-type kinase. When the two blots (Fig. 4, upper and lower panels) were overlaid, the anti-phosphotyrosine cross-reactive species aligned perfectly with the upper, hyperphosphorylated form of eIF2alpha . These results confirmed that PKR and HRI were phosphorylating eIF2alpha -S51Y on Tyr and demonstrated that in vivo these proteins possess Tyr kinase activity. Comparison of the ratio of the hyperphosphorylated to the basal form of wild-type eIF2alpha and eIF2alpha -S51Y in strains expressing PKR (see Fig. 4, lanes 4 and 5; also Fig. 3C, upper panel, 1st and 5th lanes) may suggest that eIF2alpha -S51Y is a poorer substrate for PKR than is wild-type eIF2alpha ; however, alternative interpretations of these results are provided below. The inability to detect Tyr phosphorylation by GCN2 may suggest that GCN2 is an inherently less active kinase than HRI or PKR or that the GCN2 active site cannot accommodate a Tyr residue. Alternatively, it is likely that HRI and PKR were expressed at higher levels than GCN2 in these experiments, so it may be possible to detect Tyr kinase activity if we express GCN2 at higher levels in yeast cells.


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4.   IEF gel electrophoresis of eIF2alpha and eIF2alpha -S51Y from yeast strains expressing PKR and HRI. Plasmids expressing wild-type HRI (p1246) and PKR (p1420) as well as the inactive mutants HRI-K199R (p1247) and PKR-K296R (p1421) under the control of a GAL-CYC1 hybrid promoter were introduced into gcn2Delta yeast strains expressing either wild-type eIF2alpha (lanes 1-4) or eIF2alpha -S51Y (lanes 5-8) from high copy number plasmids. For strains expressing HRI, extracts were prepared from cells grown overnight under inducing conditions in SR medium. For strains expressing PKR, extracts were prepared from cells grown exponentially in SD medium and then shifted to SGR medium to induce PKR expression. In the upper panel the blot was probed with anti-eIF2alpha antiserum, and in the lower panel the same blot was probed with affinity purified anti-phosphotyrosine antibodies.

The expression of PKR is subject to negative translational autoregulation in both yeast (7, 10) and mammalian (1) cells such that kinase expression is inversely related to its effects on cellular translational activity. For example, although PKR was expressed at lower levels than PKR-K296R in strains expressing wild-type eIF2alpha (Fig. 3C, lower panel, 1st 2 lanes), this autoregulation was relieved in eIF2alpha -S51T strains and abolished in eIF2alpha -S51A and eIF2alpha -S51Y strains (Fig. 3C, lower panel, last 6 lanes). In addition, whereas the GCN2c-513 kinase appeared to phosphorylate wild-type eIF2alpha and eIF2alpha -S51T to the same extent (Fig. 3B), the eIF2alpha -S51T strain grew significantly better (Fig. 1). This lack of correlation between eIF2alpha phosphorylation and growth rate was also observed in eIF2alpha -S51Y strains expressing PKR as noted previously. These results are consistent with a model in which the phosphorylated forms of eIF2alpha -S51T and eIF2alpha -S51Y are weaker inhibitors of eIF2B than phosphorylated wild-type eIF2alpha . Finally, whereas the eIF2alpha , eIF2alpha -S51T, and eIF2alpha -S51Y proteins were phosphorylated to similar levels, it cannot be concluded that PKR and HRI are equally efficient at phosphorylating these three different amino acids at residue 51, because in vivo phosphorylation levels are dependent on the balance between kinase and phosphatase activities. As we do not know the identity or the efficiency of the phosphatases that dephosphorylate these three eIF2alpha proteins, we cannot at this time evaluate the relative efficiencies of PKR and HRI to phosphorylate eIF2alpha in vivo on Ser versus Thr or Tyr at residue 51.

Immunodetection of Human PKR Using Anti-phosphotyrosine Antibodies-- Previously it has been reported that mouse PKR, also known as TIK, can be detected in immunoblot assays using anti-phosphotyrosine antibodies (25). To determine if human PKR is also immunoreactive with anti-phosphotyrosine antibodies, wild-type human PKR and the inactive PKR-K296R proteins were expressed in E. coli, and crude protein extracts were separated by SDS-PAGE followed by immunoblotting with anti-PKR or anti-phosphotyrosine antibodies. As shown in Fig. 5A (right panel), both wild-type PKR and the PKR-K296R mutant proteins were expressed in E. coli. When the same membrane was probed with affinity purified anti-phosphotyrosine antibodies, cross-reactive species were detected in extracts prepared from cells expressing wild-type PKR but not PKR-K296R (Fig. 5A, left panel). Similar results demonstrating that recombinant wild-type human and mouse PKR (TIK), but not catalytic mutants, cross-react with anti-phosphotyrosine antibodies were recently published during the course of these experiments (26). In addition to the prominent anti-phosphotyrosine antibody cross-reactive species co-migrating with PKR at approx 70 kDa, several larger proteins were detected (Fig. 5A, left panel). This result suggests that PKR expressed in E. coli may autophosphorylate on Tyr and can also phosphorylate certain bacterial proteins on Tyr.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 5.   Immunodetection of human PKR using anti-phosphotyrosine antibodies. A, immunoblot analysis of PKR expressed in E. coli using anti-PKR and anti-phosphotyrosine antibodies. Lysates of E. coli BL21(DE3)pLysS cells expressing wild-type PKR, PKR-K296R, or no PKR (vector) were subjected to 10% SDS-PAGE followed by immunoblotting with polyclonal anti-phosphotyrosine antibodies (left panel). Immune complexes were detected by chemiluminescence. The blot was then stripped according to the vendor's instructions, probed with anti-PKR monoclonal antibodies (right panel), and immune complexes were again detected by chemiluminescence. The migration of molecular mass markers (kDa) is indicated on the left. B, immunoprecipitation of PKR using anti-phosphotyrosine antibodies. Crude protein extracts from the indicated cells, described below, were incubated with anti-phosphotyrosine antibodies prebound to protein A-Sepharose beads or with beads alone (no antibody, no Ab). Immunoprecipitated proteins were eluted in SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting with anti-PKR monoclonal antibodies. Lanes 1-3, a derivative of the gcn2Delta yeast strain H2507 expressing eIF2alpha -S51A and transformed with the PKR expression vector p1420 was grown in SGR medium to induce PKR expression. For immunoprecipitation reactions 200 µg of whole cell extract was used; the loading control was 4 µg of crude extract. Lanes 4-6, expression of wild-type PKR in the E. coli strain BL21(DE3)pLysS was induced by addition of isopropyl-1-thio-beta -D-galactopyranoside to the culture medium. For immunoprecipitation (IP) reactions 200 µg of extract was used; the loading control was 8 µg of crude extract. Lanes 7-9, HeLa cells were treated overnight with interferon (alpha ) to induce PKR expression. For immunoprecipitation reactions 2.5 mg of whole cell extract was used; the loading control was 50 µg of crude extract. C, immunoprecipitation of PKR, but not PKR-K296R, using antiphosphotyrosine antibodies. Crude protein extracts from E. coli cells expressing PKR or PKR-K296R were incubated with anti-phosphotyrosine antibodies prebound to protein A-Sepharose beads. Immunoprecipitated proteins were eluted and analyzed by SDS-PAGE as described above. For the immunoprecipitation reactions 500 µg of extract was used; the loading control was 2 µg of crude extract.

To confirm that PKR was the major species cross-reacting with anti-phosphotyrosine antibodies, we performed immunoprecipitation reactions. As shown in Fig. 5B (lanes 3 and 6), recombinant PKR could be immunoprecipitated from both yeast and bacterial cell extracts using affinity purified anti-phosphotyrosine antibodies. PKR was not precipitated when the anti-phosphotyrosine antibodies were omitted from the reactions (Fig. 5B, compare lanes 3 versus 2 and 6 versus 5). In addition, the precipitation was specific for functional PKR because the catalytically inactive PKR-K296R protein expressed in E. coli could not be precipitated using anti-phosphotyrosine antibodies (Fig. 5C, lanes 3 and 4). As shown in Fig. 5B (lanes 7-9), the endogenous PKR expressed in interferon-treated HeLa cells could also be immunoprecipitated using the anti-phosphotyrosine antibodies. Whereas these results suggest that PKR can autophosphorylate on Tyr, phosphoamino acid analyses of PKR isolated from in vivo labeled yeast and bacterial cells revealed phosphoserine and phosphothreonine, but not phosphotyrosine (data not shown). This latter result is consistent with the results of Icely et al. (25), who could only find phosphoserine and phosphothreonine in mouse PKR despite the fact that mouse PKR also cross-reacted with anti-phosphotyrosine antibodies. Icely et al. (25) speculated that the anti-phosphotyrosine antibodies may have recognized an unusual epitope on mouse PKR (25); however, negative results in phosphoamino acid analysis may reflect a low phosphorylation stoichiometry or a labile phosphotyrosine residue (27). In addition, it has been proposed that anti-phosphotyrosine antibodies may be over 100-fold more efficient at detecting phosphotyrosine than is phosphoamino acid analysis (28). Due to these conflicting results and the plausible explanations for failure to detect phosphotyrosine in the phosphoamino acid analyses, we are unable to conclude whether human PKR autophosphorylates on Tyr.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown that the kinases PKR, HRI, and GCN2 can phosphorylate eIF2alpha on Ser or Thr at residue 51. In addition both PKR and HRI can phosphorylate eIF2alpha on Tyr at residue 51 in vivo. It is generally accepted that Ser and Thr kinases are structurally similar, and many kinases are known to phosphorylate both residues, so the finding that the eIF2alpha kinases could phosphorylate Thr in place of Ser-51 is not surprising. However, the phosphorylation of Tyr at residue 51 in eIF2alpha by PKR and HRI is unexpected.

Two proposals can account for the Tyr phosphorylation activity by PKR and HRI. In the first proposal PKR and HRI would recognize eIF2alpha with high affinity and simply phosphorylate any hydroxyl group present at residue 51. Based on a mutational analysis of the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR with homology to eIF2alpha , we have proposed that PKR utilizes a sequence element over 30 residues from the site of phosphorylation to recognize eIF2alpha (29). According to this model, the ability of PKR to phosphorylate Tyr in place of Ser-51 in eIF2alpha simply reflects the strong contribution of this remote sequence for substrate recognition by PKR and the lack of specificity determinants around residue 51. Consistent with this idea, it has ben reported that the PKR and HRI phosphorylation of intact eIF2 is roughly 3 orders of magnitude more efficient than phosphorylation of a 12-residue synthetic peptide containing the Ser-51 phosphorylation site (30). However, regardless of how PKR initially recognizes eIF2alpha , it is important to note that the kinase active site must be able to accommodate both the alkyl hydroxyl groups of Ser and Thr and the phenolic hydroxyl of Tyr. The crystal structures solved to date for both Ser/Thr and Tyr kinases suggest that these enzymes would be unable to phosphorylate substrates on the alternate phospho-accepting residue due principally to steric limitations (20). Indeed, it has previously been shown that the p130gag-fps Tyr kinase could not phosphorylate Ser or Thr in place of an authentic Tyr phosphorylation site (18). Therefore, it is reasonable to expect that PKR and HRI possess a unique and more flexible structure as compared with the traditional Ser/Thr or Tyr kinases.

The second proposal to account for the Tyr kinase activity of PKR and HRI is that these proteins are members of the class of dual specificity protein kinases. A number of kinases have been proposed to have dual specificity; however, the criteria used in making this assignment has not been standardized (27, 31). Several kinases autophosphorylate on both Tyr and Ser/Thr residues (27, 31), and the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase (32) phosphorylates the extracellular signal-regulated kinases on both Tyr and Thr. The wee1 kinase, although structurally related to the Ser/Thr class of protein kinases, phosphorylates the cdc2 kinase on Tyr (27). The Myt1 kinase, identified in both Xenopus and humans and a member of the wee1 family of kinases, phosphorylates Cdc2 on both Thr and Tyr (33); and recently, Myt1 has also been shown to autophosphorylate in vitro on Ser, Thr, and Tyr (34). In contrast to these kinases in which phosphotyrosine and phosphoserine or phosphothreonine was readily detected in substrate phosphorylation or autophosphorylation reactions, other proposed dual specificity kinases, including TIK (25) and PYT/ESK/TTK (28), were identified based primarily on cross-reactivity with anti-phosphotyrosine antibodies. These kinases structurally resemble Ser/Thr kinases; however, when expressed in bacteria they cross-reacted with anti-phosphotyrosine antibodies. In this report we have demonstrated that human PKR (the TIK homolog) will also cross-react with anti-phosphotyrosine antibodies. Whereas these results may suggest that PKR is a dual specificity kinase, it will be necessary to identify an in vivo substrate that PKR phosphorylates on Tyr to conclude convincingly that PKR is a member of the class of dual specificity protein kinases.

The identification of Tyr and Thr kinase activity by PKR is very interesting in regards to alternative substrates and biological roles proposed for PKR. Although eIF2alpha is the only well characterized PKR substrate, and the regulation of translation is thought to be the primary function of PKR, recent reports suggest additional substrates and roles for PKR. PKR has been reported to phosphorylate Ikappa B (13), HIV Tat (14, 15), and NF90 (16) in vitro. In addition, overexpression of catalytically inactive mutants of PKR leads to heightened viral sensitivity (35), altered gene regulation (Ref. 36 and references therein) and malignant transformation (12). At least some of the effects of PKR on gene regulation do not appear to be mediated by changes in eIF2alpha phosphorylation, suggesting that phosphorylation of other proteins may mediate these effects (36). Finally, mice deficient in PKR are impaired in cell signaling pathways including their interferon-gamma and double-stranded RNA-induced antiviral response (11), although these effects are not observed in all PKR null mice (37). Our studies on eIF2alpha phosphorylation by PKR raise the possibility that PKR may have other substrates that it naturally phosphorylates on Thr or Tyr. As Tyr phosphorylation is a common step in cellular signal transduction pathways, it is tempting to speculate that the defects in signal transduction pathways and transcriptional regulation of gene expression associated with reduced PKR function are due to a loss of the PKR Tyr kinase activity. It will be interesting to identify these alternative PKR substrates and to determine if they are phosphorylated on Tyr, and thereby provide a physiological role for this unexpected Tyr kinase activity of PKR.

    ACKNOWLEDGEMENTS

We are grateful to Bob Friesel for the anti-phosphotyrosine antibodies, Scott Shors for the HeLa cells, Mike Mathews for PKR polyclonal antiserum, and Julie Watson and Ribogene, Inc., for PKR monoclonal antibodies. We thank members of the Dever and Hinnebusch laboratories for helpful discussions, and especially Alan Hinnebusch, Graham Pavitt, Minerva Garcia-Barrio, and Jim Anderson for comments on the manuscript. Finally, we thank an anonymous reviewer for several critical insights and helpful suggestions.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom reprint requests should be addressed: Bldg. 6A, Rm. B1A-02, 6 Center Dr. MSC 2716, National Institutes of Health, Bethesda, MD 20892-2716. Tel.: 301-496-4519; Fax: 301-496-8576; E-mail: tdever@box-t.nih.gov.

    ABBREVIATIONS

The abbreviations used are: eIF, eukaryotic initiation factor; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; 3-AT, 3-aminotriazole.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Clemens, M. J. (1996) in Translational Control (Hershey, J. W. B. , Mathews, M. B. , and Sonenberg, N., eds) , pp. 139-172, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
2. Hinnebusch, A. G. (1994) Semin. Cell Biol. 5, 417-426[CrossRef][Medline] [Order article via Infotrieve]
3. Santoyo, J., Alcalde, J., Mendez, R., Pulido, D., and de Haro, C. (1997) J. Biol. Chem. 272, 12544-12550[Abstract/Free Full Text]
4. Olsen, D. S., Jordan, B., Chen, D., Wek, R. C., and Cavener, D. R. (1998) Genetics 149, 1495-1509[Abstract/Free Full Text]
5. Harding, H. P., Zhang, Y., and Ron, D. (1999) Nature 397, 271-274[CrossRef][Medline] [Order article via Infotrieve]
6. Shi, Y., Vattem, K. M., Sood, R., An, J., Liang, J., Stramm, L., and Wek, R. C. (1998) Mol. Cell. Biol. 18, 7499-7509[Abstract/Free Full Text]
7. Dever, T. E., Chen, J. J., Barber, G. N., Cigan, A. M., Feng, L., Donahue, T. F., London, I. M., Katze, M. G., and Hinnebusch, A. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4616-4620[Abstract/Free Full Text]
8. Chong, K. L., Feng, L., Schappert, K., Meurs, E., Donahue, T. F., Friesen, J. D., Hovanessian, A. G., and Williams, B. R. G. (1992) EMBO J. 11, 1553-1562[Medline] [Order article via Infotrieve]
9. Ramirez, M., Wek, R. C., Vazquez de Aldana, C. R., Jackson, B. M., Freeman, B., and Hinnebusch, A. G. (1992) Mol. Cell. Biol. 12, 5801-5815[Abstract/Free Full Text]
10. Romano, P. R., Green, S. R., Barber, G. N., Mathews, M. B., and Hinnebusch, A. G. (1995) Mol. Cell. Biol. 15, 365-378[Abstract]
11. Yang, Y.-L., Reis, L. F. L., Pavlovic, J., Aguzzi, A., Schafer, R., Kumar, A., Williams, B. R. G., Aguet, M., and Weissmann, C. (1995) EMBO J. 14, 6095-6106[Medline] [Order article via Infotrieve]
12. Koromilas, A. E., Roy, S., Barber, G. N., Katze, M. G., and Sonenberg, N. (1992) Science 257, 1685-1689[Abstract/Free Full Text]
13. Kumar, A., Haque, J., Lacoste, J., Hiscott, J., and Williams, B. R. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6288-6292[Abstract/Free Full Text]
14. McMillan, N. A. J., Chun, R. F., Siderovski, D. P., Galabru, J., Toone, W. M., Samuel, C. E., Mak, T. W., Hovanessian, A. G., Jeang, K., and Williams, B. R. G. (1995) Virology 213, 413-424[CrossRef][Medline] [Order article via Infotrieve]
15. Brand, S., Kobayashi, R., and Mathews, M. (1997) J. Biol. Chem. 272, 8388-8395[Abstract/Free Full Text]
16. Langland, J., Kao, P., and Jacobs, B. (1999) Biochemistry 38, 6361-6368[CrossRef][Medline] [Order article via Infotrieve]
17. Mellor, H., and Proud, C. G. (1991) Biochem. Biophys. Res. Commun. 178, 430-437[CrossRef][Medline] [Order article via Infotrieve]
18. Weinmaster, G., and Pawson, T. (1986) J. Biol. Chem. 261, 328-333[Abstract/Free Full Text]
19. Hubbard, S. R., Wei, L., Ellis, L., and Hendrickson, W. A. (1994) Nature 372, 746-754[CrossRef][Medline] [Order article via Infotrieve]
20. Taylor, S. S., Radzio-Andzelm, E., and Hunter, T. (1995) FASEB J. 9, 1255-1266[Abstract]
21. Christianson, T. W., Sikorski, R. S., Dante, M., Shero, J. H., and Hieter, P. (1992) Gene (Amst.) 110, 119-122[CrossRef][Medline] [Order article via Infotrieve]
22. Dever, T. E., Feng, L., Wek, R. C., Cigan, A. M., Donahue, T. D., and Hinnebusch, A. G. (1992) Cell 68, 585-596[CrossRef][Medline] [Order article via Infotrieve]
23. Rose, M. D., Winston, F., and Hieter, P. (1989) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
24. Barber, G. N., Tomita, J., Hovanessian, A. G., Meurs, E., and Katze, M. G. (1991) Biochemistry 30, 10356-10361[CrossRef][Medline] [Order article via Infotrieve]
25. Icely, P. L., Gros, P., Bergeron, J. J. M., Devault, A., Afar, D. E. H., and Bell, J. C. (1991) J. Biol. Chem. 266, 16073-16077[Abstract/Free Full Text]
26. Abraham, N., Jaramillo, M., Duncan, P., Methot, N., Icely, P., Stojdl, D., Barber, G., and Bell, J. (1998) Exp. Cell Res. 244, 394-404[CrossRef][Medline] [Order article via Infotrieve]
27. Lindberg, R. A., Quinn, A. M., and Hunter, T. (1992) Trends Biochem. Sci. 17, 114-119[CrossRef][Medline] [Order article via Infotrieve]
28. Lindberg, R. A., Fischer, W. H., and Hunter, T. (1993) Oncogene 8, 351-359[Medline] [Order article via Infotrieve]
29. Kawagishi-Kobayashi, M., Silverman, J. B., Ung, T. L., and Dever, T. E. (1997) Mol. Cell. Biol. 17, 4146-4158[Abstract]
30. Mellor, H., and Proud, C. G. (1991) Biochem. Biophys. Res. Commun. 178, 430-437
31. Douville, E., Duncan, P., Abraham, N., and Bell, J. C. (1994) Cancer Metatasis Rev. 13, 1-7
32. Crews, C. M., Alessandrini, A., and Erikson, R. L. (1992) Science 258, 478-480[Abstract/Free Full Text]
33. Mueller, P. R., Coleman, T. R., Kumagai, A., and Dunphy, W. G. (1995) Science 270, 86-90[Abstract/Free Full Text]
34. Liu, F., Stanton, J. J., Wu, Z., and Piwnica-Worms, H. (1997) Mol. Cell. Biol. 17, 571-583[Abstract]
35. Der, S. D., and Lau, A. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8841-8845[Abstract/Free Full Text]
36. Koromilas, A. E., Cantin, C., Craig, A. W. B., Jagus, R., Hiscott, J., and Sonenberg, N. (1995) J. Biol. Chem. 270, 25426-25434[Abstract/Free Full Text]
37. Abraham, N., Stojdl, D. F., Duncan, P. I., Methot, N., Ishii, T., Dube, M., Vanderhyden, B. C., Atkins, H. L., Gray, D. A., McBurney, M. W., Koromilas, A. E., Brown, E. G., Sonenberg, N., and Bell, J. C. (1999) J. Biol. Chem. 274, 5953-5962[Abstract/Free Full Text]

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Eukaryot CellHome page
M. C. S. Moraes, T. C. L. Jesus, N. N. Hashimoto, M. Dey, K. J. Schwartz, V. S. Alves, C. C. Avila, J. D. Bangs, T. E. Dever, S. Schenkman, et al.
Novel Membrane-Bound eIF2{alpha} Kinase in the Flagellar Pocket of Trypanosoma brucei
Eukaryot. Cell, November 1, 2007; 6(11): 1979 - 1991.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Q. Su, S. Wang, D. Baltzis, L.-K. Qu, A. H.-T. Wong, and A. E. Koromilas
Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2alpha} RNA-dependent protein kinase.
PNAS, January 3, 2006; 103(1): 63 - 68.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Dey, B. Trieselmann, E. G. Locke, J. Lu, C. Cao, A. C. Dar, T. Krishnamoorthy, J. Dong, F. Sicheri, and T. E. Dever
PKR and GCN2 Kinases and Guanine Nucleotide Exchange Factor Eukaryotic Translation Initiation Factor 2B (eIF2B) Recognize Overlapping Surfaces on eIF2{alpha}
Mol. Cell. Biol., April 15, 2005; 25(8): 3063 - 3075.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, J.
Right arrow