Protein synthesis and protein phosphorylation during heat stress, recovery, and adaptation.

Incubating cells at elevated temperatures causes an inhibition of protein synthesis. Mild heat stress at 41-42 degrees C inhibits the fraction of active, polysomal ribosomes from greater than 60% (preheating) to less than 30%. A return to 37 degrees C leads to an increase in protein synthesis, termed "recovery." Continuous incubation at 41-42 degrees C also leads to a gradual restoration of protein synthesis (greater than 70% of ribosomes reactivated by 2-4 h), termed "adaptation". Protein synthesis inhibition and reactivation is prestressed, recovered cells that contain elevated levels of the heat stress proteins occur to the same extent and at the same rate as in "naive" cells. The adaptation response requires transcription of new RNA whereas recovery does not. A large number of phosphorylation changes are induced by severe heat stress and occur with kinetics similar to the inhibition of protein synthesis. These include phosphorylation of eukaryotic protein synthesis initiation factor (eIF)-2 alpha and dephosphorylation of eIF-4B and eIF-4Fp25 (eIF-4E). However, the extent to which the modification occurs is proportional to the severity of the stress, and, under mild (41-42 degrees C) heat stress conditions, these initiation factor phosphorylation changes do not occur. Similarly, under conditions of severe heat stress eIF-2 alpha and eIF-4B frequently recover to their prestress phosphorylation state before the recovery of protein synthesis. eIF-4E dephosphorylation likewise does not occur under mild heat stress conditions. Therefore, these changes in phosphorylation states, which are thought to be sufficient cause, are not necessary for the inhibition of protein synthesis observed.

Sleep: the effect of electroconvulsive shock in cats deprived of REM sleep.

Three cats were deprived of rapid-eye-movement (REM) sleep for 10 days, and three were deprived for 12 days. All cats received an electrically induced convulsion on each of the last 3 days of deprivation, as well as on the 1st recovery day just prior to sleep onset. As controls, four cats were deprived of REM sleep for 12 days and one was deprived for 10 days; the controls received no convulsions. Compensatory increases in REM sleep during recovery days were present in the convulsed animals, but were substantially lower than the recovery increases of control animals. During recovery REM sleep, convulsed cats did not display the exaggerated bursts of eye movements and body twitches seen in the nonconvulsed controls.

Initiation factor protein modifications and inhibition of protein synthesis.

The protein covalent modification state of eucaryotic initiation factors eIF-2 and eIF-4B in HeLa cells was examined after they were exposed to a variety of conditions or treatments that regulate protein synthesis. A few factors (e.g., variant pH and sodium fluoride) altered the phosphorylation state of the initiation factor proteins, but the majority (hypertonic medium, ethanol, dimethyl sulfoxide sodium selenite, sodium azide, and colchicine) had no effect on either protein. While initiation factor phosphorylation may regulate protein synthesis in response to many physiological situations, other pathways can regulate protein synthesis under nonphysiological circumstances.

Protein synthesis initiation factor modifications during viral infections: implications for translational control.

Infection of tissue culture cells with certain viruses results in the shutoff of host cell protein synthesis. We have examined virally infected cell lysates using two-dimensional gel electrophoresis and immunoblotting to ascertain whether initiation factor protein modifications are correlated with translational repression. Moderate increases in eukaryotic initiation factor (eIF)-2 alpha phosphorylation are detected in reovirus- and adenovirus-infected cells, as reported previously (Samuel et al., 1984; O'Malley et al., 1989). Neither vesicular stomatitis virus, vaccinia virus, frog virus III, rhinovirus, nor encephalomyocarditis virus caused significantly increased 2 alpha phosphorylation. There were no reproducible, significant changes in eIF-4A, eIF-4B, or eIF-2 beta in cells infected by any of these viruses. The cleavage of eIF-4F subunit p220, such as has been previously demonstrated to occur in poliovirus (Etchison et al., 1982) and rhinovirus (Etchison and Fout, 1985), was not detected in any of the other virus infections analyzed.

Cordycepin blocks recovery of non-heat-shock mRNA translation following heat shock in Drosophila.

Treatment of cells with cordycepin (3-deoxyadenosine), an inhibitor of cytoplasmic adenylation, blocks the restoration of normal translation following heat shock. Cordycepin also reduces heat-shock protein 70 (Hsp70) protein synthesis greater than 10-fold, while having little to no effect on mRNA accumulation. Parallel analysis of the poly(A)-binding protein detects no change in its abundance during heat shock or subsequent recovery. These results suggest that normal, non-heat-shock mRNA translational repression during heat shock may be caused by deadenylation, and that readenylation is required for restoration of activity. However, three independent analyses of the adenylation status of mRNAs during heat shock and recovery indicate that no significant changes in polyadenylation occur. (a) The total poly(A) content decreases by only about 10% during heat shock; (b) the size of the poly(A) tract decreases only marginally, from an average length of 75-90 nucleotides in non-heated cells to 45-60 nucleotides during heat shock; (c) virtually all mRNAs bind to oligo d(T)-cellulose, whether extracted from normal-temperature, heat-shock or recovered cells. Our results are most consistent with a model where the process of readenylation, rather than the specific poly(A) tail length, influences translational activation during recovery, paralleling a proposed model for the activation of translation during Xenopus oocyte maturation.

Changes in eIF-4D hypusine modification or abundance are not correlated with translational repression in HeLa cells.

Initiation factor eIF-4D is represented by about 11 X 10(6) molecules/HeLa cell (0.45% of the cytoplasmic protein molecules). The fraction of eIF-4D that contains the post-translational modification of lysine converted to hypusine is not regulated with respect to translation rate in HeLa cells. It is proportional to the rate of eIF-4D synthesis in exponentially growing cells (maximal protein synthesis rates) as well as in serum-depleted cells (protein synthesis rates depressed about 6-8-fold). In cells in which protein synthesis is arrested by cycloheximide, no hypusine addition or exchange is detected. During rapid repressions of protein synthesis due to either heat shock or hypertonic shock there is no change in the extent of eIF-4D containing hypusine. These results are most consistent with an eIF-4D biogenesis in which all molecules are modified to contain hypusine during or shortly after the translation process itself, and the modification state is not regulated thereafter.

RNA/protein interactions in the 5'-untranslated leader of HSP70 mRNA in Drosophila lysates. Lack of evidence for specific protein binding.

We have investigated the hypothesis that preferential translation of the heat stress mRNAs requires the binding of a trans-acting protein factor. 32P-Labeled RNA probes covering the 5'-untranslated region (5'-UTR) of HSP70 mRNA were synthesized, and gel retardation and UV cross-linking assays performed to identify trans-acting sequence-specific RNA-binding factors. The results indicate that no HSP70 5'-UTR RNA-specific binding proteins exist. Reducing the "stringency" in the gel retardation binding analyses revealed several non-sequence-specific RNA-binding complexes. The proteins bind RNA more strongly than DNA, show minor preferences for specific sequences, but none binds more strongly to the HSP70 5'-UTR than to other non-heat stress RNAs. Ultraviolet cross-linking analysis identifies two principal HSP70 5'-UTR binding proteins, of approximately 50 and 70 kDa. The p50 binding activity is increased severalfold for all mRNAs in heat-stressed lysates, and its binding produces the primary gel-retarded complex. Further detailed analyses were performed to probe for any possible heat-influenced changes. RNA/protein interactions are not affected by capping. Neither the kinetics nor the salt sensitivity of protein binding is affected by heat. Binding analyses using partial or complete HSP70 5'-UTR give qualitatively similar conclusions. Binding analyses were also carried out with several "normal" 5'-UTRs to investigate whether a heat-induced repressor might be activated by heating. No normal mRNA-specific heat-induced binding changes are detected. We conclude that heat-induced alterations in RNA-binding proteins do not mediate preferential translation of heat stress mRNAs or repression of normal mRNAs.

Transient acquired thermotolerance in Drosophila, correlated with rapid degradation of Hsp70 during recovery.

Acquired thermotolerance, measured either as increased cell viability following a lethal heat shock or by translational thermotolerance, appears rapidly following a 'priming' heat treatment, but also decays rapidly. 4 hours after priming heating thermotolerance is reduced by > 50% and by 9 hours it is virtually undetectable. Heat-shock protein 70 (Hsp70) turns over with a half-life of approximately 2 hours, and the decline in its intracellular abundance parallels the loss of acquired thermotolerance. Continuous heat shock extends the half-life of Hsp70 to approximately 7 hours. When Hsp70 is expressed at normal temperature using a metallothionein promoter, only partial acquired translational thermotolerance results. The results suggest that acquired thermotolerance is tightly regulated in Drosophila and partly, but not wholly, determined by post-translational regulation of Hsp70 levels.

Translational repression by chemical inducers of the stress response occurs by different pathways.

The mechanism by which chemical inducers of the stress response inhibit protein synthesis was examined. All the chemicals tested principally inhibit the initiation phase of translation. Covalent modification of the initiation factor proteins does not constitute a common mechanism. Eukaryotic initiation factor (eIF)-2 alpha phosphorylation is moderately to strongly induced by Na arsenite and diamide, but only slightly to imperceptibly affected by iodoacetamide, azetidine carboxylic acid, and canavanine. eIF-4B dephosphorylation does not occur in any case. The only consistent change detected is the hyperphosphorylation of the 28,000 Da heat stress protein. These results indicate that these diverse chemicals, all of which enhance the transcription of the stress mRNAs, do not inhibit translation by a common, recognized mechanism; it is likely that several distinct pathways leading to inhibition exist.

Striking multiplicity of eIF4E-BP1 phosphorylated isoforms identified by 2D gel electrophoresis regulation by heat shock.

Eukaryotic initiation factor eIF4E-binding protein 1 (eIF4E-BP1), or PHAS-I, is multiply phosphorylated by insulin-stimulated protein kinase(s). Estimates for the number of phosphorylation sites range from two to greater than eight. IEF/SDS/PAGE can precisely differentiate protein isoforms based on their differences in charge (phosphorylation) and molecular mass. In this study, the diversity of eIF4E-BP1 isoforms was determined using IEF/SDS/PAGE/immunoblotting of unfractionated cell lysates. To investigate the molecular regulation of phosphorylation, alterations in eIF4E-BP1 in response to heat shock in HeLa cells were determined. In exponentially growing cells, 8-10 prominent eIF4E-BP1 isoforms were detected. Following heat shock, a rapid, temperature-dependent dephosphorylation of eIF4E-BP1 occurs roughly concurrent with protein synthesis inhibition; during recovery from heat shock rephosphorylation of eIF4E-BP1 parallels restoration of protein synthesis. However, eIF4E-BP1 and eIF4E kinases remain highly active during heat shock, as okadaic acid treatment restores phosphorylation of both factors in heat shocked cells. eIF4E-BP1 dephosphorylation is associated with eIF4E dissociation from large molecular mass complexes and increased binding to eIF4E-BP1. The amount of eIF4E-BP1 converted to the dephosphorylated state is sufficient to titrate all the eIF4E present. eIF4E-BP1 phosphorylation changes regulated by heat shock also occur in Drosophila. Of the 10 isoforms of eIF4E-BP1 resolved by IEF/SDS/PAGE, at least seven are labelled with [32P] and all 10 are recognized by (eIF4E-BP1)-specific antibodies. These results identify a complex set of eIF4E-BP1 phosphorylation isoforms; changes in the expression of these isoforms in response to stresses such as heat shock may contribute to translation repression.

Heat shock-induced repression of proteolysis: poly(A)-binding protein degradation patterns can illusorily suggest its specific loss during heat shock.

Poly(A)-binding protein (PABP) is highly susceptible to proteolysis during cell lysis of Drosophila tissue culture cells unless substantial amounts of proteolysis inhibitors are included in the extraction buffer. This intrinsic proteolytic activity is substantially reduced during heat shock. An artifactual appearance that poly(A)-binding protein is specifically degraded by heat shock can result. Several contradictory descriptions of PABP may also be related to its proteolysis. Repression of proteolysis is likely to reflect a physiologically significant regulatory event, based on recent examinations of HSP70 stability during and after heat shock.

Sequence and structure determinants of Drosophila Hsp70 mRNA translation: 5'UTR secondary structure specifically inhibits heat shock protein mRNA translation.

Preferential translation of Drosophila heat shock protein 70 (Hsp70) mRNA requires only the 5'-untranslated region (5'-UTR). The sequence of this region suggests that it has relatively little secondary structure, which may facilitate efficient protein synthesis initiation. To determine whether minimal 5'-UTR secondary structure is required for preferential translation during heat shock, the effect of introducing stem-loops into the Hsp70 mRNA 5'-UTR was measured. Stem-loops of -11 kcal/mol abolished translation during heat shock, but did not reduce translation in non-heat shocked cells. A -22 kcal/mol stem-loop was required to comparably inhibit translation during growth at normal temperatures. To investigate whether specific sequence elements are also required for efficient preferential translation, deletion and mutation analyses were conducted in a truncated Hsp70 5'-UTR containing only the cap-proximal and AUG-proximal segments. Linker-scanner mutations in the cap-proximal segment (+1 to +37) did not impair translation. Re-ordering the segments reduced mRNA translational efficiency by 50%. Deleting the AUG-proximal segment severely inhibited translation. A 5-extension of the full-length leader specifically impaired heat shock translation. These results indicate that heat shock reduces the capacity to unwind 5-UTR secondary structure, allowing only mRNAs with minimal 5'-UTR secondary structure to be efficiently translated. A function for specific sequences is also suggested.

Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor eIF-2. Characterization of the protein and its messenger RNA.

A clone encoding the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha) was isolated from a lambda gt11 expression library of rat brain cDNAs. The fusion protein expressed by the recombinant phage reacts with eIF-2 alpha antiserum except when the serum is preadsorbed with pure eIF-2. The translation of hybrid-selected HeLa cell mRNA produces two proteins which are indistinguishable from authentic HeLa eIF-2 alpha and its phosphorylated form when analyzed by electrophoresis in two-dimensional isoelectrofocusing/sodium dodecyl sulfate-polyacrylamide gels and by partial protease digestion. HeLa cell eIF-2 alpha mRNA migrates as a single band of about 1600 nucleotides. The rat cDNA insert was sequenced, and the region coding for eIF-2 alpha was identified. A human cDNA clone was obtained by hybridization screening with the rat cDNA, and its sequence was determined also. Both rat and human eIF-2 alpha proteins comprise 315 amino acids (36.1 kDa) and differ by only three amino acids. The eIF-2 alpha mRNA is found exclusively in polysomes containing 10 or more ribosomes in exponentially growing HeLa cells. In serum-depleted cells which synthesize eIF-2 and bulk protein more slowly than exponential cells, the level of eIF-2 alpha mRNA is not changed, the average polysome size is reduced to 7, and little or no eIF-2 alpha mRNA is detected in the ribonucleoprotein fraction. These results are consistent with the view that eIF-2 alpha mRNA translation is very efficient compared to other mRNAs in the cell.

Immunoblot analysis of the structure of protein synthesis initiation factor eIF3 from HeLa cells.

Translational initiation factor eIF3 is a large, multisubunit protein complex involved in early steps of the initiation pathway. Affinity-purified polyclonal antibodies were used to analyze by immunoblotting the mass and charge characteristics of the subunits in HeLa cell lysates and in purified eIF3 preparations. The evidence indicates that eIF3 comprises at least seven antigenically distinct subunits: p170, p115, p66, p47, p44, p40, and p36. During purification, p170, p115, and p66 are partially degraded to smaller forms, which appear to be the major cause of variable subunit composition among preparations of eIF3.

Heat shock effects on phosphorylation of protein synthesis initiation factor proteins eIF-4E and eIF-2 alpha in Drosophila.

Heat shock of mammalian cells causes changes in initiation factor phosphorylation that likely contribute to or cause the translation reprogramming characteristic of heat shock. In these investigations we have carried out a parallel analysis of Drosophila, focusing on eIF-4E and eIF-2 alpha. eIF-4E plus associated proteins was purified from lysates by m7GTP-Sepharose chromatography. A minor fraction (< 10%) of eIF-4E is phosphorylated under normal growth conditions, and phosphorylation decreases during heat shock. Drosophila eIF-2 alpha has been identified by in vitro translation of T7 RNA polymerase-transcribed mRNA, and immunoblotting with anti-Drosophila eIF-2 alpha antiserum. 32P-labeling analysis (unfractionated cell lysates and immunoprecipitates) detects phosphorylated eIF-2 alpha, whose amount increases approximately 2-3-fold upon heat shock. Immunoblotting analysis of two-dimensional gel-resolved proteins to determine the mass fraction of eIF-2 alpha phosphorylated detects a single eIF-2 alpha spot in both normal temperature and heat shocked cells, indicating less than 5% phosphorylation after and before heat shock. Staining quantification is consistent with this low prevalence. A major phosphoprotein which copurifies with eIF-4E on m7GTP-Sepharose shows decreased overall phosphorylation and decreased association with eIF-4E following heat shock. Several distinctive characteristics of this phosphoprotein suggest it is Drosophila eIF-4B.

Activation of mitogen-activated protein kinase by heat shock treatment in Drosophila.

Heat shock treatment of Drosophila melanogaster tissue culture cells causes increased tyrosine phosphorylation of several 44 kDa proteins, which are identified as Drosophila mitogen-activated protein (MAP) kinases. Tyrosine phosphorylation occurs within 5 min, and is maintained at high levels during heat shock. It decreases to basal levels during recovery, concurrent with the repression of heat shock transcription and heat-shock-protein synthesis. The increased MAP kinase tyrosine phosphorylation is parallelled by increased MAP kinase activity. At least two MAP kinases, DmERK-A and DmERK-B, are identified whose tyrosine phosphorylation increases during heat shock. Thus MAP kinase activation is an immediate early response to heat shock, and its increased activity is maintained throughout heat shock treatment. Protracted MAP kinase activation may contribute to heat shock transcription factor phosphorylation and the numerous metabolic alterations that constitute the heat-shock response.

m7GpppG cap dependence for efficient translation of Drosophila 70-kDa heat-shock-protein (Hsp70) mRNA.

To investigate whether preferential translation of the heat-shock mRNAs occurs via cap-independent translation, the requirement for the m7GpppG cap structure for efficient translation of 70-kDa heat-shock-protein (Hsp70) mRNA was quantified by in vitro translation and by in vivo translation following electroporation. Hsp70 mRNA was transcribed in vitro with and without a cap structure. Translation in the rabbit reticulocyte or wheat germ lysate was reduced about 70% when the cap was absent. For comparison, translation of uncapped encephalomyocarditis virus 5'-untranslated-region-containing mRNA was equal to or greater than capped mRNA, whereas translation of several non-heat-shock mRNAs was reduced by 85-95% when capping was omitted. Cap-dependent translational stimulation of Hsp70 is not due to increased stability, is not a kinetic effect, and requires the methylated GpppG. To confirm the in vitro analyses, capped and uncapped mRNA were introduced into Drosophila tissue culture cells by electroporation, followed by heat shock. Paralleling the in vitro results, uncapped Hsp70 mRNA translation was 70-80% reduced relative to the capped form. Complementary experiments in which eIF-4 was inactivated in vitro using either m7GTP cap analogue or foot-and-mouth-disease virus L protease expression likewise indicated that the cap-dependent translation pathway is required for optimal Hsp mRNA translation. Since cellular Hsp70 mRNA translation during heat shock is very efficient, it is unlikely that translation via a cap-independent pathway is the principal basis for preferential translation.

Modification of protein synthesis initiation factors and the shut-off of host protein synthesis in adenovirus-infected cells.

A substantial body of data, largely derived from study of cell extracts, indicates that protein synthesis in adenovirus-infected cells requires VA RNAI at late times of infection to prevent the activation of a protein kinase known as DAI, and the consequent phosphorylation of the alpha-subunit of initiation factor eIF-2. To verify this conclusion, we have measured the steady-state levels of eIF-2 alpha phosphorylation in cells infected with wild-type virus (Ad2) and a mutant that produces no VA RNAI (Ad5dl331). Consistent with the proposed mechanism, the alpha-subunit was very highly phosphorylated (approximately 90%) at late times of infection with Ad5dl331. Surprisingly, eIF-2 alpha phosphorylation also increased (to approximately 30%) at late times of infection with Ad2, suggesting that VA RNA and DAI might be involved in the selective translation of viral mRNA and the shut-off of host cell protein synthesis during the late phase. In agreement with this model, host protein synthesis shut-off is defective in cells expressing low levels of DAI.

Ca(2+)-dependent p47phox translocation in hydroperoxide modulation of the alveolar macrophage respiratory burst.

Oxidative stress produces dual effects on the respiratory burst of rat alveolar macrophages. Preincubation with hydroperoxide concentrations [H2O2 or tert-butyl hydroperoxide (t-BOOH); < 50 microM] enhances stimulation of the respiratory burst, whereas higher concentrations inhibit stimulation. Both the enhancement and inhibition are markedly attenuated by buffering t-BOOH-induced changes in intracellular Ca2+ concentration ([Ca2+]i). Phosphorylation of the NADPH oxidase component p47phox and its translocation from cytoplasm to plasma membrane are essential in respiratory burst activation. Phorbol 12-myristate 13-acetate (PMA)-stimulated p47phox phosphorylation was negligibly affected by 25 or 100 microM t-BOOH. Nonetheless, 25 microM t-BOOH increased PMA-stimulated p47phox translocation, whereas 100 microM t-BOOH decreased PMA-stimulated translocation. In unstimulated cells, however, neither phosphorylation nor translocation of p47phox was affected by t-BOOH. Buffering of the t-BOOH-mediated changes of [Ca2+]i abolished the effects of t-BOOH on PMA-stimulated translocation in parallel to effects upon the respiratory burst. The results suggest that the dual effects of hydroperoxides are mediated, in part, by Ca(2+)-dependent processes affecting the assembly of the respiratory burst oxidase at steps that are separate from p47phox phosphorylation.