Growth-related expression of a double-stranded RNA-dependent protein kinase in 3T3 cells

Cultured mouse 3T3-F442A and 3T3-C2 fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). This phosphoprotein is similar but not identical to the dsRNA-dependent eukaryotic initiation factor-2 (eIF-2) alpha protein kinase (dsI), which regulates protein synthesis in rabbit reticulocytes. We have studied the relationship between cell growth and phosphorylation of the 67K protein (designated 3T3-dsRNA-dependent eIF-2 alpha kinase). A low level of dsRNA-dependent phosphorylation of 3T3-dsI was detectable in extracts prepared from cells not treated with IFN and grown at a low cell density. The phosphorylation of dsI and the phosphorylation of a 38K protein identified as the alpha-subunit (38K) of 3T3-eIF-2 (eIF-2 alpha) occurred concomitantly; the levels of these phosphorylations confluent and thereafter decreased markedly. Treatment of cells with IFN at all stages of growth resulted in an increase in phosphorylation of dsI. 3T3-F442A and 3T3-C2 fibroblasts were found to produce and secrete IFN at levels sufficient to induce an elevated dsI activity.     

Partial characterization of a cellular factor that regulates the double-stranded RNA-dependent eIF-2 alpha kinase in 3T3-F442A fibroblasts.

The interferon-induced double-stranded RNA-dependent eIF-2 alpha kinase (dsI) has an established role in mediating part of interferon's antiviral effects. Numerous studies have suggested that dsI also has regulatory functions in cells not infected with virus. Our previous results have indicated that the activation of this kinase may be an important regulatory signal in controlling growth arrest of mouse 3T3-F442A fibroblasts prior to their subsequent differentiation to adipocytes. Here, we report that extracts from 3T3-F442A cells cultured under conditions nonpermissive for differentiation exhibit significantly reduced dsI activity and that this reduction is due, at least in part, to the presence of elevated levels of a novel inhibitor of dsI activation (dRF). This inhibitor is also detected in reduced amounts in extracts from cells cultured under conditions which are permissive for differentiation. We have achieved a 1,000-fold purification of dRF activity, and highly purified dRF preparations were found to be greatly enriched for a 15-kDa protein that was greater than 90% pure. Our results indicate that dRF is not a protein phosphatase or protease but a reversible inhibitor of dsI autophosphorylation. In addition, our results imply that dRF is a physiologic regulator of dsI, since dRF activity correlates with the ability of 3T3-F442A cells to undergo adipose conversion.     

Control of protein synthesis in human reticulocytes by heme-regulated and double-stranded RNA dependent eIF-2 alpha kinases

Heme-deficiency and double-stranded RNA (dsRNA) activate distinct cyclic 3':5'-AMP independent protein kinases (HRI and dsI, respectively) in rabbit reticulocyte lysates. These kinases inhibit protein synthesis by phosphorylating the 38,000 daltons (38K) subunit of the initiation factor eIF-2 (eIF-2 alpha). Using separation techniques to obtain a reticulocyte enriched fraction and reticulocyte-free erythrocytes, we have prepared lysates of these fractions from normal human whole blood. Human reticulocyte-enriched lysates contain the hemin-regulated and dsRNA-dependent protein kinases which inhibit protein synthesis and which phosphorylate rabbit eIF-2 alpha. An endogenous 38K polypeptide which co-migrates with rabbit eIF-2 alpha is also phosphorylated. In contrast, human mature erythrocytes contain little or no heme-regulated or dsRNA-dependent eIF-2 alpha kinase activities which are inhibitory of protein synthesis.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

Tissue distribution and immunoreactivity of heme-regulated eIF-2 alpha kinase determined by monoclonal antibodies.

A highly purified preparation of heme-regulated inhibitor (HRI), an eIF-2 alpha kinase, from rabbit reticulocyte lysates has been used for generating monoclonal antibodies (mAB). Two hybridoma clones secreting HRI-specific antibodies (mAB A and mAB F) were obtained. Both antibodies immunoprecipitated biosynthetically labeled as well as phosphorylated HRI in reticulocyte lysates and also recognized denatured HRI in a Western blot. In in vitro protein kinase assays, preincubation of HRI with the antibodies significantly diminished both autokinase and eIF-2 alpha kinase activities. HRI from reticulocyte lysates could be quantitatively removed by immunoprecipitation with mAB F, and such HRI-depleted lysates were able to maintain protein synthesis under conditions of heme deficiency. With these monoclonal antibodies, HRI was detected only in the reticulocytes and bone marrow of anemic rabbits, among several rabbit tissues tested. The antibodies did not detect cross-reacting HRI in rat or human reticulocytes or in mouse erythroleukemic cells or human K562 cells even after induction of differentiation, although eIF-2 alpha kinase activity was detected in them. Polyclonal anti-rabbit HRI antibody detected HRI in rat reticulocytes. However, no cross-reacting HRI was detected by polyclonal antibody in human reticulocytes or other cell types tested. These findings suggest that HRI is not ubiquitous, and may be erythroid-specific, and that it is antigenically different in different species.     

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.     

Vaccinia specific kinase inhibitory factor prevents translational inhibition by double-stranded RNA in rabbit reticulocyte lysate

Mouse L-cells infected with vaccinia virus produce a specific kinase inhibitory factor (SKIF) which inhibits the activation of the interferon-induced, double-stranded (ds)RNA-dependent, eukaryotic initiation factor (eIF)-2 alpha-specific protein kinase in L-cell extracts (Whitaker-Dowling, P., and Younger, J. S., (1984) Virology 137, 171). The effects of a partially purified preparation of SKIF have been examined in cell-free extracts of rabbit reticulocytes. Both the phosphorylation state of eIF-2 and protein synthetic activity have been determined. SKIF inhibits the phosphorylation of the alpha subunit of eIF-2 by dsRNA-dependent eIF-2 alpha-kinase in reticulocyte lysate, but does not affect phosphorylation of eIF-2 by the heme-sensitive kinase. In addition to its effects on eIF-2 alpha-PKds activity, SKIF prevents dsRNA-induced inhibition of protein synthesis in reticulocyte lysate. In contrast, SKIF does not prevent the translational inhibition caused by hemin depletion. These data provide a direct correlation between the effects of SKIF on eIF-2 alpha phosphorylation and on protein synthetic activity and demonstrate the specificity of SKIF. The results also show that SKIF does not abolish dsRNA sensitivity, but increases the concentration of dsRNA required to activate the kinase and phosphorylate eIF-2.     

Purification and properties of protein kinase C from rabbit reticulocyte lysates

We have previously reported that addition of Ca2+ and phospholipid (PL) inhibits translation in hemin-containing reticulocyte lysates through activation of a eukaryotic protein synthesis initiation factor (eIF-2) kinase. The possibility that this activation was mediated by a Ca2+-PL-dependent protein kinase (protein kinase C, PKC) appeared unlikely by the observation that it was prevented or reversed by NADPH-generating systems. Nevertheless, reticulocyte lysates contain a potent PKC activity and we deemed it desirable to isolate this enzyme to answer unequivocally the question whether it does or does not activate eIF-2 alpha kinase. We have purified reticulocyte PKC to near homogeneity with Mr 95,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme absolutely depended upon both Ca2+ and phosphatidylserine for activity on histone H1 or the beta-subunit of initiation factor eIF-2 and underwent autophosphorylation in a Ca2+- and PL-dependent manner. Mild treatment with trypsin yielded an Mr 82,000 polypeptide that still required Ca2+ and PL for activity. This Mr agrees with that reported for other PKCs, suggesting that these enzymes may undergo limited degradation during isolation. Further proteolytic treatment converted the reticulocyte enzyme into a Ca2+- and PL-dependent form, as is known for PKCs from other sources. The highly purified PKC had no effect on translation in hemin-supplemented reticulocyte lysates.     

Lack of inhibition of protein synthesis by double-stranded RNA in a cell-free system prepared from human reticulocytes

There are two inhibitors of protein synthesis which are related to the activity of interferon. One is a protein kinase which phosphorylates the α subunit of the eucaryotic initiation factor 2 (eIF-2). The other is an enzyme which synthesizes an unusual oligonucleotide that in turn activates a RNA endonuclease. In nucleated cells the synthesis of the inhibitors is induced by interferon but they must be activated in a subsequent lysate by double-stranded RNA (dsRNA). Rabbit reticulocytes, however, contain the inactive forms of the inhibitors in a constitutive manner and require only dsRNA activation. We report here the effect of dsRNA on protein synthesis and the generation of ribosomal eIF-2α kinase and heat-stable (oligonucleotide) inhibitory activity in human reticulocyte lysates. Our findings indicate that human reticulocytes, in contrast to rabbit reticulocytes, do not contain the interferon-related inhibitors of protein synthesis in a constitutive manner. Addition of dsRNA to the human reticulocyte cell-free system does not result in significant inhibition. Furthermore, no generation of ribosomal eIF-2α kinase or heatstable inhibitory activity could be detected. Direct addition of oligonucleotide or eIF-2α kinase (of rabbit origin), however, does result in inhibition of the human system. Thus, the ultimate inhibition mechanisms do appear operative in the human reticulocyte lysates. The differences between the rabbit and human systems may be due to either basic differences in the mechanism of interferon action or simply to variation in the history or maturity of the cells studied.     

Inhibition of rabbit reticulocyte lysate protein synthesis by heavy metal ions involves the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2

The effect of heavy metal ions (in particular Cd2+, Hg2+, and Pb2+) on protein synthesis in hemin-supplemented reticulocyte lysates was investigated. Heavy metal ions were found to inhibit protein synthesis in hemin-supplemented lysates with biphasic kinetics. The shut off of protein synthesis occurred in conjunction with the phosphorylation of the alpha-subunit of the eukaryotic initiation factor (eIF) 2, the loss of reversing factor (RF) activity, and the disaggregation of polyribosomes. Addition of eIF-2 or RF to heavy metal ion-inhibited lysates restored protein synthesis to levels observed in hemin-supplemented controls. The stimulation of protein synthesis observed upon the addition of cAMP to heavy metal ion-inhibited lysates correlated with the inhibition of eIF-2 alpha phosphorylation and the restoration of RF activity. The partial restoration of protein synthesis observed upon the addition of MgGTP to heavy metal ion-inhibited lysates correlated with a partial inhibition of eIF-2 alpha phosphorylation. Addition of glucose 6-phosphate was found to have no effect on protein synthesis of eIF-2 alpha phosphorylation under these conditions. Antiserum raised to the reticulocyte heme-regulated eIF-2 alpha kinase inhibited the phosphorylation of eIF-2 alpha catalyzed by Hg2+-inhibited lysate. The inhibition of protein synthesis observed in the presence of heavy metal ions correlated with the relative biological toxicity of the ions. Highly toxic ions (AsO-2, Cd2+, Hg2+, Pb2+) inhibited protein synthesis by 50% at concentrations of 2.5-10 microM. Cu2+, Fe3+, and Zn2+, which are moderately to slightly toxic ions, inhibited protein synthesis by 50% at concentrations of 40, 250, and 300 microM, respectively. The data presented here indicate that heavy metal ions inhibit protein chain initiation in hemin-supplemented lysates by stimulating the phosphorylation of eIF-2 alpha apparently through the activation of the heme-regulated eIF-2 alpha kinase rather than through inhibition of the rate of eIF-2 alpha dephosphorylation.     

Effects of glucose 6-phosphate and hemin on activation of heme-regulated eIF-2 alpha kinase in gel-filtered reticulocyte lysates

In heme-deficient reticulocyte lysates, protein synthesis initiation is inhibited due to the activation of a heme-regulated protein kinase which blocks protein synthesis by the specific phosphorylation of the alpha-sub-unit of eukaryotic initiation factor 2 (eIF-2 alpha). The restoration of synthesis requires both hemin and glucose-6-P (Ernst, V., Levin, D. H., and London, I. M. (1978) J. Biol. Chem. 253, 7163-7172). The sugar phosphate fulfills two functions in initiation: (i) the generation of NADPH, and (ii) an effector function in some step in initiation. This latter effect is readily demonstrated in lysates depleted of low molecular weight components by filtration in dextran gels. In gel-filtered lysates, linear protein synthesis is sustained only by the addition of both hemin (20 microM) and glucose-6-P (or 2-deoxyglucose-6-P) (50-500 microM). The omission of either component gives rise to inhibitions which are characterized by the activation of heme-regulated eIF-2 alpha kinase and the concomitant phosphorylation of both endogenous heme-regulated eIF-2 alpha kinase and endogenous eIF-2 alpha, indicating that glucose-6-P is involved in the regulation of heme-regulated eIF-2 alpha kinase. In support of this, we find (a) that gel-filtered lysates incubated with hemin but depleted of glucose-6-P produce sufficient heme-regulated eIF-2 alpha kinase to inhibit protein synthesis when mixed with normal hemin-supplemented lysates; (b) the inhibitions of protein synthesis produced by heme-regulated eIF-2 alpha kinase generated either in glucose-6-P-depleted lysates or heme-deficient lysates are reversed by added eIF-2; and (c) the eIF-2 alpha kinase activities formed in the absence of either hemin or glucose-6-P are both neutralized by an anti-heme-regulated eIF-2 alpha kinase antiserum. We conclude that the physiological activation of heme-regulated eIF-2 alpha kinase is controlled by both hemin and glucose-6-P.     

Phosphorylation of protein synthesis initiation factor-2. Identification of the site in the alpha-subunit phosphorylated in reticulocyte lysates.

The data presented here show that serine-51 of the alpha-subunit of eukaryotic initiation factor eIF-2 is the only residue phosphorylated by the eIF-2 alpha-specific kinases HCR (haem-controlled repressor) and dsI (double-stranded RNA-activated inhibitor) in vitro. This confirms our earlier finding that serine-48 is not labelled by either kinase. Methodology appropriate for the examination of phosphorylation sites in eIF-2 alpha in whole cells and their extracts has been developed, and used to study the site(s) in eIF-2 alpha labelled in reticulocyte lysates. Only serine-51 became phosphorylated under conditions of haem-deficiency or in the presence of double-stranded RNA. No evidence for a second phosphorylation site on the alpha-subunit was obtained with the lysates and conditions used here.     

Phosphorothioated binary complex of eukaryotic initiation factor eIF-2 and GDP inhibits protein synthesis in hemin-supplemented reticulocyte lysates

The rabbit reticulocyte heme-regulated eIF-2 alpha kinase (HRI) utilizes adenosine-5'-0-(3-thiotriphosphate) (ATP-gamma-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [eIF-2(alpha-[35S]P) . GDP], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2 . [3H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF-2(alpha-[35S]P) . GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2 . GDP alone, through the ability of eIF-2(alpha-P) . GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate.     

The relationship between protein synthesis and heat shock proteins levels in rabbit reticulocyte lysates.

Besides heme deficiency, protein synthesis in rabbit reticulocyte lysates becomes inhibited upon exposure to a variety of agents that mimic conditions which induce the heat shock response in cells. This inhibition has been demonstrated to be due primarily to the activation of the heme-regulated eIF-2 alpha kinase (HRI) which causes an arrest in the initiation of translation. In this report, the sensitivity of protein synthesis in hemin-supplemented lysates to inhibition by Hg2+, GSSG, methylene blue, and heat shock was examined in six different reticulocyte lysate preparations. The extent to which translation was inhibited in response to Hg2+, GSSG, methylene blue, and heat shock correlated inversely with the relative levels of the 70-kDa heat shock proteins (hsp 70) and a 56-kDa protein (p56) present in the lysates determined by Western blotting. The ability of hemin to restore protein synthesis upon addition to heme-deficient lysates was also examined. While the restoration of protein synthesis correlated roughly with the levels of hsp 90 present, the results also suggest that the heme regulation of HRI probably involves the interaction of HRI with several factors present in the lysate besides hsp 90. A comparison of two lysate preparations, which had a 2-fold difference in their protein synthesis rates, indicated that the slower translational rate of the one lysate could be accounted for by its low level of constitutive eIF-2 alpha phosphorylation, with its accompanying decrease in the eIF-2B activity and lower level of polyribosome loading. The present study supports the notion that the previously demonstrated interaction of HRI with hsp 90, hsp 70, and p56 in reticulocyte lysates may play a direct role in regulating HRI activation or activity. We hypothesize that the competition of denatured protein and HRI for the binding of hsp 70 may be a molecular signal that triggers the activation of HRI in reticulocyte lysates in response to stress. Possible functions for p56 in the regulation of HRI activity are also discussed.     

Double-stranded RNA dependent protein kinase (s) in rabbit reticulocyte lysates analogous to kinase from interferon-treated cells

A double-stranded (ds) RNA-dependent protein kinase(s) from haemin-supplemented rabbit reticulocyte lysates is purified by chromatography on columns of poly(I).poly(C)-Sepharose. This method provides a more than 1000-fold purifications of the kinase(s) in a single step and in high yield. The purified kinase is responsible for the phosphorylation of (a) a polypeptide of molecular weight 67000, (b) the smallest subunit eIF-2 and (c) added histones. These kinase activities are comparable to the dsRNA-dependent protein kinase activities from interferon-treated cells.     

Mechanism of action of a cellular inhibitor of the dsRNA-dependent protein kinase from 3T3-F442A cells.

When mouse 3T3-F442A preadipocyte fibroblasts reach confluence in the appropriate culture medium, their growth is arrested, and the cells undergo terminal differentiation to adipocytes. Two proteins that may be involved in this process are interferon and the interferon-induced double-stranded RNA (dsRNA)-dependent protein kinase (DAI). In 3T3-F442A cells, interferon and DAI are transiently expressed with a maximum level of active kinase appearing at confluence. Interestingly, the level of active DAI was found to be low when cells were maintained under conditions nonpermissive for differentiation. This reduction in DAI was at least partly because of the presence of elevated levels of a specific inhibitor of DAI, termed dRF, which appeared to be a reversible inhibitor of the autophosphorylation (activation) of DAI. In the present study, the mechanism of action of dRF was investigated. Photocross-linking experiments indicated that dRF prevented the binding of ATP to DAI. Since the binding of ATP to DAI is dsRNA-dependent, we examined the effect of dRF on the binding of dsRNA to the kinase using RNA mobility shift assays. dRF was found to prevent the formation of DAI-dsRNA complexes without a direct effect on the dsRNA. This suggests that dRF exerts its effect through an interaction with DAI.     

The alpha subunit of eucaryotic initiation factor 2 is phosphorylated in mengovirus-infected mouse L cells.

Infection of mouse L cells with mengovirus resulted in the activation of a protein kinase (PK) that selectively phosphorylated the small, 38,000-molecular-weight alpha subunit of eucaryotic initiation factor 2 (eIF-2) in vitro. The mengovirus-activated kinase was detected in vitro approximately 3 h after virus adsorption. The ratio of phosphorylated to unphosphorylated eIF-2 also increased in vivo between 3 and 7 h after adsorption. The virus-activated kinase fractionated with the ribosomal pellet and had a high affinity for DEAE-cellulose and Mono Q ion-exchange columns. Gel electrophoresis of the kinase activity eluting from the Mono Q column and silver staining of the gel revealed only one protein band with a molecular mass of 70 kilodaltons. The optimal assay conditions for the mengovirus-activated kinase paralleled those of the double-stranded RNA-activated PK (dsRNA-PK). Lysates from infected cells contained elements capable of activating partially purified dsRNA-PK. These elements were identified as double-stranded RNA by their sensitivity to double-stranded RNase. The phosphorylation of the alpha subunit of eIF-2 coincided with the synthesis of dsRNA in infected cells, suggesting that the mengovirus-activated kinase is the dsRNA-PK. The phosphorylation of the alpha subunit of eIF-2 correlated with the global inhibition of protein synthesis that occurs at late times after infection.     

Dexamethasone-dependent expression of beta 1-24 corticotropin stimulated adenylate cyclase during adipose conversion of 3T3-F442A cells

When 3T3-F442A preadipocytes were grown in culture media supplemented with corticosteroid poor fetal calf serum and insulin they differentiated into adipocytes. Glycerophosphate dehydrogenase, a marker of terminal differentiation, developed a 600-fold increase of activity whereas the adenylate cyclase system remained unresponsive to the synthetic ACTH(1-24) analog. In contrast, 3T3-F442A adipocytes, differentiated in the presence of dexamethasone, exhibited an adenylate cyclase activity which was stimulated 4-fold by ACTH(1-24). The stimulation of the adenylate cyclase activity by GTP gamma S remained unchanged (about 20-25-fold) suggesting that the G regulatory coupling protein was not functionally modified by dexamethasone. Binding studies with 125I-ACTH revealed that specific cellular binding could be evidenced in dexamethasone-treated cells while control adipocytes did not exhibit any specific binding of 125I-ACTH. These findings lend support to the hypothesis that the setting off of this ACTH responsiveness in 3T3-F442A cells is regulated by dexamethasone after cells are committed to adipose differentiation.     

The vaccinia virus K3L gene product potentiates translation by inhibiting double-stranded-RNA-activated protein kinase and phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

Interferon resistance of vaccinia virus is mediated by specific inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) by the double-stranded-RNA-activated (DAI) protein kinase. Vaccinia virus encodes a homolog of eIF-2 alpha, K3L, the deletion of which renders the virus sensitive to interferon treatment. We have studied the mechanism by which this protein product elicits interferon resistance in a transient DNA transfection system designed to evaluate regulators of eIF-2 alpha phosphorylation. In this system, translation of a reporter gene mRNA is inefficient because of eIF-2 phosphorylation mediated by the DAI protein kinase. Cotransfection of the K3L gene enhances translation of the reporter mRNA in this system. The K3L protein inhibits eIF-2 alpha phosphorylation and DAI kinase activation, apparently without being phosphorylated itself. Inhibition of protein synthesis, elicited by expression of a mutant Ser-51----Asp eIF-2 alpha designed to mimic a phosphorylated serine, is not relieved by the presence of K3L, suggesting that K3L cannot bypass a block imposed by eIF-2 alpha phosphorylation. The results suggest that K3L acts as a decoy of eIF-2 alpha to inhibit DAI kinase autophosphorylation and activation. Another vaccinia virus gene product, K1L, which is required for growth of vaccinia virus on human cells, does not enhance translation in this assay.