Mechanism of interferon action. Kinetics of interferon action in mouse L929 cells: Phosphorylation of protein synthesis initiation factor eIF-2 and ribosome-associated protein P1

VIROLOGY 93, 281-285 (197’9)

Mechanism of Interferon Phosphorylation

Action. Kinetics of Interferon Action in Mouse Lsz9 Cells: of Protein Synthesis Initiation Factor elF-2 and Ribosome-Associated Protein P,

CHARLES E. SAMUEL Section of Biochemistry

and Molecular

Biology, Department of Biological Santa Barbara, Calijowzia 98106 Accepted November

Sciences, University

of California,

1, 1978

Ribosomal salt-wash fractions prepared from interferon-treated mouse LB9 fibroblast cells catalyze the phosphorylation of the small subunit ((w)of protein synthesis initiation factor eIF-2. The effect of the duration of interferon treatment of La, cells on the ability of the ribosomal salt-wash fractions to catalyze the phosphorylation of the 38,000-dalton 01eIF-2 subunit, as well as the phosphorylation of the ribosome-associated protein P,, correlates with the appearance of the ribosome-associated inhibitor of translation. Protein P, is also detectable in interferon-treated human amnion U cells, however the apparent molecular weight of P, from interferon-treated U cells (-69,000 daltons) is slightly greater than that of P, from interferon-treated L,,, cells (-67,000 daltons).

Post-translational modifications such as protein phosphorylation provide an important mechanism by which the functional activity of proteins can be controlled and hence biological processes regulated (1, 2). For example, in rabbit reticulocytes during hemin deficiency, protein synthesis is inhibited by a cyclic AMP-independent protein kinase that catalyzes the phosphorylation of the small subunit of the protein synthesis factor eIF-2l (3, 4). The phosphorylation of eIF-2 significantly reduces its ability to function efficiently at the level of initiation of protein synthesis (5-9). In several animal virus-host cell systems, the primary level of virus genome expression inhibited as a result of interferon treatment is the translation of viral mRNA into protein (IO, 11). The inhibition of protein synthesis observed in cell-free extracts prepared from most types of inter-

feron-treated murine cells is facilitated by an inhibitor that can be separated from ribosomes by either washing with buffers containing concentrated salt (12-14) or by incubation (15). Interferon treatment also mediates an enhanced specific protein phosphorylation (16-19). As the duration of interferon treatment of murine LgZ9cells is increased, the phosphorylation of certain proteins present in the ribosomal salt-wash fraction prepared from interferon-treated cells is increased; the increase in protein phosphorylation correlates with the appearance of a ribosome-associated inhibitor of translation (19). The inhibition of translation in interferon-treated cell-free systems occurs both at the apparent level of initiation and of elongation of polypeptide chain synthesis (20-23). The inhibition at the level of initiation of protein synthesis in interferon-treated murine systems is characterized by a reduction in the association of virus mRNA to ribosomes (24) and a ’ Abbreviations and trivial names used: dsRNA, reduction in the formation of virus-specific double-stranded RNA; eIF, eukaryotic initiation factor; EF, elongation factor; (Y, the small (38,000 methionyl-X initiation peptides (22), and is dalton) subunit of eIF-2; p- the medium (53,000 accompanied by a reduction in protein syndalton) subunit of eIF-2; GTP, guanosine 5’-triphosphate. thesis initiation factor eIF-2 activity (23, 281

0042-6822/79/030281-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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The results reported in this communication establish that the small subunit of protein synthesis initiation factor eIF-2 is phosphorylated by a protein kinase present in interferon-treated cells, and that the enhanced phosphorylation of eIF-2cr is dependent upon the duration of interferon treatment in a manner that correlates with the previously established kinetics of ex-

25).

Human Wash dsRNA

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Murine

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pf FIG. 1. Autoradiogram showing the effect of interferon treatment on [yJ2P]ATP-mediated phosphorylation of 0.8 M KC1 rihosomal salt-wash fractions (wash) prepared from untreated (C) and interferon-treated (IF) human amnion U and mouse fibroblast L,,, cells. Conditions of the in vitro phosphorylation assay, carried out in the presence (+) or absence (-) of 1 pg/ml reovirus genome dsRNA, were as previously described (19). The incubation mixture (26 ~1) contained 7.5 pg protein of the indicated ribosomal salt wash. Incubation was for 30 min at 30”. Ribosomal salt-wash fractions were prepared as previously described from C or IF cells; interferon treatment, 100 units/ml human leukocyte (U cells) or murine km CL929cells) interferon (28). REO, [Wlleucinelabeled reoviron marker proteins. U and L, phosphoprotein P, in human U and murine L,, cells, respectively. Arrows indicate the positions of 32P-phosphorylated proteins P, and P,.

pression of the interferon-mediated inhibition of protein synthesis. As shown in Fig. 1, phosphorylation of a component designated P, present in the 0.8 M ribosomal wash fraction prepared both from human and from murine cells treated with interferon for 18 hr was greatly enhanced as compared to the phosphorylation obtained with the comparable wash fractions prepared from untreated cells. The relative mobility on sodium dodecyl sulfate (SDS)-polyacrylamide gels of P, from U cells is slightly slower than that of P, from L,,, cells. Interferon-treated human cells also possesseda smaller ribosomeassociated phosphorylated protein, designated Pf, that was not observed either in untreated human cells or in interferontreated murine cells. The phosphorylation of both human and murine P, was dependent upon the presence of dsRNA. Phosphorylation of Pr was not affected by dsRNA. Since protein synthesis is affected at both the initiation and elongation levels in interferon-treated systems, initiation factor eIF-2 as well as elongation factors EF-1 and EF-2 were examined for possible modification by protein phosphorylation. Purified protein synthesis factors eIF-2, EF-1, and EF-2 were incubated with the ribosomal salt-wash fraction prepared from L,,, cells treated with interferon for varying times, and the reaction mixtures were analyzed by SDS-polyacrylamide gel electrophoresis. The autoradiogram is shown in Fig. 2. eIF-2 is composed of three nonidentical subunits (26). The smallest subunit of eIF-2, (Y, was phosphorylated by the interferonmediated murine kinase. The phosphorylation of eIF-Za, as well as the phosphorylation of the endogenous ribosome-associated protein P,, was dependent upon the duration of interferon treatment as indicated by the relative peak heights of the optical density scans in the positions corresponding to P, and eIF-2a (Fig. 3). The phosphorylation of eIF-2a and protein P, was detectable when catalyzed by ribosomal salt-wash fractions prepared from untreated cells and cells treated for 2 hr. However, an increase in the apparent phosphorylation of both eIF-2a and protein P, was readily detect-

SHORT COMMUNICATIONS

able after 6 hr of interferon treatment and was maximally enhanced after 12 hr of treatment. We have previously shown (19) that ribosomal salt-wash fractions prepared from L,,, cells treated for 12 or 18 hr significantly inhibit the translation of reovirus mRNA catalyzed by cell-free protein synthesizing systems from untreated cells, whereas translation is slightly stimulated Time (h) eIF-2 EF-1 EF-2

6 2 -II-+---+---f---f---f---+- - - +---+---+

283

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IF2

.” IFS IF12

12

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,a 0

F;PCZ-

FIG. 2. Autoradiogram showing the effect of duration of interferon treatment on the [y-3ZP]ATP-mediated phosphorylation of purified protein synthesis factors eIF-2, EF-1, and EF-2, and of endogenous protein P,. The incubation mixture contained reovirus dsRNA (1 pg/ml), 0.8 M ribosomal salt-wash fraction (2.7 gg protein) prepared from mouse L,,, cells treated with interferon for either 2, 6, or 12 hr, and where indicated, eIF-2 (0.5 pg protein), EF-1 (0.5 pg protein), or EF-2 (1.0 pg protein). Conditions and symbols as for Fig. 1, except incubation was for 15 min at 30”. (Yand p indicate the positions of the small (38,000) and medium (53,000) subunits, respectively, of eIF-2 (26). Homogeneous rabbit reticulocyte initiation factor eIF-2 was generously provided by Dr. R. Ranu, M.I.T., and purified rat liver elongation factors EF-1 and EF-2 were generously provided by Dr. K. Moldave, University of California, Irvine.

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FIG. 3. Optical density scan of autoradiogram showing the effect of duration of interferon treatment on the [y-32P]ATP-mediated phosphorylation of eIF-2a! and protein P, catalyzed by mouse L,,% cell ribosomal salt-wash fractions prepared from untreated cells (C) or cells treated with interferon for 2 hr (IF,), 6 hr (IF,), or 12 hr (IF,,). Conditions as described for Fig. 2. Densitometer scanning of autoradiographs was carried out with a Gilford 250 spectrophotometer equipped with a 20-cm linear transport accessory and recorder.

by salt-wash fractions from untreated cells and cells treated for 2 hr and slightly inhibited by salt-wash fractions from cells treated for 6 hr. Neither EF-1 nor EF-2 was phosphorylated by the interferon-mediated murine kinase (Fig. 2). The purified eIF-2 preparation contained a low level of protein kinase activity that phosphorylates the p subunit of eIF-2 but not the (Ysubunit (27); the interferon-mediated ribosome-associated mu&e kinase did not significantly phosphorylate eIF-2/? (Fig. 2). The phosphorylation of eIF-2a catalyzed by the ribosomeassociated kinase activity was dependent upon both interferon treatment and the presence of RNA with double-stranded character (30). The apparent molecular weight estimated by SDS-polyacrylamide gel electrophoresis of the interferon-mediated phosphoprotein P, from human U cells was slightly larger than that of P, from mouse LgZ9cells. The extrapolated value for U cell P, is 69,OOQ

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the value for L,,, cell P, is 67,000 (Fig. 4). The extrapolated value for the (Y subunit of eIF-2 is 38,500, in good agreement with the value of 38,000 previously reported for CYin the Laemmli system (26). It has been recognized for some time that interferon-treated intact cells show a reduced ability to support the initiation of viral protein synthesis in wivo (24). In vitro studies with cell-free systems have likewise revealed that the initiation of translation is affected by interferon treatment (20-23). It is well established that in the case of the reticulocyte system the phosphorylation of eIF-2a is a necessary condition for the inhibition of protein synthesis at the initiation level (5 -9). The results reported here establish that the activity of a ribosomeassociated protein kinase capable of catalyzing the phosphorylation of the small

I51

subunit (a) of protein synthesis initiation factor eIF-2 is dependent upon the duration of interferon treatment. These results (Figs. 2 and 3), together with observations that: the appearance of the interferon-mediated protein kinase correlates with the appearance of a ribosome-associated inhibitor of reovirus mRNA translation (19), the inhibition of translation caused by the interferon-mediated kinase can be reversed, in part, by the addition of eIF-2 (25), the apparent ability of initiation factor(s) to form a ternary complex with initiator tRNA and GTP is decreased by interferon treatment (23), and the phosphorylation of eIF-2 catalyzed by the interferon-mediated human kinase occurs at a site on the (Ysubunit that is structurally indistinguishable from the site of phosphorylation catalyzed by the heme-regulated reticulocyte kinase (SO), suggest that in interferon-treated cells the reduction in initiation of translation (20-24) may be due, in part, to the phosphorylation of eIF-2. ACKNOWLEDGMENTS This work was supported in part by Research Grant AI-12520 from the National Institute of Allergy and Infectious Diseases and American Cancer Society Grant VC-192B. The expert technical assistance of Debbie Farris is gratefully acknowledged. REFERENCES

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FIG. 4. Determination by SDS-polyacrylamide gel electrophoresis (29) of the apparent molecular weight of the proteins phosphorylated by the interferonmediated human and murine kinase activities. The arrows indicate the mobility of P, U, human amnion U cell protein P,; P, L, mouse fibroblast L,,, cell protein P,; and Q, small subunit of initiation factor eIF-2. The standard marker proteins were phosphorylase a (92,500), reovirion polypeptides plc (75,000), and (TV (41,500), transketolase (70,000), leucine aminopeptidase (53,000), fumarase (48,500), peroxidase (40,000), carbonic anhydrase (31,000), and a-chymotrypsinogen (25,700).

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