Abstract
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.
Highlights
Cultured mouse3T3-F442A and 3T3-CZ fibroblasts exhibit a transient double-stranded RNA-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN)
AgadsRNA-dependent Phosphoprotein Profile in Cultured 3T3 rose-poly(r1).poly(rC)was obtained from P-L Biochemicals; poly(r1)
Cells-In initial experiments, we observed a double-stranded RNA (dsRNA)-dependpoly(rC) was obtained fromMiles, and [y-32P]ATP(20-40 Ci/mmol) ent phosphorylationof a 67K phosphoprotein in S10 fractions was obtained from New England Nuclear. eIF-2 (80% pure) was prepared from 3T3-F442A and 3T3-C2 cells
Summary
Cultured mouse3T3-F442A and 3T3-CZ fibroblasts exhibit a transient double-stranded RNA (dsRNA)-dependent phosphorylation of a 67,000-dalton protein (67K) without prior treatment with interferon (IFN). The dsRNA-dependentphosphorylation of the 67K protein increased from nonconfluent cells a t low cell density (-3 day) (Fig. 2, track 2) to a maximal level are as indicated.
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