Inhibition of protein synthesis in insect cells by baculovirus-expressed heme-regulated eIF-2 alpha kinase.

P J Chefalo,J M Yang,K V Ramaiah,L Gehrke,J J Chen

doi:10.1016/s0021-9258(18)47317-6

Abstract

To study further the regulation of the heme-regulated eIF-2 alpha kinase (HRI), we have produced functional wild type HRI using the baculovirus expression system. The amount of recombinant HRI protein expressed in insect cells is approximately 10 times higher than levels in reticulocytes. Baculovirus-expressed HRI (BV-HRI) is indistinguishable from HRI purified from rabbit reticulocytes. It is active both as an autokinase and an eIF-2 alpha kinase. BV-HRI is regulated by heme in vitro as well as in intact insect cells. Coexpression of the wild type HRI with the inactive K199R HRI, S51A eIF-2 alpha, or interleukin-1 beta (IL-1 beta) results in diminished expression of these proteins. Expression of wild type HRI also results in severe inhibition of general protein synthesis in Sf9 cells when compared with cells expressing K199R HRI or IL-beta. In addition, the guanine nucleotide exchange activity of eIF-2B is suppressed in Sf9 cells expressing wild type HRI but not in cells expressing the K199R HRI or IL-1 beta. Furthermore, expression of wild type HRI is increased by coexpression with the nonphosphorylatable S51A eIF-2 alpha or by the addition of hemin, which inhibits HRI activity. These results provide evidence that translational regulation by phosphorylation of eIF-2 alpha and sequestration of eIF-2B can operate in insect cells.

Highlights

From the Haruard-Massachusetts Instituteof Technology Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Expression of wild type heme-regulated inhibitor of protein synthesis (HRI) results in severe inhibition of generalprotein synthesis in Sf9 cells when compared with cells expressing K199R HRI or IL-p
Expression of wild type HRI is increased by coexpression with the nonphosphorylatable S51A eIF-2a or by the addition of hemin, which inhibits HRI activity.These results provide evidence

Summary

MATERIALSAND METHODS

Lated region of the HRI cDNA and ligated into the polylinker region of the pVL1392 recombination vector. Cells were subcultured three times weekly to a density of 1 x 106cells/ml.For the expression of HRI proteins, Sf9 (4 x lo6) cells were incubated with 2 ml of high titer viral halves of HRI cDNA were amplified separately (primers1and 2, primers 3 and 4) in the firstPCR reactions This infected culture wasincubated for 48 hat 27 "C. x 2 min, and 72 "C x 5 rnin), and the first round PCR products were Cells were lysed in 300 pl of a buffer containing 1% Triton X-100 purified by agarose gel electrophoresis and electroelution.

RESULTS

12 I f 1 4

Findings

DISCUSSION