Inhibition of protein synthesis in insect cells by baculovirus-expressed heme-regulated eIF-2 alpha kinase.
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.
Chefalo
P J
PJ
Harvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Cambridge 02139.
Yang
J M
JM
Ramaiah
K V
KV
Gehrke
L
L
Chen
J J
JJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D020662:Guanine Nucleotide Exchange Factors; D006418:Heme / Q000494:pharmacology*; D007375:Interleukin-1 / Q000096:biosynthesis; D009154:Mutation; D014176:Protein Biosynthesis / Q000187:drug effects*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D019892:eIF-2 Kinase
D020662:Guanine Nucleotide Exchange Factors; D007375:Interleukin-1; D011994:Recombinant Proteins; D006418:Heme; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase