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

Chefalo, Peter J.; Yang, Jane; Ramaiah, Kolluru V A; Gehrke, Lee; Chen, J.-J.

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

Cited by 29 publications

(13 citation statements)

References 43 publications

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“…To the lysate supernatant were added PMSF (1 mM) and KCl (80 mM), and the mixture was immediately aliquoted and stored in liquid N 2 . Samples of the concentrated extracts were separated on 10% SDS−PAGE and were also transferred to a nitrocellulose membrane to determine the level of expression of eIF2α based on its migration on SDS−PAGE and also on its ability to interact with a monoclonal eIF2α antibody ( , ).…”

Section: Methodsmentioning

confidence: 99%

“…Maintenance of Insect Cells, Cotransfection, and Identification of Recombinant BaculoViruses. The Spodoptera frugiperda (Sf9) cell line was maintained in complete TNM-FH medium containing 10% fetal calf serum and antimycotic and antibiotic solutions as described previously (35). Recombinant baculoviruses were generated in vitro by transfecting Bsu36I-digested AcNPV virus DNA (Clonetech) into Sf9 cells as described previously (37).…”

Section: Preparation Of the Recombinant Baculovirus Transfermentioning

confidence: 99%

“…The availability of site-specific mutants of eIF2R like the 48A or 51A mutant in which the serine residues in the respective positions of eIF2R have been replaced with alanine has advanced our understanding in identifying that (a) the serine 51 residue in eIF2R is the only site for phosphorylation in mammalian eIF2R (28), (b) the translational block caused by adenoviral mRNAs, plasmid-derived mRNAs, heat shock, or calcium release from the endoplasmic reticulum is due to an increased level of eIF2R phosphorylation (29)(30)(31)(32) or to localizing the translational inhibition caused by eIF2R phosphorylation, and (c) phosphorylation of eIF2R plays a critical role in cell proliferation and development (33,34). In addition, the coexpression of a mutant eIF2R which cannot be phosphorylated has facilitated the expression of mammalian eIF2R kinases such as the heme-regulated kinase in insect cells (35).…”

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confidence: 99%

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Serine 48 in Initiation Factor 2α (eIF2α) Is Required for High-Affinity Interaction between eIF2α(P) and eIF2B

et al. 1999

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Phosphorylation of the serine 51 residue in the alpha-subunit of translational initiation factor 2 in eukaryotes (eIF2 alpha) impairs protein synthesis presumably by sequestering eIF2B, a rate-limiting pentameric guanine nucleotide exchange protein which catalyzes the exchange of GTP for GDP in the eIF2-GDP binary complex. To further understand the importance of eIF2 alpha phosphorylation in the interaction between eIF2 alpha(P) and eIF2B proteins and thereby the regulation of eIF2B activity, we expressed the wild type (wt) and a mutant eIF2 alpha in which the serine 48 residue was replaced with alanine (48A mutant) in the baculovirus system. The findings reveal that the expression of both of these recombinant subunits was very efficient (15-20% of the total protein) and both proteins were recognized by an eIF2 alpha monoclonal antibody and were phosphorylated to the same extent by reticulocyte eIF2 alpha kinases. However, partially purified recombinant subunits (wt or 48A mutant) were not phosphorylated as efficiently as the eIF2 alpha subunit present in the purified reticulocyte trimeric eIF2 complex and were also found to inhibit the phosphorylation of eIF2 alpha of the trimeric complex. Furthermore, the extents of inhibition of eIF2B activity and formation of the eIF2 alpha(P)-eIF2B complex that occurs due to eIF2 alpha phosphorylation in poly(IC)-treated rabbit reticulocyte lysates were decreased significantly in the presence of insect cell extracts expressing the 48A mutant eIF2 alpha compared to those for wt. These findings support the hypothesis that the serine 48 residue is required for high-affinity interaction between eIF2 alpha(P) and eIF2B.

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Section: Methodsmentioning

confidence: 99%

Section: Preparation Of the Recombinant Baculovirus Transfermentioning

confidence: 99%

mentioning

confidence: 99%

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Serine 48 in Initiation Factor 2α (eIF2α) Is Required for High-Affinity Interaction between eIF2α(P) and eIF2B

et al. 1999

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“…The baculovirus expression system in Sf9 insect cells is a useful model to study the regulation of cPLA 2 . This system has been used extensively to study the function of expressed proteins and their roles in signal transduction (Mancini and Evans, 1993;Stancato et al, 1993Stancato et al, , 1997Chefalo et al, 1994). The effect of cPLA 2 mutations on arachidonic acid release and translocation can be directly compared in intact, living cells.…”

Section: Sf9 Cells As a Model For Cpla 2 Structure-function Studiesmentioning

confidence: 99%

“…We have used the baculovirus expression system in insect cells to study regulation of cPLA 2 (de Carvalho et al, 1996). This model is now being used not only as a source of recombinant proteins but also to study the function of proteins and their role in signal transduction (Stancato et al, 1993(Stancato et al, , 1997Chefalo et al, 1994). Similar to macrophages, Spodoptera frugiperda (Sf9) cells expressing cPLA 2 release arachidonic acid in response to A23187 and okadaic acid (de Carvalho et al, 1996).…”

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confidence: 99%

Role of Phosphorylation Sites and the C2 Domain in Regulation of Cytosolic Phospholipase A2

Gijón

Spencer

Kaiser

et al. 1999

The Journal of Cell Biology

193

164

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Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.

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Viral Infection

Saha

Bose

et al. 1997

Archives of Biochemistry and Biophysics

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

Cited by 29 publications

References 43 publications

Serine 48 in Initiation Factor 2α (eIF2α) Is Required for High-Affinity Interaction between eIF2α(P) and eIF2B

Serine 48 in Initiation Factor 2α (eIF2α) Is Required for High-Affinity Interaction between eIF2α(P) and eIF2B

Role of Phosphorylation Sites and the C2 Domain in Regulation of Cytosolic Phospholipase A2

Viral Infection

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