Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Thorsten Gantke,Elke Mühlberger,Steven C Ley,Nicholas A Morrice,Julia Janzen,Steven Howell,Sabrina Boussouf

doi:10.1042/bj20121873

Thorsten Gantke, Elke Mühlberger + Show 5 more

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https://doi.org/10.1042/bj20121873

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Abstract

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells.

Highlights

tumour progression locus 2 (TPL-2) [ known as MAP3K8 and COT] mediates activation of extracellular-signal-regulated kinase (ERK) 1 and 2 mitogen-activated protein kinase (MAPK) by Toll-like receptors and the receptors for tumour necrosis factor (TNF) and interleukin 1β in macrophages [1]
TPL-2 phosphorylation of MKK1/2 and activation of ERK1/2 MAPK signalling requires TPL-2 release from NF-κB1 p105, which is triggered by IKK [inhibitor of nuclear factor κB (NF-κB) (IκB) kinase]-induced proteolysis of NF-κB1 p105 by the proteasome following agonist stimulation [10,11]
Cells were adapted to growth in suspension using Pro293s-CDM medium (Lonza), supplemented with 1.5 % fetal bovine serum, 2 mM Lglutamine, 50 units/ml penicillin and 50 units/ml streptomycin, Abbreviations used: ABIN-2, A20-binding inhibitor of nuclear factor κB 2; BMDM, bone-marrow-derived macrophage; DM, decyl β-D-maltopyranoside; DTT, dithiothreitol; ERK, extracellular-signal-regulated kinase; GST, glutathione transferase; HA, haemagglutinin; HEK, human embryonic kidney; Hsp, heat-shock protein; IκB, inhibitor of NF-κB; IKK, IκB kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MKK, MAPK kinase; NF-κB, nuclear factor κB; PKR, double-stranded RNA-dependent protein kinase; TBK1, TANK [TRAFassociated NF-κB activator]-binding kinase 1; TCEP, tris-(2-carboxyethyl)phosphine; TNF, tumour necrosis factor; TPL-2, tumour progression locus 2; VP35, virus protein 35

Summary

INTRODUCTION

TPL-2 (tumour progression locus 2) [ known as MAP3K8 (mitogen-activated protein kinase kinase kinase 8) and COT (cancer Osaka thyroid)] mediates activation of ERK (extracellular-signal-regulated kinase) 1 and 2 MAPKs (mitogenactivated protein kinases) by Toll-like receptors and the receptors for TNF (tumour necrosis factor) and interleukin 1β in macrophages [1]. Analyses of Nfkb1SSAA/SSAA macrophages, in which the IKK target serine residues on NF-κB1 p105 are mutated to alanine, revealed that TPL-2 regulates TNF production independently of IKKinduced NF-κB1 p105 proteolysis and ERK1/2 activation, while still associated with NF-κB1 p105 and ABIN-2 [12] These data indicate that the TPL-2–ABIN-2–NF-κB1 p105 complex phosphorylates substrates other than MKK1/2 to stimulate TNF production, and that small-molecule inhibitors should target this complex to block TNF production. Cells were adapted to growth in suspension using Pro293s-CDM medium (Lonza), supplemented with 1.5 % fetal bovine serum, 2 mM Lglutamine, 50 units/ml penicillin and 50 units/ml streptomycin, Abbreviations used: ABIN-2, A20-binding inhibitor of nuclear factor κB 2; BMDM, bone-marrow-derived macrophage; DM, decyl β-D-maltopyranoside; DTT, dithiothreitol; ERK, extracellular-signal-regulated kinase; GST, glutathione transferase; HA, haemagglutinin; HEK, human embryonic kidney; Hsp, heat-shock protein; IκB, inhibitor of NF-κB; IKK, IκB kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MKK, MAPK kinase; NF-κB, nuclear factor κB; PKR, double-stranded RNA-dependent protein kinase; TBK1, TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)associated NF-κB activator]-binding kinase 1; TCEP, tris-(2-carboxyethyl)phosphine; TNF, tumour necrosis factor; TPL-2, tumour progression locus 2; VP35, virus protein 35

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RESULTS AND DISCUSSION

Conclusions