The role of the death-domain kinase RIP in tumour-necrosis-factor-induced activation of mitogen-activated protein kinases

Abstract

The death-domain kinase RIP (receptor-interacting protein) is an important effector of tumour necrosis factor (TNF) signalling and is essential for TNF-induced nuclear factor-kappaB activation. However, the function of RIP in the TNF-induced activation of mitogen-activated protein kinases (MAPKs) has not been fully investigated. In this report, using Rip null (Rip(-/-)) mouse fibroblast cells, we investigated whether RIP is required for TNF-induced activation of the MAPKs extracellular-signal-related kinase (ERK), p38 and c-Jun amino-terminal kinase (JNK). We found that TNF-induced activation of ERK, p38 and JNK is decreased in Rip(-/-) cells. The activation of these kinases by interleukin-1 is normal in Rip(-/-) cells. More importantly, we showed that the kinase activity of RIP is needed for ERK activation.

Figure 1

TNFR1-mediated p38, ERK and JNK activation require both TRAF2 and RIP. (A) Human-TNF-induced p38, ERK and JNK activation in wild-type, Rip^−/− and Traf2^−/− fibroblasts. Mouse fibroblasts were treated with human TNF (40 ng ml⁻¹) for various durations or were left untreated as a control. Cell extracts were used for western blotting and in in vitro kinase assays to measure the activation of p38, ERK and JNK. (B) JNK activation in wild-type, Rip^−/−, and Traf2^−/− cells was measured using an anti-phospho-JNK antibody. (C) Interleukin-1 (IL-1)-induced p38, ERK and JNK activation. Wild-type, Rip^−/− and Traf2^−/− fibroblasts were either treated with IL-1 (4 ng ml⁻¹) for various durations or were left untreated as a control. The activation of p38, ERK and JNK was measured as described in (A). These experiments were repeated three times. ERK, extracellular-signal-related kinase; GST, glutathione-S-transferase; hTNF, human TNF; JNK, c-Jun amino-terminal kinase; P-ERK, phospho-ERK; P-JNK, phospho-JNK; P-p38; phospho-p38; RIP, receptor-interacting protein; TNF, tumour necrosis factor; TNFR1, TNF receptor 1; TRAF2, TNFR-associated factor 2.

Figure 2

TNF-induced activation of p38, ERK and JNK are restored in Rip^−/− or Traf2^−/− cells by ectopically expressing MYC–RIP or FLAG–TRAF2. (A) Reconstitution of TNF-induced p38, ERK and JNK activation in Rip^−/− fibroblasts. Wild-type or Rip^−/− cells were transfected with 2 µg of either the MYC–RIP expression plasmid or an empty vector, in 100-mm dishes. Twenty-four hours after transfection, the transfected cells were treated with 40 ng ml⁻¹ of human TNF for various durations or were left untreated as a control. The amounts of protein in cell extracts were quantified by performing protein assays and were then either immunoprecipitated with anti-JNK1 antibody to enable a kinase assay to be performed, or were resolved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blotting with anti-JNK1, anti-phospho-p38, anti-p38, anti-phospho-ERK, anti-ERK or anti-MYC antibodies. (B) Reconstitution of TNF-induced p38, ERK and JNK activation in Traf2^−/− fibroblasts. Traf2^−/− cells and Flag–Traf2 stably transfected cells were treated with human TNF-α (40 ng ml⁻¹) for various durations or were left untreated as a control. The amounts of protein in cell extracts were quantified by performing protein assays and were then resolved by SDS–PAGE for western blotting with anti-JNK, anti-phospho-p38, anti-p38, anti-phospho-ERK, anti-ERK and anti-MYC antibodies. To measure JNK activity, the cell extracts were used in in vitro kinase assays. These experiments were repeated three times. ERK, extracellular-signal-related kinase; GST, glutathione-S-transferase; JNK, c-Jun amino-terminal kinase; P-ERK, phospho-ERK; P-p38, phospho-p38; RIP, receptor-interacting protein; TNF, tumour necrosis factor; Traf2, TNFR-associated factor 2.

Figure 3

The RIP kinase domain is necessary for activating ERK. (A) Rip^−/− cells were co-transfected in 60-mm dishes with 1 µg of the haemagglutinin (HA)–ERK1 expression plasmid and 0.5 µg of either the MYC–RIP expression plasmid, the Xpress–RIP(K45A) expression plasmid or an empty vector. Cells were collected 24 h after transfection. The HA–ERK1 content was quantified by western blotting (WB) using an anti-HA antibody, cell extracts were immunoprecipitated (IP) using an anti-HA antibody, and the immunoprecipitates were resolved by SDS–polyacrylamide gel electrophoresis for western blotting using anti-phospho-ERK1. The expression levels of MYC–RIP or Xpress–RIP(K45A) were detected using an anti-RIP antibody. (B) Experiments similar to those described in (A) were performed to measure ERK activity using an in vitro kinase assay with MBP as the substrate. In these experiments, Xpress-tagged RIP and RIP(K45A) were used, and these were detected using an anti-Xpress antibody. (C) Rip^−/− cells were transfected in 60-mm dishes with 1 µg of the HA–JNK1 expression plasmid and 0.5 µg of either the MYC–RIP expression plasmid, the Xpress–RIP(A45K) expression plasmid or an empty vector. Twenty-four hours after transfection, cells were collected and the HA–JNK1 content was quantified by western blotting using an anti-HA antibody. Cell extracts were then immunoprecipitated with an anti-HA antibody for use in a kinase assay. The expression levels of RIP and RIP(K45A) were detected using an anti-RIP antibody. (D) Similar experiments to those described in (C) were performed using an HA–p38 plasmid, and HA–p38 activity was measured by performing an in vitro kinase assay with glutathione-S-transferase–ATF2 as the substrate. These experiments were repeated three times. ERK, extracellular-signal-related kinase; JNK, c-Jun amino-terminal kinase; RIP, receptor-interacting protein.