MKK7 is an essential component of the JNK signal transduction pathway activated by proinflammatory cytokines - PubMed (original) (raw)

MKK7 is an essential component of the JNK signal transduction pathway activated by proinflammatory cytokines

C Tournier et al. Genes Dev. 2001.

Abstract

Mitogen-activated protein kinases (MAPK) are activated by phosphorylation on Thr and Tyr by MAPK kinases. Two MAPK kinases (MKK4 and MKK7) can activate the c-Jun NH(2)-terminal kinase (JNK) group of MAPK in vitro. JNK is phosphorylated preferentially on Tyr by MKK4 and on Thr by MKK7. Targeted gene-disruption studies in mice were performed to examine the role of MKK4 and MKK7 in vivo. Simultaneous disruption of the Mkk4 and Mkk7 genes was required to block JNK activation caused by exposure of cells to environmental stress. In contrast, disruption of the Mkk7 gene alone was sufficient to prevent JNK activation caused by proinflammatory cytokines. These data demonstrate that MKK4 and MKK7 serve different functions in the JNK signal transduction pathway.

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Figures

Figure 1

Figure 1

Biochemical characterization of MKK4 and MKK7. (A) JNK is preferentially phosphorylated on Tyr by MKK4 and on Thr by MKK7. Epitope-tagged MKK4 and MKK7 were isolated from COS cells exposed to UV-C radiation. In vitro protein kinase assays were performed using [γ-32P]ATP and purified bacterially expressed p38α or JNK1 as substrates for MKK4 and MKK7. The phosphorylated JNK and p38α were examined by phosphoamino acid analysis and autoradiography (upper panel). The relative phosphorylation on Thr and Tyr was examined by PhosphorImager analysis (lower panel). P-Ser, phosphothreonine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine. (B) MKK4 and MKK7 are selectively activated by extracellular stimuli. Wild-type MEF were untreated (Cont.) or treated with UV-C (60 J/m2; UV) or anisomycin (1 μg/mL; ANISO) for 1 h, or with TNFα (10 ng/mL) or IL1α (15 ng/mL) for 15 min. Endogenous MKK4 and MKK7 were immunoprecipitated using an anti-rabbit polyclonal antibody to MKK4 (K18, Santa Cruz) and a goat polyclonal antibody to MKK7 (T19, Santa Cruz), respectively. MKK activity was measured in the immune complex by a coupled protein kinase assay with JNK1 and c-Jun as the substrates. Phosphorylated c-Jun was detected after SDS-PAGE by autoradiography (upper panel) and was quantitated by PhosphorImager analysis (lower panel).

Figure 2

Figure 2

Phosphorylation on Thr alone causes partial JNK activation, but maximal JNK activation requires dual phosphorylation on Thr and Tyr. Cotransfection assays were performed using COS cells expressing MKK4 or MKK7 together with epitope-tagged JNK1. The effect of replacement of the dual phosphorylation site motif Thr–Pro–Tyr (TPY) with Ala–Pro–Phe (APF), Thr–Pro–Phe (TPF), or Ala–Pro–Tyr (APY) was examined. The activity of JNK1 was examined in immune complex kinase assays with the substrate c-Jun. The JNK1 proteins were detected by immunoblot analysis (IB; middle panel). JNK1 protein kinase activity was examined using the substrate c-Jun. Phosphorylated c-Jun was detected by autoradiography (KA; upper panel) and was quantitated by PhosphorImager analysis (lower panel).

Figure 3

Figure 3

Isolation of MKK4- and MKK7-deficient mouse embryo fibroblasts. (A) Extracts were prepared from wild-type (WT), Mkk4−/−, Mkk7−/−, and Mkk4−/− Mkk7−/− MEF. The expression of MKK4, MKK7, JNK, p38 MAPK, and ERK was examined by protein immunoblot analysis. (B) The saturation growth density of MEF in different concentrations of serum was examined by crystal violet staining (mean OD590 ± SD; n = 3) following the addition of 1 × 104 cells to 20 mm tissue culture dishes and culture in medium supplemented with different concentrations of fetal calf serum. Relative cell numbers were measured at day 0 (D = 0) and after culture for 9 d (D = 9).

Figure 4

Figure 4

MKK4 and MKK7 are selectively required for stress-induced apoptosis. (A) Wild-type (WT), Mkk4−/−, Mkk7−/−, and Mkk4−/− Mkk7−/− MEF were untreated (0) or exposed to UV-C radiation (60 J/m2) and incubated in culture medium for 12 or 16 h. The expression of p53, ARF, and JNK was examined by protein immunoblot analysis. (B) The subcellular distribution of p53 (green) was examined by immunofluorescence analysis. MEF were untreated (Cont.) or treated with UV-C radiation (60 J/m2; UV) and incubated in medium with serum for 16 h. DNA was visualized by staining with 4,6-diamidino-2-phenylindole (blue). (C) MEF were treated without and with UV-C (60 J/m2, 15 h; UV), methymethanesulfonate (0.5 μM, 12 h; MMS), or anti-Fas (1 μg/mL Jo2 antibody plus 0.3 μg/mL cycloheximide, 15 h; FAS). The amount of apoptosis was measured by analysis of DNA fragmentation (mean OD ± SD; n = 3) using the cell death detection Elisa method (Roche). (D) Extracts were prepared from cells treated without and with UV-C (60 J/m2; UV) or taxol (5 μM) for the indicated time. Bcl2 was detected by immunoblot analysis (IB, upper panel) and JNK activity was measured by an in vitro kinase assay. Phosphorylated c-Jun was detected by autoradioography (KA, middle panel) and was quantitated by PhosphorImager analysis (lower panel).

Figure 5

Figure 5

MKK4 and MKK7 cooperate to activate JNK in vivo. Wild-type (WT), Mkk4−/−, Mkk7−/−, and Mkk4−/− Mkk7−/− MEF were untreated or treated with UV-C (60 J/m2; UV), anisomycin (1 μg/mL; ANISO), TNFα (10 ng/mL; TNF), or IL1α (15 ng/mL; IL1) and then incubated for the indicated times. JNK (A) and p38 MAP kinase (B) activity was measured by in vitro protein kinase assay with the substrates c-Jun and ATF2, respectively. Phosphorylated c-Jun and ATF2 were detected after SDS-PAGE by autoradiography (upper), quantitated by PhosphorImager analysis (Molecular Dynamics), and presented in arbitrary units (lower).

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