Mechanism of p38 MAP kinase activation in vivo

Abstract

The p38 mitogen-activated protein kinase (MAPK) is activated in vitro by three different protein kinases: MKK3, MKK4, and MKK6. To examine the relative roles of these protein kinases in the mechanism of p38 MAP kinase activation in vivo, we examined the effect of disruption of the murine Mkk3, Mkk4, and Mkk6 genes on the p38 MAPK signaling pathway. We show that MKK3 and MKK6are essential for tumor necrosis factor-stimulated p38 MAPK activation. In contrast, ultraviolet radiation-stimulated p38 MAPK activation was mediated by MKK3, MKK4, and MKK6. Loss of p38 MAPK activation in the mutant cells was associated with defects in growth arrest and increased tumorigenesis. These data indicate that p38 MAPK is regulated by the coordinated and selective actions of three different protein kinases in response to cytokines and exposure to environmental stress.

Figure 1.

Characterization of Mkk3^–/– Mkk6^–/– mice. (A) The morphology of wild-type and Mkk3/6^–/– embryos at E11.0 is illustrated. The mutant embryos are anemic and hypovascular. (B) The morphology of the placenta of wild-type and Mkk3/6^–/– embryos at E11.0 is illustrated (ventral view). The placenta of the mutant embryos is pale with decreased vascularization. (C) Sections of the placenta from wild-type and Mkk3/6^–/– embryos at E10.5 were stained with hematoxylin and eosin (H&E). The developing labyrinth and spongiotrophoblast layers are markedly decreased in the mutant compared with the wild type. (Ch) chorionic plate; (la) labyrinth; (sp) spongiotrophoblast; (ma) maternal decidual tissue; (Gi) trophoblast giant cells.

Figure 2.

Isolation of fibroblasts from MKK3- and MKK6-deficient mice. (A) Wild-type (WT), Mkk3^–/–, Mkk6^–/–, and Mkk3^–/– Mkk6^–/– fibroblasts were cultured in vitro. Extracts prepared from these cells were examined by immunoblot analysis using antibodies to JNK, ERK, p38 MAPK, MKK3, MKK4, MKK6, MKK7, and α-tubulin. (B) Cultures of wild-type (WT), Mkk3^–/–, Mkk6^–/–, and Mkk3^–/– Mkk6^–/– fibroblasts were examined by phase contrast microscopy. (C) The proliferation of wild-type (WT), Mkk3^–/–, Mkk6^–/–, and Mkk3^–/– Mkk6^–/– fibroblasts cultured in medium supplemented with 10% fetal calf serum was examined. The relative cell number was measured by staining with crystal violet (OD_{595 nm}). The normalized data presented are the mean of triplicate determinations and are representative of three independent experiments.

Figure 3.

Targeted disruption of Mkk3 and Mkk6 prevents activation of p38 MAPK by tumor necrosis factor. (A) Wild-type (WT), Mkk3^–/–, Mkk6^–/–, and Mkk3^–/– Mkk6^–/– fibroblasts were treated without and with 10 ng/mL TNFα (10 min). Extracts prepared from these cells were examined by immunoblot analysis using antibodies to phospho-MKK3/6 (P-MKK3, P-MKK4, P-MKK6), MKK3, MKK4, MKK6, phospho-p38 MAPK (P-p38), p38 MAPK, phospho-JNK (P-JNK), JNK, phospho-ERK (P-ERK), ERK, and α-Tubulin. The phospho-MKK3/6 antibody binds activated MKK3 and MKK4 and can also bind more weakly to activated MKK6. Two exposures of the phospho-MKK3/6 immunoblot are presented to show activated MKK3, MKK4, and MKK6. (B) The activation of p38 MAPK was examined by in vitro kinase assays using ATF2 as the substrate.

Figure 4.

Targeted disruption of Mkk3 and Mkk6 does not prevent UV-stimulated activation of p38 MAPK. (A) Wild-type (WT), Mkk3^–/–, Mkk6^–/–, and Mkk3^–/– Mkk6^–/– fibroblasts were treated without and with 60 J/m² of UV radiation and incubated (30 min). Extracts prepared from these cells were examined by immunoblot analysis using antibodies to phospho-MKK3/6 (P-MKK3, P-MKK4, P-MKK6), MKK3, MKK4, MKK6, phospho-p38 MAPK (P-p38), p38 MAPK, phospho-JNK (P-JNK), JNK, phospho-ERK (P-ERK), ERK, and α-Tubulin. (B) The activation of p38 MAPK was examined by in vitro kinase assays using ATF2 as the substrate.

Figure 5.

MKK4 deficiency causes decreased activation of JNK and p38 MAPK. (A,B) Wild-type (WT) and Mkk4^–/– fibroblasts were treated without and with 10 ng/mL TNFα (15 min) or 60 J/m² of UV radiation (30 min). The expression of JNK (A) and p38 MAPK (B) was examined by immunoblot analysis (top panels). Activated JNK (P-JNK) and p38 (P-p38) were detected by immunoblot analysis (middle panels). JNK and p38 MAPK activity was also measured with an in vitro kinase assay using c-Jun and ATF2 as substrates, respectively (bottom panels). The amount of phosphorylated c-Jun and ATF2 was quantitated by PhosphorImager analysis (Molecular Dynamics). The data are presented in arbitrary units. (C) Wild-type (WT) and Mkk3^–/– Mkk6^–/– fibroblasts were transfected with siRNA duplexes targeting MKK4 (+siRNA) or luciferase (–siRNA). The cells were treated without and with 60 J/m²of UV radiation (30 min) at 48 h posttransfection. The expression of MKK4, p38 MAPK, and activated p38 MAPK (P-p38) was examined by immunoblot analysis. p38 MAPK activity was examined in an in vitro kinase assay using the substrate ATF2. The amount of phosphorylated ATF2 was quantitated by PhosphorImager analysis.

Figure 6.

Mkk3^–/– Mkk6^–/– fibroblasts exhibit defects in growth arrest. (A) Wild-type (WT) and Mkk3^–/– Mkk6^–/– fibroblasts were serum starved (24 h). The effect of addition of 10% serum (30 min) to the serum-starved cells is presented. The expression of p38 MAPK and activated p38 MAPK (P-p38) was examined by immunoblot analysis. (B) Wild-type (WT), Mkk3^–/–, Mkk6^–/–, and Mkk3^–/– Mkk6^–/– fibroblasts were cultured in different concentrations of fetal calf serum (7 d). The relative cell number was measured by staining with crystal violet (OD_{595 nm}). The normalized data presented are the mean of triplicate determinations and are representative of three independent experiments. (C) Cyclin and L32 mRNAexpression in wild-type (WT) and Mkk3^–/– Mkk6^–/– fibroblasts was examined in a ribonuclease protection assay. Cells growing in 10% fetal calf serum were compared with cells cultured (24 h) in serum-free medium. (D) Extracts prepared from wild-type (WT) and Mkk3^–/– Mkk6^–/– fibroblasts were examined by immunoblot analysis using antibodies to cyclin D1, cyclin D2, Rb (detects Rb and phospho-Rb), hypo-pRb (detects hypo-phosphorylated Rb), and α-Tubulin. Cells growing in 10% fetal calf serum were compared with cells cultured (24 h) in serum-free medium. (E) c-Jun, JunB, and JunD mRNAexpression in wild-type (WT) and Mkk3^–/– Mkk6^–/– fibroblasts incubated without and with 10 ng/mL TNFα or 10% serum (24 h) was examined in a ribonuclease protection assay.

Figure 7.

Compound disruption of Mkk3 and Mkk6 causes increased tumorigenesis. Wild-type (WT) and Mkk3^–/– Mkk6^–/– fibroblasts immortalized with SV40 large T antigen were injected subcutaneously in athymic nude mice. Representative mice with tumors are illustrated. The mice were euthanized, and the tumors were fixed and processed for histological analysis. Sections of the tumors stained with H&E are shown. The tumor volume was measured and is presented graphically (mean ± S.D.; n = 5).