Silencing of DGKη expression inhibits HeLa cell proliferation. HeLa cells were transfected with control or DGKη-specific siRNA 1. After 48 and 96 h, the cells were harvested. A, cell lysates prepared from HeLa cells transfected with siRNAs and COS7 cells transfected with pcDNA3.1-DGKη1 or -η2 were separated by SDS-PAGE. The protein levels of DGKη and actin in cell lysates were analyzed using Western blot (WB). B, cells were counted with a hemocytometer. The cell numbers are shown as means ± S.D. (n = 3). Statistical significance was determined using Student's t test (*, p < 0.05). Results are representative of three independent experiments.

Activation of ERK1/2 by EGF requires DGKη but not its catalytic activity. A, HeLa cells were transfected with control siRNA or DGKη siRNA 1. After 72 h, the cells were serum-starved for 5 h and stimulated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation and total ERK1/2 in the cell lysates were analyzed by Western blot (WB). Upper panels, representative results of Western blot analysis are shown. Bottom panel, the phospho-ERK1/2 levels were quantified by densitometry. Phospho-ERK1/2 levels in cells, which were treated with control siRNA and stimulated with EGF for 2 min, were set to 100. The data are shown as means ± S.D. of three independent experiments. B, HeLa cells were transfected with control siRNA or DGKη siRNA 1. After 24 h, the cells were further transfected with p3xFLAG vector or p3xFLAG-DGKη1-WT (wild-type) or -G392D (a kinase-dead mutant), which were introduced with siRNA-resistant mutations. After 48 h of transfection with the expression plasmids, the cells were serum-starved for 5 h and stimulated with EGF for 2 min. Phospho-ERK1/2, ERK1/2, 3xFLAG-tagged DGKη1, and DGKη in the cell lysates were detected by Western blot. Upper panels, representative results of Western blot analysis are shown. Bottom panel, visualized bands of phospho-ERK1/2 were quantified by densitometry. Phospho-ERK1/2 levels in the cells that were transfected with control siRNA and p3xFLAG vector were set to 100. The data are shown as means ± S.D. of three independent experiments.

Activation of MEK1/2 by EGF requires DGKη. The HeLa cells were transfected with control siRNA or DGKη siRNA 1. After 72 h, the cells were serum-starved for 5 h and stimulated with 100 ng/ml EGF for the indicated times. Phospho-MEK1/2 at Ser-217/221, total MEK1/2, and DGKη in the cell lysates were detected using Western blot (WB). Upper panels, representative results of Western blot analysis are shown. Bottom panel, phospho-MEK1/2 levels were quantified by densitometry. Phospho-MEK1/2 levels in the cells, which were treated with control siRNA and stimulated with EGF for 2 min, were set to 100. The data are shown as means ± S.D. of three independent experiments.

DGKη does not affect the EGF-induced activation of Ras but regulates the MEK1/2 activities in a Ras-dependent manner. A, HeLa cells were transfected with control siRNA or DGKη siRNA 1. After 72 h, the cells were serum-starved for 5 h and stimulated with 100 ng/ml EGF for the indicated times. Ras-GTP precipitated with GST-Raf-RBD and total Ras in the cell lysates were detected using Western blot (WB). Upper panels, representative results of Western blot analysis are shown. Bottom panel, visualized bands of Ras-GTP were quantified by densitometry. Ras-GTP levels in the cells that were treated with control siRNA and stimulated with EGF for 2 min were set to 100. The data are shown as means ± S.D. of three independent experiments. B, HeLa cells were transfected with control siRNA or DGKη siRNA 1. After 24 h, the cells were transfected with pcDNA3.1 or pCMV-H-RasV12. After 48 h of transfection with the expression plasmids, the cells were serum-starved for 5 h. The cells transfected with pcDNA3.1 were stimulated with 100 ng/ml EGF as controls (EGF) for 2 min. Phospho-MEK1/2, MEK1/2, and Ras were detected by Western blot (WB). Upper panels, representative results of Western blot analysis are shown. Bottom panel, visualized bands of phospho-MEK1/2 were quantified by densitometry. Phospho-MEK1/2 levels in the cells that were transfected with control siRNA and pcDNA3.1 and stimulated with EGF for 2 min were set to 100. The data are shown as means ± S.D. of three independent experiments.

DGKη is required for kinase activation of C-Raf in response to EGF but not B-Raf. HeLa cells were transfected with control siRNA or DGKη siRNA 1. After 72 h, the cells were serum-starved for 5 h and stimulated with 100 ng/ml EGF for 2 min. Endogenous C-Raf and B-Raf in the cell lysates were immunoprecipitated with anti-C-Raf antibody (A) and anti-B-Raf antibody (B), respectively. A, C-Raf kinase activities of the immunoprecipitants were measured in vitro using His₆-tagged MEK1-K97R as a substrate in the presence or absence of ATP. Phosphorylation of His₆-tagged MEK1-K97R was analyzed by Western blot (WB) using anti-phospho-MEK1/2 antibody. Upper panels, representative results of Western blot analysis are shown. Bottom panel, C-Raf activities were measured in the presence of ATP, and visualized bands of phospho-MEK1 were quantified by densitometry. C-Raf activities of the cells that were transfected with control siRNA and stimulated with EGF for 2 min were set to 100. The data are shown as means ± S.D. of three independent experiments. B, B-Raf kinase activities of immunoprecipitants were measured and quantified using the same methods as the C-Raf activity assay described in A. Upper panels, representative results of Western blot analysis are shown. Bottom panel, B-Raf activities of the cells that were transfected with control siRNA and stimulated with EGF for 2 min were set to 100. The data are shown as means ± S.D. of three independent experiments.

Heterodimerization of B-Raf with C-Raf induced by EGF requires DGKη. A, HeLa cells were transfected with control siRNA or DGKη siRNA 1. After 72 h, the cells were serum-starved for 5 h and stimulated with 100 ng/ml EGF for 2 min. Endogenous C-Raf in the cell lysates was immunoprecipitated with anti-C-Raf antibody. B-Raf associated with C-Raf was detected by Western blot (WB). Normal rabbit IgG was used as a negative control. Upper panels, representative results of Western blot analysis are shown. Bottom panel, visualized bands of co-precipitated B-Raf were quantified by densitometry. Relative intensities of the interaction are shown as means ± S.D. of three independent experiments. B, HeLa cells were transfected with control and B-Raf, C-Raf, and DGKη-specific siRNAs. After 72 h, the cells were serum-starved for 5 h and stimulated with 100 ng/ml EGF for 2 min. Phospho-MEK1/2, MEK1/2, B-Raf, C-Raf, and DGKη were detected by Western blot using their specific antibodies. Upper panels, representative results of Western blot analysis are shown. Bottom panel, phospho-MEK1/2 levels were quantified by densitometry. Phospho-MEK1/2 levels in the cells, which were treated with control siRNA and stimulated with EGF for 2 min, were set to 100. The data are shown as means ± S.D. of three independent experiments. TCL, total cell lysates; IP, immunoprecipitate.

DGKη1 interacts with B-Raf and C-Raf. A, HeLa cells were transiently transfected with p3xFLAG vector or p3xFLAG-DGKη1. After 48 h, the cells were lysed. 3xFLAG-tagged DGKη1 in the cell lysates was immunoprecipitated using anti-FLAG M2 affinity gel. The cell lysates (Input) were also subjected to Western blot (WB) analysis. B-Raf, C-Raf, and FLAG-tagged protein in the cell lysates and immunoprecipitants were detected using their specific antibodies as indicated. Similar results were obtained in three independent experiments. B, schematic diagrams of the DGKη1 truncated mutants used are shown. C, HeLa cells were transfected with plasmids encoding 3xFLAG-tagged DGKη1 mutants. After 48 h, the cells were lysed. 3xFLAG-tagged DGKη1 in the cell lysates (Input) was immunoprecipitated using anti-FLAG M2 affinity gel. Co-immunoprecipitation of C-Raf with DGKη1 mutants was detected by Western blot (WB). The cell lysates (Input) were also subjected to Western blot analysis. Similar results were obtained in three independent experiments. D, HEK293 cell extracts were immunoprecipitated with anti-DGKη antibody or pre-immune serum. Endogenous C-Raf and DGKη were detected by Western blot using anti-C-Raf and anti-DGKη antibodies, respectively. With longer exposures, DGKη was detectable in the Input lane. Input represents 2% of the starting materials. IP, immunoprecipitate; PH, pleckstrin homology.

DGKη is necessary for recruitment of B-Raf and C-Raf to membranes induced by EGF and active Ras. HeLa cells were transfected with control siRNA or DGKη siRNA 1. A, after 72 h, the cells were serum-starved for 5 h and stimulated with 100 ng/ml EGF for 2 min. B, after 24 h of transfection with siRNAs, the cells were transiently transfected with either pcDNA3.1 or pCMV-H-RasV12. After 48 h of transfection with the expression plasmids, the cells were serum-starved for 5 h. The cytosol and membrane fractions were isolated using ProteoExtract subcellular proteome extraction kit. Each fraction (10 μl) was analyzed by Western blot (WB) using anti-B-Raf and anti-C-Raf antibodies. Upper panels, representative results of Western blot analysis are shown. Bottom panel, intensity of the each band was quantified by densitometry, and the percentages of B-Raf and C-Raf in the membrane fractions are shown as the means ± S.D. of three independent experiments. Statistical significance was determined using Student's t test (*, p < 0.05; **, p < 0.01). C, co-localization of DGKη1 with RasV12 and C-Raf. HeLa cells grown on type I collagen-coated glass coverslips were co-transfected with plasmids encoding AcGFP1-tagged DGKη1, DsRed-monomer-tagged C-Raf, and either ECFP, ECFP-tagged H-RasV12, or ECFP-tagged H-RasN17. After 24 h of transfection, the cells were serum-starved for 5 h. The cells were fixed with 4% paraformaldehyde and observed by confocal laser scanning microscopy. ECFP-, AcGFP1-, and DsRed-monomer fusion proteins are shown in blue, green, and red, respectively. Scale bar, 10 μm.

A working model of DGKη-mediated C-Raf activation. The data are consistent with a model in which DGKη physically interacts with B-Raf and C-Raf and enhances EGF-dependent heterodimerization between B-Raf and C-Raf. As B-Raf activates C-Raf via the heterodimerization with C-Raf, DGKη augments C-Raf activity. Black and white arrows indicate “activation” and “interaction,” respectively.