Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling

doi: 10.1073/pnas.0800750105

Figure Lengend Snippet: Elk1-mediated regulation of gene transcription is stimulated by hBVR-activating MEK1/ERK1 signaling. (a) hBVR induces Elk1 activation by ERK. Cells cotransfected with hBVR, in addition to the Elk1 reporter plasmids, were starved and treated with IGF1 (16 h). Lysates were assayed by dual luciferase as described in Materials and Methods. *, P < 0.01 vs. untreated. (b) hBVR enhances constitutively active MEK1-induced Elk1 activity. Cells transfected with constitutively active MEK1 were starved and treated with 10 μM inhibitors or cotransfected with hBVR. *, P < 0.01 vs. untreated; †, P < 0.01 vs. wt hBVR alone. (c) hBVR increases ERK1-dependent Elk1 phosphorylation. ERK1 kinase activity using GST-Elk1 and hBVR as substrates was analyzed as in Fig. 1b. (d) hBVR does not phosphorylate Elk1. hBVR kinase activity using Elk1 as the substrate was analyzed as in Fig. 1b.

Article Snippet: Sources of ERK1 and ERK2 active proteins and antibodies were EMD and BD Biosciences, respectively, while monoclonal anti-ERK2, anti-phospho-ERK1/2, and Elk1 fusion protein (amino acids 307–428) were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Luciferase, Activity Assay, Transfection, Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling

doi: 10.1073/pnas.0800750105

Figure Lengend Snippet: hBVR C-box ERK docking site is necessary for the activation of ERK and Elk1 in the cell. (a) Flow-cytometry analysis. Cells were transfected with wt- or C-box mutant hBVR, starved, treated with IGF1, fixed, permeabilized, and treated with FITC-conjugated anti-phospho-ERK1/2 antibody. The proportion of stained cells was determined by flow cytometry. *, P < 0.01 vs. control. †, P < 0.01 vs. IGF1. (b) hBVR D-box mutant does not stimulate ERK. Cells were transfected with hBVR or D-box mutant plasmids or were infected with si-hBVR or sc-hBVR, starved, and treated with IGF1. IP obtained with anti-ERK2 was assayed for ERK activity. The blot was reprobed by using ERK2 antibody. (c) C- and D-boxes are required for hBVR-mediated induction of ELK. Cells were cotransfected with Elk1 reporters and wt hBVR, or C- or D-box mutants, and lysates were assayed for luciferase. *, P < 0.01 vs. control; †, P < 0.01 vs. wt hBVR. (d) hBVR D- and C-box peptides regulate ERK activity in vitro. Phosphorylation of MBP by ERK1 was measured in the presence of 10 μM C- or D-box peptide. *, P < 0.01 vs. control. (e) hBVR C-box peptide prevents IGF1-dependent activation of Elk1 by ERK. Cells transfected with the Elk1 reporters were treated with C-box peptide before IGF1 treatment. *, P < 0.01 vs. untreated; †, P < 0.01 vs. IGF1-treated. Experimental details are provided in Materials and Methods.

Article Snippet: Sources of ERK1 and ERK2 active proteins and antibodies were EMD and BD Biosciences, respectively, while monoclonal anti-ERK2, anti-phospho-ERK1/2, and Elk1 fusion protein (amino acids 307–428) were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Flow Cytometry, Transfection, Mutagenesis, Staining, Control, Infection, Activity Assay, Luciferase, In Vitro, Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling

doi: 10.1073/pnas.0800750105

Figure Lengend Snippet: hBVR is required for MEK1/ERK/Elk1-mediated transcription. (a) The hBVR ATP-binding domain is needed for Elk1 activation. Cells were cotransfected with Elk1 reporters and wt- or kinase-deficient hBVR, or infected with si-hBVR, treated with IGF1, and assayed for luciferase reporter activity. *, P < 0.001 vs. untreated; †, P < 0.01 vs. wt. (b) si-hBVR prevents MEK1 binding to ERK. Cells infected with si-hBVR or sc-hBVR were treated with IGF1 and IPed by using MEK1 antibodies. The blot was consecutively probed with ERK1/2 and MEK1 antibodies. Experimental details are provided in Materials and Methods.

Article Snippet: Sources of ERK1 and ERK2 active proteins and antibodies were EMD and BD Biosciences, respectively, while monoclonal anti-ERK2, anti-phospho-ERK1/2, and Elk1 fusion protein (amino acids 307–428) were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Activation Assay, Infection, Luciferase, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling

doi: 10.1073/pnas.0800750105

Figure Lengend Snippet: hBVR NLS and NES motifs are involved in the transport of ERK. (a) Localization of hBVR and pERK in IGF1-treated cells. HEK293A cells were transfected with pEGFP-hBVR or its mutants and then treated with IGF1 15 min before fixation and treatment with the pERK1/2T185Y187 primary and Rhodamine-conjugated secondary antibodies. Confocal microscopy was performed as described previously (5, 6). (b) hBVR NES and NLS are required for the activation of Elk1 by IGF1. Cells cotransfected with Elk1 reporters and wt hBVR or mutants were starved before treatment with IGF1. After 18 h, luciferase was assayed as described in Materials and Methods. *, P < 0.01 vs. untreated cells; †, P < 0.001 vs. wt.

Article Snippet: Sources of ERK1 and ERK2 active proteins and antibodies were EMD and BD Biosciences, respectively, while monoclonal anti-ERK2, anti-phospho-ERK1/2, and Elk1 fusion protein (amino acids 307–428) were from Santa Cruz Biotechnology.

Techniques: Transfection, Confocal Microscopy, Activation Assay, Luciferase

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Human biliverdin reductase is an ERK activator; hBVR is an ERK nuclear transporter and is required for MAPK signaling

doi: 10.1073/pnas.0800750105

Figure Lengend Snippet: Proposed functions of hBVR in the MAPK-signaling pathway. The C- and D-boxes of hBVR, MEK, and Elk1 are indicated by the rounded and triangular protuberances, respectively.

Article Snippet: Sources of ERK1 and ERK2 active proteins and antibodies were EMD and BD Biosciences, respectively, while monoclonal anti-ERK2, anti-phospho-ERK1/2, and Elk1 fusion protein (amino acids 307–428) were from Santa Cruz Biotechnology.

Techniques: