Human biliverdin reductase suppresses Goodpasture antigen-binding protein (GPBP) kinase activity: the reductase regulates tumor necrosis factor-alpha-NF-kappaB-dependent GPBP expression

Abstract

The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-alpha). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-kappaB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-alpha-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-kappaB show the hBVR role in the initial stimulation of GPBP expression by TNF-alpha-activated NF-kappaB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the (281)CX(10)C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC(281), corresponding to the core of the consensus D(delta)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-alpha dependent NF-kappaB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-alpha-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis.

FIGURE 1.

GPBP binds to hBVR in HEK293A cells. a, a GST pulldown assay is shown. HEK293A cells were transfected with the pcDNA3-expression plasmid encoding GPBP. One day after DNA addition, cell lysates were prepared and subjected to a GST pulldown assay using either GST-hBVR or GST alone. The membrane was sequentially probed with anti-GPBP and anti-hBVR antibodies. b, co-IP of GPBP and hBVR detected by hBVR antibody is shown. Lysate prepared from cells co-transfected with pcDNA3-hBVR and pcDNA3-GPBP was immunoprecipitated with an anti-hBVR antibody or with control rabbit IgG. The precipitate was subjected to Western blot analysis by sequentially probing the membrane with anti-GPBP and anti-hBVR antibodies. c, co-IP of GPBP and hBVR as detected by GPBP antibody is shown. The same cell lysates were immunoprecipitated using anti-GPBP antibody or control rabbit IgG and analyzed as in b. d, cross-linking of GPBP and hBVR is shown. HEK293A cells were cross-linked with formaldehyde, and cell extracts were immunoprecipitated with protein A-Sepharose after incubation with or without the indicated GPBP-specific antibodies. The input lane shows the presence of both GPBP (and/or the closely migrating GPBPΔ26) and BVR. The immunoprecipitates were resolved by gel electrophoresis, and the Western blot was probed with biotinylated N27 antibody or anti-BVR. In the competition experiment, anti-BVR binding was blocked by the addition of recombinant hBVR. In a–d, detection was by secondary antibody conjugate followed by ECL. Purified preparations of GST-hBVR (BVR-st) and GPBP were included as controls (GPBP-st).

FIGURE 2.

hBVR suppresses the kinase activity of GPBP. a, TNF-α-stimulated phosphorylation of GPBP is suppressed by hBVR. HEK293A cells transfected with GPBP alone or co-transfected with hBVR-pcDNA expression plasmids were metabolically labeled with [³²P]H₃PO₄. Cell lysates were obtained, and GPBP was immunoprecipitated. The precipitate was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and autoradiographed. The blot was subsequently probed with anti-GPBP antibody followed by ECL detection as the control for loading. Immunoprecipitates with mouse IgG and anti-mouse IgG-ECL was used as the control for specificity of binding. b, hBVR decreases GPBP autophosphorylation in a concentration-dependent manner. Cells were transfected with FLAG-GPBP expression plasmid together with increasing concentrations of pcDNA3-hBVR. One day later cells were starved overnight and thereafter treated with TNF-α (20 ng/ml, 15 min). GPBP was isolated with anti-FLAG beads and assayed in the kinase reaction as described under “Experimental Procedures” (6). c, hBVR suppresses GPBP-dependent phosphorylation of its substrate, the GPA-derived peptide. Cells were transfected with expression plasmid for FLAG-GPBP alone or co-transfected with pcDNA3-hBVR followed 24 h later by starvation overnight, treatment with TNF-α as in b, and subsequent lysis. GPBP was isolated from the lysates using anti-FLAG beads. The N-terminal peptide of GPA was isolated as a GST fusion polypeptide from E. coli and used as the substrate in the GPBP kinase reaction assay. The reaction mixture was resolved by SDS-PAGE and transferred to nitrocellulose membrane, and phosphorylated GPBP and GPA (GPBP^P and GPA^P) was visualized by autoradiography. The membrane was subsequently probed with anti-GPBP antibody followed by ECL detection.

FIGURE 3.

Both NF-κB and hBVR are required for TNF-α induction of GPBP. a, treatment of cells with si-hBVR prevents TNF-α induction of GPBP expression. Cells were infected with si-hBVR retrovirus 16 h before the addition of 20 ng/ml TNF-α; RNA was prepared after the indicated intervals and used as a template for random hexamer-primed cDNA synthesis. The GPBP mRNA content was determined by quantitative RT-PCR by the ΔΔCt method using 18 S rRNA as control. A representative experiment is shown; the errors were determined from the differences between the relative mRNA calculated by ΔΔC_T and ΔΔC_T ± S.E. (“Experimental Procedures”). b, Western blot of cells treated with siRNA for hBVR. Total cell lysates shown in a were subjected to immunoblotting. The nitrocellulose membrane was sequentially probed with anti-hBVR and anti β-actin antibodies. c, treatment of cells with si-p65 prevents TNF-α induction of GPBP expression. Cells were transfected with hBVR or with siRNA against the p65 NF-κB subunit. Eighteen hours later the cells were transferred to low serum medium and treated with 20 ng/ml TNF-α. Sample preparation and RT-PCR analysis were as in a. d, a Western blot of cells treated with siRNA for p65 is shown. Total cell lysates shown in c were subjected to immunoblotting. The nitrocellulose membrane was sequentially probed with anti-p65 and anti β-actin antibodies. e, activation of NF-κB is enhanced by elevated hBVR in the cell. HEK293A cells seeded in 6-well plates were co-transfected with the pNF-κB luciferase reporter plasmid, β-galactosidase reporter, and increasing amounts of either pEGFP-hBVR or the empty pEGFP vector as indicated. 12 h after DNA addition the cells were treated with TNF-α, and after a further 12 h the cells were harvested and lysed, and the luciferase and β-galactosidase activities were measured. Luciferase activity was normalized to that of β-galactosidase to correct for differences in transfection efficiency. f, hBVR does not alter GPBP mRNA stability. Cells were pretreated with si-hBVR retrovirus for 12 h followed by 20 ng/ml TNF-α for 6 h. The cells were then treated with 2.5 μg/ml actinomycin D (Act-D), and samples were withdrawn at the indicated times. Quantification of GPBP mRNA was as in a; the data were fitted by nonlinear regression to the first order exponential decay equation, enabling calculation of the half-life of the mRNA.

FIGURE 4.

hBVR binds to GPBP through its C terminus. a, GST pulldown of GPBP with hBVR fragments is shown. Lysate was prepared from cells transfected with the FLAG-GPBP expression plasmid. Truncated hBVR proteins were expressed as GST fusions and purified as described under “Experimental Procedures.” These were then used in the GST pulldown assay as described in Fig. 1a; full-length hBVR was used as the control. b, the C-terminal cysteine residues are essential for binding of GPBP to hBVR. Similar GST pulldown experiments to those in a were performed using either the full-length wt hBVR, hBVR aa 272–296 fragment, or a mutant form of the aa 292–296 fragment with Cys^281,292,293 changed to Ala. ST, standard.

FIGURE 5.

A 7-residue long peptide corresponding to hBVR aa 275–281 is as effective as the full-length hBVR polypeptide in blocking TNF-α-stimulated GPBP autophosphorylation. a, HEK293A cells were transfected with FLAG-tagged GPBP expression construct. 24 h later cells were starved overnight and treated for 2 h with the 10 μm myristoylated hBVR-based peptides indicated followed by TNF-α (20 ng/ml for 15 min). Cell lysate was obtained, immunoprecipitated with anti-FLAG antibody, and used for analysis of GPBP autophosphorylation. Analysis of kinase activity was carried out as described (6). b, quantification of GPBP phosphorylation signals is shown. The graph represents the ratio of band intensities of phosphorylated GPBP to GPBP protein shown in a, determined by densitometry of autoradiograph and ECL films, respectively.