Formation of ternary complex of human biliverdin reductase-protein kinase Cδ-ERK2 protein is essential for ERK2-mediated activation of Elk1 protein, nuclear factor-κB, and inducible nitric-oxidase synthase (iNOS)

Abstract

Growth factors, insulin, oxidative stress, and cytokines activate ERK1/2 by PKCδ and MEK1/2. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase and intracellular scaffold/bridge/anchor, is a nuclear transporter of MEK1/2-stimulated ERK1/2 (Lerner-Marmarosh, N., Miralem, T., Gibbs, P. E., and Maines, M. D. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 6870-6875). hBVR, PKCδ, and MEK1/2 overlap in their tissue expression profile and type of activators. Presently, we report on formation of an hBVR-PKCδ-ERK2 ternary complex that is essential for ERK2 signal transduction and activation of genes linked to cell proliferation and cancer. MEK1/2 and the protein phosphatase PP2A were also present in the complex. When cells were stimulated with insulin-like growth factor-1 (IGF-1), an increased interaction between hBVR and PKCδ was detected by FRET-fluorescence lifetime imaging microscopy. hBVR and ERK2 were phosphorylated by PKCδ; however, the PKC was not a substrate for either ERK2 or hBVR. IGF-1 and phorbol ester increased hBVR/PKCδ binding; hBVR was required for the activation of PKCδ and its interaction with ERK2. The C-terminal phenylalanine residues of PKCδ (Phe(660), Phe(663), and Phe(665)) were necessary for binding to ERK2 but not for hBVR binding. Formation of the hBVR-PKCδ-ERK2 complex required the hBVR docking site for ERK, FXFP (DEF, C-box) and D(δ)-box (ILXXLXL) motifs. The hBVR-based peptide KKRILHCLGLA inhibited PKC activation and PKCδ/ERK2 interaction. Phorbol ester- and TNF-α-dependent activation of the ERK-regulated transcription factors Elk1 and NF-κB and expression of the iNOS gene were suppressed by hBVR siRNA; those activities were rescued by hBVR. The findings reveal the direct input of hBVR in PKCδ/ERK signaling and identify hBVR-based peptide regulators of ERK-mediated gene activation.

FIGURE 1.

Schematic presentation of consensus sequences of hBVR for which functions have been ascribed. The numbers indicated for each consensus sequence are those of the hBVR primary structure. The N-terminal segment of 99 residues is the catalytic domain of hBVR; it houses a sequence of four valines followed by the consensus for the ATP/adenine ring-binding site. The kinase activity of hBVR is responsible for its autophosphorylation (22, 38). hBVR is a kinase for serine phosphorylation of IRS-1, the phosphorylation of which halts glucose uptake (60). hBVR is also a likely kinase for Thr⁵⁰⁰ in the activating loop of PKCβII (14); the PKC is a key component of cell growth and differentiation. The reductase domain catalyzes reduction of biliverdin to bilirubin, a component of cellular defense mechanisms protecting against reactive oxygen species (ROS) (61) and apoptosis (32). The sequences designated by one or two asterisks closely resemble sites in the primary sequence of repeats V (QAMLWDLNE) and VI (SIKIWDLE) of the receptor for activated C-kinase-1 (RACK1). RACK1 is a 36-kDa protein that is similar in size to hBVR (62). Activation of PKCs, including the β, δ, and ϵ isoforms (62), is associated with conformational change that exposes their RACK-binding sites. We predict that the presence of RACK1-like sequences in hBVR may allow its binding to PKCs. The binding would not require kinase activity of hBVR. The basic leucine zipper motif binds to 7- and 8-bp AP-1 and AP-2 sites. Stress-response genes are activated by AP-1, and cAMP-responsive genes are regulated by AP-2 regulatory elements. hBVR regulates expression of stress-responsive HO-1, c-Fos, c-Jun, and ATF2/CREB (14, 31, 32). Within this sequence is a motif that strongly resembles a conserved protein kinase motif (63). The high affinity ERK-binding site, known either as C-box or DEF (19), is the site of interaction of ERK1/2 and hBVR, positioning ERK in proximity to its kinase (7). Nuclear localization of hBVR is also critical for transport of the transcriptional regulators ERK1/2 and heme into the nucleus (7, 13). Reentry of ERK into the cytoplasm requires the intact hBVR nuclear export signal (NES) (7). hBVR is directly phosphorylated by IRK upon activation by insulin or IGF-1 (22). The tyrosine in the SH2 recognition motif of hBVR, as with other SH2 recognition motif-containing proteins, is predicted to form a platform for formation of signaling complexes (64). hBVR is phosphorylated by ERK, and MotifScan predicts serine in the SP sequence as the phosphorylation target site of ERK1/2. A second SH2 recognition motif follows the nuclear localization signal (NLS) and is involved in activation of PKCζ by TNF-α (15). The low affinity D-box-like sequence is the binding site for kinases and substrates in the MAPK signaling cascade. The C-terminal six residues are the Zn²⁺-binding domain of hBVR (28). Based on the reported role of Zn²⁺ for plasma membrane translocation of PKCs and nuclear translocation of NF-κB (65) we predict that the function of hBVR in translocation of PKCs -β and -ζ to the cell membrane may involve its associated Zn. Notably, hBVR under resting conditions is found in the cytoplasm and membrane caveolae (66). The C-terminal lysine 296 is critical for the hBVRs catalytic activity (M. D. Maines, unpublished data.); although it lies in a disordered region of the BVR molecule (16), this does not preclude a catalytic function.

FIGURE 2.

Interaction between PKCδ and hBVR is enhanced by extracellular stimulus. a, IGF-1 treatment promotes intracellular co-localization of PKCδ and hBVR. HeLa cells were co-transfected with EGFP-PKCδ and pDsRed2-hBVR and treated with IGF-1 as described in the text. The fluorescence images from live cells maintained at 37 °C and 5% CO₂ were collected as described under “Experimental Procedures.” b, stimulation with IGF-1 enhances PKCδ/hBVR binding. Cells expressing the above constructs were treated with IGF-1 as described in the text. Representative fluorescence intensity (panels i, iii, and v) and FLIM (panels ii, iv, and vi) images of EGFP-PKCδ (panels i and ii), EGFP-PKCδ/DsRed2-hBVR (panels iii and iv), and EGFP-PKCδ/DsRed2-hBVR + IGF 1 (panels v and vi) in HeLa cells are shown. The scale bar ranges from 1 to 4 ns. c, fluorescence lifetime histograms of EGFP-PKCδ (○), EGFP-PKCδ/DsRed2-hBVR (●), and EGFP-PKCδ/DsRed2-hBVR+ IGF-1 (▴) in HeLa cells. Curves represent FLIM data recorded from ∼25 cells per condition; frequency of events is shown in arbitrary units (a.u.).

FIGURE 3.

PKCδ autophosphorylation and activity are increased by hBVR. a, stimulation with IGF-1 or PMA enhances PKCδ/hBVR binding. HEK293A cells were transfected with pcDNA-HA-hBVR and pcDNA-PKCδ plasmids and then treated with IGF-1 or PMA as described (see under “Experimental Procedures”). Cell lysates were prepared, immunoprecipitated (IP) with anti-HA antibodies, and followed by sequential immunoblotting with antibodies to the C-terminal domain of PKCδ and anti-hBVR antibodies. WB, Western blot. b, hBVR increases PKCδ autophosphorylation. Increasing concentrations (0, 0.06, and 0.3 μm) of hBVR were preincubated with active recombinant human PKCδ for 5 min at room temperature prior to the kinase assay. The PKCδ assay was carried out as described under “Experimental Procedures.” The reaction products were processed for autoradiography as described in the text. c, hBVR increases PKCδ kinase activity. WT PKCδ or its kinase-inactive mutant were overexpressed either alone or together with hBVR in cells. Lysates prepared from PMA-treated cells were immunoprecipitated with anti-PKCδ antibodies, as in a. PKCδ activity of the immunoprecipitates was then determined using the PKCδ peptide substrate ARRKRKGSFFYGG, and the incorporated phosphate was measured by the P81 method. *, p < 0.001. d, PKCδ is not a substrate for hBVR kinase activity. PKCδ kinase activity was measured in the presence or absence of hBVR, under assay conditions optimal for hBVR kinase activity. Reaction products were analyzed as in b. e, hBVR is required for activation of PKCδ in metabolically labeled cells. HEK293A cells transfected with pcDNA-PKCδ were infected with virus expressing hBVR-siRNA for 24 h. Cells were starved (24 h) and metabolically labeled with [³²P]H₃PO₄ for 4 h prior to treatment with PMA as in a. Cell lysates were immunoprecipitated with anti-PKCδ antibodies. The immunoprecipitates were processed for autoradiography, as described in the text. After decay of radioactivity, the membrane was probed with anti-PKCδ antibodies as a reference for loading. f, phosphatase PP2A is present in the hBVR-PKCδ complex. Cells were co-transfected with pcDNA-HA-hBVR and pcDNA-PKCδ and treated with IGF-1 or PMA. Cell lysates were processed for immunoprecipitation with anti-HA antibodies, and analyzed by Western blotting. The blot was probed sequentially with anti-PP2A C-subunit and anti-hBVR antibodies.

FIGURE 4.

hBVR, PKCδ, and ERK2 form a ternary complex. a, hBVR, PKCδ, and ERK2 co-immunoprecipitate. HEK293A cells were co-transfected with pcDNA-PKCδ- and pcDNA-HA-tagged hBVR and treated with either PMA or IGF-1 as described in the text. Immunoprecipitates (IP) obtained with anti-HA antibodies were subjected to Western blotting (WB). The membrane was sequentially probed with anti-PKCδ, anti-ERK2, and anti-hBVR antibodies. b, depletion of hBVR attenuates PKCδ and ERK2 binding. Cells were transfected with pcDNA-PKCδ and infected with the viral constructs described in the text to express either siRNA for hBVR or scRNA. Lysates prepared from IGF-1-treated cells were immunoprecipitated with anti-ERK2 antibodies and analyzed by Western blotting, using anti-PKCδ antibodies. IgG was used as a loading reference. c, hBVR, PKCδ, and ERK2 form a ternary complex in the cell. HEK cells were serum-starved and treated with IGF-1 as described in the text. Cell lysates were immunoprecipitated with anti-ERK2 antibodies, and the immunoprecipitates were processed for Western blotting; the blot was sequentially probed with anti-hBVR, anti-PKCδ, and anti-ERK2 antibodies. d, MEK1 also associates with the hBVR-ERK-PKCδ complex. Cells were transfected with pcDNA-PKCδ and treated with PMA or IGF-1 as in a. Cell lysates were immunoprecipitated with anti-PKCδ antibodies and analyzed by Western blotting. The blot was sequentially probed with anti-hBVR, anti-MEK1, and anti-ERK2 antibodies. ST, protein standards.

FIGURE 5.

Activities affected by protein/protein interaction in the complex. a, activation of PKCδ is not required for ERK binding. Cells were transfected with WT- or kinase-inactive pcDNA-PKCδ, starved, and treated with IGF-1. Cell lysate was immunoprecipitated (IP) with anti-ERK2 antibodies, and the precipitate was analyzed by Western blotting (WB), using antibodies to the C-terminal domain of PKCδ. IgG served as a loading reference. b, PKCδ phosphorylates ERK2 in vitro. Kinase-inactive GST-ERK2 was used as the substrate for PKCδ as described in Fig. 3b. Control reactions contained only one of the proteins. Reaction products were detected by autoradiography. c, ERK2 does not phosphorylate PKCδ in vitro. GST-ERK kinase activity was assessed using GST-PKCδ kinase-inactive mutant or hBVR as substrates. Control reactions included either the GST-PKCδ kinase-inactive mutant or hBVR. The reaction products were processed for autoradiography. d, hBVR is activated by ERK2. GST-hBVR was phosphorylated in vitro by GST-ERK2 as in c, using unlabeled ATP; the reaction product was then assayed for reductase activity. The basal activity was measured for GST-hBVR incubated with ATP in the absence of ERK2. *, p < 0.001 compared with basal activity. Experimental details are provided in the text.

FIGURE 6.

hBVR ERK docking motif and D-box-like sequence participate in PKCδ-ERK binding. a, two hBVR motifs are required for PKCδ and ERK2 binding. Cells co-transfected with pcDNA-PKCδ and plasmids expressing hBVR protein carrying mutations in either ERK docking motif (FGFPAF → VGAPAV) or D-box-like sequence (KKRILHCLGLA → KKRAAHCAGAA) were treated with either IGF-1 or PMA as in Fig. 3. Cell lysates were immunoprecipitated (IP) with anti-ERK2 antibodies. The immunoprecipitated proteins were subjected to Western blotting (WB) using anti-PKCδ antibodies as the probe. IgG served as a loading reference. b, hBVR-based KKRILHCGLA peptide inhibits PKCδ autophosphorylation in a concentration-dependent manner, although the peptide FGFPAFSG, corresponding to the hBVR ERK-docking sequence, does not. PKCδ autophosphorylation was measured in vitro in the presence of increasing concentrations of hBVR-based D-box peptide (KKRILHCGLA). The hBVR-based DEF-like FGFPAFSG peptide was also tested at a concentration of 1.0 μm. The reaction was carried as described in the text; products were detected by autoradiography. c, KKRILHCLGLA hBVR-based peptide inhibits ERK1/2 and PKCδ binding. Cells transfected with PKCδ were starved and loaded with myristoylated hBVR-based D-box-like peptide. After treatment with IGF-1, cell lysates were immunoprecipitated with anti-ERK2 antibody followed by Western blotting, using anti-PKCδ antibody as the probe.

FIGURE 7.

Intact C-terminal hydrophobic motif (FXXF(S/T)(F/Y)) of PKCδ is essential for binding to and signaling by ERK2. a, C-terminal PKCδ phenylalanine residues are not required for binding to hBVR. Cells were transfected with WT PKCδ or its hydrophobic site mutant (⁶⁶⁰FAGFSF → VAGASV) plasmids and treated with IGF-1 or PMA. Proteins immunoprecipitated (IP) from cell lysates by anti-HA antibodies were examined by Western blotting (WB); the blot was sequentially probed with antibodies to the N-terminal domain of PKCδ and anti-hBVR antibodies. b, PKCδ hydrophobic sequence is not required for binding to hBVR. Cells were co-transfected with pcDNA-HA-hBVR and WT PKCδ or its C-terminal truncated mutant and treated with IGF-1 or PMA. Cell lysates were immunoprecipitated with anti-HA antibodies and subjected to Western blotting, which was sequentially probed with anti-PKCδ N-terminal domain and anti-BVR antibodies. c, intact PKCδ hydrophobic sequence is required for binding to ERK2. Cells overexpressing either WT or hydrophobic site mutant PKCδ plasmids were treated as described in a. Cell lysates were immunoprecipitated with anti-ERK2 antibodies and subjected to Western blotting; the blot was probed with anti-PKCδ N-terminal domain antibodies. Immunoprecipitate inputs were adjusted to correct for differential expression of the WT PKCδ and PKCδ hydrophobic mutant. PKCδ-ST = PKCδ standard. d, intact PKCδ is required for full activation of Elk1. Cells were co-transfected with the Elk1 reporter, β-galactosidase, and either WT pcDNA-PKCδ or its kinase-inactive (K378R), truncated (Δ656–676) or hydrophobic site mutants. Cells were serum-starved for 12 h and treated with 100 nm PMA for 10 h, and the lysates were assayed for luciferase activity. The activity was normalized against that of β-galactosidase. Experimental details are provided in the text. *, p < 0. 01 compared with WT PKCδ.

FIGURE 8.

hBVR is a determinant in both PKCδ and MEK1/2 ERK-1/2/Elk1 signaling. a, depletion of PKCδ and hBVR suppresses PMA-dependent Elk1 activation. Cells were co-transfected with the Elk1 reporter system (as used in Fig. 7d) and PKCδ, either alone or together with expression viruses for hBVR siRNA. Controls were transfected with schBVR virus. The cells were treated with 100 nm PMA for 10 h, and the cell lysates were assayed for luciferase activity as in Fig. 7d. *, p < 0.001 compared with scRNA control. b, restoration of siRNA-depleted hBVR or PKCδ recovers PMA-dependent induction of Elk1. Cells were transfected with PKCδ siRNA or infected with hBVR siRNA expression virus as in a. 18 h later, the cells were co-transfected with the Elk1 reporter system together with either pcDNA-PKCδ or pcDNA-hBVR. siRNA or scRNA treatment was continued in the cells that were not rescued. After 12 h, cells were treated with 100 nm PMA or vehicle for an additional 12 h. Harvested cells were lysed, and the lysates were assayed for luciferase activity. c, depletion of hBVR increases suppression of PMA-dependent Elk1 activation by MEK1/2 siRNAs. Cells were co-transfected with Elk1 luciferase reporter and, where indicated, with siRNAs for MEK1/2 and/or hBVR. Cells were processed as in a. d, siRNA for PKCδ decreases PKCδ level in cells. Cells were transfected with either siRNA for PKCδ or its control scRNA, as described in the text, and cell lysates were subjected to immunoblotting with antibodies to the C-terminal segment of PKCδ followed by anti-β-actin antibodies. WB, Western blot. e, PKCδ-driven induction of NF-κB promoter is blocked by depletion of hBVR. Cells co-transfected with NF-κB and β-galactosidase reporters and pcDNA-PKCδ were also transfected with siRNA for hBVR or with control scRNA, as indicated. 24 h after transfection, cells were harvested, and cell lysates were assayed for luciferase activity, as in a. f, replacement of hBVR or PKCδ expression restores TNF-α-stimulated NF-κB activation. Cells were transfected with PKCδ siRNA or infected with hBVR siRNA expression virus. 18 h later, the cells were co-transfected with the NF-κB reporter and either pcDNA-PKCδ or pcDNA-hBVR, as indicated. Where indicated, siRNA treatment was continued. After 12 h, the cells were treated with 20 ng/ml TNF-α or vehicle for an additional 12 h and then harvested, and cell lysates were assayed for expression of luciferase, as in a. *, p < 0.001 compared with scRNA control.

FIGURE 9.

hBVR regulates PKCδ-mediated NF-κB and iNOS induction. a, iNOS promoter activity is restored by replenishing siRNA-depleted protein. Cells were pretreated with siRNAs for 18 h as in Fig. 8b. Thereafter, cells were co-transfected with the iNOS-luciferase reporter and either pcDNA-PKCδ or pcDNA-hBVR. 12 h later, cells were treated with 20 ng/ml TNF-α for an additional 12 h, and cell lysates were assayed for luciferase activity. *, p < 0.001 for TNF-α-treated samples compared with scRNA + TNF-α control. b, hBVR and p65 are necessary for PKCδ-dependent iNOS induction by TNF-α. Cells were transfected with the indicated siRNAs or expression plasmids. 24 h later, cells were treated with TNF-α for 3 h. iNOS mRNA was measured using quantitative PCR, relative to 18 S rRNA. *, p < 0.01 for bracketed values.

FIGURE 10.

Proposed mechanism for hBVR, PKCδ, and ERK2 ternary complex formation. Sites of interaction between proteins are represented by boxed sequences. IGF-1 stimulates the activation and phosphorylation of PKCδ by upstream kinases such as PDK1 and mammalian TOR; the activated PKC is then tightly associated with hBVR. Two sequence motifs are believed to be critical for association as follows: an ERK D-domain in PKCδ and the D-box-like sequence of hBVR. Activation and binding of PKCδ to hBVR allow PKCδ to phosphorylate the reductase, at Ser¹⁴⁹, which is in a consensus protein kinase motif (63), and Ser²³⁰ in the SH2 domain that is activated as a consequence of stimulation of cells with insulin/IGF-1 (22). Because the two serine residues are closely juxtaposed on adjacent antiparallel strands of a β-sheet (Ref. and also PDB code 2H63), electrostatic forces are predicted to trigger a conformational change resulting in exposure of the C-box-like sequence of hBVR (FGFPAFS, amino acids 162–168) and formation of a ternary complex, with hBVR functioning as the bridge between PKCδ and ERK2. The noted sequence is the high affinity ERK-binding motif (19); the sequence motif in ERK2 responsible for this binding reaction is not known. hBVR/ERK2 interaction positions PKCδ and ERK2 domains in close proximity, allowing their interaction. Because a peptide based on the hBVR D-box inhibits interaction between PKCδ and ERK2, the scheme predicts displacement of hBVR from PKCδ by ERK2. However, there is a distinct possibility that a combination of PKCδ/hBVR, hBVR/ERK, and PKCδ/ERK also occurs.