Insulin’s stimulation of glucose uptake by binding to the IRK extracellular domain is compromised in diabetes. We have recently described an unprecedented approach to stimulating glucose uptake. KYCCSRK (P2) peptide, corresponding to the C-terminal segment of hBVR, was effective in binding to and inducing conformational change in the IRK intracellular kinase domain. Although myristoylated P2, made of L-amino acids, was effective in cell culture, its use for animal studies was unsuitable. We developed a peptidase-resistant formulation of the peptide that was efficient in both mice and cell culture systems. The peptide was constructed of D-amino acids, in reverse order, and blocked at both termini. Delivery of the encapsulated peptide to HepG2 and HSKM cells was confirmed by its prolonged effect on stimulation of glucose uptake (>6 h). The peptide improved glucose clearance in both wild-type and Ob/Ob mice; it lowered blood glucose levels and suppressed glucose-stimulated insulin secretion. IRK activity was stimulated in the liver of treated mice and in cultured cells. The peptide potentiated function of IRK’s downstream effector, Akt-GSK3-(α,β) axis. Thus, P2-based approach can be used for improving glucose uptake by cells. Also, it allows for screening peptides in vitro and in animal models for treatment of diabetes.

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner-Marmarosh, N., Gibbs, P. E. M., Jenkins, J. L., Heimiller, C., Maines, M. D. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Reactive thiols of cysteine (cys) residues in proteins play a key role in transforming chemical reactivity into a biological response. The heme oxygenase-2 (HO-2) isozyme contains two cys residues that have been implicated in binding of heme and also the regulation of its activity. In this paper, we address the question of a role for cys residues for the HO-2 inhibitors or activators designed in our laboratory. We tested the activity of full length recombinant human heme oxygenase-2 (FL-hHO-2) and its analog in which cys265 and cys282 were both replaced by alanine to determine the effect on activation by menadione (MD) and inhibition by QC-2350. Similar inhibition by QC-2350 and almost identical activation by MD was observed for both recombinant FL-hHO-2s. Our findings are interpreted to mean that thiols of FL-hHO-2s are not involved in HO-2 activation or inhibition by the compounds that have been designed and identified by us. Activation or inhibition of HO-2 by our compounds should be attributed to a mechanism other than altering binding affinity of HO-2 for heme through cys265 and cys282.

Biliverdin reductase (BVR) is a multifunctional protein that is the primary source of the potent antioxidant, bilirubin. BVR regulates activities/functions in the insulin/IGF-1/IRK/PI3K/MAPK pathways. Activation of certain kinases in these pathways is/are hallmark(s) of cancerous cells. The protein is a scaffold/bridge and intracellular transporter of kinases that regulate growth and proliferation of cells, including PKCs, ERK and Akt, and their targets including NF-κB, Elk1, HO-1 and iNOS. The scaffold and transport functions enable activated BVR to relocate from the cytosol to the nucleus or to the plasma membrane, depending on the activating stimulus. This enables the reductase to function in diverse signaling pathways. And, its expression at the transcript and protein levels are increased in human tumors and the infiltrating T-cells, monocytes and circulating lymphocytes, as well as the circulating and infiltrating macrophages. These functions suggest that the cytoprotective role of BVR may be permissive for cancer/tumor growth. In this review, we summarize the recent developments that define the pro-growth activities of BVR, particularly with respect to its input into the MAPK signaling pathway and present evidence that BVR-based peptides inhibit activation of protein kinases, including MEK, PKCδ and ERK as well as downstream targets including Elk1 and iNOS, and thus offers a credible novel approach to reduce cancer cell proliferation.

Insulin binding changes conformation of the insulin receptor kinase (IRK) domain and initiates glucose uptake through the insulin, IGF-1, phosphatidyl inositol 3-kinase (PI3K), and MAPK pathways; human biliverdin reductase (hBVR) is an IRK substrate and pathway effector. This is the first report on hBVR peptide-mediated IRK activation and conformational change. (290)KYCCSRK, which increased IRK V(max) without changing K(m), stimulated glucose uptake and potentiated insulin and IGF-1 stimulation in 4 cell lines. KYCCSRK in native hBVR was necessary for the hBVR and IRK cross-activation. Peptide treatment also activated PI3K downstream effectors, Akt and ERK, phosphorylation, and Elk transcriptional activity. In cells transfected with CMV-regulated EGFP-VP-peptide plasmid, C(292)→A mutant did not stimulate glucose uptake; K(296)→A decreased uptake and kinase activity. KEDQYMKMTV, corresponding to hBVR's SH2-binding domain, was a potent inhibitor of glucose uptake and IRK. The mechanism of action of peptides was examined using cells expressing IRK (aa 988-1263) activated by coexpressed KYCCSRK. Three active cys-mutants of IRK, with fluorophore coupled to cysteines, C(1056), C(1138), or C(1234), were examined for changes in fluorescence emission spectra in the presence of peptides. KYCCSRK and KEDQYMKMTV bound to different sites in IRK. The findings identify novel agents for activating or inhibiting insulin signaling and offer a new approach for treatment of type 2 diabetes and hypoglycemia.

The expanse of human biliverdin reductase (hBVR) functions in the cells is arguably unmatched by any single protein. hBVR is a Ser/Thr/Tyr-kinase, a scaffold protein, a transcription factor, and an intracellular transporter of gene regulators. hBVR is an upstream activator of the insulin/IGF-1 signaling pathway and of protein kinase C (PKC) kinases in the two major arms of the pathway. In addition, it is the sole means for generating the antioxidant bilirubin-IXα. hBVR is essential for activation of ERK1/2 kinases by upstream MAPKK-MEK and by PKCδ, as well as the nuclear import and export of ERK1/2. Small fragments of hBVR are potent activators and inhibitors of the ERK kinases and PKCs: as such, they suggest the potential application of BVR-based technology in therapeutic settings. Presently, we have reviewed the function of hBVR in cell signaling with an emphasis on regulation of PKCδ activity.

PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066-1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser(230) and Ser(237)), flanking the ATP-binding motif (Ser(21)) and in PHPS sequence (Ser(33)) as targets of PKCδ. Ser(21) and Ser(230) were also phosphorylated in hBVR-based peptides. The Ser(230)-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell.

EDITORIAL article Front. Pharmacol., 13 July 2012Sec. Drug Metabolism and Transport Volume 3 - 2012 | https://doi.org/10.3389/fphar.2012.00136

Growth factors, insulin, oxidative stress, and cytokines activate ERK1/2 by PKC delta 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, PKC delta, and MEK1/2 overlap in their tissue expression profile and type of activators. Presently, we report on formation of an hBVR-PKC delta-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 PKC delta was detected by FRET-fluorescence lifetime imaging microscopy. hBVR and ERK2 were phosphorylated by PKC delta; however, the PKC was not a substrate for either ERK2 or hBVR. IGF-1 and phorbol ester increased hBVR/PKC delta binding; hBVR was required for the activation of PKC delta and its interaction with ERK2. The C-terminal phenylalanine residues of PKC delta (Phe(660), Phe(663), and Phe(665)) were necessary for binding to ERK2 but not for hBVR binding. Formation of the hBVR-PKC delta-ERK2 complex required the hBVR docking site for ERK, FXFP (DEF, C-box) and D(delta)-box (ILXXLXL) motifs. The hBVR-based peptide KKRILHCLGLA inhibited PKC activation and PKC delta/ERK2 interaction. Phorbol ester-and TNF-alpha-dependent activation of the ERK-regulated transcription factors Elk1 and NF-kappa B 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 PKC delta/ERK signaling and identify hBVR-based peptide regulators of ERK-mediated gene activation.

HO-2 oxidizes heme to CO and biliverdin; the latter is reduced to bilirubin by biliverdin reductase (BVR). In addition, HO-2 is a redox-sensitive K/Ca(2)-associated protein, and BVR is an S/T/Y kinase. The two enzymes are components of cellular defense mechanisms. This is the first reporting of regulation of HO-2 by BVR and that their coordinated increase in isolated myocytes and intact heart protects against cardiotoxicity of β-adrenergic receptor activation by isoproterenol (ISO). The induction of BVR mRNA, protein, and activity and HO-2 protein was maintained for ≥ 96 h; increase in HO-1 was modest and transient. In isolated cardiomyocytes, experiments with cycloheximide, proteasome inhibitor MG-132, and siBVR suggested BVR-mediated stabilization of HO-2. In both models, activation of BVR offered protection against the ligand's stimulation of apoptosis. Two human BVR-based peptides known to inhibit and activate the reductase, KKRILHC(281) and KYCCSRK(296), respectively, were tested in the intact heart. Perfusion of the heart with the inhibitory peptide blocked ISO-mediated BVR activation and augmented apoptosis; conversely, perfusion with the activating peptide inhibited apoptosis. At the functional level, peptide-mediated inhibition of BVR was accompanied by dysfunction of the left ventricle and decrease in HO-2 protein levels. Perfusion of the organ with the activating peptide preserved the left ventricular contractile function and was accompanied by increased levels of HO-2 protein. Finding that BVR and HO-2 levels, myocyte apoptosis, and contractile function of the heart can be modulated by small human BVR-based peptides offers a promising therapeutic approach for treatment of cardiac dysfunctions.

hBVR is a Ser/Thr/Tyr kinase/scaffold protein/transcription factor/intracellular transporter of regulators. hBVR is an upstream activator of the insulin/IGF-1/MAPK/PI3K signaling pathway, and of NF-kappaB. As a reductase, it converts biliverdin to the antioxidant, bilirubin. hBVR gene has 8 exons; exon 1 is not translated. We report the characterization of hBVR promoter and its negative and positive regulation, respectively, by TNF-alpha and hypoxia. The 5' end of exon 1 was defined by primer extension analyses; deletion of an inhibitor sequence 350-425 bp upstream of this exon enhanced the promoter activity. One of two NF-kappaB binding sites in the 836-bp promoter was functional; the P65 subunit of NF-kappaB and TNF-alpha acted as inhibitors. On the basis of EMSA and ChIP assays, TNF-alpha treatment increases binding of NF-kappaB to its regulatory element. Overexpression of IkappaB increased hBVR mRNA. Biliverdin, but not bilirubin, was as effective as TNF-alpha in inhibiting hBVR promoter activity. Only one of 4 hypoxia responsive elements (HREs) bound to HIF-1alpha and ARNT expressed in HEK293A cells. An abasic site was introduced at the 3' G of the HRE. This element bound HIF-1 in the gel shift and in in-cell luciferase assays. hBVR was detected in the nucleus at 1, 2, and 4 h after hypoxia (1% O(2)), at which times its kinase and reductase activities were increased. Because hypoxia positively influences hBVR promoter and phosphorylation and TNF-alpha activated NF-kappaB inhibits the promoter, while biliverdin inhibits both NF-kappaB activity and hBVR promoter, we propose a regulatory mechanism for NF-kappaB by hypoxia and TNF-alpha centered on hBVR/biliverdin.

The range and diversity of functions of biliverdin reductase (BVR) is unmatched by any enzyme characterized to date. BVR is the sole catalyst for the conversion of biliverdin-IXα the activity product of the stress-inducible HO-1 and the constitutive HO-2, to bilirubin-IXα. Bilirubin is both cytoprotective and cytotoxic, quenches reactive oxygen species (ROS) and inhibits inflammatory and mitogen-induced ROS-mediated responses, and its elevated levels in the newborn adversely effects neuronal cells. Thus, BVR occupies a center stage in cellular defense mechanisms. As a dual specificity (serine/threonine/tyrosine) kinase the human (h) BVR influences transduction of extracellular stimuli to kinases downstream of the insulin/IGF-1(insulin-like growth factor-1)/MAPK/PI3-K signaling pathways. As a bZip-type transcription factor it binds to AP-1 (activator protein-1) and CRE (cAMP response element) sites and stimulates stress-inducible gene expression; as a scaffold protein, it is a platform for interaction of kinases; while acting as an intracellular shuttle, it transports regulatory factors to their target sites. hBVR promoter has consensus sequences with several regulatory elements. The gene is subject to negative and positive regulation, respectively, by TNF-α (tumor necrosis factor-α) and oxidative stress/hypoxia. Small human BVR-based peptides effectively duplicate polypeptide's activating influence on kinases, or mimic inhibitors of cell signaling. This, points to a realistic prospect of their use in clinical settings. The present review will briefly update cytoprotective activity and cytotoxicity of bile pigments and will focus on findings that link hBVR to cell signaling.