Peter E. M. Gibbs

Evidence for a second function for Saccharomyces cerevisiae Rev1p.

The function of the Saccharomyces cerevisiae REV1 gene is required for translesion replication and mutagenesis induced by a wide variety of DNA‐damaging agents. We showed previously that Rev1p possesses a deoxycytidyl transferase activity, which incorporates dCMP opposite abasic sites in the DNA template, and that dCMP insertion is the major event during bypass of an abasic site in vivo. However, we now find that Rev1p function is needed for the bypass of a T–T (6–4) UV photoproduct, a process in which dCMP incorporation occurs only very rarely, indicating that Rev1p possesses a second function. In addition, we find that Rev1p function is, as expected, required for bypass of an abasic site. However, replication past this lesion was also much reduced in the G‐193R rev1‐1 mutant, which we find retains substantial levels of deoxycytidyl transferase activity. This mutant is, therefore, presumably deficient principally in the second, at present poorly defined, function. The bypass of an abasic site and T–T (6–4) lesion also depended on REV3 function, but neither it nor REV1 was required for replication past the T–T dimer; bypass of this lesion presumably depends on another enzyme.

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

Activation of the MEK/ERK/Elk-signaling cascade is a mechanism for relaying mitogenic and stress stimuli for gene activation. MEK1 is the proximate kinase for activation of ERK1/2, and nuclear targeting of ERK1/2 is obligatory for Elk1 transcriptional activity. Human biliverdin reductase (hBVR) is a recently described Ser/Thr/Tyr kinase in the MAPK insulin/insulin-like growth factor 1 (IGF1)-signaling cascade. Using 293A cells and in vitro experiments, we detail the formation of a ternary complex of MEK/ERK/hBVR, activation of MEK1 and ERK1/2 kinase activities by hBVR, and phosphorylation of hBVR by ERK1/2. hBVR is nearly as effective as IGF1 in activating ERK; intact hBVR ATP-binding domain is necessary for Elk1 activation, whereas protein–protein interaction is the basis for hBVR activation of MEK1 and ERK. The two MAPK docking consensus sequences present in hBVR, F162GFP and K275KRILHCLGL (C- and D-box, respectively), are ERK interactive sites; interaction at each site is critical for ERK/Elk1 activation. Transfection with mutant hBVR-P165 or peptides corresponding to the C- or D-box blocked activation of ERK by IGF1. Transfection with D-box mutant hBVR prevented the activation of ERK by wild-type protein and dramatically decreased Elk1 transcriptional activity. hBVR is a nuclear transporter of ERK; experiments with hBVR nuclear export signal (NES) and nuclear localization signal (NLS) mutants demonstrated its critical role in the nuclear localization of IGF-stimulated ERK for Elk1 activation. These findings, together with observations that si-hBVR blocked activation of ERK and Elk1 by IGF1 and prevented formation of ternary complex between MEK/ERK/hBVR, define the critical role of hBVR in ERK signaling and nuclear functions of the kinase.

Biliverdin reductase is a transporter of haem into the nucleus and is essential for regulation of HO-1 gene expression by haematin.

hBVR (human biliverdin reductase) is an enzyme that reduces biliverdin (the product of haem oxygenases HO-1 and HO-2 activity) to the antioxidant bilirubin. It also functions as a kinase and as a transcription factor in the MAPK (mitogen-activated protein kinase) signalling cascade. Fluorescence correlation spectroscopy was used to investigate the mobility of hBVR in living cells and its function in the nuclear transport of haematin for induction of HO-1. In transiently transfected HeLa cells only kinase-competent hBVR translocates to the nucleus. A reduced mobility in the nucleus of haematin-treated cells suggests formation of an hBVR-haematin complex and its further association with large nuclear components. The binding of haematin is specific, with the formation of a 1:1 molar complex, and the C-terminal 7-residue fragment KYCCSRK(296) of hBVR contributes to the binding. The following data suggest formation of dynamic complexes of hBVR-haematin with chromatin: (i) the reduction of hBVR mobility in the presence of haematin is greater in heterochromatic regions than in euchromatic domains and (ii) hBVR mobility is not retarded by haematin in nuclear lysates that contain only soluble factors. Moreover, hBVR kinase activity is stimulated in the presence of double-stranded DNA fragments corresponding to HO-1 antioxidant and HREs (hypoxia response elements), as well as by haematin. Experiments with nuclear localization, export signal mutants and si-hBVR [siRNA (small interfering RNA) specific to hBVR] indicate that nuclear localization of hBVR is required for induction of HO-1 by haematin. Because gene regulation is energy-dependent and haematin regulates gene expression, our data suggest that hBVR functions as an essential component of the regulatory mechanisms for haem-responsive transcriptional activation.

Human Biliverdin Reductase, a Previously Unknown Activator of Protein Kinase C βII

Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) βII, activated by insulin and free radicals (Miralem, T., Hu, Z., Torno, M. D., Lelli, K. M., and Maines, M. D. (2005) J. Biol. Chem. 280, 17084–17092; Lerner-Marmarosh, N., Shen, J., Torno, M. D., Kravets, A., Hu, Z., and Maines, M. D. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 7109–7114). Here, by using 293A cells co-transfected with pcDNA3-hBVR and PKC βII plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC βII, but not the reductase and PKC ζ, transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC βII K371R mutant protein (“kinase-dead”) was also a substrate for hBVR. The reductase increased the Vmax but not the apparent Km values of PKC βII for myelin basic protein; activation was independent of phospholipids and extended to the phosphorylation of S2, a PKC-specific substrate. The increase in substrate phosphorylation was blocked by specific inhibitors of conventional PKCs and attenuated by sihBVR. The effect of the latter could be rescued by subsequent overexpression of hBVR. To a large extent, the activation was a function of the hBVR N-terminal chain of valines and intact ATP-binding site and the cysteine-rich C-terminal segment. The cobalt protoporphyrin-activated hBVR phosphorylated a threonine in a peptide corresponding to the Thr500 in the human PKC βII activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC βII nor PKC ζ activation loop-derived peptides were substrates. The phosphorylation of Thr500 was confirmed by immunoblotting of hBVR·PKC βII immunocomplex. The potential biological relevance of the hBVR activation of PKC βII was suggested by the finding that in cells transfected with the PKC βII, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC βII, as well as in untransfected cells, upon treatment with phorbol myristate acetate, the PKC translocated to the plasma membrane and co-localized with hBVR. hBVR activation of PKC βII underscores its potential function in propagation of signals relayed through PKCs.

Regulation of TNF-α-activated PKC-ζ signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase

Human biliverdin reductase (hBVR) is a dual function enzyme: a catalyst for bilirubin formation and a S/T/Y kinase that shares activators with protein kinase C (PKC) ‐ζ, including cytokines, insulin, and reactive oxygen species (ROS). Presently, we show that hBVR increases PKC‐ζ autophosphorylation, stimulation by TNF‐α, as well as cytokine stimulation of NF‐κB DNA binding and promoter activity. S149 in hBVR S/T kinase domain and S230 in YLS230F in hBVRs docking site for the SH2 domain of signaling proteins are phosphorylation targets of PKC‐ζ. Two hBVR‐based peptides, KRNRYLS230F (#1) and KKRILHC281 (#2), but not their S→AorC→A derivatives, respectively, blocked PKC‐ζ stimulation by TNF‐α and its membrane translocation. The C‐terminal‐based peptide KY‐CCSRK296 (#3), enhanced PKC‐ζ stimulation by TNF‐α; for this, Lys296 was essential. In metabolically 32P‐labeled HEK293 cells transfected with hBVR or PKC‐ζ, TNF‐α increased hBVR phosphorylation. TNF‐α did not stimulate PKC‐ζ in cells infected with small interfering RNA for hBVR or transfected with hBVR with a point mutation in the nucleotide‐binding loop (G17), S149,orS230; this was similar to the response of “kinase‐dead” PKC‐ζK281R. We suggest peptide #1 blocks PKC‐ζ‐docking site interaction, peptide #2 disrupts function of the PKC‐ζ C1 domain, and peptide #3 alters ATP presentation to the kinase. The findings are of potential significance for development of modulators of PKC‐ζ activity and cellular response to cyto‐kines.— Lerner‐Marmarosh, N., Miralem, T., Gibbs, P. E. M., Maines, M. D. Regulation of TNF‐α‐activated PKC‐ζ signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase. FASEB J. 21, 3949–3962 (2007)

The Molecular Clock Runs at Different Rates Among Closely Related Members of a Gene Family

Abstract. The serum albumin gene family is composed of four members that have arisen by a series of duplications from a common ancestor. From sequence differences between members of the gene family, we infer that a gene duplication some 580 Myr ago gave rise to the vitamin D–binding protein (DBP) gene and a second lineage, which reduplicated about 295 Myr ago to give the albumin (ALB) gene and a common precursor to α-fetoprotein (AFP) and α-albumin (ALF). This precursor itself duplicated about 250 Myr ago, giving rise to the youngest family members, AFP and ALF. It should be possible to correlate these dates with the phylogenetic distribution of members of the gene family among different species. All four genes are found in mammals, but AFP and ALF are not found in amphibia, which diverged from reptiles about 360 Myr ago, before the divergence of the AFP-ALF progenitor from albumin.Although individual family members display an approximate clock-like evolution, there are significant deviations—the rates of divergence for AFP differ by a factor of 7, the rates for ALB differ by a factor of 2.1. Since the progenitor of this gene family itself arose by triplication of a smaller gene, the rates of evolution of individual domains were also calculated and were shown to vary within and between family members. The great variation in the rates of the molecular clock raises questions concerning whether it can be used to infer evolutionary time from contemporary sequence differences.

The Saccharomyces cerevisiae rev6-1 Mutation, Which Inhibits Both the Lesion Bypass and the Recombination Mode of DNA Damage Tolerance, Is an Allele of POL30, Encoding Proliferating Cell Nuclear Antigen

The rev6-1 allele was isolated in a screen for mutants deficient for UV-induced reversion of the frameshift mutation his4-38. Preliminary testing showed that the rev6-1 mutant was substantially deficient for UV-induced reversion of arg4-17 and ilv1-92 and markedly UV sensitive. Unlike other REV genes, which encode DNA polymerases and an associated subunit, REV6 has been found to be identical to POL30, which encodes proliferating cell nuclear antigen (PCNA), the subunit of the homotrimeric sliding clamp, in which the rev6-1 mutation produces a G178S substitution. This substitution appears to abolish all DNA damage-tolerance activities normally carried out by the RAD6/RAD18 pathway, including translesion replication by DNA polymerase ζ/Rev1 and DNA polymerase η, and the error-free, recombination-dependent component of this pathway, but has little effect on the growth rate, suggesting that G178S may prevent ubiquitination of lysine 164 in PCNA. We also find that rev6-1 mutation can be fully complemented by a centromere-containing, low copy-number plasmid carrying POL30, despite the presumed occurrence in the mutant of sliding clamp assemblies that contain between one and three G178S PCNA monomers as well as the fully wild-type species.

Biliverdin Reductase: More than a Namesake – The Reductase, Its Peptide Fragments, and Biliverdin Regulate Activity of the Three Classes of Protein Kinase C

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.

The coordinated increased expression of biliverdin reductase and heme oxygenase-2 promotes cardiomyocyte survival: a reductase-based peptide counters β-adrenergic receptor ligand-mediated cardiac dysfunction

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/Ca2‐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 cyclohex‐imide, proteasome inhibitor MG‐132, and siBVR suggested BVR‐mediated stabilization of HO‐2. In both models, activation of BVR offered protection against the ligands stimulation of apoptosis. Two human BVR‐based peptides known to inhibit and activate the reduc‐tase, KKRILHC281 and KYCCSRK296, 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.—Ding, B., Gibbs, P. E. M., Brookes, P. S., Maines, M. D. The coordinated increased expression of biliverdin reductase and heme oxygenase‐2 promotes cardiomyo‐cyte survival; a reductase‐based peptide counters β‐ad‐renergic receptor ligand‐mediated cardiac dysfunction. FASEB J. 25, 301–313 (2011). www.fasebj.org

Control of feather keratin synthesis by the availability of keratin mRNA

Abstract The relative timing of the synthesis of keratin and its mRNA in the developing chick embryo feather has been examined. Study of both active mRNA in polysomes, and of the total number of mRNA sequences in the tissue, leads to the conclusion that the rate-limiting step in the synthesis of keratin is the accumulation in the cytoplasm of its mRNA.