Nicole Lerner-Marmarosh

Regulation of Epidermal Growth Factor-induced Connexin 43 Gap Junction Communication by Big Mitogen-activated Protein Kinase 1/ERK5 but Not ERK1/2 Kinase Activation

The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5α, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.

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)

Bcr Kinase Activation by Angiotensin II Inhibits Peroxisome Proliferator-Activated Receptor γ Transcriptional Activity in Vascular Smooth Muscle Cells

Bcr is a serine/threonine kinase activated by platelet-derived growth factor that is highly expressed in the neointima after vascular injury. Here, we demonstrate that Bcr is an important mediator of angiotensin (Ang) II and platelet-derived growth factor–mediated inflammatory responses in vascular smooth muscle cells (VSMCs). Among transcription factors that might regulate Ang II–mediated inflammatory responses we found that ligand-mediated peroxisome proliferator-activated receptor (PPAR)&ggr; transcriptional activity was significantly decreased by Ang II. Ang II increased Bcr expression and kinase activity. Overexpression of Bcr significantly inhibited PPAR&ggr; activity. In contrast, knockdown of Bcr using Bcr small interfering RNA and a dominant-negative form of Bcr (DN-Bcr) reversed Ang II–mediated inhibition of PPAR&ggr; activity significantly, suggesting the critical role of Bcr in Ang II–mediated inhibition of PPAR&ggr; activity. Point-mutation and in vitro kinase analyses showed that PPAR&ggr; was phosphorylated by Bcr at serine 82. Overexpression of wild-type Bcr kinase did not inhibit ligand-mediated PPAR&ggr;1 S82A mutant transcriptional activity, indicating that Bcr regulates PPAR&ggr; activity via S82 phosphorylation. DN-Bcr and Bcr small interfering RNA inhibited Ang II–mediated nuclear factor &kgr;B activation in VSMCs. DN-PPAR&ggr; reversed DN-Bcr–mediated inhibition of nuclear factor &kgr;B activation, suggesting that PPAR&ggr; is downstream from Bcr. Intimal proliferation in low-flow carotid arteries was decreased in Bcr knockout mice compared with wild-type mice, suggesting the critical role of Bcr kinase in VSMC proliferation in vivo, at least in part, via regulating PPAR&ggr;/nuclear factor &kgr;B transcriptional activity.

Role of p90 Ribosomal S6 Kinase–Mediated Prorenin-Converting Enzyme in Ischemic and Diabetic Myocardium

Background— Epidemiological data strongly indicate that diabetes increases the incidence of heart failure. Although the benefit of angiotensin-converting enzyme inhibitor (ACE-I) treatment during and after myocardial infarction has been found to be greater in diabetics than nondiabetics and activation of the renin-angiotensin system (RAS) has been implicated, the molecular basis of these actions remains unclear. Methods and Results— We generated transgenic mice with cardiac-specific overexpression of wild-type p90 ribosomal S6 kinase (WT-p90RSK-Tg) and a dominant-negative form of p90RSK (DN-p90RSK-Tg). Recovery of cardiac function after ischemia/reperfusion in WT-p90RSK-Tg isolated mouse hearts was significantly impaired. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed specific induction of prorenin-converting enzyme (PRECE) in WT-p90RSK-Tg mice. mRNA induction of PRECE was confirmed with serial angiotensinogen protein reduction after perfusion in WT-p90RSK-Tg mice, suggesting an increase of angiotensinogen cleavage and subsequent RAS activation in WT-p90RSK-Tg mice. We investigated the role of the RAS in WT-p90RSK-Tg animals after ischemia/reperfusion with the use of an ACE-I (captopril) and an angiotensin II type 1 receptor blocker (olmesartan). We did not observe any effect of these inhibitors in non-Tg littermate controls, thus corroborating other reports in rodents. In contrast, both captopril and olmesartan significantly improved cardiac function and reduced infarct size in WT-p90RSK-Tg mice. At 8 months of age, WT-p90RSK-Tg mice developed cardiac dysfunction. p90RSK activity and PRECE mRNA were both increased by streptozotocin-induced hyperglycemia in non-Tg littermate controls, whereas DN-p90RSK-Tg animals exposed to streptozotocin did not have PRECE induction. Conclusions— This study demonstrates the critical role of p90RSK in hyperglycemia-mediated myocardial PRECE induction, which may explain the augmentation of the RAS in diabetic hearts and provide an alternative therapeutic approach to treat diabetic cardiomyopathy.

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).

Background: ERK2 activation by PKCδ relays cell growth signals. hBVR is a bridge/scaffold protein and nuclear transporter of ERK. Results: hBVR forms a ternary complex with PKCδ and ERK2; this requires specific hBVR sequences. Corresponding peptides inhibit PKCδ/ERK2 interaction. PKCδ/ERK-mediated transcriptional activation is hBVR-dependent. Conclusion: hBVR is essential for ERK2 activation by PKCδ and MEK1/2. Significance: hBVR-based peptides are useful in regulating PKCδ/ERK signaling. 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 (Phe660, Phe663, and Phe665) 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.

The novel role of the C-terminal region of SHP-2. Involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation.

SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198–593 (SHP-2-(198–593)), but not the smaller SHP-2-(399–593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198–593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399–593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198–593) mutant did not associate with Gab1. SHP-2-(1–202) or SHP-2-(198–593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399–593) increased Elk-1 activation. Co-expression of SHP-2-(1–202) with SHP-2-(399–593) inhibited SHP-2-(399–593)/Gab1 interaction, and the SHP-2-(399–593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.

Structure–activity relationships for nicotine analogs comparing competition for [3H]nicotine binding and psychotropic potency†

The structure–activity relationships were established for nicotine analogs and related agents comparing their competition for [3H]nicotine binding to rat brain membranes, Torpedo membranes, and transfected insect cells with their ability to produce prostration in rats following administration into the rat lateral ventricles. A total of 59 compounds were investigated, consisting of pyridine‐ and pyrrolidine‐substituted analogs of nicotine, other tobacco alkaloids and related molecules, various aminoalkylpyridines, and ring‐shifted analogs of nicotine. Some of the compounds were also evaluated for [3H]nicotine binding to Torpedo electroplax membranes and to Sf9 cells expressing an α4β2 nicotinic cholinergic receptor subtype. Linear regression plots of Ki vs ED50 values for prostration of the various classes of the compounds yielded correlation of determination (R2) of 0.923 for the pyridine‐substituted analogs, 0.725 for the pyrrolidine‐substituted analogs, 0.947 for other tobacco alkaloids and related compounds, and 0.537 for all 59 compounds. An excellent correlation was observed comparing Torpedo Ki values with both prostration ED50 values and rat brain Ki values. Within the pyridine‐substituted series, methyl substiuents in the 6‐position resulted in over three‐fold greater potency compared to nicotine, whereas potency decreased markedly with bulkier alkyl and cycloalkyl substituents. Within the pyrrolidine‐substituted series, methyl groups in the 2′‐position only slightly reduced potency compared to nicotine, whereas 3′‐ and 5′‐addition markedly reduces potency. A linear regression plot of the Ki values of brain vs. those of Sf9 cells expressing the α4β2 nicotinic cholinergic receptor subtype yielded a coefficient of correlation of 0.981; a finding which is consistent with the notion that the α4β2 subtype comprises over 90% of total rat brain receptor. Drug Dev. Res. 45:10–16, 1998.