humanbiliverdinreductase,apreviouslyunknownactivator ... · but not all 12. the bvr kinase motifs...

Human Biliverdin Reductase, a Previously Unknown Activatorof Protein Kinase C �II*

Received for publication, December 16, 2005, and in revised form, November 20, 2006 Published, JBC Papers in Press, January 16, 2007, DOI 10.1074/jbc.M513427200

Mahin D. Maines1, Tihomir Miralem2, Nicole Lerner-Marmarosh2, Jenny Shen, and Peter E. M. GibbsFrom the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14624

Human biliverdin reductase (hBVR), a dual specificity kinase(Ser/Thr/Tyr) is, as protein kinase C (PKC) �II, activated byinsulin 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 cellsco-transfected with pcDNA3-hBVR and PKC �II plasmids, wereport the co-immunoprecipitation of the proteins and co-puri-fication in the glutathione S-transferase (GST) pulldown assay.hBVR andPKC�II, but not the reductase andPKC �, transphos-phorylated in assay systems supportive of activity of only one ofthe kinases. PKC�II K371Rmutant protein (“kinase-dead”)wasalso a substrate for hBVR. The reductase increased theVmax butnot the apparent Km values of PKC �II for myelin basic protein;activation was independent of phospholipids and extended tothe phosphorylation of S2, a PKC-specific substrate. Theincrease in substrate phosphorylation was blocked by specificinhibitors of conventional PKCs and attenuated by sihBVR. Theeffect of the latter could be rescued by subsequent overexpres-sion of hBVR. To a large extent, the activation was a function ofthe hBVR N-terminal chain of valines and intact ATP-bindingsite and the cysteine-rich C-terminal segment. The cobalt pro-toporphyrin-activated hBVR phosphorylated a threonine in apeptide corresponding to the Thr500 in the humanPKC�II acti-vation loop. Neither serine nor threonine residues in peptidescorresponding toother phosphorylation sites of thePKC�II norPKC � activation loop-derived peptides were substrates. Thephosphorylation of Thr500was confirmed by immunoblotting ofhBVR�PKC �II immunocomplex. The potential biological rele-vance of the hBVR activation of PKC �II was suggested by thefinding that in cells transfected with the PKC �II, hBVR aug-mented phorbol myristate acetate-mediated c-fos expression,and infection with sihBVR attenuated the response. Also, incells overexpressing hBVR and PKC �II, as well as in untrans-fected cells, upon treatment with phorbol myristate acetate, thePKC translocated to the plasma membrane and co-localizedwith hBVR. hBVR activation of PKC �II underscores its poten-tial function in propagation of signals relayed through PKCs.

Biliverdin reductase catalyzes the final step in the hememet-abolic pathway, the reduction of biliverdin IX� to bilirubin.The enzyme remains unique among all biological catalystsdescribed to date in having a dual pH/cofactor-dependentactivity profile (3). The protein displays microheterogeneitybecause of post-translational phosphorylation that is requiredfor its activity (4, 5). Free radical generators such as H2O2 andNa2AsO3, as well as insulin and the metalloporphyrin Co-PP,3activate BVR and increase its phosphorylation (1, 2, 4, 6).Reduction of biliverdin IX� to lipophilic bilirubin serves severalfunctions that include trafficking of the heme degradationproduct through the cell membrane, inactivation of a potentkinase inhibitor, biliverdin, and formation of bilirubin, aneffective quencher of free radicals (7, 8). BVR is presentacross metazoan species, and its homologue is found in uni-cellular cyanobacteria (9–11). Plants use biliverdin IX� pro-duced by ferredoxin-dependent heme oxygenase (HO) tosynthesize phytochromes, the sensory photoreceptors (9, 12).BVR is a small soluble protein (296 residues) foundmainly in

the cytoplasm. If activated, the BVR leads to nuclear transloca-tion and association with the nucleolus (13). Notably, smallproteins, similar in molecular weight to hBVR, have beenshown to bind to and activate PKCs (14). TheN terminus of thereductase is composed of hydrophobic and charged residues,which include a chain of four valines flanking the consensusWalker A homology ATP-binding motif, GXGXXG (15, 16),and a notable degree of sequence similarity to IRK and IRS (2).In this domain is the AQELWE sequence (amino acids 107–112) that shares identity of sequence and composition with theconserved six-residue RACK1 sequence in PKC �, SVEIWD(pseudo-RACK), and PKC pseudosubstrateAVEIWD. Trypto-phan at position 5 and the negatively charged residue at posi-tion 3 characterize these sequences (14, 17). A syntheticpseudo-RACK1 has an effect on PKC �, activating the kinase inthe absence of activators by inducing structural changes in theprotein to expose the catalytic site (14, 17).Traditionally, the presence of 12 motifs characterizes a pro-

tein as a kinase (15); the primary structure of hBVR predicts itssharing several of those motifs, including the aforementioned,

* This work was supported by Grants ES12187 and ES004066 from theNational Institutes of Health. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

1 To whom correspondence should be addressed. Tel.: 585-275-5383; Fax:585-275-6007; E-mail: [email protected].

2 Both authors contributed equally to this work.

3 The abbreviations used are: Co-PP, cobalt protoporphyrin; ATF-2, activatingtranscription factor-2; CREB, cAMP-regulatory element-binding protein;DAG, diacylglycerol; GST, glutathione S-transferase; IRK, insulin receptorkinase; IRS, insulin receptor substrate; HO, heme oxygenase; hBVR, humanbiliverdin reductase; MAPK, mitogen-activated protein kinase; MBP, mye-lin basic protein; PMA, phorbol myristate acetate; PKCi, PKC inhibiting pep-tide; PS, phosphatidylserine; RACK, receptor for activated C-kinase; siRNA,small interfering RNA; BVR, biliverdin reductase; WT, wild type; LDH, lactatedehydrogenase; PBS, phosphate-buffered saline; sihBVR, small interfer-ence RNA for hBVR.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 11, pp. 8110 –8122, March 16, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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but not all 12. The BVR kinase motifs are conserved amongmammalian species (2, 4, 10, 18, 19). Notably, not all kinaseshave a complete set of 12 motifs. Recently, a number of non-conventional protein kinases have been identified. For instance,the Goodpasture antigen-binding protein has only a modifiedWalker A domain and no other motifs, but nonetheless hasserine/threonine kinase activity (20). Similarly, the catalyticdomain of myosin heavy chain kinase A does not resemble thecatalytic domains of protein kinases (21).The carboxyl domain of hBVR, as predicted by the crystal

structures of rat BVR, consists of six strands that form a large�-sheet, an ideal interface for protein-protein interaction (22).The predicted basic leucine zipper protein domain, which linksthe two termini of the protein, has been shown to bind to theconsensus sequences of AP-1 (activator protein-1) and ATF-2/CREB-binding sites (1, 23, 24).The human enzyme, in strictly Mn2�-dependent assay con-

ditions, phosphorylates known serine/threonine and tyrosinekinase substrates e.g.MBP, casein, IRS-1 (insulin receptor sub-strate), and Raytide (a PTK substrate). In addition, hBVR auto-phosphorylates several serines, at least one threonine, and twoof its six tyrosine residues (2, 4). The remaining four tyrosineresidues are phosphorylated by the insulin receptor kinase(IRK). This includes Tyr198 in the YMXMmotif that, in insulinreceptor interactive proteins, is the binding site for proteinswith Src homology domain, such as phosphatidylinositol 3-ki-nase (25). hBVR tyrosine kinase activity was demonstrated bythe recombinant human protein expressed in Escherichia coli.Notably the E. coli genome does not encode protein-tyrosinekinases, which are a multigenic family of Mn2�-dependentkinases exclusive to higher organisms (15). Although there ismuch information available on identification of IRK function inhBVR tyrosine phosphorylation, to date there is no informationavailable on the identity of the serine/threonine kinase thatphosphorylates hBVR.The MAPK and IRS/phosphatidylinositol 3-kinases are con-

sidered the major arms of the insulin/insulin-like growth fac-tor-1 signaling pathway; signaling through the two arms is“linked” by the family of PKC isozymes that includes PKC �II,classified as a conventional PKC. hBVR prominently figures inoxidative stress response of the cell by its being amember of thebasic leucine zipper protein family of transcription factors thatregulate expression of stress-responsive genes, such as ho-1,ATF-2/CREB, and c-jun in theMAPK signaling pathway (1, 23,24). PKC enzymes are activated by oxidative stress, insulin, andgrowth factors (26–30). PKC� isozymes (I and II) stimulate celldivision and differentiation by regulating the expression of sev-eral oncogenes, including c-fos (31). Spatial localization withinthe cell is a component of the biological function of many pro-tein kinases, including the PKC enzymes whose catalytic com-petence and localization are regulated by serine/threoninephosphorylation (14, 32, 33). In the case of PKC �II, when acti-vated, the kinase translocates to the plasmamembrane from thecytoplasm(34).This kinase is amemberof theMg2�- andCa2�-dependent, phospholipid/phorbol ester-activated family of theconventional PKCs.Presently, we have identified hBVR as a substrate for PKC�II

kinase activity. In the course of the study, the reciprocal phos-

phorylation and activation of PKC �II by hBVRwas uncovered.Collectively, the present findings and past reports define hBVRnot only as an enzyme with a unique activity profile but also asone with the possibility of having input at multiple stages in cellsignaling pathways.

EXPERIMENTAL PROCEDURES

Materials and Constructs—Recombinant human PKC �II waspurchased from Calbiochem. [�-32P]ATP and [�-32P]dCTPwere from PerkinElmer Life Sciences. Monoclonal and poly-clonal anti-PKC �II antibodies were from Zymed LaboratoriesInc. and Abgent (San Diego, CA), respectively. Polyclonal anti-phospho-Thr500 PKC �II antibodies were from Abcam (Cam-bridge,MA). Biotrace polyvinylidene difluoridemembrane wasa product of Pall Science Corp. (Pensacola, FL). PKC �II-basedpeptides referred to as Thr500 (MCKENIWDGVTTKTFCG),Thr500mut (MAKENIWDGVTTKAFAG), Thr641 (VLTPP-DQEVIRNIDQ), Ser661 (FEGFSFVNSEFLKPEVKS), and thecontrol peptide Smut (FEGFAFVNAEFLKPEVKA) were syn-thesized byAnaspec Inc. (San Jose, CA). PKC-�-based peptides,PKC�281 (DQIYAMKVVKKE), PKC�410 (GDTTSTF-CGTPN), PKC�560 (EPVQLTPDDEDA), and PKC�585(EFEGFEYINPLLL), were custom-synthesized by Synpep(Dublin, CA). The numbered residues in PKC�II are detrimen-tal to its kinase activity (35). The mutant constructs, G17A andV11A/V12A/V13A/V14A, were generated by site-directedmutagenesis of the wild-type hBVR cDNA (19) and used tocreate expression plasmids in pcDNA3 (used for transfectioninto 293A cells) and pGEX4-T2 (used for transfection intoE. coli) hBVR. Expression plasmids for PKC�II and PKC �wereconstructed by subcloning cDNA from pSP65-PKC �II orpCO2-PKC � (generous gift from Peter Parker, LondonResearch Institute, London, UK) into pcDNA3 and pGEX4-T2.Site-directedmutagenesis of the pGEX4-T2 constructwas usedto replace Lys371 in the ATP-binding site with arginine toexpress a “kinase-dead” protein (36, 37). hBVR truncations1–108, 109–175, 272–296, 272–296 C/A, and 272–296 Y/Fwere constructed from existing pGEX4-T2-hBVR by appro-priate deletion and site-directed mutagenesis of WT-hBVR.pSuper-Retro-siBVR was constructed as described previously(1). The primers 5�-GATCCCCTCCTCAGTCCGTTCGAAC-CTGTTCAAGAGACAGGTTGCTGCAACGGACTGAGG-ATTTTTGGAAA-3�, and 5�-AGCTTTTCCAAAAATCCTC-AGTCCGTTCGAACCTGTCTCTTGAACAGGTTGCAAC-GGACTGAGGAGGG-3� were designed as a scrambled formof the hBVR siRNAandwere used tomake the siBVR-sc controlconstruct. The PKC-specific substrate S2 (VRKRTLRRL) waspurchased from Anaspec Inc. The PKC �-specific inhibitorLY333531 was obtained from A.G. Scientific, Inc. (San Diego);and the inhibitor of conventional PKCs, Go-6976, was fromCalbiochem. PKCi, MBP, and PKC lipid activator (PS: DAG)were purchased fromUpstate (Charlottesville, VA). Co-PP wasobtained from Porphyrin Products Inc. (Logan, UT).Cell Culture, Transfection, Co-immunoprecipitation, and

GST Pulldown—293A cells were grown in Dulbecco’s modifiedEagle’smedium (Invitrogen) containing 10% fetal bovine serumand 1% penicillin G/streptomycin for 24 h or until the cellsreached 70% confluency. Depending on the experiment, cells

Transphosphorylation of hBVR and PKC �II

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were subsequently transfected with up to 5 �g of pcDNA3-hBVRor pcDNA3-PKC �II plasmid using transfectin lipid reagent (Bio-Rad) in 10-cm plates, according to the manufacturer’s instruc-tions. Western blotting confirmed overexpression of hBVR orPKC �II. To prepare hBVR siRNA or siBVR-sc, pSuper-Retro-siBVR or siBVR-sc was transfected into 293A cells for packaging,and the siBVR or siBVR-sc retrovirus was then titrated usingNIH3T3 cells. 293A cells were infected with 4 plaque-formingunits/cell to inhibit hBVR synthesis (1). For the protein-proteininteraction experiments, cells were seeded into 10-cm dishes andco-transfected with bothWTpcDNA3-hBVR and pcDNA3-PKC�II for co-immunoprecipitation, whereas for GST pulldownpcDNA3-PKC�II andpEGFP-HO2wereused. Prior to treatmentwith 100 nM PMA, the cells were serum-starved in growthmedium containing 0.1% fetal bovine serum for 24 h and thenlysed in RIPA buffer.For immunoprecipitation experiments, cell lysate (500 �g of

protein) was incubatedwithmonoclonal anti-PKC�II antibod-ies or normal mouse serum overnight at 4 °C. A/G-agarosebeads (Santa Cruz Biotechnology, Santa Cruz, CA) were addedto select antibody-bound protein. The agarose beads werewashed three times in lysis buffer, and the samples were boiledin Laemmli gel loading buffer, separated by SDS-PAGE, anddetected by Western blotting using rabbit polyclonal anti-hBVR antibodies. For the GST pulldown assay, cell lysate wasincubated with 10 �g of GST-hBVR fusion protein or GSTimmobilized on GSH-agarose beads (Amersham Biosciences)at 4 °C for 2 h. The beads were washed three times and boiled inLaemmli buffer to release the boundproteins. Resolved by SDS-PAGE, proteins were detected by immunoblotting usingmousemonoclonal anti-PKC �II antibodies.Cell Fractionation—After a cold wash in PBS, cells were

scraped and collected by centrifuging at 500 � g for 5 min.Intact cell pellets were resuspended in homogenizationmedium containing 0.28 M sucrose, 50 mM Tris-HCl (pH 7.5),25 mM KCl, 5 mM MgCl2, 1 mM EDTA and 1 �g/ml each of theprotease inhibitors leupeptin, pepstatin, and aprotinin and 1mM phenylmethylsulfonyl fluoride and homogenized by pass-ing five times through a 25-gauge needle. Cell homogenateswere centrifuged for 25 min at 120 � g at 4 °C to remove largecellular debris (most nuclei and larger), and collected superna-tants were centrifuged 15 min at 13,000 � g, 4 °C, to pelletmitochondria. Collected supernatants were centrifuged at25,000� g, 4 °C, for 60min to separate plasmamembrane (pel-let) from cytoplasm (supernatant). Both fractions were col-lected, dissolved inRIPAbuffer�1%TritonX-100 (membrane)or RIPA buffer (cytoplasm), processed for protein determina-tion, and stored at �20 °C for further examination. To test thepurity of extracted cell fractions, aliquots of cell fractions wereexamined for LDH activity as described earlier (3).PKC �II Activity in Vitro—PKC �II kinase activity was

assayed in vitro as recommended by the manufacturer (Calbio-chem). MBP was used as the substrate, as it is commonly usedfor PKC isozymes and for BVR serine/threonine kinase activity(4). Phosphorylation of MBP can be detected by SDS-PAGE orby trapping on P81 phosphocellulose filters. The design of theassay system was modified, depending on whether hBVR orPKC �II was used as the enzyme or substrate. Unless otherwise

specified, for PKC �II activity 5 ng of PKC �II was incubated ina 50-�l assay containing 20mMHEPES (pH 7.2), 15mMMgCl2,0.2 mMCaCl2, 12.5 �MMBP, 10mM �-glycerophosphate, and 1mM dithiothreitol in the presence of a sonicated lipid activatorat final concentrations of 0.05mg/ml PS and 0.005mg/mlDAG,or PS alone. The addition of 100 �M ATP containing 5 �Ci of[�-32P]ATP initiated the reaction. To examine the effect ofWTormutant hBVRonPKC�II activity, theywere incubated for 10minwith PKCprior to addition ofMBP. IfWTormutant hBVRwas used as substrate, MBP was omitted. If used, the inhibitorPKCi was added to PKC �II 2 min prior to hBVR orMBP addi-tion. The incubation lasted for 10 min at 30 °C when MBP wasthe substrate and 20minwith hBVRas the sole substrate, unlessotherwise stated. The reaction was terminated on ice, either bythe addition of Laemmli buffer for SDS-PAGE followed bytransfer to polyvinylidene difluoride membrane and autora-diography, or by the addition of 1 volume of 10% phosphoricacid for the P81 phosphocellulose binding assay. An aliquot ofthe samples was directly applied to the center of the filter,washed six times in 0.75% phosphoric acid, and dried with ace-tone prior to measurement for radioactivity.PKC Assay in Situ—The assay was performed by a modifica-

tion of procedures detailed by Williams and Schrier (38). Cellswere seeded into 48-well plates and transfected with 0.5�g/well pcDNA3-hBVR or with G17A or V11A/V12A/V13A/V14A mutants. 24 h later, the medium was replaced with star-vation medium (0.1% serum), and the incubation was contin-ued for another 24 h to synchronize cells. In some cases, cellswere pretreated with the PKC inhibitors Go-6976 (200 nM) forconventional PKCs or LY333531 (30 nM) for PKC� (39–41) for30 min before addition of PMA (100 nM, 15 min). Cells werewashedwithmedium and incubated for 10min at 30 °C in 50�lof kinase assay buffer (137mMNaCl, 5.4mMKCl, 10mMMgCl2,0.3 mM Na2HPO4, 0.4 mM KH2PO4, 25 mM �-glycerophos-phate, 5.5 mM D-glucose, 5 mM EGTA, 1 mM CaCl2, 20 mM

HEPES (pH 7.2), 50 �g/ml digitonin, 120 �g/ml S2 substrate,and 100 �M ATP labeled with 10 �Ci/ml [�-32P]ATP). Thereaction stopped with the addition of 25 �l of ice-cold 30%(w/v) trichloroacetic acid on ice. The trichloroacetic acid-solu-ble fraction samples were transferred to P81 phosphocellulosefilters. After 15 min at room temperature, the filters werewashed three times in 75 mM phosphoric acid, once in 2.75 mM

sodium phosphate (pH 7.5), and once with acetone before liq-uid scintillation counting. Kinase activity was normalized toprotein content.hBVR Kinase Activity—The activity was measured as

described recently (2). For routine assays, purified hBVR, atconcentrations noted in the appropriate figure legends, wasincubated at 30 °C in a 50-�l reaction mixture containing 50mM HEPES (pH 8.4), 30 mM MnCl2, 0.2 mM dithiothreitol, 10�M ATP labeled with 10 �Ci of [�-32P]ATP, and substrate; theconcentration and duration of incubation of the substrate arespecified in the appropriate figure legends. Incorporation of 32Pwas detected either by autoradiography or by the P81Whatmanfilter method, as described above for PKC �II activity in vitro.To re-establish the Mn2� dependence of the hBVR kinaseactivity, both Mn2� and Mg2� were tested in autophosphoryl-




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ation reactions; to examine kinase activity of hBVR under PKCassay conditions, 10 mM concentration of Mg2� was used.Northern and Western Blot Analyses—For Northern blot

analysis, RNA was extracted with the RNeasy kit (Qiagen,Valencia, CA) from 293A cells treated with PMA. RNA wasseparated by electrophoresis on agarose gels containing form-aldehyde and transferred to Hybond-N� membrane (Amer-sham Biosciences). The membranes were probed with full-length hBVR cDNA, full-length c-fos cDNA (generous gift fromD. Templeton, University of Toronto, Canada (42)) or a 1.1-kbfragment of human �-actin cDNA. Probes were labeled using[�-32P]dCTP and a random primer labeling system (Invitro-gen). Pre-hybridization, hybridization, and autoradiographywere performed as described previously (43). For Western blotanalysis, proteins were resolved by SDS-PAGE, transferredto nitrocellulose membrane, probed with either anti-hBVRor anti-PKC �II antibodies, and visualized by enhancedchemiluminescence.Confocal Microscopy—These experiments were based on

those of Edwards et al. (44). 293A cells were grown in a chamberslide system (Nalge Nunc International Corp., Naperville, IL).Cells were transfectedwith either pcDNA3-PKC�II or pEGFP-hBVR, or both. After a 24-h starvation period, cells were fixedfor 10min in 3.7% formaldehyde in PBS and permeabilizedwith1% Triton X-100 in PBS. After washing three times with ice-cold PBS, cells were pretreated with 3% bovine serum albuminin PBS for 2 h followed by overnight incubation with a 1:150dilution of polyclonal anti-PKC �II antibodies. PBS-washedcells were then treated with Rhodamine Red-conjugated don-key anti-rabbit IgG antibodies (Jackson ImmunoResearch,West Grove, PA) for 30 min and washed before being used forimage analysis by confocal microscopy. If nontransfected cellswere permeabilized as above, they were treated with mono-clonal anti-PKC �II antibodies, washed, blocked, and thentreated with polyclonal anti-hBVR antibodies. After washingaway the excess primary antibodies, the cells were treated withfluorescein isothiocyanate-conjugated goat anti-mouse IgG(Jackson ImmunoResearch) and Rhodamine Red-conjugateddonkey anti-rabbit IgG antibodies, followedby a treatmentwith2 �MTO-PRO-3 (Molecular Probes, Eugene, OR) for 10min inorder to visualize the nuclei. A Leica TCS SP, model DMRE,confocal microscope was used.

RESULTS

hBVR Binds to PKC �II, Increases Its Phosphorylation, and Isa Substrate for the Kinase—Physical interaction between hBVRand PKC�II was examined using immunoprecipitation andGST pulldown approaches. Data shown in Fig. 1, a and b, dem-onstrate binding of the two proteins in cells co-transfectedwithpcDNA3 expression constructs for hBVR and PKC �II. 24 hafter transfection, cells were starved (0.1% serum) and eithertreated with PMA (15 min) or left untreated. For the immuno-precipitation experiment, the cell lysate was incubated witheither antibody to PKC �II normal mouse serum. The immu-noprecipitate was subjected to Western blot analysis andprobed with polyclonal antibodies to hBVR. As shown, hBVRwas found in the anti-PKC �II immunoprecipitates obtainedfrom both PMA-treated and untreated cells, but it was not

found in control IgG. Binding of the two proteins was con-firmed using a GST pulldown assay (Fig. 1b). In this experi-ment, the cell lysate, obtained 24 h after transfection with PKC�II, was incubated with GST-hBVR fusion protein and immo-bilized on GSH-agarose beads. Bound proteins were eluted andsubjected toWestern blot analysis usingmonoclonal antibodiesto PKC �II. PKC �II has a better affinity for hBVR in PMA-treated cells. To assess the specificity of hBVR binding, PKC �(72 kDa), a protein of similarmolecular weight, size, and chargeto PKC � II was ectopically expressed in 293 cells, and its bind-ing to hBVR after treatment with PMAwas tested. As shown inFig. 1c, GST-hBVR was not able to pull down PKC � from celllysates. Additionally, binding of HO-2 (36 kDa), a protein ofsimilar size to hBVR, was tested. This protein was also notrecovered from cell lysates that are mixed with GST-hBVR.These findings suggest that BVR is discriminating in its associ-ation with other proteins.Next, the possibility of transphosphorylation of the kinases

was examined. This examination required differentiationbetween contribution of the two kinases to phosphotransferactivity. hBVR differs frommost serine/threonine kinases in itsstrict requirement for Mn2�, whereas Mg2� and/or Ca2� areoften required for activity of this class of kinases, includingPKCs (15, 16, 45). Also, the pH optimum for hBVR and PKCkinase activities differs (8.0–8.4 and 7.0–7.2, respectively).Data in Fig. 1e confirm the previously reported Mn2� require-ment of hBVR and show this property can be used to differen-tiate hBVR kinase activity from that of PKC �II.First, whether hBVR is a substrate for PKC �II was examined

under optimum PKC assay conditions. As shown in Fig. 1f,there was a time-dependent increase in hBVRphosphorylation.Next, the ability of hBVR to phosphorylate PKC �II was exam-ined. Surprisingly, in the presence of hBVR and under hBVRkinase assay conditions, a striking increase in PKC �II phos-phorylation was observed, whereas in the absence of hBVR,there was a very modest phosphorylation of PKC �II detected(Fig. 1g). In addition, hBVR autophosphorylation showed aremarkable increase in the presence of PKC �II, which sug-gested the possibility that hBVR was an activator of PKC �II.A series of studies described below define hBVR as a PKCactivator.It was essential to substantiate that the observed increase in

phosphorylation of PKC �II was related to hBVR activity orreflected autophosphorylation by the kinase. Therefore, amutation was introduced into a key lysine residue that resideswithin the ATP-binding site of PKC �II. The K371R replace-ment reportedly leads to complete loss of both autophospho-rylation and substrate phosphorylation (37). A similar phenom-enon was observed when Lys368 was mutated to Arg in PKC �(36).The mutant PKC �II K371R was constructed, expressed,

purified, and used as the substrate in the hBVR kinase assaysystem. As shown in Fig. 1h, the ability of the PKC �II mutantprotein to autophosphorylate, in the absence of hBVR andunder hBVR kinase assay conditions, was minimal. However,the protein shows a prominent level of phosphorylation in thepresence of the reductase. To confirm this observation, thetime course of phosphorylation of the PKC �II-K371R mutant




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by hBVR was examined. The kinase-dead PKC �II was phos-phorylated within 5 min of incubation at 30 °C. This graduallyincreased in a time-dependent manner (Fig. 1i). These obser-vations suggest that phosphorylation of PKC �II mutant pro-tein is a consequence of the kinase activity of hBVR. Increasedphosphorylation of PKC �II is linked to stimulation of itsenzyme activity (46, 47). Proteins canmodulate PKC activity bydirect interaction with the kinase, leading to the conforma-tional change in the latter (48). Because PKC �II autophospho-rylation was increased in the presence of hBVR, under condi-tions where hBVR is not an effective kinase (Fig. 1f), thereappeared to be two components to hBVR activation of the PKC,with the second one to involve protein-protein interaction, anda change in conformation of the PKC. Protein-protein interac-tion also appears to be involved in hBVR activation by PKC �II.Finding that phosphorylation of hBVR was increased in thepresence of the kinase-dead PKC �II indicates this interactionand raises the possibility that protein-protein interactionresults in a change in conformation of the reductase to an acti-vated form. Kinase activation through change in conformationis not limited to PKCs but has been reported for others. Also, aninactive kinase can, through protein-protein interaction, influ-ence the response of the interacting kinase. For instance, theinactive (kinase-dead) PDK1 mutant permits normal proteinkinase B activation by insulin (49). The phosphorylation of PKC�II by hBVR is specific, because PKC � in the same kinase assayshowed little to no phosphorylation (Fig. 1j). These observa-tions suggest that the interaction between hBVR and PKC�II isspecific and that phosphorylation of the PKC �II mutant pro-tein is directly related to the hBVR kinase activity.Characterization of hBVR-mediated Augmentation of PKC

�II Activity—The following experiment was conducted toexamine whether increased phosphorylation of PKC �IIaffected its activity to transfer phosphate to a second phos-phoacceptor substrate. The substrate-dependent analysis of theinfluence of hBVR on PKC �II phosphotransfer activity isshown in Fig. 2a. In this experiment, constant amounts of eitherPKC �II alone or PKC �II together with hBVR were incubatedin the PKC assay system with increasing amounts of the MBP.The incorporation of [32P] was analyzed by the Michaelis-Menten nonlinear regression equation. Both experimental con-

FIGURE 1. Binding and transphosphorylation of hBVR and PKC �II.a, hBVR and PKC �II co-immunoprecipitate. 293A cells were co-transfectedwith pcDNA3-hBVR and pcDNA3-PKC �II and starved for 24 h. Thereafter, cellswere treated with 100 nM PMA and subsequently lysed as described under“Experimental Procedures.” Cell lysates were incubated with either mono-clonal anti-PKC �II antibodies (lanes 1 and 2) or with normal mouse IgG (lane3) at 4 °C. Lane 4 contained purified hBVR. Antigen-antibodies complexeswere precipitated as detailed in the text, resolved on 10% SDS-PAGE, fol-lowed by immunoblotting with anti-hBVR antibodies. Enhanced chemilumi-nescence visualized immunocomplex formation. The membrane wasstripped and re-probed with anti-PKC �II antibodies. Data shown are repre-sentative of three independent experiments. b, PKC �II and GST-hBVR asso-ciate in a pulldown assay. 293A cells were transfected with pcDNA3-PKC �IIfor 24 h and treated with PMA as in a. The cell lysate was incubated withGST-hBVR fusion protein (lanes 1 and 2) or with GST (lane 3) immobilized onglutathione-agarose (Amersham Biosciences). Lane 4 contained the PKC �IIstandard. Bound proteins were resolved by SDS-PAGE, transferred to mem-brane, and probed sequentially with monoclonal anti-PKC �II antibodies (top)and with polyclonal anti-hBVR antibodies (bottom). The GST pulldown assaywas repeated twice. c, GST-BVR does not pull down PKC �. Cells were trans-fected with plasmid containing PKC �. The cell lysates �btained after treat-ment with PMA were subjected to pull-down assay with GST-hBVR fusionprotein or with GST alone as described under “Experimental Procedures.”Precipitated complex was separated on SDS-PAGE along with standards forhBVR and PKC � on sides and transferred onto nitrocellulose, and membraneswere subsequently probed with anti-PKC � and anti-hBVR antibodies. Theblots are representative of two separate experiments. d, GST-BVR does notpull down HO-2 protein. Cell extract containing overexpressed HO-2 wasmixed with 10 �g of GST-BVR or GST alone as described under “ExperimentalProcedures.” The GST beads were processed as in c. Membranes were probedwith either an anti-HO-2 antibody or with an anti-BVR antibody. HO-2 andhBVR standards are on sides. e, metal ion specificity of hBVR kinase activity.Autophosphorylation of purified hBVR was analyzed in BVR kinase bufferdescribed under “Experimental Procedures” (pH 8.4) containing indicatedconcentrations of MnCl2, MgCl2, or both. The reaction was initiated by theaddition of [32P]ATP and was terminated after 1 h by the addition of Laemmlibuffer. Samples were resolved on SDS-polyacrylamide gel, transferred topolyvinylidene difluoride membrane, and autoradiographed. The data arerepresentative of three independent experiments. f, hBVR is a substrate for

PKC �II kinase activity. The reaction mixture was optimal for PKC �II kinaseactivity (i.e. pH 7.2, Mg2�), and contained 5 ng of PKC �II and 5 �g of hBVR.Autophosphorylation of each protein was assessed in parallel reactions in thesame experiment. The reaction was initiated as in e and was terminated at theindicated times. Phosphorylated products were detected as described in e.The data are representative of three independent experiments. g, hBVR phos-phorylates PKC �II. Under conditions that preferentially support hBVR kinaseactivity (i.e. pH 8.4, Mn2�), hBVR, PKC �II, or an equimolar mixture of the twowere assayed for 32P incorporation. Reactions were initiated by addition of[32P]ATP, and terminated after 1 h by the addition of Laemmli buffer and thereaction products were analyzed as in e. The data are representative of threeindependent experiments. h, hBVR phosphorylates PKC �II-K371R mutantprotein. hBVR kinase activity was analyzed as described in e using the inactivemutant protein (PKC �II-K371R) instead of WT PKC �II as a substrate. The dataare representative of three independent experiments. i, time course of phos-phorylation of the PKC �II-K371R mutant. The same preparations of hBVR andPKC �II mutant protein as in h were used in a hBVR kinase assay. At indicatedtime points, aliquots were removed and processed for autoradiography as inh. The data are representative of two independent experiments. j, hBVR doesnot phosphorylate PKC �. hBVR kinase activity was analyzed as described in gusing PKC � as a substrate instead of PKC �II.




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ditions yield the same Km value for the reaction, indicating thathBVR does not change the affinity of PKC �II for its substrate.However, the data reveal that hBVRmediates an increased for-mation of a complex between PKC �II and its substrate asdenoted by the 1.64-fold increase in theVmax value (from 423.6;�hBVR to 695.7; �hBVR). The increased incorporation ofphosphate into MBP was not related to hBVR kinase activity,because under PKC kinase assay conditions minimal phospho-rylation of the substrate by hBVR was observed (Fig. 2a).Because hBVR increased PKC�II activity, it was of interest to

dissect out the region of the protein that contributed to theactivation of the PKC. For this, plasmids that expressed a num-ber of truncated forms of hBVR were constructed. The trun-

cated proteins were expressed, purified, and used in the PKCassay system. The truncated proteins are defined and resultsobtained using them are shown in Fig. 2b. The N terminus(amino acids 1–108) and theC terminus (amino acids 272–296)were nearly as effective as theWThBVR,when used in equimo-lar concentration in activating the PKC, whereas the midsec-tion of hBVR (amino acids 109–175) had minimum effect onPKC �II kinase activity. In the C-terminal sequence (aminoacids 272–296), both cysteine and tyrosine residues were cru-cial in potentiation of PKC �II activity by the amino acids 272–296 peptide. As noted in the panel, the mutations of C281A/C293A or Y291F suppressed the effect of this peptide on theactivity of the enzyme.Increased phosphorylation of PKC �II has been linked to

stimulation of its enzyme activity (46, 47). Moreover, proteinscan modulate PKC activity by direct interaction with a proteinleading to its conformational change (48). Because PKC �IIautophosphorylation was increased in the presence of hBVRunder conditions where hBVR is not an effective kinase(Fig. 1f ), it is possible that hBVR not only activates PKC �II byphosphorylating it but also by direct effect on its conformation.As shown in Fig. 3, PKC �II activity, as assessed by MBP phos-phorylation, was nearly doubled in the presence of hBVR. PKCi,a specific PKC inhibitor, essentially blocked phosphorylation,indicating that PKC �II activity was mainly responsible for theincrease and that hBVR was inactive. These observations sug-gest that hBVRbindingmay increase PKC� II activity by induc-ing a conformation change in the PKC molecule. Although,hBVR is a substrate for PKC �II, it is relatively poor by compar-ison toMBP (Fig. 3). Therefore, we conclude that PKC �II acti-vation by hBVR is not merely an additive effect of two kinases.The next experiment further examined the basis for activa-

tion of PKC�II by hBVR. Because lipids are known activators ofPKCs, we questioned whether the hBVR-mediated increase in

FIGURE 2. a, kinetic analysis of PKC �II activity. Kinase activity of PKC �II wasmeasured as a function of increasing concentration of MBP, in the presence orabsence of a constant amount of hBVR (5 �g/50 �l reaction mixture). Controlreactions represent kinase assay in presence of hBVR and absence of PKC �II.Incorporation of 32P into MBP was measured using the P81 filter method asdescribed in the text. The experiment was done three times in triplicate. Thedata were fitted to the Michaelis-Menten equation by nonlinear regression.b, effect of hBVR residues on PKC �II activity. PKC �II activity assay was per-formed in the presence of 1.5 �M hBVR or hBVR truncation mutants. Thereaction was started with the addition of radiolabeled ATP as described under“Experimental Procedures.” The incorporated radioactive phosphate wasdetermined by using the P81 method. The values are expressed as % ofchange of a sample containing PKC �II and MBP taken as 100% � S.D. of threeseparate experiments.

FIGURE 3. In vitro hBVR activation of PKC �II is blocked by PKC inhibitorypeptide. PKC �II activity was measured in the presence or absence of hBVRand/or PKCi. The peptide was added 2 min prior to addition of hBVR whenapplicable. PKC activity was determined as described under “ExperimentalProcedures.” 32P incorporation into MBP was measured as above. PKC �IIactivity in the absence of hBVR was 346 pmol/min/�g for triplicate samples.




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PKC � II activity is a reflection of its substitution for lipid acti-vators. The data shown in Fig. 4 negate this possibility; it isnoted that a 10-fold increase in phosphatidylserine concentra-tion (0.05 to 0.5 mg/ml) did not stimulate PKC �II activity tothe same extent as did hBVR. In this experiment, PKC�II activ-ity wasmeasured in the presence of a constant amount of hBVR(5�g). The data indicate that hBVR directly influences PKC�IIkinase activity and is not merely a substitute for phospholipid.A similar observation was made using DAG (data not shown).hBVR Increases PKCActivity in the Cell—Human embryonic

kidney 293A cells and PMA (a PKC activator) were used. Cellstransfected with pcDNA3 or with different amounts ofpcDNA3-hBVRwere either treatedwith PMAor left untreated.As noted in Fig. 5a, hBVR potentiates PMA-mediated PKCactivation. The effect of hBVR on PMA-mediated PKC activa-tion is significantwhen cells are transfectedwith 0.3�g ormore(p � 0.01; 0.3 �g versus control) of expression plasmid. hBVRhas a similar effect on basal PKC activity without PMA treat-ment (not shown), with a maximum increase of about 35%(from 280 to 378 pmol/min/�g cell protein, p � 0.02; 3 �gversus nontransfected). Clearly, this approach did not distin-guish between PKC �II and other isozymes. The measuredactivity thus reflects the aggregate activity of the four classes ofPKC isozymes as follows: conventional (�, �I, �II, and �), novel(�, �,/L, and ), atypical (� and �/�), and protein kinaseD (�/ )(33, 50–53).The effects of siRNA for hBVR and of two different PKC

inhibitors with differential isozyme specificities were examinedto confirm that the increase in PKC �II activity and incorpora-tion 32P into the substrate is dependent upon hBVR. LY333531is a PKC �-specific inhibitor (39–41), whereas Go-6976 pri-marily inhibits the conventional isoforms (54). Cells transfectedwith hBVR expression plasmids or empty vectors were treatedwith PMA. Measuring the incorporation of 32P into S2, a PKC-specific peptide substrate, assessed PKC activity. To enable S2entry into cells, the membrane was permeabilized using kinasebuffer containing digitonin to treat the cells (38). The effective-ness of the inhibitors was first established in control cellstreated with PMA in the presence or absence of Go-6976 or

LY333531 (Fig. 5b). When compared with the nontransfectedcells, the hBVR-mediated increase in phosphate incorporationinto S2 was more than doubled. Significant attenuation of thiseffect was observed on the addition of the PKC inhibitors (p �0.01; Go-6976 or LY333531 versus PMA � hBVR). About 30%of kinase activity was retained when LY333531 was used as theinhibitor, whereas less than 11% of activity was left withGo-6976. When more general PKC inhibitors such as stauro-

FIGURE 4. hBVR-mediated increase in PKC �II activity is independent ofphospholipid. PKC �II activity was measured in the presence of the indicatedconcentration of phosphatidylserine (PS) with or without hBVR. 32P incorpo-ration into MBP was determined as described for Fig. 2a. The average base-line PKC �II activity in triplicate samples was 116 pmol/min/�g. All values arenormalized, taking this base line as 1.

FIGURE 5. hBVR augments the PMA-induced increase in PKC activity in293A cells. a, hBVR concentration-dependent activation of PKC activity.Human embryonic kidney 293A cells were transfected with the increasingconcentrations of pcDNA3-hBVR or with empty vector. After starvation cellswere treated with 100 nM PMA or with vehicle for 15 min. The PKC-specificsubstrate S2 was added to permeabilized cells, and PKC activity was assessedbased on the incorporation of 32P into the substrate. PKC activity of cellstransfected with empty vector was 286 � 42 pmol/min/�g protein (triplicatesamples). b, effect of PKC �-specific inhibitor on hBVR-mediated increase in S2phosphorylation. Control 293A cells or cells transfected with pcDNA3-hBVRwere starved. Cells were treated with the PKC � inhibitor LY333531 (30 nM) orwith the general inhibitor of conventional PKCs (Go-6976, 200 nM) for 30 minprior to PMA treatment (100 nM, 15 min). Incorporation of 32P into the S2substrate was determined as in a. PKC activity of control cells treated with thePMA solvent Me2SO was 365 � 43 pmol/min/�g proteins. The data representthe results of 8 –12 determinations. c, the expression of BVR is suppressed byhBVR siRNA. Cells were seeded into 6-well plates and treated with retroviralconstruct containing siBVR or control siBVR-sc that was constructed by scram-bling the existing siBVR as described under “Experimental Procedures.” Somecells that were treated with siBVR were transfected with plasmidpCDNA3-hBVR to rescue depletion of hBVR. At the end of the treatment, cellswere harvested, and cell lysates were subjected to Western blotting. Anti-BVRand anti-actin antibodies sequentially probed the nitrocellulose membrane.The experiment was repeated three times. d, sihBVR attenuates PMA-medi-ated activation of PKC, which is rescued by hBVR overexpression. 293A cellswere seeded into 48-well plates and transfected with plasmid containing PKC�II or either co-transfected with hBVR, infected with construct containingsiBVR, or infected with its scrambled control siBVR-sc. The next day, some cellsin plates infected with siBVR were transfected with hBVR, as indicated. 24 hlater cells were starved for another 24 h, then treated with PMA as indicated,and processed for determination of PKC activity as in a. PKC activity ofuntreated cells was 272 � 40 pmol/min/�g cell proteins. Data presented aremeans � S.D. of quadruple wells of two experiments.




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sporine (classical and nonclassical isoforms, 150 nM) or chel-erythrine (general inhibitor of PKCs, 20 �M) were used, mini-mal PKC activity was observed (data not shown). Because theinhibitors were used at maximally effective concentrations, theresidual activities seen in Fig. 5bmay reflect activation of otherPKC isoforms by hBVR.To assess the effect of hBVR on stimulating PKC activity, a

retroviral construct containing siRNA for hBVR and a scram-bled version (siBVR-sc, control) of the same construct wereused to deplete endogenous hBVR in 293A cells. Supportingour previous observation (1), hBVR siRNA almost abolishedendogenous BVRproteinwhen comparedwith nontreated cellsor to cells treated with control sihBVR-sc (Fig. 5c). The hBVRdepletion was successfully reversed when sihBVR-treated cellswere transfected with plasmid containing hBVR (Fig. 5c).Removing endogenous hBVR affected PKC activity in the pres-ence and absence of PMA treatment (Fig. 5d). In the presence ofsihBVR, the PKC basal activity decreased significantly by 45%(272 to 151 pmol/min/�g protein, p � 0.02; sihBVR treatmentversus control), whereas PMA-dependent PKC activity wasdecreased by 36% (1.09 to 0.81 nmol/min/�g of protein, p �0.01; PMA versus PMA � sihBVR). The hBVR depletion-de-pendent decrease in PKC activity was effectively rescued by thesubsequent overexpression of hBVR in the sihBVR-treated cells(Fig. 5d). This finding confirmed the effect of hBVR on PKCactivity in 293A cells.Both Intact ATP Binding Domain and the Chain of Four

Valines in the N-terminal Segment of hBVR Are Involved inActivation of PKC �II—Two sequences in the N-terminaldomain of hBVRwere selected for examination of their possibleimportance in mediating the effects of hBVR on PKC activity.Gly17 lies within the candidate ATP binding domain, andimmediately N-terminal to this is a sequence of four valine res-idues (Val11–14), a sequence that is found only in proteins thatassociate with membranes and cell surface constituents. Thissequence is suspected of being a myristoylation site. The mem-brane association of conventional PKCs is a determinant factorin their activation by membrane lipids and Ca2� (32). Muta-tions were introduced into the hydrophobic and ATP adeninebinding domains in the N terminus of hBVR by changingVal11–14 and Gly17 to alanine. These mutants were used to gen-erate pcDNA- and/or pGEX-hBVR constructs for expression ofthe proteins in 293A cells and in E. coli, respectively. Cellstransfected with expression constructs for 4, 12, or 20 h weresynchronized (24 h), treated with PMA (100 nM, 15 min), andanalyzed for PKC activity by measuring 32P incorporation intothe PKC �II substrate. Data shown in Fig. 6a indicate that bothN-terminal sequences are involved in potentiation of PKC �IIactivity. As shown, in the presence of theWT hBVR resulted ina significant increase in PKC activity when compared with thatof cells transfected with the empty vector. The Gly17 mutant,which does not effectively bind ATP, hence kinase-dead (4),was not as effective as theWThBVR in potentiating PKC activ-ity. The mutant hBVR caused a 21% increase in PKC activitywhenmeasured at the 20-h time point, whereas a near doublingof activity was observed with the WT hBVR at this point. TheVal11–14 hBVR mutant not only failed to activate PKC but

also decreased its activity by 22% during the course of theexperiment.The experiment shown in Fig. 6a indicated that hBVR could

modulate PKC �II activity over an extended time scale. Apotential mechanism for this effect could involve stabilizationof PKC �II by hBVR. Therefore, whether elevated levels of PKC�II were present in cells transfected with hBVR was examinedin cells co-transfected with hBVR and PKC �II expression plas-mid or with PKC �II alone. The cell lysates were subjected toWestern blot analysis; blots were probed with anti-PKC �IIantibodies for assessment of the PKCprotein at 4, 8, 12 and 24 hafter transfection. The experimental results did not show a dif-

FIGURE 6. The N-terminal hydrophobic chain of valines and an intact ATP-binding site are involved in hBVR-mediated increase in PKC activity.a, mutation of Gly17 decreases and Val11–14 prevents hBVR-mediated increasein PKC activity in the cell. 293A cells grown in 48-well plates were transfectedwith pcDNA3 plasmid expressing WT, Gly17, or Val11–14 hBVR, or pcDNA3empty plasmid. Cells grown in serum-enriched medium for the indicatedtime points were synchronized by removing serum. Cells were treated withPMA (100 nM, 15 min) and processed for determination of PKC activity usingS2 as the substrate as described in the text. The values are mean � S.D. of sixmeasurements and are presented as a fold change in activity compared withthat of cells transfected with empty vector, which measured 4.18 � 0.12nmol/min/mg cell proteins. b, concentration-dependent effect of hBVRV11–14

mutant on activation of PKC. Cells were transfected with the indicatedconcentrations of pcDNA3-hBVRV11–14 mutant or with 1 �g of eitherpcDNA3-hBVR or empty vector; after 24 h of growth in serum suppliedmedium, they were synchronized and treated with PMA as in a. c, hBVRV11–14

mutant binds PKC �II. 293 cells were transfected with PKC �II and co-trans-fected with either pcDNA3 WT-hBVR or with pcDNA3-hBVRV11–14 mutant.After starvation cells were treated with PMA for 15 min, and cell lysates weresubjected to immunoprecipitation (IP) with an anti-PKC �II antibody. Precip-itated proteins were separated on SDS-PAGE and transferred to the nitrocel-lulose membrane. The membrane was sequentially probed with anti-hBVRand anti-PKC �II antibodies. The experiment was repeated twice. d, intactN-terminal hydrophobic domain is essential for in vitro augmentation of PKC�II activity by hBVR. Under optimum conditions for PKC �II, kinase activity wasmeasured using MBP as the substrate in the presence of purified WT hBVR orthe V11A/V12A/V13A/V14A mutant protein. MBP phosphorylation by PKC �IIin the absence of the hBVR proteins served as the control. The activity wasassessed as described in the text by autoradiography, and the data are repre-sentative of three independent experiments.




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ference in PKC �II levels between the two conditions at theindicate time points, therefore suggesting that hBVR does notmodulate stability of PKC �II (data not shown). It is thereforeplausible that the prolonged activation of PKC �II reflects themultiplicity of input of hBVR in signaling cascades. Further-more, because the kinase-inactive form of hBVR is less effectivethan the native form in activating the PKC�II, the possibilitymust be considered that the hBVR input, in part, requires theATP-binding competent protein.The observation that expression of the Val11–14 mutant

caused a reduction in PKC expression was extended to cellstreated with PMA. As depicted in Fig. 6b, overexpression ofintact hBVR caused an increase in PKC activity. The mutationof the protein at Val11–14 did not affect its binding to PKC�II(Fig. 6C); however it caused a statistically significant inhibitionof kinase activity, whichwas dependent on the concentration ofthe expression construct.Support for the possibility that the observations with

Val11–14 in the cells were, at least in part, a reflection of its effecton PKC �II activity, was sought by examining phosphorylationof MBP in vitro in the presence of the either WT or Val11–14mutant hBVR protein in a PKC kinase assay system. The resultsof this experiment are shown in Fig. 6d. Consistent with find-ings in whole cells, the presence of the WT hBVR stimulatedphosphorylation of the substrate, whereas the Val11–14 mutantinhibited the reaction.A Threonine Residue in PKC �II Activation Domain-based

Peptide Is a Potential Target of hBVR—Because hBVR has beenshown to phosphorylate PKC �II, a preliminary investigationwas made into the identification of those PKC �II residue(s)that might be target(s) of hBVR phosphorylation. PKC �II acti-vation requires phosphorylation at three distinct serine andthreonine residues (Thr500, Thr641, and Ser661) (35). Three pep-tides of 15–18 residues were synthesized based on the PKC �IIsequence surrounding each of the specific phosphorylationsites. They were then used as substrates for hBVR under con-ditions favorable to hBVR kinase activity in the presence of theactivator, Co-PP. An 18- amino acid variant of one of these PKC�II peptides, having no potential phosphorylation sites(Ser661mut), was used to test the specificity of the effect of hBVR.Co-PP, an activator of the reductase function of BVR (6) and anenhancer of hBVR autophosphorylation, was used in the fol-lowing experiment. Phosphorylation is necessary for the reduc-tase activity of BVR (4). Activators and/or a substrate are oftenrequired to enhance activity of kinases; these can be nonphysi-ological, such as the phorbol esters used to activate PKCs. Datapresented in Fig. 7a indicate that the PKC �II peptide contain-ing Thr500 was phosphorylated by the activated hBVR, but theserine and the threonine residues in the other two peptideswere not appreciably phosphorylated. A modest transfer ofphosphate to the Thr500 peptide was detected in the absence ofCo-PP (Fig. 7a). Also, the control peptides Thr500mut andSer661mut (Fig. 7a), and four peptides, identified under “Exper-imental Procedures,” derived from PKC � activation loop wereminimally phosphorylated (data with PKC � peptides are notshown). Collectively, the data identify the specificity of Thr500as a substrate for hBVR. To test whether the presence of hBVRwould cause an increase in Thr500 phosphorylation of PKC �II

in the cell, 293A cells were transfected with PKC �II alone ortogether with hBVR. After 24 h of starvation, cells were treatedwith 100 nM PMA for 20 min, and total cell lysates were immu-noprecipitated with anti-PKC �II antibodies. Immunoprecipi-tates were subjected to gel electrophoresis, transferred tomembrane, and probed with anti-phospho-Thr500-PKC �IIantibodies. As noted in Fig. 7b, PMA caused a pronouncedincrease in the phosphorylation of theThr500 of PKC�II in cellstransfected with hBVR, compared with cells that were nottransfected with the reductase. This observation is consistentwith the possibility that hBVR has a role in the phosphorylationof PKC �II on Thr500.hBVR Increases PMA-dependent c-fos Activation and PKC

�II Translocation to the Membrane in 293A Cells—The reduc-tase activity of hBVR in cells treated with H2O2 or insulin islinked to an increase in its kinase activity, and an increase inoxidative stress response gene expression and glucose uptake(1, 2, 23). Presently, whether PMA could stimulate hBVRreductase activity and, if so, whether there is a correspondingincrease in PKC-related gene activation, e.g. the c-fos oncogene,were examined. First, activation of endogenousBVRby phorbol

FIGURE 7. PKC �II T500 is a potential target of hBVR kinase activity. a, hBVRdisplays selectivity for phosphorylation of serine/threonine residues in PKC�II-based peptides in vitro. Four peptides based on the PKC �II activation loop(MCKENIWDGVTTKTFCG, where underline indicates position 500), itsmutated control (Thr500mut, MAKENIWDGVTTKAFAG), and autophosphoryla-tion sites (VLTPPDQEVIRNIDQ and FEGFSFVNSEFLKPEVKS, where underlinesindicate positions 641 and 661, respectively), and a serine-free variant(Ser661mut; FEGFAFVNAEFLKPEVKA) were synthesized. The positions ofknown phosphorylation sites in the PKC �II sequence are indicated by bold-face type. hBVR (64 nM) was incubated with 1 �M peptide substrate in thepresence of the hBVR activator, Co-PP. To show the effect of activator, Co-PPwas omitted (lane 1). Phosphate incorporation into the peptides was assessedusing the P81 procedure. The experiments were repeated three times and thevalues represent mean � S.D. b, in cells transfected with hBVR and PKC �II,Thr500 phosphorylation is increased. 293A cells were co-transfected withpcDNA constructs for PKC �II and hBVR (lanes 1 and 4) or with the PKC �IIexpression construct alone (lanes 2 and 3) and starved. Thereafter, cells wereleft untreated (lane 2) or treated with 100 nM PMA (lanes 1, 3, and 4) for 15 min.Cell lysates were immunoprecipitated with anti-PKC �II antibodies or withanti-mouse IgG (control lane 1), resolved on SDS-PAGE, transferred to nitro-cellulose membrane, and probed with anti-phospho-Thr500-PKC �II antibod-ies followed with either anti-PKC �II or anti-BVR antibodies. The data are rep-resentative of two separate experiments.




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esters in 293Acellswas examined.A significant increase inBVRreductase activity in 293A cells was detected within 5 min oftreatment, which was followed by a gradual increase for thenext 60 min (data not shown).Next, influence of hBVR on the oncogene, c-fos, and the

expression in response to PMA exposure of cells were exam-ined. In cells transfected with hBVR expression plasmid, thePMA-mediated increase in c-fos mRNA levels was strikinglyincreased when compared with those observed in cells treated

with PMA alone (Fig. 8a). Anincrease was not detected whensiBVR was used. In addition, over-expression of hBVR did not affectc-fosmRNA levels in the absence ofPMA. Results are consistent withthe contribution of the reductase tothe regulation of gene expression byactivated PKC. To discern whetherhBVR did in fact contribute toPMA-activated PKC �II c-fosexpression, the following analyseswere performed. In one, cells weretransfected with PKC �II expressionconstruct, infected with sihBVR, andtreated with PMA; the secondinvolved pcDNA3-PKC �II trans-fection and PMA treatment. Asshown in Fig. 8a, c-fosmRNA levelsincreased under the latter condi-tions, but the magnitude of theincrease was substantially reducedwhen infected with sihBVR. Theresults are consistent with thelikelihood of hBVR contributing tothe regulation of stress-responsegene expression by activated PKC.To ascertain the biological rele-

vance of the hBVR-PKC�II interac-tion, additional experiments exam-ining the cellular localization ofPKC �II in the presence of hBVRwere performed (Fig. 8, b, c and d).First, we examined the co-localiza-tion of the endogenous proteins.PKC �II and hBVR were visualizedby green fluorescence of fluoresceinisothiocyanate-conjugated and redfluorescence of rhodamine-conju-gated secondary antibodies, respec-tively (Fig. 8b). As indicated bymerged images (Fig. 8b, panels 3and 6) of multiple cell (panels 1–3)and single cell (panels 4–6) slidesections, PKC �II and hBVR co-localize. To closer examine theirinteraction, both proteins wereoverexpressed in 293A cells. ApEGFP-hBVR construct was used to

visualize hBVR, whereas an antibody conjugated with rhoda-mine red detected PKC�II. In the image shown in Fig. 8c, panel1 is a 293A cell transfected with pcDNA-PKC �II, and panels2–4 are cells co-transfected with both expression plasmids. Itis apparent that PKC �II expressed alone is dispersedthroughout the cytoplasm. The image in panel 2 of Fig. 8cshows the effective expression of green fluorescent protein-tagged hBVR in the transfected cell. As visualized in panel 3 ofFig. 8c, PKC �II is relocated to the membrane of the same cell.

FIGURE 8. hBVR influences PKC �II-mediated gene expression and cellular localization of the kinase.a, hBVR potentiates PMA-dependent PKC-regulated c-fos gene expression. 293A cells transfected withpcDNA3-hBVR or pcDNA3-PKC �II expression plasmids were starved and treated with 100 nM PMA or withMe2SO for 30 min. Some nontransfected cells or cells transfected with pcDNA3-PKC �II expression plasmidwere infected with sihBVR. Total cellular RNA was isolated and analyzed by Northern blotting, as described inthe text. The membranes were sequentially probed with c-fos and �-actin cDNAs and used for Northern blotanalysis of c-fos mRNA levels. The blots are representative of three separate experiments. b, endogenous hBVRand PKC �II co-localize. Cells were seeded in 4-well chamber slides and starved with growth media containing0.1% fetal bovine serum. After the treatment with PMA (100 nM) for 15 min, hBVR and PKC �II were subjected tohistochemistry as detailed in the text and visualized by confocal microscopy. Green fluorescence (panels 1 and4) represents PKC � II; red fluorescence (panels 2 and 5) represents hBVR, and yellow-orange fluorescence (panels3 and 6) is from co-localized proteins of merged images. The nuclei were stained with TO-PRO-3 (blue fluores-cence). c, hBVR triggers membrane localization of PKC �II when both are overexpressed. Cells grown in cham-ber slides were transfected with either pcDNA3-PKC �II alone (panel 1) or with both pcDNA3-PKC �II andpEGFP-hBVR (panels 2– 4) 24 h after transfection. hBVR and PKC �II were visualized as above. Red fluorescencerepresents PKC �II expression; green fluorescence is the hBVR; and the merger of the two shows yellow fluores-cence. A merge of the images in panels 2 and 3 is shown in panel 4. Images are representative of three inde-pendent experiments. d, BVR induces translocation of PKC �II to the membrane. Cells were transfected withplasmid containing PKC �II or co-transfected with hBVR (�). After starvation for 24 h, cells were collected, andcytoplasmic (C) and membranous (M) fractions were extracted as described under “Experimental Procedures.”The same amounts of fractions were loaded and separated on SDS-PAGE along with standards for hBVR (leftside) and PKC �II (right side). e, LDH activity of cytoplasmic and membranous cellular fractions. To test the purityof cytoplasmic fractions the activity of lactate dehydrogenase (LDH) was determined in both cytoplasmic (C)and membranous (M) fractions and presented as a bar graph. The values of LDH activity are expressed asmicrounits/min/�g of proteins and are average � S.D. of triplicate measurements. �, overexpression of hBVR.




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When these images are merged, the appearance of yellow-or-ange fluorescence is observed (Fig. 8c, panel 4), indicating thatat least some of the green fluorescent protein-tagged hBVR isalso membrane-associated. The effect of hBVR on PKC �IItranslocation was confirmed by cell fractionation followed byWestern blotting. As indicated in Fig. 8d, the increased pres-ence of hBVR in a membrane cellular fraction leads to anincrease in the translocation of PKC �II to the membrane sup-porting the observation made by confocal imaging. About 95%of cellular LDH activity was detected from cytoplasmic frac-tions, and the rest was observed in the cell membrane, suggest-ing a high purity in both fractions (Fig. 8e). Collectively, theseimages are supportive of a role for hBVR in intracellular trafficof PKC �II and its membrane translocation.

DISCUSSION

This study demonstrates phosphorylation of hBVR by theserine/threonine kinase PKC �II and describes activation ofPKC�II by hBVR in vitro as well as potentiation of PKC activityin the cell. Two sequences in the N terminus of hBVR,11VVVV14 and 15GVGRAG, are relevant to the latter. Further-more, the data suggest two components to hBVR action: kinaseactivity and protein-protein interaction perhaps leading to con-formational changes. The former is suggested by phosphoryla-tion of the kinase-dead PKC �II Arg371 protein in the hBVRkinase assay system, and the latter by hBVR-stimulated PKC�IItranslocation and co-localization of the two proteins to theplasma membrane. Results of co-immunoprecipitation andGST pulldown experiments suggest a physical interaction. Theintegrated function of the two components is suggested bydirect correspondence of either the inability of hBVR to bindATP or the reduced hydrophobic character of the N-terminalsequence, with its inability to activate PKC.There are several mechanisms by which the phospho-

transferase activity of protein kinases, including PKCs, canbe modulated by a binding partner. Protein-protein interac-tions can act to aid substrate presentation, to activate theenzyme, or to cause a change in the secondary structure ofthe kinase. In the cell, the interaction with binding partner(s)confers specificity to individual PKCs and, by directing thekinases to subcellular targets, regulates their function atdefined target site (55–57). Additionally, a change in theconformation of PKCs initiated by ligand binding can pro-mote activation.Largely, activation of PKCs reflects their structural features.

The PKC isozymes contain a conserved sequence in the regu-latory domain, the pseudosubstrate, that is responsible formaintaining the enzyme in an inactive form (33, 58) and aRACK-binding site for association with intracellular receptor-interacting proteins (14). Mochly-Rosen and co-workers (17,59) have identified, in conventional PKC isozymes, RACK1-likesequences that are located within the C2 region of the regula-tory domain; RACK1 binds to PKC � forms I and II better thanto other PKC isozymes. The conserved RACK1-like six-residuesequence in PKC �, SVEIWD (pseudo-RACK) has a conservedtryptophan at position 5 and a negatively charged residue atposition 3. This sequence resembles a sequence in the hBVR,amino acids 107–112 AQELWE, which is, in turn, similar to

that of the PKC pseudosubstrate AVEIWD, with an alanine atposition 1 rather than serine. A synthetic pseudo-RACK1 has ademonstrable effect on PKC � and activates the kinase, in theabsence of other activators, by inducing structural changes inthe protein to expose the catalytic site (14, 17). The presence ofa pseudosubstrate/RACK1-like sequence in hBVR supportsthe possibility of hBVR functioning in a similar way to acti-vate PKC �II. The involvement of protein-protein interac-tions in the activation of kinases is not specific to PKC �II,given the similarity of structure among PKC family mem-bers. Our findings may apply to other kinases that sharestructural similarity with PKC �II. Therefore, it is reasonableto suspect that the observation with PKC �II is not specific tothis form.The N-terminal domain of hBVR is significantly involved

in the activation of PKC �II. Specifically, the composition ofthe first 28 residues includes 30% hydrophobic and 30%charged residues, including 6 valines within and flanking theadenine-binding site. Based on the solved crystal structure ofrat BVR, they are located in an �/� dinucleotide-bindingmotif or Rosen-fold (22, 60). Although the sequence of thefour valine residues does not contribute to the binding of adinucleotide, it likely play a significant role in stabilizing thefold. For instance, the rat BVR residues Val11, Val13, Leu24(human Met24), Leu27, and Val42 (human Val43) form ahydrophobic core structure that strengthens the interactionbetween sheet strand S1 (amino acids 9–14) and helix 1(amino acids 18–27). It is therefore likely that mutatingVal11–14 to all alanine weakens the structure of the hydro-phobic core, perhaps by increasing the flexibility of theS1-H1 interaction. Such changes could greatly interfere withthe folding of the protein, allowing the adoption of an alter-native conformation, thus disturbing normal interaction ofBVR with its usual partners, in this case PKC �II, or promot-ing abnormal protein-protein interactions.Destabilization of the N-terminal domain of hBVR could

result in an inhibition of PKC activity as the result of theincreased accessibility of the hBVR, AQELWE motif, which, asnoted above, shares identity with the pseudosubstrate domainof PKC � SVELWE (61). It is plausible that an intermolecularinteraction between the two proteins at this domain hindersPKC-substrate complex formation. This interpretation is con-sistent with the findings that a mutation of these valines exertsa dominant negative effect on PKC activity, whereas mutationof Gly17 in the ATP-binding site attenuates the BVR-mediatedactivation of PKC (Fig. 6a). Although hBVR is bound to thePKC, it may also bind to a kinase substrate and/or ATP, whichincreases the enzyme-complex concentration. This possibilityis consistent with the observed increase in Vmax of PKC �II forMBP in the absence of a significant change in the Km value ofthe kinase (Fig. 2a).Subsequent to the change in conformation, activation of

PKC �II involves three functionally distinct phosphorylationsites (35) as follows: Thr500 in the activation loop and Thr641and Ser661 in the C terminus. The latter two are autophos-phorylated, whereas Thr500 is phosphorylated by anotherkinase; autophosphorylation of the two residues is critical toPKC � activity (35). Presently, whether phosphorylation of




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the key threonine in PKC �II, Thr500, may be a factor in themechanism of its activation of by hBVRwas considered. Dataobtained for phosphorylation by hBVR of a synthetic peptidewith a sequence derived from the PKC �II activation loopand the apparent influence of BVR in the cell on Thr500 phos-phorylation support this consideration. The preference ofhBVR for phosphorylation of the Thr500 peptide is consistentwith this hypothesis. Notably, Thr500 is conserved in otherconventional PKCs (47). Therefore, hBVR may potentiallyinfluence other conventional PKC activities. Our findingsmay apply to other kinases that are structurally similar toPKC �II. Whether BVR could be one of the kinases thatinitiate PKC activation is an intriguing and not unreasonablepossibility.Evidence for the potential relevance of hBVR to the acti-

vation of conventional PKCs, including PKC �II in the cell, isprovided by data presented in Fig. 8. As noted in Fig. 8a, thePMA-mediated increase in c-fos mRNA levels in cells trans-fected with pcDNA-PKC �II was attenuated by siRNA forhBVR, which suggests a role for hBVR in the regulation ofgene expression by the kinase. Additional evidence for thepotential contribution of BVR to PKC-mediated functions isprovided by data in Fig. 8, b and c, which presents images ofco-localization of hBVR and PKC �II membrane obtained byconfocal microscopy of cells nontransfected or co-trans-fected with expression plasmids for pcDNA3-PKC �II andpEGFP-hBVR. The visualization of PKC-bound fluorescenceprovides an efficient tool for assessing PKC II cellular local-ization in response to the presence of different proteins (44).Such an approach was utilized in this study by using a Rho-damine Red antibody conjugate to stain expressed PKC �II.The images of the PKC �II indicate that hBVR activates PKC�II by facilitating its membrane translocation. Similar obser-vations have been interpreted to suggest activation of PKC�II by phorbol ester (44). Collectively, these data togetherwith previous observations from this laboratory with c-junand ATF-2/CREB activation (1, 24) predict that expressionof genes receiving input from PKC-activated signaling path-ways could be influenced by activation of hBVR.

Acknowledgments—We thank Esther Liu for preparation of themanuscript and Brigette Brown-Kipphut for technical support.

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E. M. GibbsMahin D. Maines, Tihomir Miralem, Nicole Lerner-Marmarosh, Jenny Shen and Peter

IIβC Human Biliverdin Reductase, a Previously Unknown Activator of Protein Kinase

doi: 10.1074/jbc.M513427200 originally published online January 16, 20072007, 282:8110-8122.J. Biol. Chem.

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humanbiliverdinreductase,apreviouslyunknownactivator ... · but not all 12. the bvr kinase motifs...

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