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.

Biliverdin reductase catalyzes the final step in the heme metabolic pathway, the reduction of biliverdin IX␣ to bilirubin. The enzyme remains unique among all biological catalysts described to date in having a dual pH/cofactor-dependent activity profile (3). The protein displays microheterogeneity because of post-translational phosphorylation that is required for its activity (4,5). Free radical generators such as H 2 O 2 and Na 2 AsO 3 , as well as insulin and the metalloporphyrin Co-PP, 3 activate BVR and increase its phosphorylation (1,2,4,6). Reduction of biliverdin IX␣ to lipophilic bilirubin serves several functions that include trafficking of the heme degradation product through the cell membrane, inactivation of a potent kinase inhibitor, biliverdin, and formation of bilirubin, an effective quencher of free radicals (7,8). BVR is present across metazoan species, and its homologue is found in unicellular cyanobacteria (9 -11). Plants use biliverdin IX␣ produced by ferredoxin-dependent heme oxygenase (HO) to synthesize phytochromes, the sensory photoreceptors (9,12).
BVR is a small soluble protein (296 residues) found mainly in the cytoplasm. If activated, the BVR leads to nuclear translocation and association with the nucleolus (13). Notably, small proteins, similar in molecular weight to hBVR, have been shown to bind to and activate PKCs (14). The N terminus of the reductase is composed of hydrophobic and charged residues, which include a chain of four valines flanking the consensus Walker 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 the conserved six-residue RACK1 sequence in PKC ␤, SVEIWD (pseudo-RACK), and PKC pseudosubstrate AVEIWD. Tryptophan at position 5 and the negatively charged residue at position 3 characterize these sequences (14,17). A synthetic pseudo-RACK1 has an effect on PKC ␤, activating the kinase in the absence of activators by inducing structural changes in the protein to expose the catalytic site (14,17).
Traditionally, the presence of 12 motifs characterizes a protein as a kinase (15); the primary structure of hBVR predicts its sharing several of those motifs, including the aforementioned, but not all 12. The BVR kinase motifs are conserved among mammalian species (2,4,10,18,19). Notably, not all kinases have a complete set of 12 motifs. Recently, a number of nonconventional protein kinases have been identified. For instance, the Goodpasture antigen-binding protein has only a modified Walker A domain and no other motifs, but nonetheless has serine/threonine kinase activity (20). Similarly, the catalytic domain of myosin heavy chain kinase A does not resemble the catalytic 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 links the two termini of the protein, has been shown to bind to the consensus sequences of AP-1 (activator protein-1) and ATF-2/ CREB-binding sites (1,23,24).
The human enzyme, in strictly Mn 2ϩ -dependent assay conditions, phosphorylates known serine/threonine and tyrosine kinase substrates e.g. MBP, casein, IRS-1 (insulin receptor substrate), and Raytide (a PTK substrate). In addition, hBVR autophosphorylates several serines, at least one threonine, and two of its six tyrosine residues (2,4). The remaining four tyrosine residues are phosphorylated by the insulin receptor kinase (IRK). This includes Tyr 198 in the YMXM motif that, in insulin receptor interactive proteins, is the binding site for proteins with Src homology domain, such as phosphatidylinositol 3-kinase (25). hBVR tyrosine kinase activity was demonstrated by the recombinant human protein expressed in Escherichia coli.
Notably the E. coli genome does not encode protein-tyrosine kinases, which are a multigenic family of Mn 2ϩ -dependent kinases exclusive to higher organisms (15). Although there is much information available on identification of IRK function in hBVR tyrosine phosphorylation, to date there is no information available on the identity of the serine/threonine kinase that phosphorylates hBVR.
The MAPK and IRS/phosphatidylinositol 3-kinases are considered the major arms of the insulin/insulin-like growth factor-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 in oxidative stress response of the cell by its being a member of the basic leucine zipper protein family of transcription factors that regulate expression of stress-responsive genes, such as ho-1, ATF-2/CREB, and c-jun in the MAPK signaling pathway (1,23,24). PKC enzymes are activated by oxidative stress, insulin, and growth factors (26 -30). PKC ␤ isozymes (I and II) stimulate cell division and differentiation by regulating the expression of several oncogenes, including c-fos (31). Spatial localization within the cell is a component of the biological function of many protein kinases, including the PKC enzymes whose catalytic competence and localization are regulated by serine/threonine phosphorylation (14,32,33). In the case of PKC ␤II, when activated, the kinase translocates to the plasma membrane from the cytoplasm (34). This kinase is a member of the Mg 2ϩ -and Ca 2ϩdependent, phospholipid/phorbol ester-activated family of the conventional 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 hBVR was uncovered. Collectively, the present findings and past reports define hBVR not only as an enzyme with a unique activity profile but also as one with the possibility of having input at multiple stages in cell signaling pathways.
Cell Culture, Transfection, Co-immunoprecipitation, and GST Pulldown-293A cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum and 1% penicillin G/streptomycin for 24 h or until the cells reached 70% confluency. Depending on the experiment, cells were subsequently transfected with up to 5 g of pcDNA3-hBVR or pcDNA3-PKC ␤II plasmid using transfectin lipid reagent (Bio-Rad) in 10-cm plates, according to the manufacturer's instructions. Western blotting confirmed overexpression of hBVR or PKC ␤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 using NIH3T3 cells. 293A cells were infected with 4 plaque-forming units/cell to inhibit hBVR synthesis (1). For the protein-protein interaction experiments, cells were seeded into 10-cm dishes and co-transfected with both WT pcDNA3-hBVR and pcDNA3-PKC ␤II for co-immunoprecipitation, whereas for GST pulldown pcDNA3-PKC ␤II and pEGFP-HO2 were used. Prior to treatment with 100 nM PMA, the cells were serum-starved in growth medium containing 0.1% fetal bovine serum for 24 h and then lysed in RIPA buffer.
For immunoprecipitation experiments, cell lysate (500 g of protein) was incubated with monoclonal anti-PKC ␤II antibodies or normal mouse serum overnight at 4°C. A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added to select antibody-bound protein. The agarose beads were washed three times in lysis buffer, and the samples were boiled in Laemmli gel loading buffer, separated by SDS-PAGE, and detected by Western blotting using rabbit polyclonal anti-hBVR antibodies. For the GST pulldown assay, cell lysate was incubated with 10 g of GST-hBVR fusion protein or GST immobilized on GSH-agarose beads (Amersham Biosciences) at 4°C for 2 h. The beads were washed three times and boiled in Laemmli buffer to release the bound proteins. Resolved by SDS-PAGE, proteins were detected by immunoblotting using mouse monoclonal anti-PKC ␤II antibodies.
Cell Fractionation-After a cold wash in PBS, cells were scraped and collected by centrifuging at 500 ϫ g for 5 min. Intact cell pellets were resuspended in homogenization medium containing 0.28 M sucrose, 50 mM Tris-HCl (pH 7.5), 25 mM KCl, 5 mM MgCl 2 , 1 mM EDTA and 1 g/ml each of the protease inhibitors leupeptin, pepstatin, and aprotinin and 1 mM phenylmethylsulfonyl fluoride and homogenized by passing five times through a 25-gauge needle. Cell homogenates were centrifuged for 25 min at 120 ϫ g at 4°C to remove large cellular debris (most nuclei and larger), and collected supernatants were centrifuged 15 min at 13,000 ϫ g, 4°C, to pellet mitochondria. Collected supernatants were centrifuged at 25,000 ϫ g, 4°C, for 60 min to separate plasma membrane (pellet) from cytoplasm (supernatant). Both fractions were collected, dissolved in RIPA buffer ϩ1% Triton X-100 (membrane) or RIPA buffer (cytoplasm), processed for protein determination, and stored at Ϫ20°C for further examination. To test the purity of extracted cell fractions, aliquots of cell fractions were examined 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 (Calbiochem). MBP was used as the substrate, as it is commonly used for PKC isozymes and for BVR serine/threonine kinase activity (4). Phosphorylation of MBP can be detected by SDS-PAGE or by trapping on P81 phosphocellulose filters. The design of the assay system was modified, depending on whether hBVR or PKC ␤II was used as the enzyme or substrate. Unless otherwise specified, for PKC ␤II activity 5 ng of PKC ␤II was incubated in a 50-l assay containing 20 mM HEPES (pH 7.2), 15 mM MgCl 2 , 0.2 mM CaCl 2 , 12.5 M MBP, 10 mM ␤-glycerophosphate, and 1 mM dithiothreitol in the presence of a sonicated lipid activator at final concentrations of 0.05 mg/ml PS and 0.005 mg/ml DAG, or PS alone. The addition of 100 M ATP containing 5 Ci of [␥-32 P]ATP initiated the reaction. To examine the effect of WT or mutant hBVR on PKC ␤II activity, they were incubated for 10 min with PKC prior to addition of MBP. If WT or mutant hBVR was used as substrate, MBP was omitted. If used, the inhibitor PKCi was added to PKC ␤II 2 min prior to hBVR or MBP addition. The incubation lasted for 10 min at 30°C when MBP was the substrate and 20 min with hBVR as the sole substrate, unless otherwise stated. The reaction was terminated on ice, either by the addition of Laemmli buffer for SDS-PAGE followed by transfer to polyvinylidene difluoride membrane and autoradiography, or by the addition of 1 volume of 10% phosphoric acid for the P81 phosphocellulose binding assay. An aliquot of the samples was directly applied to the center of the filter, washed six times in 0.75% phosphoric acid, and dried with acetone prior to measurement for radioactivity.
PKC Assay in Situ-The assay was performed by a modification of procedures detailed by Williams and Schrier (38). Cells were 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 starvation medium (0.1% serum), and the incubation was continued for another 24 h to synchronize cells. In some cases, cells were pretreated with the PKC inhibitors Go-6976 (200 nM) for conventional PKCs or LY333531 (30 nM) for PKC ␤ (39 -41) for 30 min before addition of PMA (100 nM, 15 min). Cells were washed with medium and incubated for 10 min at 30°C in 50 l of kinase assay buffer (137 mM NaCl, 5.4 mM KCl, 10 mM MgCl 2 , 0.3 mM Na 2 HPO 4 , 0.4 mM KH 2 PO 4 , 25 mM ␤-glycerophosphate, 5.5 mM D-glucose, 5 mM EGTA, 1 mM CaCl 2 , 20 mM HEPES (pH 7.2), 50 g/ml digitonin, 120 g/ml S2 substrate, and 100 M ATP labeled with 10 Ci/ml [␥-32 P]ATP). The reaction stopped with the addition of 25 l of ice-cold 30% (w/v) trichloroacetic acid on ice. The trichloroacetic acid-soluble fraction samples were transferred to P81 phosphocellulose filters. After 15 min at room temperature, the filters were washed three times in 75 mM phosphoric acid, once in 2.75 mM sodium phosphate (pH 7.5), and once with acetone before liquid scintillation counting. Kinase activity was normalized to protein content.
hBVR Kinase Activity-The activity was measured as described recently (2). For routine assays, purified hBVR, at concentrations noted in the appropriate figure legends, was incubated at 30°C in a 50-l reaction mixture containing 50 mM HEPES (pH 8.4), 30 mM MnCl 2 , 0.2 mM dithiothreitol, 10 M ATP labeled with 10 Ci of [␥-32 P]ATP, and substrate; the concentration and duration of incubation of the substrate are specified in the appropriate figure legends. Incorporation of 32 P was detected either by autoradiography or by the P81 Whatman filter method, as described above for PKC ␤II activity in vitro. To re-establish the Mn 2ϩ dependence of the hBVR kinase activity, both Mn 2ϩ and Mg 2ϩ were tested in autophosphoryl-ation reactions; to examine kinase activity of hBVR under PKC assay conditions, 10 mM concentration of Mg 2ϩ 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 was separated by electrophoresis on agarose gels containing formaldehyde and transferred to Hybond-N ϩ membrane (Amersham Biosciences). The membranes were probed with fulllength hBVR cDNA, full-length c-fos cDNA (generous gift from D. Templeton, University of Toronto, Canada (42)) or a 1.1-kb fragment of human ␤-actin cDNA. Probes were labeled using [␣-32 P]dCTP and a random primer labeling system (Invitrogen). Pre-hybridization, hybridization, and autoradiography were performed as described previously (43). For Western blot analysis, proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane, probed with either anti-hBVR or anti-PKC ␤II antibodies, and visualized by enhanced chemiluminescence.
Confocal Microscopy-These experiments were based on those of Edwards et al. (44). 293A cells were grown in a chamber slide system (Nalge Nunc International Corp., Naperville, IL). Cells were transfected with either pcDNA3-PKC ␤II or pEGFP-hBVR, or both. After a 24-h starvation period, cells were fixed for 10 min in 3.7% formaldehyde in PBS and permeabilized with 1% Triton X-100 in PBS. After washing three times with icecold PBS, cells were pretreated with 3% bovine serum albumin in PBS for 2 h followed by overnight incubation with a 1:150 dilution of polyclonal anti-PKC ␤II antibodies. PBS-washed cells were then treated with Rhodamine Red-conjugated donkey anti-rabbit IgG antibodies (Jackson ImmunoResearch, West Grove, PA) for 30 min and washed before being used for image analysis by confocal microscopy. If nontransfected cells were permeabilized as above, they were treated with monoclonal anti-PKC ␤II antibodies, washed, blocked, and then treated with polyclonal anti-hBVR antibodies. After washing away the excess primary antibodies, the cells were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) and Rhodamine Red-conjugated donkey anti-rabbit IgG antibodies, followed by a treatment with 2 M TO-PRO-3 (Molecular Probes, Eugene, OR) for 10 min in order to visualize the nuclei. A Leica TCS SP, model DMRE, confocal microscope was used.

RESULTS
hBVR Binds to PKC ␤II, Increases Its Phosphorylation, and Is a Substrate for the Kinase-Physical interaction between hBVR and PKC␤II was examined using immunoprecipitation and GST pulldown approaches. Data shown in Fig. 1, a and b, demonstrate binding of the two proteins in cells co-transfected with pcDNA3 expression constructs for hBVR and PKC ␤II. 24 h after transfection, cells were starved (0.1% serum) and either treated with PMA (15 min) or left untreated. For the immunoprecipitation experiment, the cell lysate was incubated with either antibody to PKC ␤II normal mouse serum. The immunoprecipitate was subjected to Western blot analysis and probed with polyclonal antibodies to hBVR. As shown, hBVR was found in the anti-PKC ␤II immunoprecipitates obtained from both PMA-treated and untreated cells, but it was not found in control IgG. Binding of the two proteins was confirmed using a GST pulldown assay (Fig. 1b). In this experiment, the cell lysate, obtained 24 h after transfection with PKC ␤II, was incubated with GST-hBVR fusion protein and immobilized on GSH-agarose beads. Bound proteins were eluted and subjected to Western blot analysis using monoclonal antibodies to PKC ␤II. PKC ␤II has a better affinity for hBVR in PMAtreated cells. To assess the specificity of hBVR binding, PKC (72 kDa), a protein of similar molecular weight, size, and charge to PKC ␤ II was ectopically expressed in 293 cells, and its binding to hBVR after treatment with PMA was tested. As shown in Fig. 1c, GST-hBVR was not able to pull down PKC from cell lysates. Additionally, binding of HO-2 (36 kDa), a protein of similar size to hBVR, was tested. This protein was also not recovered from cell lysates that are mixed with GST-hBVR. These findings suggest that BVR is discriminating in its association with other proteins.
Next, the possibility of transphosphorylation of the kinases was examined. This examination required differentiation between contribution of the two kinases to phosphotransfer activity. hBVR differs from most serine/threonine kinases in its strict requirement for Mn 2ϩ , whereas Mg 2ϩ and/or Ca 2ϩ are often required for activity of this class of kinases, including PKCs (15,16,45). Also, the pH optimum for hBVR and PKC kinase activities differs (8.0 -8.4 and 7.0 -7.2, respectively). Data in Fig. 1e confirm the previously reported Mn 2ϩ requirement of hBVR and show this property can be used to differentiate 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 hBVR phosphorylation. Next, the ability of hBVR to phosphorylate PKC ␤II was examined. Surprisingly, in the presence of hBVR and under hBVR kinase assay conditions, a striking increase in PKC ␤II phosphorylation 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 a remarkable increase in the presence of PKC ␤II, which suggested the possibility that hBVR was an activator of PKC ␤II. A series of studies described below define hBVR as a PKC activator.
It was essential to substantiate that the observed increase in phosphorylation of PKC ␤II was related to hBVR activity or reflected autophosphorylation by the kinase. Therefore, a mutation was introduced into a key lysine residue that resides within the ATP-binding site of PKC ␤II. The K371R replacement reportedly leads to complete loss of both autophosphorylation and substrate phosphorylation (37). A similar phenomenon was observed when Lys 368 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 assay system. As shown in Fig. 1h, the ability of the PKC ␤II mutant protein to autophosphorylate, in the absence of hBVR and under hBVR kinase assay conditions, was minimal. However, the protein shows a prominent level of phosphorylation in the presence of the reductase. To confirm this observation, the time course of phosphorylation of the PKC ␤II-K371R mutant by hBVR was examined. The kinase-dead PKC ␤II was phosphorylated within 5 min of incubation at 30°C. This gradually increased in a time-dependent manner (Fig. 1i). These observations suggest that phosphorylation of PKC ␤II mutant protein is a consequence of the kinase activity of hBVR. Increased phosphorylation of PKC ␤II is linked to stimulation of its enzyme activity (46,47). Proteins can modulate PKC activity by direct interaction with the kinase, leading to the conformational change in the latter (48). Because PKC ␤II autophosphorylation was increased in the presence of hBVR, under conditions where hBVR is not an effective kinase (Fig. 1f), there appeared to be two components to hBVR activation of the PKC, with the second one to involve protein-protein interaction, and a change in conformation of the PKC. Protein-protein interaction also appears to be involved in hBVR activation by PKC ␤II. Finding that phosphorylation of hBVR was increased in the presence of the kinase-dead PKC ␤II indicates this interaction and raises the possibility that protein-protein interaction results in a change in conformation of the reductase to an activated form. Kinase activation through change in conformation is not limited to PKCs but has been reported for others. Also, an inactive kinase can, through protein-protein interaction, influence the response of the interacting kinase. For instance, the inactive (kinase-dead) PDK1 mutant permits normal protein kinase B activation by insulin (49). The phosphorylation of PKC ␤II by hBVR is specific, because PKC in the same kinase assay showed little to no phosphorylation (Fig. 1j). These observations suggest that the interaction between hBVR and PKC ␤II is specific and that phosphorylation of the PKC ␤II mutant protein is directly related to the hBVR kinase activity.
Characterization of hBVR-mediated Augmentation of PKC ␤II Activity-The following experiment was conducted to examine whether increased phosphorylation of PKC ␤II affected its activity to transfer phosphate to a second phosphoacceptor substrate. The substrate-dependent analysis of the influence of hBVR on PKC ␤II phosphotransfer activity is shown in Fig. 2a. In this experiment, constant amounts of either PKC ␤II alone or PKC ␤II together with hBVR were incubated in the PKC assay system with increasing amounts of the MBP. The incorporation of [ 32 P] 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-transfected with pcDNA3-hBVR and pcDNA3-PKC ␤II and starved for 24 h. Thereafter, cells were treated with 100 nM PMA and subsequently lysed as described under "Experimental Procedures." Cell lysates were incubated with either monoclonal anti-PKC ␤II antibodies (lanes 1 and 2) or with normal mouse IgG (lane 3) at 4°C. Lane 4 contained purified hBVR. Antigen-antibodies complexes were precipitated as detailed in the text, resolved on 10% SDS-PAGE, followed by immunoblotting with anti-hBVR antibodies. Enhanced chemiluminescence visualized immunocomplex formation. The membrane was stripped and re-probed with anti-PKC ␤II antibodies. Data shown are representative of three independent experiments. b, PKC ␤II and GST-hBVR associate in a pulldown assay. 293A cells were transfected with pcDNA3-PKC ␤II for 24 h and treated with PMA as in a. The cell lysate was incubated with GST-hBVR fusion protein (lanes 1 and 2) or with GST (lane 3) immobilized on glutathione-agarose (Amersham Biosciences). Lane 4 contained the PKC ␤II standard. Bound proteins were resolved by SDS-PAGE, transferred to membrane, and probed sequentially with monoclonal anti-PKC ␤II antibodies (top) and with polyclonal anti-hBVR antibodies (bottom). The GST pulldown assay was repeated twice. c, GST-BVR does not pull down PKC . Cells were transfected with plasmid containing PKC . The cell lysates ⌷btained after treatment with PMA were subjected to pull-down assay with GST-hBVR fusion protein or with GST alone as described under "Experimental Procedures." Precipitated complex was separated on SDS-PAGE along with standards for hBVR and PKC on sides and transferred onto nitrocellulose, and membranes were subsequently probed with anti-PKC and anti-hBVR antibodies. The blots are representative of two separate experiments. d, GST-BVR does not pull down HO-2 protein. Cell extract containing overexpressed HO-2 was mixed with 10 g of GST-BVR or GST alone as described under "Experimental Procedures." The GST beads were processed as in c. Membranes were probed with either an anti-HO-2 antibody or with an anti-BVR antibody. HO-2 and hBVR standards are on sides. e, metal ion specificity of hBVR kinase activity. Autophosphorylation of purified hBVR was analyzed in BVR kinase buffer described under "Experimental Procedures" (pH 8.4) containing indicated concentrations of MnCl 2 , MgCl 2 , or both. The reaction was initiated by the addition of [ 32 P]ATP and was terminated after 1 h by the addition of Laemmli buffer. Samples were resolved on SDS-polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and autoradiographed. The data are representative of three independent experiments. f, hBVR is a substrate for PKC ␤II kinase activity. The reaction mixture was optimal for PKC ␤II kinase activity (i.e. pH 7.2, Mg 2ϩ ), and contained 5 ng of PKC ␤II and 5 g of hBVR. Autophosphorylation of each protein was assessed in parallel reactions in the same experiment. The reaction was initiated as in e and was terminated at the indicated times. Phosphorylated products were detected as described in e. The data are representative of three independent experiments. g, hBVR phosphorylates PKC ␤II. Under conditions that preferentially support hBVR kinase activity (i.e. pH 8.4, Mn 2ϩ ), hBVR, PKC ␤II, or an equimolar mixture of the two were assayed for 32 P incorporation. Reactions were initiated by addition of [ 32 P]ATP, and terminated after 1 h by the addition of Laemmli buffer and the reaction products were analyzed as in e. The data are representative of three independent experiments. h, hBVR phosphorylates PKC ␤II-K371R mutant protein. hBVR kinase activity was analyzed as described in e using the inactive mutant protein (PKC ␤II-K371R) instead of WT PKC ␤II as a substrate. The data are representative of three independent experiments. i, time course of phosphorylation of the PKC ␤II-K371R mutant. The same preparations of hBVR and PKC ␤II mutant protein as in h were used in a hBVR kinase assay. At indicated time points, aliquots were removed and processed for autoradiography as in h. The data are representative of two independent experiments. j, hBVR does not phosphorylate PKC . hBVR kinase activity was analyzed as described in g using PKC as a substrate instead of PKC ␤II. ditions yield the same K m value for the reaction, indicating that hBVR does not change the affinity of PKC ␤II for its substrate. However, the data reveal that hBVR mediates an increased formation of a complex between PKC ␤II and its substrate as denoted by the 1.64-fold increase in the V max value (from 423.6; ϪhBVR to 695.7; ϩhBVR). The increased incorporation of phosphate into MBP was not related to hBVR kinase activity, because under PKC kinase assay conditions minimal phosphorylation 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 the activation of the PKC. For this, plasmids that expressed a number of truncated forms of hBVR were constructed. The trun-cated proteins were expressed, purified, and used in the PKC assay system. The truncated proteins are defined and results obtained using them are shown in Fig. 2b. The N terminus (amino acids 1-108) and the C terminus (amino acids 272-296) were nearly as effective as the WT hBVR, when used in equimolar concentration in activating the PKC, whereas the midsection of hBVR (amino acids 109 -175) had minimum effect on PKC ␤II kinase activity. In the C-terminal sequence (amino acids 272-296), both cysteine and tyrosine residues were crucial 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 the activity of the enzyme.
Increased phosphorylation of PKC ␤II has been linked to stimulation of its enzyme activity (46,47). Moreover, proteins can modulate PKC activity by direct interaction with a protein leading to its conformational change (48). Because PKC ␤II autophosphorylation was increased in the presence of hBVR under conditions where hBVR is not an effective kinase (Fig. 1f ), it is possible that hBVR not only activates PKC ␤II by phosphorylating it but also by direct effect on its conformation. As shown in Fig. 3, PKC ␤II activity, as assessed by MBP phosphorylation, 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 the increase and that hBVR was inactive. These observations suggest that hBVR binding may increase PKC ␤ II activity by inducing a conformation change in the PKC molecule. Although, hBVR is a substrate for PKC ␤II, it is relatively poor by comparison to MBP (Fig. 3). Therefore, we conclude that PKC ␤II activation by hBVR is not merely an additive effect of two kinases.
The next experiment further examined the basis for activation of PKC ␤II by hBVR. Because lipids are known activators of PKCs, we questioned whether the hBVR-mediated increase in FIGURE 2. a, kinetic analysis of PKC ␤II activity. Kinase activity of PKC ␤II was measured as a function of increasing concentration of MBP, in the presence or absence of a constant amount of hBVR (5 g/50 l reaction mixture). Control reactions represent kinase assay in presence of hBVR and absence of PKC ␤II. Incorporation of 32 P into MBP was measured using the P81 filter method as described in the text. The experiment was done three times in triplicate. The data were fitted to the Michaelis-Menten equation by nonlinear regression. b, effect of hBVR residues on PKC ␤II activity. PKC ␤II activity assay was performed in the presence of 1.5 M hBVR or hBVR truncation mutants. The reaction was started with the addition of radiolabeled ATP as described under "Experimental Procedures." The incorporated radioactive phosphate was determined by using the P81 method. The values are expressed as % of change of a sample containing PKC ␤II and MBP taken as 100% Ϯ S.D. of three separate experiments. FIGURE 3. In vitro hBVR activation of PKC ␤II is blocked by PKC inhibitory peptide. PKC ␤II activity was measured in the presence or absence of hBVR and/or PKCi. The peptide was added 2 min prior to addition of hBVR when applicable. PKC activity was determined as described under "Experimental Procedures." 32 P incorporation into MBP was measured as above. PKC ␤II activity in the absence of hBVR was 346 pmol/min/g for triplicate samples.
PKC ␤ II activity is a reflection of its substitution for lipid activators. The data shown in Fig. 4 negate this possibility; it is noted that a 10-fold increase in phosphatidylserine concentration (0.05 to 0.5 mg/ml) did not stimulate PKC ␤II activity to the same extent as did hBVR. In this experiment, PKC ␤II activity was measured in the presence of a constant amount of hBVR (5 g). The data indicate that hBVR directly influences PKC ␤II kinase activity and is not merely a substitute for phospholipid. A similar observation was made using DAG (data not shown).
hBVR Increases PKC Activity in the Cell-Human embryonic kidney 293A cells and PMA (a PKC activator) were used. Cells transfected with pcDNA3 or with different amounts of pcDNA3-hBVR were either treated with PMA or left untreated. As noted in Fig. 5a, hBVR potentiates PMA-mediated PKC activation. The effect of hBVR on PMA-mediated PKC activation is significant when cells are transfected with 0.3 g or more (p Յ 0.01; 0.3 g versus control) of expression plasmid. hBVR has a similar effect on basal PKC activity without PMA treatment (not shown), with a maximum increase of about 35% (from 280 to 378 pmol/min/g cell protein, p Յ 0.02; 3 g versus nontransfected). Clearly, this approach did not distinguish between PKC ␤II and other isozymes. The measured activity thus reflects the aggregate activity of the four classes of PKC isozymes as follows: conventional (␣, ␤I, ␤II, and ␥), novel (␦, ⑀, /L, and ), atypical ( and /), and protein kinase D (/) (33, 50 -53).
The effects of siRNA for hBVR and of two different PKC inhibitors with differential isozyme specificities were examined to confirm that the increase in PKC ␤II activity and incorporation 32 P into the substrate is dependent upon hBVR. LY333531 is a PKC ␤-specific inhibitor (39 -41), whereas Go-6976 primarily inhibits the conventional isoforms (54). Cells transfected with hBVR expression plasmids or empty vectors were treated with PMA. Measuring the incorporation of 32 P into S2, a PKCspecific peptide substrate, assessed PKC activity. To enable S2 entry into cells, the membrane was permeabilized using kinase buffer containing digitonin to treat the cells (38). The effectiveness of the inhibitors was first established in control cells treated with PMA in the presence or absence of Go-6976 or LY333531 (Fig. 5b). When compared with the nontransfected cells, the hBVR-mediated increase in phosphate incorporation into S2 was more than doubled. Significant attenuation of this effect was observed on the addition of the PKC inhibitors (p Յ 0.01; Go-6976 or LY333531 versus PMA ϩ hBVR). About 30% of kinase activity was retained when LY333531 was used as the inhibitor, whereas less than 11% of activity was left with Go-6976. When more general PKC inhibitors such as stauro- . hBVR-mediated increase in PKC ␤II activity is independent of phospholipid. PKC ␤II activity was measured in the presence of the indicated concentration of phosphatidylserine (PS) with or without hBVR. 32 P incorporation into MBP was determined as described for Fig. 2a. The average baseline PKC ␤II activity in triplicate samples was 116 pmol/min/g. All values are normalized, taking this base line as 1.

FIGURE 5. hBVR augments the PMA-induced increase in PKC activity in 293A cells. a, hBVR concentration-dependent activation of PKC activity.
Human embryonic kidney 293A cells were transfected with the increasing concentrations of pcDNA3-hBVR or with empty vector. After starvation cells were treated with 100 nM PMA or with vehicle for 15 min. The PKC-specific substrate S2 was added to permeabilized cells, and PKC activity was assessed based on the incorporation of 32 P into the substrate. PKC activity of cells transfected with empty vector was 286 Ϯ 42 pmol/min/g protein (triplicate samples). b, effect of PKC ␤-specific inhibitor on hBVR-mediated increase in S2 phosphorylation. Control 293A cells or cells transfected with pcDNA3-hBVR were starved. Cells were treated with the PKC ␤ inhibitor LY333531 (30 nM) or with the general inhibitor of conventional PKCs (Go-6976, 200 nM) for 30 min prior to PMA treatment (100 nM, 15 min). Incorporation of 32 P into the S2 substrate was determined as in a. PKC activity of control cells treated with the PMA solvent Me 2 SO was 365 Ϯ 43 pmol/min/g proteins. The data represent the results of 8 -12 determinations. c, the expression of BVR is suppressed by hBVR siRNA. Cells were seeded into 6-well plates and treated with retroviral construct containing siBVR or control siBVR-sc that was constructed by scrambling the existing siBVR as described under "Experimental Procedures." Some cells that were treated with siBVR were transfected with plasmid pCDNA3-hBVR to rescue depletion of hBVR. At the end of the treatment, cells were harvested, and cell lysates were subjected to Western blotting. Anti-BVR and anti-actin antibodies sequentially probed the nitrocellulose membrane. The experiment was repeated three times. d, sihBVR attenuates PMA-mediated activation of PKC, which is rescued by hBVR overexpression. 293A cells were seeded into 48-well plates and transfected with plasmid containing PKC ␤II or either co-transfected with hBVR, infected with construct containing siBVR, or infected with its scrambled control siBVR-sc. The next day, some cells in plates infected with siBVR were transfected with hBVR, as indicated. 24 h later 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 of untreated cells was 272 Ϯ 40 pmol/min/g cell proteins. Data presented are means Ϯ S.D. of quadruple wells of two experiments. sporine (classical and nonclassical isoforms, 150 nM) or chelerythrine (general inhibitor of PKCs, 20 M) were used, minimal PKC activity was observed (data not shown). Because the inhibitors were used at maximally effective concentrations, the residual activities seen in Fig. 5b may reflect activation of other PKC isoforms by hBVR.
To assess the effect of hBVR on stimulating PKC activity, a retroviral construct containing siRNA for hBVR and a scrambled version (siBVR-sc, control) of the same construct were used to deplete endogenous hBVR in 293A cells. Supporting our previous observation (1), hBVR siRNA almost abolished endogenous BVR protein when compared with nontreated cells or to cells treated with control sihBVR-sc (Fig. 5c). The hBVR depletion was successfully reversed when sihBVR-treated cells were transfected with plasmid containing hBVR (Fig. 5c). Removing endogenous hBVR affected PKC activity in the presence and absence of PMA treatment (Fig. 5d). In the presence of sihBVR, the PKC basal activity decreased significantly by 45% (272 to 151 pmol/min/g protein, p Յ 0.02; sihBVR treatment versus control), whereas PMA-dependent PKC activity was decreased by 36% (1.09 to 0.81 nmol/min/g of protein, p Յ 0.01; PMA versus PMA ϩ sihBVR). The hBVR depletion-dependent decrease in PKC activity was effectively rescued by the subsequent overexpression of hBVR in the sihBVR-treated cells (Fig. 5d). This finding confirmed the effect of hBVR on PKC activity in 293A cells.

Both Intact ATP Binding Domain and the Chain of Four Valines in the N-terminal Segment of hBVR Are Involved in
Activation of PKC ␤II-Two sequences in the N-terminal domain of hBVR were selected for examination of their possible importance in mediating the effects of hBVR on PKC activity. Gly 17 lies within the candidate ATP binding domain, and immediately N-terminal to this is a sequence of four valine residues (Val [11][12][13][14], a sequence that is found only in proteins that associate with membranes and cell surface constituents. This sequence is suspected of being a myristoylation site. The membrane association of conventional PKCs is a determinant factor in their activation by membrane lipids and Ca 2ϩ (32). Mutations were introduced into the hydrophobic and ATP adenine binding domains in the N terminus of hBVR by changing Val [11][12][13][14] and Gly 17 to alanine. These mutants were used to generate pcDNA-and/or pGEX-hBVR constructs for expression of the proteins in 293A cells and in E. coli, respectively. Cells transfected with expression constructs for 4, 12, or 20 h were synchronized (24 h), treated with PMA (100 nM, 15 min), and analyzed for PKC activity by measuring 32 P incorporation into the PKC ␤II substrate. Data shown in Fig. 6a indicate that both N-terminal sequences are involved in potentiation of PKC ␤II activity. As shown, in the presence of the WT hBVR resulted in a significant increase in PKC activity when compared with that of cells transfected with the empty vector. The Gly 17 mutant, which does not effectively bind ATP, hence kinase-dead (4), was not as effective as the WT hBVR in potentiating PKC activity. The mutant hBVR caused a 21% increase in PKC activity when measured at the 20-h time point, whereas a near doubling of activity was observed with the WT hBVR at this point. The Val 11-14 hBVR mutant not only failed to activate PKC but also decreased its activity by 22% during the course of the experiment.
The experiment shown in Fig. 6a indicated that hBVR could modulate PKC ␤II activity over an extended time scale. A potential mechanism for this effect could involve stabilization of PKC ␤II by hBVR. Therefore, whether elevated levels of PKC ␤II were present in cells transfected with hBVR was examined in cells co-transfected with hBVR and PKC ␤II expression plasmid or with PKC ␤II alone. The cell lysates were subjected to Western blot analysis; blots were probed with anti-PKC ␤II antibodies for assessment of the PKC protein at 4, 8, 12 and 24 h after transfection. The experimental results did not show a dif-  [11][12][13][14] mutant on activation of PKC. Cells were transfected with the indicated concentrations of pcDNA3-hBVRV 11-14 mutant or with 1 g of either pcDNA3-hBVR or empty vector; after 24 h of growth in serum supplied medium, they were synchronized and treated with PMA as in a. c, hBVRV 11-14 mutant binds PKC ␤II. 293 cells were transfected with PKC ␤II and co-transfected with either pcDNA3 WT-hBVR or with pcDNA3-hBVRV 11-14 mutant. After starvation cells were treated with PMA for 15 min, and cell lysates were subjected to immunoprecipitation (IP) with an anti-PKC ␤II antibody. Precipitated proteins were separated on SDS-PAGE and transferred to the nitrocellulose membrane. The membrane was sequentially probed with anti-hBVR and anti-PKC ␤II antibodies. The experiment was repeated twice. d, intact N-terminal hydrophobic domain is essential for in vitro augmentation of PKC ␤II activity by hBVR. Under optimum conditions for PKC ␤II, kinase activity was measured using MBP as the substrate in the presence of purified WT hBVR or the V11A/V12A/V13A/V14A mutant protein. MBP phosphorylation by PKC ␤II in the absence of the hBVR proteins served as the control. The activity was assessed as described in the text by autoradiography, and the data are representative of three independent experiments. ference in PKC ␤II levels between the two conditions at the indicate time points, therefore suggesting that hBVR does not modulate stability of PKC ␤II (data not shown). It is therefore plausible that the prolonged activation of PKC ␤II reflects the multiplicity of input of hBVR in signaling cascades. Furthermore, because the kinase-inactive form of hBVR is less effective than the native form in activating the PKC␤II, the possibility must be considered that the hBVR input, in part, requires the ATP-binding competent protein.
The observation that expression of the Val 11-14 mutant caused a reduction in PKC expression was extended to cells treated with PMA. As depicted in Fig. 6b, overexpression of intact hBVR caused an increase in PKC activity. The mutation of the protein at Val 11-14 did not affect its binding to PKC␤II (Fig. 6C); however it caused a statistically significant inhibition of kinase activity, which was dependent on the concentration of the expression construct.
Support for the possibility that the observations with Val 11-14 in the cells were, at least in part, a reflection of its effect on PKC ␤II activity, was sought by examining phosphorylation of MBP in vitro in the presence of the either WT or Val 11-14 mutant hBVR protein in a PKC kinase assay system. The results of this experiment are shown in Fig. 6d. Consistent with findings in whole cells, the presence of the WT hBVR stimulated phosphorylation of the substrate, whereas the Val 11-14 mutant inhibited the reaction.
A Threonine Residue in PKC ␤II Activation Domain-based Peptide Is a Potential Target of hBVR-Because hBVR has been shown to phosphorylate PKC ␤II, a preliminary investigation was made into the identification of those PKC ␤II residue(s) that might be target(s) of hBVR phosphorylation. PKC ␤II activation requires phosphorylation at three distinct serine and threonine residues (Thr 500 , Thr 641 , and Ser 661 ) (35). Three peptides of 15-18 residues were synthesized based on the PKC ␤II sequence surrounding each of the specific phosphorylation sites. They were then used as substrates for hBVR under conditions favorable to hBVR kinase activity in the presence of the activator, Co-PP. An 18-amino acid variant of one of these PKC ␤II peptides, having no potential phosphorylation sites (Ser 661mut ), was used to test the specificity of the effect of hBVR. Co-PP, an activator of the reductase function of BVR (6) and an enhancer of hBVR autophosphorylation, was used in the following experiment. Phosphorylation is necessary for the reductase activity of BVR (4). Activators and/or a substrate are often required to enhance activity of kinases; these can be nonphysiological, such as the phorbol esters used to activate PKCs. Data presented in Fig. 7a indicate that the PKC ␤II peptide containing Thr 500 was phosphorylated by the activated hBVR, but the serine and the threonine residues in the other two peptides were not appreciably phosphorylated. A modest transfer of phosphate to the Thr 500 peptide was detected in the absence of Co-PP (Fig. 7a). Also, the control peptides Thr 500mut and Ser 661mut (Fig. 7a), and four peptides, identified under "Experimental Procedures," derived from PKC activation loop were minimally phosphorylated (data with PKC peptides are not shown). Collectively, the data identify the specificity of Thr 500 as a substrate for hBVR. To test whether the presence of hBVR would cause an increase in Thr 500 phosphorylation of PKC ␤II in the cell, 293A cells were transfected with PKC ␤II alone or together with hBVR. After 24 h of starvation, cells were treated with 100 nM PMA for 20 min, and total cell lysates were immunoprecipitated with anti-PKC ␤II antibodies. Immunoprecipitates were subjected to gel electrophoresis, transferred to membrane, and probed with anti-phospho-Thr 500 -PKC ␤II antibodies. As noted in Fig. 7b, PMA caused a pronounced increase in the phosphorylation of the Thr 500 of PKC ␤II in cells transfected with hBVR, compared with cells that were not transfected with the reductase. This observation is consistent with the possibility that hBVR has a role in the phosphorylation of PKC ␤II on Thr 500 .
hBVR Increases PMA-dependent c-fos Activation and PKC ␤II Translocation to the Membrane in 293A Cells-The reductase activity of hBVR in cells treated with H 2 O 2 or insulin is linked to an increase in its kinase activity, and an increase in oxidative stress response gene expression and glucose uptake (1,2,23). Presently, whether PMA could stimulate hBVR reductase activity and, if so, whether there is a corresponding increase in PKC-related gene activation, e.g. the c-fos oncogene, were examined. First, activation of endogenous BVR by phorbol  1, 3, and 4) for 15 min. Cell lysates were immunoprecipitated with anti-PKC ␤II antibodies or with anti-mouse IgG (control lane 1), resolved on SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-Thr 500 -PKC ␤II antibodies followed with either anti-PKC ␤II or anti-BVR antibodies. The data are representative of two separate experiments. esters in 293A cells was examined. A significant increase in BVR reductase activity in 293A cells was detected within 5 min of treatment, which was followed by a gradual increase for the next 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 examined. In cells transfected with hBVR expression plasmid, the PMA-mediated increase in c-fos mRNA levels was strikingly increased when compared with those observed in cells treated with PMA alone (Fig. 8a). An increase was not detected when siBVR was used. In addition, overexpression of hBVR did not affect c-fos mRNA levels in the absence of PMA. Results are consistent with the contribution of the reductase to the regulation of gene expression by activated PKC. To discern whether hBVR did in fact contribute to PMA-activated PKC ␤II c-fos expression, the following analyses were performed. In one, cells were transfected with PKC ␤II expression construct, infected with sihBVR, and treated with PMA; the second involved pcDNA3-PKC ␤II transfection and PMA treatment. As shown in Fig. 8a, c-fos mRNA levels increased under the latter conditions, but the magnitude of the increase was substantially reduced when infected with sihBVR. The results are consistent with the likelihood of hBVR contributing to the regulation of stress-response gene expression by activated PKC.
To ascertain the biological relevance of the hBVR-PKC ␤II interaction, additional experiments examining the cellular localization of PKC ␤II in the presence of hBVR were performed (Fig. 8, b, c and d). First, we examined the co-localization of the endogenous proteins. PKC ␤II and hBVR were visualized by green fluorescence of fluorescein isothiocyanate-conjugated and red fluorescence of rhodamine-conjugated secondary antibodies, respectively (Fig. 8b). As indicated by merged images (Fig. 8b, panels 3 and 6) of multiple cell (panels 1-3) and single cell (panels 4 -6) slide sections, PKC ␤II and hBVR colocalize. To closer examine their interaction, both proteins were overexpressed in 293A cells. A pEGFP-hBVR construct was used to visualize hBVR, whereas an antibody conjugated with rhodamine red detected PKC ␤II. In the image shown in Fig. 8c, panel 1 is a 293A cell transfected with pcDNA-PKC ␤II, and panels 2-4 are cells co-transfected with both expression plasmids. It is apparent that PKC ␤II expressed alone is dispersed throughout the cytoplasm. The image in panel 2 of Fig. 8c shows the effective expression of green fluorescent proteintagged hBVR in the transfected cell. As visualized in panel 3 of Fig. 8c, PKC ␤II is relocated to the membrane of the same cell. a, hBVR potentiates PMA-dependent PKC-regulated c-fos gene expression. 293A cells transfected with pcDNA3-hBVR or pcDNA3-PKC ␤II expression plasmids were starved and treated with 100 nM PMA or with Me 2 SO for 30 min. Some nontransfected cells or cells transfected with pcDNA3-PKC ␤II expression plasmid were infected with sihBVR. Total cellular RNA was isolated and analyzed by Northern blotting, as described in the text. The membranes were sequentially probed with c-fos and ␤-actin cDNAs and used for Northern blot analysis of c-fos mRNA levels. The blots are representative of three separate experiments. b, endogenous hBVR and PKC ␤II co-localize. Cells were seeded in 4-well chamber slides and starved with growth media containing 0.1% fetal bovine serum. After the treatment with PMA (100 nM) for 15 min, hBVR and PKC ␤II were subjected to histochemistry as detailed in the text and visualized by confocal microscopy. Green fluorescence (panels 1 and 4) represents PKC ␤ II; red fluorescence (panels 2 and 5) represents hBVR, and yellow-orange fluorescence (panels 3 and 6) is from co-localized proteins of merged images. The nuclei were stained with TO-PRO-3 (blue fluorescence). c, hBVR triggers membrane localization of PKC ␤II when both are overexpressed. Cells grown in chamber slides were transfected with either pcDNA3-PKC ␤II alone (panel 1) or with both pcDNA3-PKC ␤II and pEGFP-hBVR (panels 2-4) 24 h after transfection. hBVR and PKC ␤II were visualized as above. Red fluorescence represents PKC ␤II expression; green fluorescence is the hBVR; and the merger of the two shows yellow fluorescence. A merge of the images in panels 2 and 3 is shown in panel 4. Images are representative of three independent experiments. d, BVR induces translocation of PKC ␤II to the membrane. Cells were transfected with plasmid containing PKC ␤II or co-transfected with hBVR (ϩ). After starvation for 24 h, cells were collected, and cytoplasmic (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 (left side) and PKC ␤II (right side). e, LDH activity of cytoplasmic and membranous cellular fractions. To test the purity of 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 as microunits/min/g of proteins and are average Ϯ S.D. of triplicate measurements. ϩ, overexpression of hBVR.
When these images are merged, the appearance of yellow-orange fluorescence is observed (Fig. 8c, panel 4), indicating that at least some of the green fluorescent protein-tagged hBVR is also membrane-associated. The effect of hBVR on PKC ␤II translocation was confirmed by cell fractionation followed by Western blotting. As indicated in Fig. 8d, the increased presence of hBVR in a membrane cellular fraction leads to an increase in the translocation of PKC ␤II to the membrane supporting the observation made by confocal imaging. About 95% of cellular LDH activity was detected from cytoplasmic fractions, and the rest was observed in the cell membrane, suggesting a high purity in both fractions (Fig. 8e). Collectively, these images are supportive of a role for hBVR in intracellular traffic of PKC ␤II and its membrane translocation.

DISCUSSION
This study demonstrates phosphorylation of hBVR by the serine/threonine kinase PKC ␤II and describes activation of PKC ␤II by hBVR in vitro as well as potentiation of PKC activity in the cell. Two sequences in the N terminus of hBVR, 11 VVVV 14 and 15 GVGRAG, are relevant to the latter. Furthermore, the data suggest two components to hBVR action: kinase activity and protein-protein interaction perhaps leading to conformational changes. The former is suggested by phosphorylation of the kinase-dead PKC ␤II Arg 371 protein in the hBVR kinase assay system, and the latter by hBVR-stimulated PKC ␤II translocation and co-localization of the two proteins to the plasma membrane. Results of co-immunoprecipitation and GST pulldown experiments suggest a physical interaction. The integrated function of the two components is suggested by direct correspondence of either the inability of hBVR to bind ATP or the reduced hydrophobic character of the N-terminal sequence, with its inability to activate PKC.
There are several mechanisms by which the phosphotransferase activity of protein kinases, including PKCs, can be modulated by a binding partner. Protein-protein interactions can act to aid substrate presentation, to activate the enzyme, or to cause a change in the secondary structure of the kinase. In the cell, the interaction with binding partner(s) confers specificity to individual PKCs and, by directing the kinases to subcellular targets, regulates their function at defined target site (55)(56)(57). Additionally, a change in the conformation of PKCs initiated by ligand binding can promote activation.
Largely, activation of PKCs reflects their structural features. The PKC isozymes contain a conserved sequence in the regulatory domain, the pseudosubstrate, that is responsible for maintaining the enzyme in an inactive form (33,58) and a RACK-binding site for association with intracellular receptorinteracting proteins (14). Mochly-Rosen and co-workers (17,59) have identified, in conventional PKC isozymes, RACK1-like sequences that are located within the C 2 region of the regulatory domain; RACK1 binds to PKC ␤ forms I and II better than to other PKC isozymes. The conserved RACK1-like six-residue sequence in PKC ␤, SVEIWD (pseudo-RACK) has a conserved tryptophan at position 5 and a negatively charged residue at position 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 at position 1 rather than serine. A synthetic pseudo-RACK1 has a demonstrable effect on PKC ␤ and activates the kinase, in the absence of other activators, by inducing structural changes in the protein to expose the catalytic site (14,17). The presence of a pseudosubstrate/RACK1-like sequence in hBVR supports the possibility of hBVR functioning in a similar way to activate PKC ␤II. The involvement of protein-protein interactions in the activation of kinases is not specific to PKC ␤II, given the similarity of structure among PKC family members. Our findings may apply to other kinases that share structural similarity with PKC ␤II. Therefore, it is reasonable to suspect that the observation with PKC ␤II is not specific to this form.
The N-terminal domain of hBVR is significantly involved in the activation of PKC ␤II. Specifically, the composition of the first 28 residues includes 30% hydrophobic and 30% charged residues, including 6 valines within and flanking the adenine-binding site. Based on the solved crystal structure of rat BVR, they are located in an ␣/␤ dinucleotide-binding motif or Rosen-fold (22,60). Although the sequence of the four valine residues does not contribute to the binding of a dinucleotide, it likely play a significant role in stabilizing the fold. For instance, the rat BVR residues Val 11 , Val 13 , Leu 24 (human Met 24 ), Leu 27 , and Val 42 (human Val 43 ) form a hydrophobic core structure that strengthens the interaction between sheet strand S1 (amino acids 9 -14) and helix 1 (amino acids 18 -27). It is therefore likely that mutating Val [11][12][13][14] to all alanine weakens the structure of the hydrophobic core, perhaps by increasing the flexibility of the S1-H1 interaction. Such changes could greatly interfere with the folding of the protein, allowing the adoption of an alternative conformation, thus disturbing normal interaction of BVR with its usual partners, in this case PKC ␤II, or promoting abnormal protein-protein interactions.
Destabilization of the N-terminal domain of hBVR could result in an inhibition of PKC activity as the result of the increased accessibility of the hBVR, AQELWE motif, which, as noted above, shares identity with the pseudosubstrate domain of PKC ␤ SVELWE (61). It is plausible that an intermolecular interaction between the two proteins at this domain hinders PKC-substrate complex formation. This interpretation is consistent with the findings that a mutation of these valines exerts a dominant negative effect on PKC activity, whereas mutation of Gly 17 in the ATP-binding site attenuates the BVR-mediated activation of PKC (Fig. 6a). Although hBVR is bound to the PKC, it may also bind to a kinase substrate and/or ATP, which increases the enzyme-complex concentration. This possibility is consistent with the observed increase in V max of PKC ␤II for MBP in the absence of a significant change in the K m value of the kinase (Fig. 2a).
Subsequent to the change in conformation, activation of PKC ␤II involves three functionally distinct phosphorylation sites (35) as follows: Thr 500 in the activation loop and Thr 641 and Ser 661 in the C terminus. The latter two are autophosphorylated, whereas Thr 500 is phosphorylated by another kinase; autophosphorylation of the two residues is critical to PKC ␤ activity (35). Presently, whether phosphorylation of the key threonine in PKC ␤II, Thr 500 , may be a factor in the mechanism of its activation of by hBVR was considered. Data obtained for phosphorylation by hBVR of a synthetic peptide with a sequence derived from the PKC ␤II activation loop and the apparent influence of BVR in the cell on Thr 500 phosphorylation support this consideration. The preference of hBVR for phosphorylation of the Thr 500 peptide is consistent with this hypothesis. Notably, Thr 500 is conserved in other conventional PKCs (47). Therefore, hBVR may potentially influence other conventional PKC activities. Our findings may apply to other kinases that are structurally similar to PKC ␤II. Whether BVR could be one of the kinases that initiate PKC activation is an intriguing and not unreasonable possibility.
Evidence for the potential relevance of hBVR to the activation of conventional PKCs, including PKC ␤II in the cell, is provided by data presented in Fig. 8. As noted in Fig. 8a, the PMA-mediated increase in c-fos mRNA levels in cells transfected with pcDNA-PKC ␤II was attenuated by siRNA for hBVR, which suggests a role for hBVR in the regulation of gene expression by the kinase. Additional evidence for the potential contribution of BVR to PKC-mediated functions is provided by data in Fig. 8, b and c, which presents images of co-localization of hBVR and PKC ␤II membrane obtained by confocal microscopy of cells nontransfected or co-transfected with expression plasmids for pcDNA3-PKC ␤II and pEGFP-hBVR. The visualization of PKC-bound fluorescence provides an efficient tool for assessing PKC II cellular localization in response to the presence of different proteins (44). Such an approach was utilized in this study by using a Rhodamine 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 observations have been interpreted to suggest activation of PKC ␤II by phorbol ester (44). Collectively, these data together with previous observations from this laboratory with c-jun and ATF-2/CREB activation (1,24) predict that expression of genes receiving input from PKC-activated signaling pathways could be influenced by activation of hBVR.