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J. Biol. Chem., Vol. 282, Issue 11, 8110-8122, March 16, 2007

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Human Biliverdin Reductase, a Previously Unknown Activator of Protein Kinase C II^*

From the Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14624

Received for publication, December 16, 2005 , and in revised form, November 20, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Human biliverdin reductase (hBVR), a dual specificity kinase (Ser/Thr/Tyr) is, as protein kinase C (PKC) II, 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 II plasmids, we report the co-immunoprecipitation of the proteins and co-purification in the glutathione S-transferase (GST) pulldown assay. hBVR and PKC II, but not the reductase and PKC , transphosphorylated in assay systems supportive of activity of only one of the kinases. PKC II K371R mutant protein ("kinase-dead") was also a substrate for hBVR. The reductase increased the V_max but not the apparent K_m values of PKC II 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 II activation loop. Neither serine nor threonine residues in peptides corresponding to other phosphorylation sites of the PKC II nor PKC activation loop-derived peptides were substrates. The phosphorylation of Thr⁵⁰⁰ was confirmed by immunoblotting of hBVR·PKC II immunocomplex. The potential biological relevance of the hBVR activation of PKC II was suggested by the finding that in cells transfected with the PKC II, hBVR augmented phorbol myristate acetate-mediated c-fos expression, and infection with sihBVR attenuated the response. Also, in cells overexpressing hBVR and PKC II, 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 II underscores its potential function in propagation of signals relayed through PKCs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 H₂O₂ and Na₂AsO₃, 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 non-conventional 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 Mn²⁺-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 Tyr¹⁹⁸ 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 Mn²⁺-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 Mg²⁺- and Ca²⁺-dependent, 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 phosphorylation 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials and Constructs—Recombinant human PKC II was purchased from Calbiochem. [-³²P]ATP and [-³²P]dCTP were from PerkinElmer Life Sciences. Monoclonal and polyclonal anti-PKC II antibodies were from Zymed Laboratories Inc. and Abgent (San Diego, CA), respectively. Polyclonal anti-phospho-Thr⁵⁰⁰ PKC II antibodies were from Abcam (Cambridge, MA). Biotrace polyvinylidene difluoride membrane was a product of Pall Science Corp. (Pensacola, FL). PKC II-based peptides referred to as Thr⁵⁰⁰ (MCKENIWDGVTTKTFCG), Thr^500mut (MAKENIWDGVTTKAFAG), Thr⁶⁴¹ (VLTPPDQEVIRNIDQ), Ser⁶⁶¹ (FEGFSFVNSEFLKPEVKS), and the control peptide S^mut (FEGFAFVNAEFLKPEVKA) were synthesized by Anaspec Inc. (San Jose, CA). PKC--based peptides, PKC281 (DQIYAMKVVKKE), PKC410 (GDTTSTFCGTPN), PKC560 (EPVQLTPDDEDA), and PKC585 (EFEGFEYINPLLL), were custom-synthesized by Synpep (Dublin, CA). The numbered residues in PKC II are detrimental to its kinase activity (35). The mutant constructs, G17A and V11A/V12A/V13A/V14A, were generated by site-directed mutagenesis of the wild-type hBVR cDNA (19) and used to create expression plasmids in pcDNA3 (used for transfection into 293A cells) and pGEX4-T2 (used for transfection into E. coli) hBVR. Expression plasmids for PKC II and PKC were constructed by subcloning cDNA from pSP65-PKC II or pCO2-PKC (generous gift from Peter Parker, London Research Institute, London, UK) into pcDNA3 and pGEX4-T2. Site-directed mutagenesis of the pGEX4-T2 construct was used to replace Lys³⁷¹ in the ATP-binding site with arginine to express a "kinase-dead" protein (36, 37). hBVR truncations 1–108, 109–175, 272–296, 272–296 C/A, and 272–296 Y/F were constructed from existing pGEX4-T2-hBVR by appropriate deletion and site-directed mutagenesis of WT-hBVR. pSuper-Retro-siBVR was constructed as described previously (1). The primers 5'-GATCCCCTCCTCAGTCCGTTCGAACCTGTTCAAGAGACAGGTTGCTGCAACGGACTGAGGATTTTTGGAAA-3', and 5'-AGCTTTTCCAAAAATCCTCAGTCCGTTCGAACCTGTCTCTTGAACAGGTTGCAACGGACTGAGGAGGG-3' were designed as a scrambled form of the hBVR siRNA and were used to make the siBVR-sc control construct. The PKC-specific substrate S2 (VRKRTLRRL) was purchased from Anaspec Inc. The PKC -specific inhibitor LY333531 was obtained from A.G. Scientific, Inc. (San Diego); and the inhibitor of conventional PKCs, Go-6976, was from Calbiochem. PKCi, MBP, and PKC lipid activator (PS: DAG) were purchased from Upstate (Charlottesville, VA). Co-PP was obtained from Porphyrin Products Inc. (Logan, UT).

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 µg 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 µg 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 µg 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 x 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 MgCl₂, 1 mM EDTA and 1 µg/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 x g at 4 °C to remove large cellular debris (most nuclei and larger), and collected supernatants were centrifuged 15 min at 13,000 x g, 4 °C, to pellet mitochondria. Collected supernatants were centrifuged at 25,000 x g, 4 °C, for 60 min to separate plasma membrane (pellet) from cytoplasm (supernatant). Both fractions were collected, dissolved in RIPA buffer +1% Triton X-100 (membrane) or RIPA buffer (cytoplasm), processed for protein determination, and stored at -20 °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-µl assay containing 20 mM HEPES (pH 7.2), 15 mM MgCl₂, 0.2 mM CaCl₂, 12.5 µM 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 µM ATP containing 5 µCi of [-³²P]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 µg/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 µl of kinase assay buffer (137 mM NaCl, 5.4 mM KCl, 10 mM MgCl₂, 0.3 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 25 mM -glycerophosphate, 5.5 mM D-glucose, 5 mM EGTA, 1 mM CaCl₂, 20 mM HEPES (pH 7.2), 50 µg/ml digitonin, 120 µg/ml S2 substrate, and 100 µM ATP labeled with 10 µCi/ml [-³²P]ATP). The reaction stopped with the addition of 25 µl 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-µl reaction mixture containing 50 mM HEPES (pH 8.4), 30 mM MnCl₂, 0.2 mM dithiothreitol, 10 µM ATP labeled with 10 µCi of [-³²P]ATP, and substrate; the concentration and duration of incubation of the substrate are specified in the appropriate figure legends. Incorporation of ³²P 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 Mn²⁺ dependence of the hBVR kinase activity, both Mn²⁺ and Mg²⁺ were tested in autophosphorylation reactions; to examine kinase activity of hBVR under PKC assay 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 was separated by electrophoresis on agarose gels containing formaldehyde and transferred to Hybond-N⁺ membrane (Amersham Biosciences). The membranes were probed with full-length 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 [-³²P]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 ice-cold 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 µM 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
hBVR Binds to PKC II, Increases Its Phosphorylation, and Is a Substrate for the Kinase—Physical interaction between hBVR and PKCII 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 PMA-treated 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 Mn²⁺, whereas Mg²⁺ and/or Ca²⁺ 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 Mn²⁺ 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 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 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.

View larger version (43K): [in this window] [in a new window]   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 Obtained 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 µg 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 MnCl2, MgCl2, or both. The reaction was initiated by the addition of [32P]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, Mg2+), and contained 5 ng of PKC II and 5 µg 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, Mn2+), hBVR, PKC II, or an equimolar mixture of the two were assayed for 32P incorporation. Reactions were initiated by addition of [32P]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.

View larger version (21K): [in this window] [in a new window]   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 µg/50 µl reaction mixture). Control reactions represent kinase assay in presence of hBVR and absence of PKC II. Incorporation of 32P 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 µM 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% ± S.D. of three separate experiments.

View larger version (14K): [in this window] [in a new window]   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." 32P incorporation into MBP was measured as above. PKC II activity in the absence of hBVR was 346 pmol/min/µg for triplicate samples.

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 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 µg). 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 µg or more (p 0.01; 0.3 µg 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/µg cell protein, p 0.02; 3 µg 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 (µ/) (33, 50–53).

View larger version (20K): [in this window] [in a new window]   FIGURE 4. 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. 32P incorporation into MBP was determined as described for Fig. 2a. The average base-line PKC II activity in triplicate samples was 116 pmol/min/µg. All values are normalized, taking this base line as 1.

View larger version (26K): [in this window] [in a new window]   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 32P into the substrate. PKC activity of cells transfected with empty vector was 286 ± 42 pmol/min/µg 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 32P into the S2 substrate was determined as in a. PKC activity of control cells treated with the PMA solvent Me2SO was 365 ± 43 pmol/min/µg 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 ± 40 pmol/min/µg cell proteins. Data presented are means ± S.D. of quadruple wells of two experiments.

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. Gly¹⁷ lies within the candidate ATP binding domain, and immediately N-terminal to this is a sequence of four valine residues (Val^11–14), 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 Ca²⁺ (32). Mutations were introduced into the hydrophobic and ATP adenine binding domains in the N terminus of hBVR by changing Val^11–14 and Gly¹⁷ 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 ³²P 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 Gly¹⁷ 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.

View larger version (29K): [in this window] [in a new window]   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 increase in PKC activity in the cell. 293A cells grown in 48-well plates were transfected with pcDNA3 plasmid expressing WT, Gly17, or Val11–14 hBVR, or pcDNA3 empty plasmid. Cells grown in serum-enriched medium for the indicated time points were synchronized by removing serum. Cells were treated with PMA (100 nM, 15 min) and processed for determination of PKC activity using S2 as the substrate as described in the text. The values are mean ± S.D. of six measurements and are presented as a fold change in activity compared with that of cells transfected with empty vector, which measured 4.18 ± 0.12 nmol/min/mg cell proteins. b, concentration-dependent effect of hBVRV11–14 mutant on activation of PKC. Cells were transfected with the indicated concentrations of pcDNA3-hBVRV11–14 mutant or with 1 µg 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, hBVRV11–14 mutant binds PKC II. 293 cells were transfected with PKC II and co-transfected with either pcDNA3 WT-hBVR or with pcDNA3-hBVRV11–14 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 PKCII, 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.

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 PKCII (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 (Thr⁵⁰⁰, Thr⁶⁴¹, and Ser⁶⁶¹) (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 Thr⁵⁰⁰ 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 Thr⁵⁰⁰ 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 Thr⁵⁰⁰ as a substrate for hBVR. To test whether the presence of hBVR would cause an increase in Thr⁵⁰⁰ 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-Thr⁵⁰⁰-PKC II antibodies. As noted in Fig. 7b, PMA caused a pronounced increase in the phosphorylation of the Thr⁵⁰⁰ 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 Thr⁵⁰⁰.

View larger version (34K): [in this window] [in a new window]   FIGURE 7. PKC II T500 is a potential target of hBVR kinase activity. a, hBVR displays 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), its mutated control (Thr500mut, MAKENIWDGVTTKAFAG), and autophosphorylation sites (VLTPPDQEVIRNIDQ and FEGFSFVNSEFLKPEVKS, where underlines indicate positions 641 and 661, respectively), and a serine-free variant (Ser661mut; FEGFAFVNAEFLKPEVKA) were synthesized. The positions of known phosphorylation sites in the PKC II sequence are indicated by bold-face type. hBVR (64 nM) was incubated with 1 µM peptide substrate in the presence of the hBVR activator, Co-PP. To show the effect of activator, Co-PP was omitted (lane 1). Phosphate incorporation into the peptides was assessed using the P81 procedure. The experiments were repeated three times and the values represent mean ± S.D. b, in cells transfected with hBVR and PKC II, Thr500 phosphorylation is increased. 293A cells were co-transfected with pcDNA constructs for PKC II and hBVR (lanes 1 and 4) or with the PKC II expression construct alone (lanes 2 and 3) and starved. Thereafter, cells were left 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 with anti-mouse IgG (control lane 1), resolved on SDS-PAGE, transferred to nitrocellulose membrane, and probed with anti-phospho-Thr500-PKC II antibodies followed with either anti-PKC II or anti-BVR antibodies. The data are representative of two separate experiments.

View larger version (67K): [in this window] [in a new window]   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 with pcDNA3-hBVR or pcDNA3-PKC II expression plasmids were starved and treated with 100 nM PMA or with Me2SO 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 (+). 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/µg of proteins and are average ± S.D. of triplicate measurements. +, overexpression of hBVR.

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 co-localize. 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 protein-tagged hBVR in the transfected cell. As visualized in panel 3 of Fig. 8c, PKC II is relocated to the membrane of the same cell. 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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, ¹¹VVVV¹⁴ and ¹⁵GVGRAG, 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 Arg³⁷¹ 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. Re