The Human Biliverdin Reductase-based Peptide Fragments and Biliverdin Regulate Protein Kinase Cδ Activity

Background: hBVR reduces biliverdin to antioxidant bilirubin. PKCδ promotes tumorigenesis and apoptosis. Results: Complex formation between PKCδ and hBVR results in transactivation. hBVR-based peptides are identified as substrates or inhibitors of the PKC in vitro and in the cell. Biliverdin inhibits PKCδ. Conclusion: A regulatory loop links PKCδ and hBVR in cell signaling. Significance: hBVR-based peptides can be used to regulate PKCδ signaling. PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066–1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser230 and Ser237), flanking the ATP-binding motif (Ser21) and in PHPS sequence (Ser33) as targets of PKCδ. Ser21 and Ser230 were also phosphorylated in hBVR-based peptides. The Ser230-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell.

In unstimulated cells, PKCs, including PKC␦, are present in an inactive conformation (1,2). Stimulation of cells with anionic lipid second messengers/cofactors, such as phorbol 12-myristate 13-acetate (PMA) or diacylglycerol, causes conformational changes in PKCs that result in exposure of the activation loop and release of the auto-inhibitory pseudosubstrate sequence in the N-terminal regulatory domain of the protein from the active site (1,2). The pseudosubstrate domain, placed between the C2-like and C1 regions, maintains the kinase in an inactive conformation by interacting with the substrate recognition site in the catalytic domain, as is the case for all PKCs (15). The two zinc finger motifs in the N-terminal regulatory C1 domain of PKCs are the recognition motifs for the second messengers (16); hBVR is also a Zn 2ϩ metalloprotein (17). PKC␦ signaling activity is a function of its phosphorylation (1,2); however, it differs from other members of the PKC family enzymes by also being activated independent of lipids and translocation to the cell membrane (18). Phosphorylation of serine or tyrosine residues influences the translocation of the PKC to organelle targets, enabling it to exert anti-apoptotic/ proliferative or pro-apoptotic effects (10,19).
Activated PKC␦ interacts with, and phosphorylates, a number of pro-apoptotic proteins. Therefore, its kinase activity plays a determining role in the regulation of cell death (20); for instance, in PMA-stimulated cells, activation of caspase-3 results in cleavage of PKC␦ between the regulatory and catalytic domains of the PKC, leading to translocation of the catalytic domain into the nucleus and hence the onset of apoptosis (21). Conversely, the proliferative effects of PKC␦ likely involve its activation of ERK1/2, which are the upstream kinases for a host of transcriptional factors, including Elk1 and NF-B, that in turn regulate cell growth, proliferation, and survival (22,23). We have recently characterized hBVR as the scaffold/bridge/ anchor for activation of Elk1 by ERK1/2 in the nucleus and also a molecular scaffold/bridge for activation of ERK1/2 by MEK1/2 and PKC␦ (7,24).
By virtue of its catalysis of the conversion of the tetrapyrrole biliverdin-IX to bilirubin-IX, a quencher of ROS, hBVR limits ROS and free radical-mediated apoptosis (25). Bilirubin-IX plays a central role in cellular defense mechanisms (26 -29), and its formation is solely dependent on hBVR activity; reportedly, bilirubin is as effective as glutathione in hindering the toxicity of free radicals (30). Biliverdin is the product of oxidative cleavage of heme (Fe 2ϩ -protoporphyrin-IX) at the mesocarbon bridge by the two active forms of heme oxygenase, the stress-inducible HO-1 and the constitutive HO-2 (31). hBVR is essential both for activation of HO-1 expression by free radicals (25) and for stabilization of HO-2 (32); HO-2 stabilization is a result of attenuation of ubiquitination and proteasomal degradation. In addition, hBVR is an activator of AP-1-and AP-2-dependent gene expression; the stress-responsive genes, FOS, JUN, and ATF-2/CREB, are downstream targets of hBVR (13,33).
Consensus phosphorylation targets of several kinases (34,35) are present in hBVR (7). Three serine residues in consensus phosphorylation targets of protein kinases (36,37) are present in hBVR. The 21 SVR (SXR) sequence flanks the ATP-binding domain of hBVR ( 15 GVGRAG), and the 294 SRK is upstream of the hBVR cysteine-rich Zn-binding domain ( 280 HCX 10 CC) (17). An RXX(S/T) motif, which includes Tyr 228 , is located in the sequence 224 KRNRYLSFHFKSGSL, a segment of hBVR that we have presently identified as a vital link between PKC␦ and hBVR in regulation of their activities. 228 YLSF and 198 YMKM, when tyrosine-phosphorylated, form SH2 protein-docking sites (38).
Because PKC␦ and hBVR both have a broad range of biological activities, their interaction could influence an array of processes that are associated with normal cellular activities, as well as those that are associated with pathophysiology of the cell (1,2,4,39,40). Accordingly, it is reasonable to postulate that the activation of either enzyme has a bearing on the other. Should this be the case, in conducting this investigation we reasoned that short peptides designed based on the hBVR primary structure could function as surrogates for the intact hBVR polypeptide, capable of modulating PKC␦ activity. Our studies have revealed coupled regulation of the activated enzymes. The investigation has led to identification of hBVR-based small peptides, derived from the 224 KRNRYLSFHFKSGSL sequence, that are highly effective inhibitors of PKC␦ kinase activity, whereas the 15-residue-long peptide itself serves as an exceptionally good substrate for the kinase. The inhibitory peptides identified here add to the small battery of peptides that have therapeutic potential for control of PKC␦ activity in the cell.

EXPERIMENTAL PROCEDURES
Materials-Recombinant activated PKC␦ for in vitro studies, TNF-␣ and PMA, were obtained from Calbiochem. The PKC␦ peptide substrate, ARRKRKGSFFYGG, was purchased from Biomol (Plymouth Meeting, PA). DTT and ATP were obtained from Sigma. Myelin basic protein, phosphatidylserine, and diacylglycerol mixture were from Millipore (Temecula, CA). hBVRbased peptides KRNRYLSF, SFHFKSGSL, and KYCCSRK were synthesized in both unmodified and N-myristoylated forms by EZBiolab (Westfield, IN); the peptides KRNRYLSFHFKSGSL, GLKRNRYLAFHFKSGSL, GLKRNRYLAFHFK, GLAANAY-LSFHFK, and RAGSVRMRDL were obtained from the same source in the unmodified forms only. [␥-32 P]ATP and [ 32 P]H 3 PO 4 (carrier-and HCl-free) were from PerkinElmer Life Sciences. Polyclonal anti-PKC␦ antibodies were from Cell Signaling. Anti-human hBVR polyclonal antibodies were obtained as described before (41).
Plasmids and Mutants-The hBVR open reading frame was cloned in the pEGFP-C1 and pDsRed-C1 vectors (Clontech) for expression of fluorescent protein-tagged hBVR in cells and as an HA-tagged species in pcDNA3. The GST-hBVR plasmid has been described elsewhere (42). Selected serine residues were mutated to alanine, using the QuikChange kit (Stratagene, Cedar Creek, TX) The human PKC␦ open reading frame was also cloned in pcDNA3 and pEGFP-C1, using PCR amplification products derived from a human brain cDNA library (Invitrogen). The constitutively active PKC␦ was generated by deletion of amino acids 151-160 in the pseudosubstrate loop (43) from the pcDNA3-PKC␦ clone. All plasmids were verified by sequencing to ensure both the integrity of inserts and placement in the correct reading frame.
Cell Culture and Transfection-Cultures of HEK293A cells were grown and transfected with plasmids, using TransFectin lipid reagent (Bio-Rad). Overexpression of proteins was verified by Western blotting. Transfected cells were serum-starved in DMEM containing 0.1% FBS for 24 h, before treatment with 100 nM PMA or 20 ng/ml TNF-␣ for 15 min. Cell lysates were immunoprecipitated as described previously (14).
PKC␦ Activity Measurements-PKC␦ assay in vitro was performed using 5 ng of purified recombinant human PKC␦ (as a GST fusion protein) in a 50-l reaction containing 50 mM HEPES, pH 7.4, 10 mM MgCl 2 , 0.2 mM DTT, sonicated lipid activators (0.5 g of phosphatidylserine and 0.05 g of diacylglycerol) or lipid activators plus PMA (as indicated in appropriate experiments), and 50 M specific PKC␦ substrate or hBVRbased peptides at concentrations indicated in the appropriate figures. The reaction was started by the addition of 50 M ATP labeled with 5 Ci of [␥-32 P]ATP and incubated for 15 min at 30°C, unless otherwise stated. The reaction was terminated either by the addition of 1 volume of 10% phosphoric acid, followed by transfer of the reaction mixture to P81 membranes for scintillation counting (12). For autophosphorylation of PKC␦, 20 M ATP was used to start a 40-min reaction.
PKC␦ activity in cells was also measured by immunoprecipitation from cell lysates with anti-PKC␦ antibody followed by protein A/G-agarose. The immunoprecipitates were used in kinase reactions, as above, containing 50 M PKC␦-specific peptide substrate; incorporation of 32 P was measured by the P81 method.
To measure the effect of biliverdin on PKC␦ activity, the PKC was preincubated in kinase buffer for 5 min first with 0.2 mM DTT, followed by addition of biliverdin (Frontier Scientific, Logan UT) as indicated in the figures, with [␥ 32 P]ATP being added last to initiate the autophosphorylation reaction. Alternatively, biliverdin was added prior to DTT, or DTT was omitted entirely. The reaction products were resolved by gel electrophoresis and detected by autoradiography.
In Vitro Assays with Recombinant PKC␤II and PKC Kinases-Recombinant PKC␤II (Ͼ800 units/mg, Calbiochem) was assayed in vitro in 20 mM HEPES, pH 7.2, 15 mM MgCl 2 , 0.2 mM CaCl 2 , 1 mM DTT, 25 mM ␤-glycerophosphate, 50 g/ml phosphatidylserine, and 5 g/ml diacylglycerol using the peptide GLKRNRYLSFHFK or myelin basic protein (12.5 M) substrates and 5 ng of enzyme per 50 l of reaction. 100 M ATP (containing [␥-32 P]ATP, as above) was used to start the reaction, and incorporation was determined as above using the P81 filter binding assay (12). Similarly, recombinant PKC (Millipore) was incubated in 20 mM MOPS, pH 7.2, 15 mM MgCl 2 , 0.2 mM EDTA, with the peptide GLKRNRYLSFHFK or myelin basic protein as substrates, again using 5 ng of enzyme per 50-l reaction (14). Otherwise, reaction conditions were as for PKC␤II.
Elk1 and NF-B Signal Transduction Assay-Elk1 and NF-B transcriptional activities were stimulated by PKC␦. Two separate luciferase reporter assay systems for measurement of their activities were as described before (7). Elk1 activity was measured using a transactivation system, where phosphorylation of Elk1 activator domain resulted in activation of the luciferase reporter (expressed from pFA2-Elk1 and pFR-Luc (Stratagene)), respectively. NF-B activity was monitored using a luciferase reporter plasmid regulated by multiple NF-B recognition elements (Stratagene). Cells were co-transfected with pcDNA-PKC␦, pCMV-␤-gal (as a control for transfection efficiency), and luciferase reporter plasmid(s), and 1 day later, they were serum-starved in medium with 0.1% FBS. Peptide (10 M), as indicated for each experiment, was added, and 2 h later, the cells were treated with 100 nM PMA (for Elk1 induction) or 20 ng/ml TNF-␣ (for NF-B) and an additional dose of the peptide. Incubation was continued for 10 h; additional peptide was added at 2-h intervals. Details of treatments are given in the figure legends. Luciferase assays for transactivation activity were normalized, using the ␤-galactosidase activity, as detailed before (13,33).
Measurement of hBVR Reductase Activity-hBVR has a dual pH/cofactor activity profile; at pH 6.7, NADH is the preferred cofactor, and at pH 8.4, NADPH is used (41). hBVR activity was measured at pH 6.7 using NADH as the cofactor; enzyme activity was measured spectrophotometrically from the rate of increase in absorption at 450 nm, reflecting reduction of biliverdin to bilirubin at 25°C. Specific activity was expressed as nanomoles of bilirubin/min/mg of protein.
PKC␦ Phosphorylation Site Mapping in Synthetic Peptides Using Mass Spectrometry-PKC␦ (10 ng) was incubated for 1 h, with 10 M peptides (GLKRNRYLSFHFK, KYCCSRK, and ARRKRKGSFFYGG) in a 50-l reaction, as above, containing 50 M ATP. After the reaction was complete, the lipids were removed by two extraction steps with 200 l of water-saturated ethyl acetate. The delipidated peptides were reduced with 2 mM dithiothreitol (60°C for 60 min) and alkylated with 10 mM iodoacetate (room temperature for 30 min in the dark), followed by addition of 10 mM cysteine to quench the reducing and alkylating reagents. For mass spectrometry analysis, 1% of this reaction was loaded on a reverse phase nanospray column/tip, packed with Magic C18 AQ resin (Michrom). This tip was installed as a nano-electrospray source on the HPLC of a Thermo LTQ mass spectrometer and equilibrated for 10 min with 5% methanol, 0.1% formic acid, at a flow rate of 400 nl/min (i.e. about 16 column volumes). Bound peptides and phosphopeptides were eluted and analyzed in a 45-min LC-MS/MS run, using 5% methanol for 2 min, 5-15% methanol gradient over 3 min, followed by a 15-60% methanol gradient for 38 min, ending with a 60% methanol isocratic step of 2 min, with all solvents containing 0.1% formic acid. The LTQ mass spectrometer was operated in the data-dependent mode to collect MS, MS/MS, and neutral loss-dependent MS 3 data. A full MS survey scan was performed every 3 s, although the seven most intense ions were sequentially isolated and fragmented in the linear ion trap. A neutral loss of 98, 49, 32.7, and 24.5 daltons (for 1-, 2-, 3-and 4-charge state peptides) among the 10 most intense peaks was programmed to trigger an MS 3 scan. The MS and fragmenta-tion spectrum data were used in a Mascot search of a custom database, containing individual entries for the three peptides. Mascot search parameters included precursor and fragment ion mass tolerance of 1.5 and 0.8 daltons, respectively, and allowed for one C 13 incorporation, fixed carbamidomethyl-cysteine modification, variable methionine oxidation, and serine/ threonine/tyrosine phosphorylation. The ion score threshold value was set for 15, with an Expect score less than 0.05. Ion peaks containing major peptide species in the four-charge state were analyzed both manually and using Mascot. LC-MS/MS analysis of kinase-treated peptide samples was compared with untreated peptides, using label-free quantification of extracted ion chromatogram analysis and ProteoIQ software.
Mass Spectrometry Analysis of Peptides from Tissue Culture-Cells were transfected with pcDNA-PKC␦ and serum-starved as described above. They were then treated with 100 nM PMA for 15 min, and the in situ PKC␦ assay (42) was used to introduce the peptides into the cells. Cells were washed 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 PKC␦ peptide substrate, and 50 M ATP). After 1 h, the reaction mix was collected (leaving the cells adhering to the plate), and debris was removed by centrifugation. The supernatant was applied to a Bio-Gel P4 (Bio-Rad) column (equilibrated with 68.5 mM NaCl, 2.7 mM KCl, 0.5 mM EGTA, 0.1 mM EDTA, and 10 mM HEPES, pH 7.2), to separate large proteins from the peptide fraction. Peptide containing fractions were pooled, concentrated, extracted with ethyl acetate and processed for mass spectrometry analysis, using the procedure described above.
Mapping of Phosphorylation Sites in Intact hBVR by Mass Spectrometry-GST-tagged hBVR was overexpressed from the plasmid pGEX-hBVR in Escherichia coli and purified by affinity chromatography using GSH-agarose. The protein was eluted either with glutathione to give the intact fusion protein or by treatment with thrombin to release intact hBVR. Both preparations were incubated with PKC␦ as described above, and the protein was resolved by SDS-gel electrophoresis. To map the complete protein, the following digests were used: chymotrypsin, complete and partial trypsin, and trypsin after treatment of GST-hBVR with acetic anhydride. Stained protein bands were cut from gels, cut into 1-mm square pieces, washed with 50 mM NH 4 HCO 3 , and dehydrated. The proteins were reduced with DTT and alkylated with iodoacetamide, and the rehydrated gels were digested with 20 g/ml trypsin or chymotrypsin (mass spectrometry grade, Promega) in bicarbonate buffer containing 10% acetonitrile for 1 h at 24°C and then at 37°C overnight, followed by a further addition of enzyme, and incubated for 3 h. The digested material was extracted from the gel and analyzed by LC-MS/MS, essentially as described for the peptide samples, above, except that 100 ng of digest peptides were loaded on the nanospray column. The Mascot search parameters were adjusted; the ion score cutoff was set at 25 for the custom database, and the Expect value cutoff was set at 0.1 for the complete human protein database. Peptides with an Expect score less than 0.05 were considered positive identification if more than one peptide was identified for a given protein and if identified as a positive spectral match by ProteoIQ software (NuSep). Phosphopeptide fragmentation spectra were accepted if fragment ions allowed for unambiguous mapping of modification sites to a hydroxyamino acid.
Confocal Microscopy-HeLa cells were maintained as described above for HEK293 cells. Transfection of HeLa cells was performed at ϳ80% confluency using FuGENE HD reagent (Promega) following the manufacturer's instructions. One day after the transfection, the cells were serum-starved for 24 h (0.1% FBS). The peptides (KRNRYLSF, SFHFKSGSL, or KKRILHC), at a concentration of 10 M, were added 2 h prior the addition of 100 nM PMA for 15 min. The fluorescence images were collected using a Cell Observer spinning disc from Zeiss. During the experiments, the cells were kept at 37°C and 5% CO 2 . GFP fluorescence was excited using a 488-nm diode laser, and the emission was collected using a 500 -550-nm band pass.
Test of Covalent Binding of Biliverdin to PKC␦-Association between PKC␦ and biliverdin was examined essentially as described by Lamparter et al. (44). Biliverdin was dissolved in 0.1 M NaOH and diluted to 2 M in PBS, pH 7.4; a 500-l sample was used to measure the absorption spectrum between 260 and 760 nm. GST-tagged PKC␦ was then added to a final concentration of 1 M and incubated for 5 min at 25°C, and the spectrum again was measured. To test for covalent association, the sample was adjusted to 1% SDS, loaded on four Sephadex G-50 spin columns equilibrated in PBS, centrifuged, and the excluded fractions were collected and pooled, and the spectrum was again recorded.
Statistical Analysis-Data as presented in bar graphs are the means with standard deviations of three experiments, unless otherwise indicated, each with triplicate samples. Data were analyzed by one-way analysis of variance from which Student's t test was calculated for all sample pairs. Differences within experiments were considered significant if p Յ 0.05. In the figures, brackets indicate the paired data, and significant differences are indicated by asterisks. Kinetic data for the peptide substrate were fitted to the Michaelis-Menten equation using Prism 3.0 software (GraphPad, San Diego).

Characterization of an hBVR-based Peptide as a PKC␦
Substrate-We had previously observed augmented PKC␦ kinase activity and autophosphorylation in IGF-1-stimulated cells (7). That study also detected increased interaction between hBVR and PKC␦ in response to IGF-1 and PMA stimulation and further formation of a complex that also included ERK2 and MEK1. We examined the consequence of the hBVR/ PKC␦ interaction on hBVR phosphorylation, aiming to identify specific targets of the PKC by evaluating several candidate phosphorylation sites on hBVR that are contained within the consensus phosphorylation motifs of PKC␦. Among these potential phosphorylation sites are the three serine residues Ser 230 , Ser 21 , and Ser 294 . The Ser 230 site is found in an RXRXX(S/T) (RYLS) motif in one of the hBVR SH2 domains. Ser 21 flanks the ATP-binding domain of hBVR ( 15 GVGRAG) in the SXR (SVR) motif; Ser 294 is in the SXK (SRK) motif and is prox-hBVR and Its Fragments Regulate PKC␦ Activity JULY 13, 2012 • VOLUME 287 • NUMBER 29 imal to a cysteine-rich Zn 2ϩ -binding domain ( 280 HCX 10 CC). Synthetic peptides were used as substrates for PKC␦ in the assay system described under "Experimental Procedures." As noted in Fig. 1a, the synthetic peptides 18 RAGSVRMRDL and 224 KRNRYLSFHFKSGSL were efficiently phosphorylated by the kinase. In addition, a peptide, including the 294 SRK sequence, was also phosphorylated in vitro; subsequent experiments, however, indicated that Ser 294 was not phosphorylated in the intact protein. Because Ser 230 is an integral part of the YLSF SH2 domain, peptides spanning Ser 230 of the hBVR were selected as substrates for more extensive phosphorylation assays. The hBVR-based peptides have a highly basic amino acid sequence; for example, the peptide that corresponds to hBVR amino acids 222-234 (GLKRNRYLSFHFK) has four basic residues and is qualitatively similar to the sequence of an accepted consensus PKC phosphorylation site in the commercial peptide ARRKRKGSFFYGG, identified by Nishikawa as being an ideal substrate for PKC␦ (45). A kinase assay, using immunoprecipitated PKC␦ from cells overexpressing the protein and stimulated with 100 nM PMA for 15 min, was used to assess the phosphorylation rate of the hBVR-based peptide, in comparison with the commercially available PKC consensus peptide substrate. As shown in Fig. 1b, at equimolar concentrations, the hBVR-based peptide was a superior substrate for the PKC, relative to the commercial standard, and this higher reaction rate was further amplified for PMA-activated PKC␦. This observation was further examined by measuring the concentration dependence of hBVR-based peptide phosphorylation. Data obtained for increasing peptide substrate concentrations were fitted to the Michaelis-Menten equation, yielding a K m of 1.59 Ϯ 0.58 M (Fig. 1c). This value compares favorably with the reported K m value for the commercial substrate (0.98 M (45)). Kinetic analysis of other PKC family members, using the hBVRderived peptide substrate, revealed that the hBVR peptide is a more favorable substrate for PKC␦, relative to other PKC family members PKC (K m 6.89 M) and PKC␤II (K m 14.03 M).
Furthermore, we examined hBVR peptide sequence requirements for PKC␦ substrates, by substitution of the serine in the KRNRYLSFHFK sequence, as well as the basic residues N-terminal to the potential Ser 230 phosphorylation site (Fig. 1d). A serine 3 alanine replacement at Ser 230 of the peptide (i.e. GLKRNRYLSFHFK 3 GLKRNRYLAFHFK) produced a peptide that was not a substrate for PKC␦. PKC␦ specifically targets Ser 230 , a longer peptide containing two additional serines (corresponding to hBVR Ser 235 and Ser 237 ), and the S230A substitution was a poor PKC␦ substrate (Fig. 1d), although there was some incorporation of 32 P above basal levels, suggesting that one of the two Ser residues might be a kinetically unfavorable target. Similarly, the positively charged residues, N-terminal to the target serine, were also critical for phosphorylation of Ser 230 in the peptide by PKC. The observed essential role of positively charged residues to render the peptide a suitable substrate is consistent with composition of the optimal substrate for PKC␦ (45).
We had observed that in IGF-1-stimulated cells, hBVR stimulated PKC␦ activity (7). Presently, we examined whether an external stimulus is required for hBVR-mediated enhancement of PKC␦ activity, using a constitutively active form of PKC␦ that was engineered by deleting 10 residues (amino acids 151-160) from the pseudosubstrate domain of the PKC (43). The results are shown in Fig. 1e. Co-expression of hBVR with the mutant FIGURE 1. PKC␦ efficiently phosphorylates hBVR-based peptides. a, in vitro, hBVR consensus phosphorylation motifs are targets of PKC␦. PKC␦ was incubated with 10 M hBVR-based peptides, as indicated, for 5 min prior to the addition of radioactive ATP. After incubation, the incorporated radioactivity was measured by the P81 method detailed in the text. b, hBVR-based peptide compares favorably with a commercial PKC␦ peptide substrate. Cells transfected with PKC␦ expression plasmid were treated with PMA (100 nM, 15 min). PKC␦ immunoprecipitated from cell lysate was assayed using the hBVRbased peptide 222 GLKRNRYLSFHFK and a commercially available peptide, ARRKRKGSFFYGG, as substrates. Experimental details are provided in the text. *, p Ͻ 0.01. c, hBVR-based peptide is a high affinity substrate for PKC␦. PKC␦ activity was determined in vitro with increasing concentrations of GLKRNR-YLSFHFK peptide as the substrate. Incorporation of phosphate was measured as in a, and data were fitted to the Michaelis-Menten equation. Identical assays for PKC and PKC␤II activity used conditions optimal for each (13,14). Raw data are expressed as a percentage of the V max for each enzyme, to allow visual comparison of the K m value for each PKC. d, serine residue in the peptide GLKRNRYLSFHFK is a specific target of PKC␦, and N-terminal positively charged residues are essential for its phosphorylation. The hBVR-based peptides with the amino acid substitutions at sites indicated in boldface were tested as substrates for PKC␦ kinase activity, as in a. e, hBVR increases kinase activity of constitutively active PKC␦ in cells. Cells were co-transfected with a constitutively active pcDNA-PKC␦⌬151-160 and the hBVR expression plasmid and treated with 100 nM PMA (15 min.). Kinase activity was measured in immunoprecipitates obtained using anti-PKC␦ antibodies. Experimental details are provided in the text.

hBVR and Its Fragments Regulate PKC␦ Activity
PKC resulted in a near doubling of PKC kinase activity. This suggests that hBVR stimulation of PKC␦ is independent of and/or synergizes the action of other mechanisms that activate the kinase. This observation further indicates that hBVR indeed interacts with and activates PKC␦; as reported before, hBVR does not phosphorylate PKC␦.
Detection of Peptide Phosphorylation by PKC␦ Using Mass Spectrometry-We extended the above observations to mapping the modification site in the peptide using mass spectrometry. In the first experiment, a mixture of three peptides, GLKRNRYLSFHFK, ARRKRKGSFFYGG, and KYCCSRK, was phosphorylated by PKC␦ in vitro. The peptide mixtures yielded high intensity signals and chromatograms that were readily interpretable using LC-MS/MS analysis, as illustrated by GLKRNRYLSFHFK (Fig. 2). A peptide having an experimental mass of 1665.9 daltons was observed in the untreated mixture ( Fig. 2A); this peptide was depleted in the PKC␦-treated sample, and a modified species with a mass of 1746.1 daltons was observed (Fig. 2B). The increase in mass is characteristic of addition of a single phosphate group, and it was apparent that this species was not present in the untreated sample. The peptide is highly basic and is protonated in the LC-MS system; as shown in Fig. 2C, the predominant species was in a 4ϩ charge state, with lesser amounts of 3ϩ and 2ϩ states. As only one serine or tyrosine could be phosphorylated in this peptide, collision-induced dissociation and neutral loss analysis were used to distinguish the less stable phosphoserine from phosphotyrosine; the peptide mass was reduced by 98 daltons (Fig. 2D), characteristic of a ␤-elimination reaction involving phosphoserine. Moreover, a Mascot search and fragmentation spectra based on the LC-MS 3 data of Fig. 2E indicated that the peptide contained phosphoserine rather than phosphotyrosine, as expected for the product of PKC␦ activity. Similar analyses were applied to the other two peptides in the mixture, ARRKRKGSFFYGG and KYCCSRK; these data are summarized in Table 1. Serine phosphorylation of both GLKRNRYLS-FHFK and ARRKRKGSFFYGG was highly efficient; both were at least 90% phosphorylated by the PKC, based on loss of signal from the unmodified peptide. Analysis of KYCCSRK was complicated by its being predominantly triply protonated, resulting in an m/z Ͻ400, below the scan range of the mass spectrometer.
Detection of Phosphorylation of Intact hBVR by PKC␦ Using Mass Spectrometry-The phosphorylation of hBVR was determined by using GST-hBVR in an in vitro kinase reaction. Table  2 lists the aggregate mass spectrometry data on kinased recombinant proteins from in vitro and in vivo preparations. GST-hBVR (2 g) was incubated in a PKC␦-driven kinase reaction, essentially as described for the peptides, as above. LC-MS analysis of a chymotrypsin digestion covered 93% of the protein (Fig. 3a) and yielded a single phosphorylated peptide; the peptide was identified from LC-MS 3 data as GVVVVGVGRAGS-VRMRDL, where the phosphoserine corresponds to Ser 21 of hBVR (Fig. 3b); the peak assignments are shown as supplemental Fig. 1. As noted in Fig. 1a, a peptide including this Ser 21 sequence was phosphorylated by PKC␦ in vitro. Ser 149 , although readily detected in both a chymotrypsin and trypsin digest, was only found in an unmodified state, suggesting that this position is not a significant substrate for PKC␦ modifica-tion. Other candidate phosphorylation sites (Ser 230 and Ser 294 ) could not be detected in this chymotrypsin digest, as some of these peptide fragments from these regions were basic and were expected to have multiple charges, producing m/z values below the range of detection of the mass spectrometer. The tryptic digest revealed three additional phosphorylation targets in the protein, Ser 33 , Ser 230 , and Ser 237 (Fig. 3, c-e, with peak assign-   Figs. 2-4). The two digests collectively cover all but two residues of GST-hBVR, including the Ser 294 site, which was not detected as a phosphopeptide, in a trypsin digest, where GST-BVR was treated with acetic anhydride just prior to trypsin digestion, to block cleave at lysine positions. Phosphorylation of Ser 237 in intact BVR was detected preferentially in kinase reactions that were incubated for extended times, confirming the kinetic assays with synthetic peptides that show a low rate of phosphorylation at this position (Fig. 1d). The phosphorylation of Ser 33 , in the 31 HPSSA sequence, was not predicted based on known sequence motifs for PKC␦ and may represent a novel specificity.
By combining multiple protease mapping strategies and LC-MS/MS runs, a total of 5,704 spectra were detected for GST-BVR (Table 3). The greatest numbers of spectra for phosphorylated peptides were identified for two serine positions in BVR, Ser 21 and Ser 230 . To demonstrate PKC␦ specificity, GST-BVR, without any prior kinase treatment, was also mapped in parallel, using chymotrypsin and trypsin digestion. In this case, the Ser 21 , Ser 230 , and Ser 237 phosphorylation sites were not detected in the negative control, by LC-MS/MS analysis.
Confirmation of both the phosphorylation of Ser 21 and Ser 230 by PKC␦ and of the inability of the PKC to phosphorylate Ser 149 and Ser 294 was obtained by using GST-hBVR carrying single mutations at each of these serine residues as substrates for PKC␦ in vitro. The Ser 230 and particularly the Ser 21 mutants both showed decreased incorporation of phosphate in this experiment (Fig. 3f), whereas there was little or no effect on incorporation by the Ser 149 or Ser 294 mutants. The Ser 149 and Ser 230 double mutant and a protein carrying mutations at all four sites also showed decreased labeling.
Substrate Peptides Are Phosphorylated in the Cell-The MS detection and mapping of PKC␦-dependent phosphorylation of substrate peptides were examined in the cell. Cells were trans-

Mass spectrometry analysis of unmodified and phosphorylated peptides in untreated and kinase-treated samples.
A mixture of the peptides GLKRNRYLSFHFK, ARRKRKGSFFYGG, and KYCCSRK was the substrate of a PKC␦ kinase reaction in vitro. Peptides recovered after removal of lipid from the reaction and from a mock-treated control were analyzed by LC/MS.

TABLE 2 Mapping of phosphopeptides in BVR sequences from phosphorylated GST-BVR
Total spectral counts are shown by SC; underlining indicates potential phosphorylation sites that were not modified. A dash indicates that no spectra were found for this site or peptide. The 4th column represents spectral data from kinased GST-BVR that was treated with acetic anhydride prior to trypsin digestion, to block cleavage of lysine residues and to facilitate mapping of position Ser 294 .

hBVR and Its Fragments Regulate PKC␦ Activity
fected with pcDNA-PKC␦, starved, and treated with PMA. The cells were permeabilized to allow entry of the same peptides as used above, together with ATP (see under "Experimental Procedures"). At the conclusion of the reaction, soluble materials were recovered and processed by size-exclusion chromatography, to remove larger proteins. The recovered peptides were analyzed by LC-MS/MS, as described for the in vitro kinase reactions. LC-MS/MS analysis revealed that the peptide GLKRNRYLS*FHFK was predominantly phosphorylated, as the spectral count for the phosphorylated form was 6-fold greater than the unmodified peptide sequence ( Table 3). The spectral signal was not as high as that seen in the in vitro assay, but it was significant. In addition, the phosphorylated peptide produced essentially the same neutral loss in the MS 2 spectra and mapped the phosphorylation site to the same serine posi-tion in the MS 3 spectra ( Table 3). The mass spectrometry data therefore indicate that peptides introduced into the cell are phosphorylated with a similar specificity as in the in vitro PKC␦ assay.
Identification of Potent hBVR-based PKC␦ Inhibitor Peptides-Because hBVR protein is a substrate for PKC␦ kinase activity and because, as established in the above experiments, the peptide GLKRNRYLSFHFK has amino acid sequence that is critical for PKC reactivity, the peptide sequence at either side of the target serine was dissected and analyzed in the tissue culture assay. The basic peptide 224 KRNRYLSF is predominantly N-terminal to the Ser 230 phosphorylation site, whereas 230 SFH-FKSGSL is C-terminal. Cells transfected with PKC␦ expression vectors were serum-starved and treated with these myristoylated peptides for 2 h prior to treatment with PMA. PKC␦ activ- FIGURE 3. Mass spectrometry mapping of PKC␦ phosphorylation sites in hBVR. a, GST-hBVR was treated with PKC␦ and then subjected to protease mapping, using chymotrypsin (solid lines), partial trypsin digestion (dashed lines), and complete trypsin digestion of chemically acetylated GST-hBVR (dotted lines). Underlining indicates sequence coverage by LC-MS/MS analysis. Only wild type hBVR sequences are shown from the recombinant GST-hBVR construct. b, fragmentation spectrum of the phosphopeptide mapping to the Ser 21 site. The intensity of the b13ϩϩ fragment ion is off-scale. c, fragmentation spectrum for the Ser 33 phosphorylation site. d, fragmentation spectrum for the Ser 230 phosphorylation site. The neutral loss peptide ion intensity is off-scale. e, fragmentation spectrum for the Ser 237 phosphorylation site. The y7ϩϩ and the neutral loss peptide ion intensity is off-scale. f, purified GST-hBVR and serine mutants, as indicated, were treated with PKC␦ and [␥-32 P]ATP (see under "Experimental Procedures"). The products were separated by gel electrophoresis and detected by autoradiography. The loading for each sample was estimated by probing the blot with anti-BVR antibody, after first allowing radioactivity to decay (7,58). JULY 13, 2012 • VOLUME 287 • NUMBER 29 ity was determined after immunoprecipitation of cell lysates with antibodies raised against the C terminus of the enzyme. Contrary to our expectation, treatment with the peptide KRN-RYLSF led to significant inhibition of PKC␦ activity (Fig. 4a). The inhibition was also observed for PKC␦ obtained from cells expressing the constitutively active PKC␦. The hBVR-based SFHFKSGSL peptide also attenuated PMA-mediated stimulation of intact and constitutively active PKC␦. The findings that the peptides were effective inhibitors of the constitutively active kinase argue for a direct interaction of the peptide with the kinase.

hBVR and Its Fragments Regulate PKC␦ Activity
Only hBVR-based Peptide SFHFKSGSL Disrupts Cell Membrane Integrity-The two hBVR-based PKC␦ inhibitory peptides in the cell were tested for their effects on translocation of the PKC in response to PMA stimulation and cell membrane integrity using confocal microscopy. Experimental details are provided in the legend to Fig. 4b. In the presence of KRNRYLSF, PKC␦ exhibited the expected response to PMA, which is localization in the cell membrane (Fig. 4b, panel i). This redistribution was similar to that observed in cells treated with PMA, where PKC␦ was observed at the periphery of the cell (Fig. 4b,  panel ii); in the absence of the phorbol ester, it was located in the Golgi apparatus (Fig. 4b, panel iii). A similar distribution was observed in cells treated with an unrelated peptide KKRILHC (Fig. 4b, panel iv). However, prior treatment with SFHFKSGSL, the sequence of which somewhat resembles PKC␦ translocation inhibitory peptide, SFNSYELGSL (46), caused a more dramatic response, manifested by extensive membrane blebbing and contraction of cell size (Fig. 4b, panel  v). The effect of the peptide appeared specific to cells expressing PKC␦ upon exposure to PMA; notably, treatment with SFH-FKSGSL did not appear to disrupt the integrity of cells expressing hBVR and treated with PMA (Fig. 4b, panel vi).
Disruption of Elk1 Activation by the Inhibitory Peptide KRNRYLSF-We next examined the consequences of KRNR-YLSF inhibition on a PKC␦-dependent signaling function, using activation of ERK/Elk1-and NF-B-dependent transcription of a luciferase reporter. In the first such experiment, cells were co-transfected with pcDNA-PKC␦ and Elk1-luciferase reporter plasmids ("Experimental Procedures") and serumstarved. They were then treated with myristoylated inhibitor peptides or with randomly selected inactive control peptide, 139 KEVVGKD, for 2 h prior to treatment with PMA for 10 h, and additional peptide was added at 2-h intervals. Experimental details are provided under "Experimental Procedures." As noted in Fig. 5a, PMA treatment resulted in a robust stimula-tion of Elk1 activity, which was attenuated in cells treated with either of the inhibitor peptides but not with the control. Similarly, in cells co-transfected with pcDNA-PKC␦ and an NF-B reporter, TNF-␣-mediated activation of NF-B was blocked by treatment with KRNRYLSF (Fig. 5b). In contrast, in cells treated with SFHFKSGSL, the expression was strikingly reduced to about 10% that of the untreated cells, an observation that is consistent with the likelihood that this peptide rapidly stimulated the onset of apoptosis and thus extensive cell loss. In the experiments shown in Fig. 5c, cells were co-transfected with constitutively active PKC␦ and the reporter plasmids for Elk1 or NF-B; in both instances, the peptide KRNRYLSF inhibited expression of the luciferase reporter gene. These observations support the above noted suggestion that the peptide directly inhibits PKC␦ activity, as opposed to preventing its stimulusdependent activation.
hBVR Reductase Activity Is Increased in the Presence of PKC␦, and Biliverdin Blocks Activation of PKC␦ by PMA in Cells-Having established that hBVR is a substrate for PKC␦ (7), we next examined the consequences of phosphorylation by PKC␦ on the reductase activity of the enzyme; as noted earlier, this activity is dependent on hBVR phosphorylation (5). hBVR was used as a substrate by PKC␦ in vitro. The recovered hBVR was analyzed for the rate of conversion of biliverdin to bilirubin. The reductase activity was compared with that of the control assay system that did not contain PKC␦. As shown in Fig. 6a, there was a significant increase in the conversion rate by the reductase subsequent to phosphorylation by PKC␦. If the preliminary phosphorylation was carried out under conditions that favor hBVR kinase activity rather than PKC␦, there was no change in the reductase activity (Fig. 6b), indicating that the stimulation in activity observed in Fig. 6a was a consequence of hBVR phosphorylation by PKC␦. Next, to examine the potential consequences of increased activation of the reductase activity on PKC␦, we examined the effect on PKC␦ autophosphorylation of biliverdin, the heme degradation product and hBVR substrate. The presence of biliverdin in the autophosphorylation reaction led to significant inhibition (Fig. 6c); the inhibition was independent of the order of addition of biliverdin and DTT. This suggested that biliverdin was not acting by interaction with sulfhydryl groups in the PKC. A spectrophotometric analysis was used to test whether biliverdin binds covalently to PKC␦. The Soret band and ␣, ␤ maxima in the absorption spectrum of biliverdin were not shifted in the presence of GST-PKC␦, suggesting that any interaction was transient (Fig. 6d). The biliverdin/GST-PKC␦ was incubated at 25°C for 5 min

LC-MS/MS analysis of GLKRNRYLSFHFK incubated with tissue culture cells or in vitro
HEK cells were transfected with pcDNA-PKC␦, serum-starved, and treated with 100 nM PMA for 15 min. The cells were permeabilized to allow uptake of GLKRNRYLS-FHFK and ATP. After 1 h, the peptide was recovered from the reaction mixture by size-exclusion chromatography and concentrated, and lipids were removed. The lipid-free peptide was analyzed by LC/MS.

Reaction
Peptide a Ϫ98 PO 4 at Ser 9 12 a The position of the phosphorylated residue is indicated by *. b Product of m/z value times peptide charge state is shown. c Mass is in daltons.

hBVR and Its Fragments Regulate PKC␦ Activity
prior to recording the spectrum. The sample was scanned four times, over a period of 30 min., and there was no discernible difference among the spectra. Addition of SDS to the biliverdin/ GST-PKC␦ sample followed by size-exclusion separation of GST-PKC␦ from low molecular weight components yielded a spectrum identical to that of SDS-treated GST-PKC␦, including the shifted ultraviolet absorbance peak, indicating that the inhibitor is removed by simple physical dissociation and is therefore not a consequence of covalent association of biliverdin with the PKC. This is in contrast to the observation of covalent binding of biliverdin to phytochrome proteins in Pseudomonas aeroginosa and Agrobacterium tumefaciens (44,47).

DISCUSSION
Although hBVR is an activator of PKC␦ (7), its substrate, biliverdin (the HO-1/HO-2 catalytic activity product), and two peptides, designed based on the primary structure of the hBVR protein, are potent inhibitors of the PKC. There are a number of ways to activate PKC␦ (18, 48 -51). The known mechanisms FIGURE 4. hBVR-based peptides, KRNRYLSF and SFHFKSGSL, inhibit PKC␦ activity; only the latter disrupts cell membrane integrity. a, peptides KRNRYLSF and SFHFKSGSL suppress PKC␦ activity in cells. Cells were transfected with either pcDNA-PKC␦ plasmid or the constitutively active pcDNA-PKC␦⌬151-160 and treated with myristoylated KRNRYLSF or SFH-FKSGSL for 2 h before treatment with PMA. Cells were processed, and PKC␦ activity was measured as in Fig. 1a. b, hBVR-based peptide SFH-FKSGSL disrupts cell membrane integrity in response to PMA. HeLa cells were transfected with pEGFP-PKC␦, pretreated with myristoylated KRNR-YLSF (panel i), KKRILHC (panel iv), or SFHFKSGSL (panels v and vi) for 2 h, followed by treatment with 100 nM PMA for 15 min. Cells in panel iii were left untreated, and those in panel ii were treated with PMA alone. Cells in panel vi were co-transfected with pDsRed2-hBVR. Expressed proteins in live cells were imaged as described under "Experimental Procedures." Scale bars, 10 m. The regimen of treatment with TNF-␣ (20 ng/ml, final concentration) was similar to that described for PMA in a. Cell lysates were assayed for luciferase activity as above. c, KRNRYLSF peptide also attenuates promoter activity induced by constitutively active PKC␦. Cells were co-transfected with pcDNA-PKC␦⌬151-160, pCMV-␤gal, and either the Elk1 or NF-B reporters. Treatment with stimulants and analysis of promoter activity were the same as described in a and b. hBVR and Its Fragments Regulate PKC␦ Activity JULY 13, 2012 • VOLUME 287 • NUMBER 29 include phosphorylation of the C-terminal Ser 645 and Ser 664 , the change in the conformation of the PKC that follows binding of second messengers, and proteolysis to remove the pseudosubstrate sequence and C1-and C2-like regulatory domains (2,52,53). Phosphorylation at Tyr 311 and Tyr 334 in response to ROS-generating stimuli is also linked to PKC␦ activation (50,54,55). Because kinase-inactive hBVR can activate PKC␦ (7), it is most likely that the mechanism of activation of PKC␦ by hBVR involves a conformational change brought about by the protein/protein interaction.
Analysis of the primary structure of hBVR suggested multiple potential PKC␦ interaction sites. As proposed previously, the hBVR D(␦)-Box-like motif 275 KKRILHCLGL, which is essential for interaction with, and activation of, PKC␦ (7), is a likely site of interaction with the sequence RLGVTGNIKIH-PFFK in the catalytic domain of PKC␦. This interaction is likely to change the PKC kinase domain structure to a more active form. The association of the two proteins can be considered to predispose them to additional forms of binding and interaction. For instance, the sequence in PKC␦ SFNSYELGSL that mediates annexin binding (56) is located in the C2-like domain; in nonactivated PKC␦ this sequence is associated with the IVLM-RAAEEPVSE sequence to maintain the kinase in an inactive conformation. We postulate that the hBVR SFHFKSGSL sequence, which closely resembles the PKC␦ motif SFNSYEL-GSL, could compete with that motif for binding to IVLM-RAAEEPVSE, thereby changing the conformation of the PKC regulatory domain. The combination of the two interactions, which are depicted in Fig. 7, could result in enhanced activity of pseudosubstrate-deleted PKC␦, which is shown in Fig. 1e. Moreover, based on our previous study with another hBVRinteractive kinase, Goodpasture antigen-binding protein, an atypical protein kinase (57,58), it is plausible that the C-terminal segment of hBVR is involved in interaction with the Zn 2ϩbinding sites in the C1 domain of PKC␦. The Zn 2ϩ -binding domain of hBVR ( 280 HCX 10 CC) is in part contained in the D(␦)-Box-like motif (17). Zn 2ϩ , as does Ca 2ϩ , targets PKCs to the cell membrane (59); it is conceivable that the metal ion may be involved in membrane translocation of an hBVR-PKC␦ complex mediated by their respective Zn 2ϩ -binding domains. The combination of interactions would be expected to maintain the conformation of the PKC in a more open form during activation in response to PMA or IGF-1, preventing binding of the pseudosubstrate to the active site.
The Ser/Thr residues in RXX(S/T) and its related motif RXRXX(S/T) are phosphorylation targets of PKC␦, as well as CaMK2 and PKB/Akt (34,36,37). The identified substrate peptide, GLKRNRYLSFHFK, presents a new type of substrate for the PKC, and it shares with the previously identified substrates, FIGURE 6. PKC␦ activates the reductase activity of hBVR, and biliverdin suppresses the PKC activity. a, phosphorylation of hBVR by PKC␦ accelerates the conversion of biliverdin to bilirubin by the enzyme. PKC␦ was used to phosphorylate hBVR in vitro under PKC␦ assay conditions. The reductase activity was measured as detailed in the text. b, phosphorylation of hBVR by PKC␦ is essential for stimulation of the reductase. hBVR was incubated with PKC␦ under hBVR kinase conditions prior to measurement of the reductase activity. c, biliverdin suppresses PKC␦ autophosphorylation. Recombinant human PKC␦ was preincubated in vitro either in standard kinase buffer containing DTT or in buffer lacking DTT but including the indicated concentrations of biliverdin. As indicated, biliverdin was added to the DTT-treated samples or DTT to those treated with biliverdin. The samples were then used in an in vitro kinase assay, and autophosphorylation of PKC␦ was detected by gel electrophoresis and autoradiography. d, biliverdin (BV) interaction with PKC␦ is noncovalent. The absorbance spectra, in PBS, pH 7.4, of biliverdin and of biliverdin together with GST-PKC␦ were measured between 260 and 760 nm. The latter spectrum was measured after 5 min of incubation at 25°C. The sample containing GST-PKC␦ and biliverdin was adjusted to 1% SDS and fractionated by size-exclusion chromatography, and the spectrum of the high molecular weight (MW) fraction was determined. Details are provided in the text.  (7). The regulatory domain of hBVR is depicted in green and brown, with the position of the D-box motif in the C-terminal helix indicated by the brace. It was proposed that the interaction site in the PKC␦ catalytic domain is the sequence RLGVTGNIKIHPFFK. The precise orientation of the domains has not been determined. b, model for activation of PKC␦ by hBVR. PKC␦ is envisioned as being in an inactive closed state, where the pseudosubstrate is bound in the active site and the annexinV-like and annexinVbinding sequences in the C2 domain are inaccessible because of the C1 domain. The hBVR sequence SFHFKSGSL could compete with the PKC␦ annexinV-binding sequence for the annexinV-like site, opening the PKC␦ regulatory domain. myristoylated alanine-rich C kinase substrate (KKKRFSFKKS-FKLSG) (60) and the commercially available peptide (ARRK-RKGSFFYGG), the density and distribution of positively charged residues. This peptide also stands in contrast to peptides derived from PKC regulatory sequences, such as those based on the pseudosubstrate sequence or the ones that resemble regions in the receptor for activated C-kinase-1 (RACK1). The pseudosubstrate sequences of PKCs are a potent inhibitor of the kinase from which they are derived (52). Also, a peptide based on the PKC␤ pseudo-RACK sequence activates the kinase (61). Here, multiple types of analyses, mass spectrometry analysis of the substrate peptide and that of the intact hBVR protein as well as in vitro and in situ kinase assays, revealed that the serine residue in the RXXS motif contained in the substrate peptide, which corresponds to Ser 230 of hBVR, is a high affinity acceptor of the PKC␦ phosphotransferase activity. A synthetic peptide that lacked the arginine and lysine residues, but contained hydrophobic residues downstream of serine, GLAAN-AYLSFHFK, was not an effective substrate in vitro for the PKC, indicating that the sequence and the composition of the peptide, as a whole, are required for its serving as a substrate for PKC␦ activity. The specificity of Ser 230 for phosphorylation by PKC␦ was suggested by the finding that the residue in the intact protein was not phosphorylated by kinase-inactive PKC␦. Collectively, the data permit consideration that the peptide GLKRNRYLSFHFK has the potential value for experimental/ therapeutic/clinical evaluation of PKC␦ activity.
Dissection of the substrate peptide composition resulted in an unexpected finding that the two related peptides, KRNR-YLSF and SFHFKSGSL, were potent inhibitors in cells toward both PMA-activated PKC␦ and a constitutively active pseudosubstrate-deleted mutant protein. The SFHFKSGSL peptide is suggested to be an inhibitor of PKC␦ binding to hBVR, which is in line with the site of action proposed above. Notably, the SFH-FKSGSL peptide, to a certain extent, resembles the PKC␦ translocation inhibitor SFNSYELGSL (46). KRNRYLSF did not cause morphological disruption of the cell membrane integrity and did not affect the membrane translocation of PKC␦. Because the SFHFKSGSL peptide promotes a morphological change in the membrane, visualized as blebbing, it is likely that inhibition of the PKC␦ activity is, in part, a manifestation of disrupted cell integrity. Blebbing is an early event in apoptosis (62). Accordingly, the decreased activity of the kinase in cells in the presence of this peptide may, in part, be a consequence of the onset of apoptosis. The effects appear to be specific to cell culture conditions with the combination of PKC␦/PMA/SFHFKSGSL, as it was neither observed in cells expressing hBVR and treated with PMA together with the peptide nor with the combination of PKC␦/PMA/KRNRYLSF or 275 KKRILHC. We propose the following chain of events underlies the SFHFKSGSL peptide-mediated membrane blebbing. In response to prolonged treatment with PMA or oxidative stress caused by ionizing radiation, activated PKC␦ can be cleaved, presumably by caspase-3, and the released catalytic domain is translocated to the nucleus (21,63), although there is a lag of several hours between treatment and PKC␦ cleavage. Nuclear PKC␦ phosphorylates and thereby inactivates both the DNA damage checkpoint protein hRad9 and DNA-dependent pro-tein kinase; the latter is essential for double strand break repair (64,65). In addition, PKC␦-dependent phosphorylation of lamin-B initiates its degradation and thus compromises the integrity of the nucleus (66). The chain of events is depicted in Fig. 8. The short term exposure of PKC␦ to SFHFKSGSL prior to PMA may predispose the cell to processes that trigger caspase-3 cleavage of the PKC.
The transactivation of hBVR and PKC␦ in the cell signaling network and pathophysiological conditions that are associated with disorders of PKC␦ activity are likely to be of biological relevance. The significance of hBVR phosphorylation by PKC␦ can be viewed in the context of its role in the cellular defense mechanisms against free radicals. As noted above, activation of hBVR can significantly influence those cellular functions that extend beyond its role in the cellular defense mechanisms (5,6,12,14,25,42,67).
Furthermore, it is not unreasonable to ponder whether in PKC␦ deficiency-related disorders, such as the autoimmune disease lupus (68), there is an associated defect in hBVR expression. However, there are those instances in which excessive activation and expression of PKC␦ result in the undesirable outcome of sustaining cell survival in certain types of tumors, such as non-small cell lung cancer cells, by promoting chemotherapeutic resistance (69). Another example is human breast tumor cells, in which the PKC functions as a survival factor (69,70). Clearly, in such instances, a therapeutic approach based on blunting PKC␦ activity would be expedient. The cell morphology nondisruptive hBVR-based inhibitory peptide may be a good candidate for this purpose. The peptides offer an intriguing possibility of their application to initiate cell death or to halt cell growth, the outcomes that are sought in the treatment of cancer and inhibiting tumorigenesis. The previous findings that hBVR and biliverdin activate and inhibit NF-B, respectively (71), together with the recently reported observation that sih-BVR is a highly effective inhibitor of PKC␦ transcriptional activation of NF-B as well as Elk1 (7), are supportive of the potential applicability of hBVR-based therapeutics in blunting PKC␦ activity. This assertion is further supported by the finding that biliverdin and the hBVR-based peptides, at low concentrations (2-10 M), were very effective inhibitors of PKC␦. The inhibitory action of biliverdin on PKC␦, which we show here to be due to noncovalent interaction between the bile pigment and the PKC, can be distinguished from its function as a chromophore in bacterial phytochromes, such as those of A. tumefaciens and P. aeruginosa (44,47). The observed noncovalent association would lend itself more to regulatory significance by being a reversible event. Hence, the inhibition could be reversed by activated hBVR. Collectively, the findings of this study and previously published observations allow us to postulate the occurrence of a regulatory loop between hBVR and PKC␦ with opposing effects on cell survival and apoptosis (Fig. 8). In cells stimulated with PMA, insulin/IGF-1, or TNF-␣, PKC and hBVR transactivate; and activation of hBVR mediates a twopronged process, removal of the inhibitory biliverdin and HO-1 gene expression (25,33). hBVR also stabilizes the HO-2 mRNA and protein, in the latter case by preventing proteasomal degradation (32). Because there are overlaps in the type of stimuli and downstream targets of hBVR and PKC␦, it is likely that the hBVR and Its Fragments Regulate PKC␦ Activity JULY 13, 2012 • VOLUME 287 • NUMBER 29 outcome of their transactivation transcends their individual signaling activities. Observations with the inhibitory peptides permit the suggestion that the two peptides, particularly KRN-RYLSF, potentially could be useful for development of a new generation of PKC␦ inhibitors. It is also reasonable to suggest that both inhibitory peptides might disrupt substrate binding by blocking the access of the substrate to the catalytic site, a variation on the manner by which the pseudosubstrate hinders PKC kinase activity (52). The ability of the hBVR-based peptides to inhibit PKC␦ activity is not unprecedented, as inhibitor peptides targeting other regions of PKC␦ have been characterized (56). However, what is unique to the presently identified inhibitory peptides is that they were not experimentally designed in the laboratory (52, 61), rather are integral segments of the interacting protein hBVR.