INTRODUCTION

Protein kinase C (PKC)¹ family members play crucial roles in the signal transduction of a variety of extracellular stimuli, such as hormones and growth factors (1). To date, twelve isozymes of PKC have been identified in mammalian tissues and subdivided into conventional PKC (cPKC) members comprising , I, II, and  isoforms (activated by calcium, acidic phospholipid, and diacylglycerol (DAG)), novel PKCs (nPKC) comprising , , , and  (activated by DAG and acidic phospholipid but insensitive to calcium), and atypical PKCs (aPKC) / and  (mechanism of regulation not clear) (1, 2, 3, 4, 5, 6). Another subgroup of PKCs may be defined by PKCµ, which has a potential signal peptide and transmembrane domain (7). Since these PKC isozymes differ in their expression in different tissues and in their mode of activation (1), each isozyme may play some specific role in signal transduction processes. Recent investigations using various approaches such as overexpression and down-regulation of specific isozymes support this idea (1, 5).

A large number of proteins have been shown to be phosphorylated by PKC in vivo and in vitro, such as growth factor receptors, ion channels, ion pumps, transcription factors, and translation factors (1, 8). Based on the sequences of the phosphorylated sites and the use of synthetic peptides based on these sites, a consensus phosphorylation site motif for PKC was determined to be RXXS/TXRX, where X indicates any amino acid (8). However, the optimal substrates have not been investigated by peptide library approaches, and relatively little information is available about differences in substrate selectivity between individual PKC isoforms. Histone IIIS, myelin basic protein, protamine, and protamine sulfate, which contain the above consensus phosphorylation site motif, are known to be efficient substrates for cPKCs, but poor substrates of the nPKC group (1). Recently, elongation factor eEF-1 was shown to be phosphorylated with much greater efficiency by nPKC than by cPKCs, nPKC or -, or aPKC (9). Heterogeneous ribonucleoprotein A1 is efficiently phosphorylated by PKC but not by cPKCs or PKC (10). These findings suggest that the substrate specificity of each PKC isozyme is quite different.

We have developed a new technique for determining the substrate specificity of protein kinases, using an oriented library of more than 2.5 billion peptide substrates (11, 12, 13). In this approach, the consensus sequence of optimal substrates is determined by sequencing the mixture of products generated during a brief reaction with the kinase of interest. This technique predicts an optimal sequence and provides information about the relative importance of each position for selectivity. Here we have used this approach to determine optimal peptide substrates for human PKC, -I, -II, -, -, -, -, -, and -µ and found that each PKC isozyme has a unique optimal substrate sequence. Furthermore, we prepared synthetic peptides based on the predicted optimal sequences for PKC, -I, -, -, and -µ and showed that these peptides are high affinity and selective substrates for the respective PKC isozymes.

EXPERIMENTAL PROCEDURES

DAG was purchased from Boehringer Mannheim. PS was purchased from Avanti Polar Lipids. Isozyme-selective antipeptide antibodies for PKC, -I, -II, -, -, -, -, and - were purchased from Santa Cruz Biotechnology, Inc. PKC substrate peptide (PRKRQGSVRRRV) was purchased from Upstate Biotechnology Inc. Anti c-myc antibody was purchased from Oncogene Sciences. P81 phosphocellulose paper was purchased from Whatman. [-³²P]ATP (3000Ci/mmol) was obtained from DuPont NEN. Liquiscint was purchased from National Diagnostics. A ferric iminodiacetic acid (IDA) beads was purchased from Pierce. All other chemicals were obtained from Sigma. Synthesis of the degenerate peptide library was accomplished according to the standard 1-benzotriazolyloxy-tris-dimethylamino-phosphonium hexafluorophosphate (BOP/HOBt) N-hydroxybenzotriazole coupling protocols using Peptide BioSynthesizer (Minipore Model) as described previously (12).

The full-length cDNAs for human PKC, -I, -II, -, -, -, -, and - were subcloned into baculovirus transfer vectors as described previously (6). Recombinant baculoviruses were produced by co-infecting Spondoptera frugiperda cells (Sf9 cells) with the purified baculovirus transfer vectors and purified genomic AcNPV viral DNA using established protocols (14). Recombinant baculovirus encoding c-myc tagged PKCµ and anti-PKCµ monoclonal antibodies was prepared as described previously (7, 15). Baculoviruses expressing each of the nine different PKC isozymes were confirmed using PKC activity screens and immunoblotting with isozyme-selective antipeptide antibodies. Purification of PKC, -I, -II, -, -, and - were performed by several steps of column chromatography as described previously (16, 17). PKC, -, and -µ were isolated by immunoprecipitation using isozyme-selective antipeptide antibodies (for PKC and -) and anti-c-myc antibody (for PKCµ), respectively. 1 unit of PKC, -I, -II, -, -, -, -, or - is defined as the amount of kinase required to incorporate 1 nmol of phosphate into the -pseudosubstrate peptide per min. 1 unit of PKCµ is defined as the amount of kinase required to incorporate 1 nmol of phosphate into the µ-peptide (see Fig. 2) per min.

Comparison of the phosphorylation efficiency of synthetic peptides by PKC and PKCµ.

Each PKC isozyme (0.2-0.5 units) was added to 300 µl of solution containing 1 mg of degenerate peptide mixture, 100 µM ATP with a trace of [-³²P]ATP (roughly 6 × 10⁵ cpm), 1 mM DTT, 10 mM MgCl₂, 50 mM Tris-HCl (pH 7.5), 20 µg/ml phosphatidylserine (PS), 10 µM DAG, 200 µM CaCl₂ (for PKC, -I, -II, and -), and 0.5 mM EGTA (for PKC, -, -, -, and -µ). Reactions were started by addition of PKC and incubated at 30 °C. Reaction conditions were adjusted to allow phosphorylation of about 1% of the total peptide mixture. After reaction, peptide separation was performed as described previously (12). Briefly, the peptide supernatant was removed and diluted with 300 µl of 30% acetic acid. This mixture was then added to a 1-ml DEAE column previously equilibrated with 30% acetic acid, and the column was eluted with 30% acetic acid. After the 600-µl void volume, the next 1 ml contained both phosphorylated and non-phosphorylated peptides but was free of [-³²P]ATP. A 0.5-ml column of ferric iminodiacetic acid beads was charged with 2.5 ml of 20 mM ferric chloride, washed with 4 ml of water, then washed with 3 ml of 500 mM NH₄HCO₃ (pH 8.0), washed again with 3 ml of water, and then equilibrated with 3 ml of buffer A (50 mM MES, 1 M NaCl (pH 5.5)). The dried sample of peptide/phosphopeptide mixture was dissolved in 200 µl of buffer A and loaded onto the ferric column. The column was then eluted with 2.5 ml of buffer A followed by 2.5 ml of buffer B (2 mM MES (pH 6.0)). The phosphopeptides were then eluted with 2 ml of 500 mM NH₄HCO₃ (pH 8.0). Control experiments, in which the peptides were subjected to a mock phosphorylation, were conducted. The same column protocol was used and the fractions in which phosphopeptides usually elute were collected.

Typically, 1-2 nmol of phosphopeptide mixture was added to the sequencer. The data analysis was performed as described previously (12). Briefly, the abundance of each amino acid at a given cycle in the sequence of the phosphopeptide mixture from the mock phosphorylation experiments was subtracted from the kinase experiments to correct for the background. To calculate the relative preference for amino acids at each degenerate position, the corrected data were then compared with the starting mixture to calculate the ratios of abundance of amino acids. The sum of the abundance of each amino acid at a given cycle was normalized to 14, 15, or 16 (the number of amino acids present at the degenerate positions) so that each amino acid would have a value of 1 in the absence of selectivity at a particular position.

PKC activity was assayed in vitro essentially as described previously using the standard PKC vesicle assay (16, 17). The reaction mixture (30 µl) contained 100 µM ATP with [-³²P]ATP (5 µCi), 1 mM DTT, 5 mM MgCl₂, 25 mM Tris-HCl (pH 7.5), 20 µg/ml PS, 10 µM DAG, 200 µM CaCl₂ (for PKC, -I, -II, and -), 0.5 mM EGTA (for PKC, -, -, -, and -µ) and indicated amount of synthetic substrate peptide. Reactions were started by addition of PKC (0.002-0.005 units) and incubated at 30 °C for 10 min. Reaction mixtures were spotted onto P81 phosphocellulose paper and washed 4 times in 500 ml of 1% phosphoric acid. Incorporation of ³²P was determined by liquid scintillation counting. For each experimental condition, values for control reactions lacking substrate peptide were subtracted as blanks. In all assays to determine K_m and V_max, reaction rates were linear with respect to time for all conditions of peptide, and less than 10% of the peptide substrate was phosphorylated.

RESULTS

In order to determine optimal substrate sequences for each of nine human PKC isozymes (, I, II, , , , , µ, and ), we used a degenerate peptide library, comprising peptides of sequence: MAXXXXRXXSXXXXXAKKK (RS-peptide library), where X indicates all amino acids except Trp, Cys, Ser, or Thr. Trp and Cys were omitted to avoid problems with sequencing and oxidation, whereas Ser and Thr were omitted to ensure that the only potential site of phosphorylation was the Ser at position 10. The Met-Ala sequence at the amino terminus was included to verify that peptides from this mixture are being sequenced and to quantify the peptides present. Ala at position 16 provides an estimate of how much peptide loss has occurred during sequencing. The poly(Lys) tail prevents wash-out during sequencing and improves the solubility of the mixture. Arg was "locked-in" at position 7 since previous studies had shown the importance of Arg at the p3 position for PKC substrates (8). The library was sequenced, and all 16 amino acids were present at similar amounts at all 11 degenerate positions (data not shown). Another Ser-kinase substrate library (12), comprising peptides of sequence MAXXXXSXXXXAKKK, was also used to investigate the 9 PKC isozymes, and was poorly phosphorylated compared with the RS-peptide library confirming the importance of Arg at the p3 position.

The RS-peptide library was incubated with each PKC isozyme under conditions in which approximately 1% of the total peptide mixture was phosphorylated. The phosphopeptide products were separated from non-phosphorylated peptides using the ferric-iminodiacetic acid column, and the mixture was sequenced. In Fig. 1, the relative abundance of amino acids at each of the 11 positions of degeneracy are presented from experiments using PKC and -µ. These two enzymes clearly selected for distinct peptide substrates. PKC preferred peptides with Arg at p5 and p4, while PKCµ selected peptides with Leu and Val, respectively, at p5 and p4. In fact, PKCµ had an extremely strong selectivity for peptides with Leu at position p5. More than 40% of the phosphopeptide products of PKCµ had Leu at this position. At the p2 position, both PKCs selected peptides with hydrophilic residues, with Gln and Lys preferred. PKC selected for peptides with Gly at p1, while PKCµ selected against peptides with Gly at p1. Both PKCs preferred peptides with hydrophobic amino acids at p+1, though PKC selected Phe while PKCµ selected Val. At p+2, p+3, and p+4 positions, PKC strongly selected for peptides with the basic amino acids Arg or Lys. In contrast, PKCµ preferred peptides with hydrophobic amino acids in both positions.

Comparison of the substrate specificities of PKC and PKCµ. Human PKC and -µ were expressed in Sf9 cells using baculovirus. A degenerated substrate library with the sequence Met-Ala-X-X-X-X-Arg-X-X-Ser-X-X-X-X-X-Ala-Lys-Lys-Lys (where X indicates any amino acid apart from Trp, Cys, Ser, or Thr) was presented to PKC and -µ. Each PKC isozyme was added to 300 µl of solution containing 1 mg of degenerate peptide mixture, 100 µM ATP with a trace of [-³²P]ATP (roughly 6 × 10⁵ cpm), 1 mM DTT, 10 mM MgCl₂, 50 mM Tris-HCl (pH 7.5), 20 µg/ml PS, 10 µM DAG, 200 µM CaCl₂ (for PKC), and 0.5 mM EGTA (for PKCµ). After 2 h at 30 °C, the phosphopeptides were separated on DEAE-Sephacel and a ferric chelation column. Reaction conditions were adjusted to allow phosphorylation of about 1% of the total peptide mixture. The phosphopeptides mixture was sequenced. Each panel indicates the relative abundance of the 15 (position 5 to 2 N-terminal to the phosphorylated Ser) or 16 (position 1 to +3 C-terminal to the Ser) amino acids at a given cycle of sequencing. The signal of Gly at position 7 to 2 and the signal of Ile for PKCµ at position +2 to +5 were not available because of a high background. The columns represent average values from two independent experiments. The one-letter amino acid code is used: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; V, Val; Y, Tyr.

The results obtained for other PKC isozymes are summarized in Table I. All PKC isozymes selected for peptides with hydrophobic amino acid at p+1 position, and all isozymes except PKCµ selected for peptides with basic amino acid at the p6, p4 and p2 positions. Interestingly, all PKC isozymes selected for peptide substrates with Gln or Glu at the p2 position, although in most cases Lys at this position was optimal. PKC, -I, -II, -, and - selected for substrates with basic amino acid at positions p+2, p+3, and p+4, but PKC, -, -, and -µ preferred substrates with hydrophobic amino acid in these regions. At p5, the selectivity was quite different among these isozymes. PKC, -, and - selected peptides with Arg; in contrast PKCII, -, and -µ selected for peptides with hydrophobic amino acids such as Phe, Leu, or Val. PKCI and - preferred substrates with either Leu or Arg.

Table I. Substrate specificities of protein kinase C isozymes Values in parentheses indicate the relative selectivities for the amino acids; amino acids with values less than 1.5 are omitted. Bold letters indicate amino acids that are strongly selected; X indicates no selectivity. The one-letter amino acid code is used. All human PKC isozymes were expressed in Sf9 cells using baculovirus. A kinase substrate library with the sequence Met-Ala-X-X-X-X-Arg-X-X-Ser-X-X-X-X-X-Ala-Lys-Lys-Lys (where X indicates any amino acid apart from Trp, Cys, Ser or Thr) was presented to each PKC isozyme. The kinase reaction was performed as described in the legend for Fig. 1. Each PKC isozyme was evaluated at least twice; average values are shown. PKC isozymes Position  7  6  5  4  3  2  1 0 +1 +2 +3 +4 +5 PKC R(2.1) R/F(1.5) R(2.1) R(2.1) R K(2.2) G(2.0) S F(2.6) R/K(3.4) R/K(3.0) K(2.3) A(1.8) L/F(1.7) F(1.6) Q(2.0) A/M(1.7) M/I/L(1.8) R(1.9) K(1.7) R(1.8) V (1.5) PKCI R/K/F(1.5) K(1.8) L/R(2.1) K/R(2.3) R K(2.7) G(2.0) S F(3.1) K(2.5) K(2.0) F(1.8) A(2.3) R(1.6) F(2.0) F(1.5) Q(2.2) A(1.9) M(1.8) R(1.9) F/R(1.7) V(1.6) F(1.8) R/A(1.7) K(1.8) I/V(1.6) F(1.8) PKCII Y(1.8) K(1.6) L(1.9) K(1.9) R K(2.7) G(2.1) S F(3.0) K(3.0) K(2.6) K(1.9) A(1.7) F(1.8) R(1.6) Q(2.0) K(1.8) M(2.1) R(2.0) Y(1.7) F(1.5) F/K(1.6) E(1.5) M(1.7) L(1.8) F/M(1.5) F/R(1.5) M(1.5) PKC R(1.9) R(1.9) R(2.7) R(3.5) R K(3.5) G(2.7) S F(2.1) K(2.8) R(3.4) K(2.0) A(2.1) K(1.6) F(1.5) K(1.6) R(1.9) K(2.4) K(1.9) R(2.3) K(2.3) R(1.9) K(1.6) Q(1.8) A(1.6) M(1.5) R(1.5) PKC A(1.6) A/R(1.7) R(2.1) K/A(1.7) R K(2.1) G(2.6) S F(2.4) F(1.8) Y/F(1.5) G(1.9) G(2.2) K(1.6) E(1.9) A(1.7) M/V(1.8) K/M(1.6) F(1.6) F(1.8) Q(1.5) K(1.5) V(1.5) PKC Y(1.9) Y/K(1.5) X K(1.7) R K(3.1) M(1.7) S F(1.9) F/A(1.7) E(1.7) F/E(1.5) D/E/F(1.5) E(1.6) A/Q(1.5) E(2.7) K(1.6) I/M(1.7) Y/D(1.6) R(2.1) V/L(1.6) F(1.5) D(1.9) PKC A(2.1) R(2.0) L/R(1.9) R(1.9) R R(2.2) R(2.3) S F(2.4) R(2.0) R(2.1) X R(1.6) R(1.8) A(1.6) K(1.5) K(2.0) G(1.8) R(1.9) Y(1.9) Y(1.6) Q(1.9) M(1.5) F(1.8) PKC R(1.8) R/K(1.6) F(2.5) K(1.8) R Q/K(1.8) G(1.7) S F(2.6) F(2.1) Y/F(1.8) F(1.9) F(1.7) L/R(1.6) F(1.6) Y(1.6) K/M/Y(1.5) M(2.0) M(2.0) K(1.7) Y/K(1.6) K/Y(1.5) A(1.5) PKCµ A(1.9) A/K(1.7) L(6.2) V(2.2) R Q(2.4) M(2.2) S V(2.0) A/M(1.7) F(1.8) F(2.2) F(1.9) Y(1.5) P(1.5) V(3.3) L(2.1) K(2.1) L/K(1.7) M(1.7) V(1.5) Y(1.5) V(2.0) P(1.6) A(2.0) E(2.0) L(1.6) A(1.6) M(1.5) M(1.8)

In Table II, the predicted optimal sequence for each PKC isozyme was compared with the pseudosubstrate region of the respective isozyme by lining up the pseudosubstrate Ala with the Ser of the substrate. The amino acid at p3 in all the pseudosubstrate sequences was Arg, consistent with our observation that peptides with Arg at p3 are preferentially phosphorylated. Furthermore, hydrophobic amino acids are present at the p+1 position in all the pseudosubstrate sequences except that of PKC. The predicted optimal sequences from p3 to p+2 for PKC, -I, -II, -, -, and - were in good agreement with pseudosubstrate sequences of the corresponding PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides.

Table II. Comparison of the optimal sequence of each PKC isozyme determined by the peptide library with the pseudosubstrate region of each isozyme The optimal sequence of each PKC isozyme determined by the peptide library (optimal.) and the pseudosubstrate region of each isozyme (pseudo.) are presented by the one-letter codes, respectively. Bold letters indicate the phosphorylated Ser in the optimal sequence and the corresponding Ala in the pseudosubstrate region. Boxed amino acids emphasize positions that are similar between optimal sequence and pseudosubstrate region. PKC isozymes Position  7  6  5  4  3  2  1 0 1 2 3 4 5 PKC   optimal. R R R R R K G S F R R K A   pseudo. N R F A R K G A L R Q K N PKCI F K L K R K G S F K K F A V R F A R K G A L R Q K N PKCII Y K L K R K G S F K K K A V R F A R K G A L R Q K N PKC R R R R R K G S F K R K A P L F C R K G A L R Q K V PKC A R R K R K G S F F Y G G P T M N R R G A I K Q A K PKC Y Y X K R K M S F F E F F R P R K R Q G A V R R R V PKC A R R R R R R S F R R X R F T R K R Q R A M R R R V PKC R R F K R Q G S F F Y F F K S I Y R R G A R R W R K

The optimal substrates predicted for the various PKC isozymes are in good agreement with known substrates. In Table III, the optimal substrate sequence of PKC is compared with known PKC substrates, most of which were determined to be PKC substrates using cPKCs or partially purified PKC isozyme mixtures (probably mixtures of PKC, -, and -). Most of these proteins have the motif R/K-X-R/K-R/K-X-S/T--R/K-R/K, where  indicates hydrophobic amino acids (F, L, V). This motif is in agreement with the predicted optimal peptide from the peptide library experiment. The strongest selectivities from the library were for hydrophobic amino acids at p+1 and R/K at p+2, and almost every protein substrate has these characteristics. As mentioned above, the presence of Arg at the p3 position is important for PKC substrates. However, some known substrates do not have Arg at this position (Table III). Using another library, comprising MAXXXXSXXXXAKKK, we found that PKC, -I, and - strongly selected for peptides with Arg at p3 (selectivity values 6.0, 4.5, and 6.5, respectively), followed by His (1.9, 1.9, and 1.7) and Lys (1.3, 2.8, and 1.4) (data not shown). Thus, although Arg is preferred, substrates with His or Lys at p3 are also selected. It is also expected that peptides lacking a basic amino acid at p3 but with optimal amino acids at the other critical positions could still be reasonable substrates. Recently, a few proteins has been shown to be isozyme-specific substrates. eEF-1 is reported to be a specific substrate for PKC (9). The sequence of 426-436 from murine eEF-1 containing Thr-431 was compared with the predicted optimal substrate of PKC (Table III). PKC strongly selects for substrates with Arg at p5, basic at p2, hydrophobic at p+1, and Gly at p+4. The site in eEF-1 meets these criteria. The failure of cPKCs to phosphorylate this site could be explained by the lack of basic residues at p+2 and p+3. PKCµ is shown to phosphorylate glycogen synthase-derived peptide (15) much better than MARCKS, which is known to be a good substrate for other PKC isozymes (18, 19). The glycogen synthase-derived peptide sequence is in good agreement with the optimal peptide sequence that we predicted for PKCµ. Hydrophobic amino acids are found at positions 1 and +1 to +5, Arg is at 3, and most importantly, Leu is at position 5, the position at which PKC shows greatest selectivity (Table III). None of the PKC phosphorylation sites in the MARCKS protein have Leu at the p5 position (Table III), explaining why MARCKS is not a good substrate for PKCµ.

Table III. Comparison of the optimal sequence of each PKC isozyme determined by the peptide library with sequences at the same regions of known PKC substrates Amino acids are represented by the one-letter code. Positions that showed great selectivity for each PKC are indicated by an asterisk. Peptide/protein position  5  4  3  2  1 0 +1 +2 +3 +4 +5 * * * * PKC optimal seq. R R R K G S F R R K A K K   MARCKS proteina K K K R F S F K K S F   MARCKS proteina F S F K K S F K L S G   MARCKS proteina K L S G F S F K K N K   Neuromodulina A K I Q A S F R G H I   GABA type A receptor 2Lb L L R M F S F K A P T   EGF receptora I V R K A T L R R L L   Ribosomal protein S6a R R R L S S L R A S T   PTP1Bc R V V G G S L R G A Q   Troponin Id K F K R P T L R R V R   Insulin receptor tyrosine kinasee N G R I L T L P R S N   P-glycoproteinf R S T R R S V R G S Q   P-glycoproteinf L I R K R S T R R S V   Kit/SCFRg A D K R R S V R I G S   Annexin IIa P S A Y G S V K P Y T * * * * * * * PKC optimal seq. R K R K G S F F Y G G   Elongation factor-1h R D M R Q T V A V G V * * * * * * * PKCµ optimal seq. L V R Q M S V A F F F   GS-peptidei L S R T L S V A A L L a  From Ref. 8. b  From Ref. 26. c  From Ref. 27. d  From Ref. 28. e  From Ref. 29. f  From Ref. 30. g  From Ref. 31. h  From Ref. 9. i  From Ref. 15.

The predicted optimal peptide substrates for PKC, PKCI, PKC, PKC, and PKCµ were synthesized (Fig. 2A) and investigated as substrates of the various PKC isozymes. For comparison, a commercially-available PKC substrate peptide based on the pseudosubstrate region of PKC was used to assay the various PKC isozymes. A set of experiments using 100 µM of the various peptides as substrates of PKC and PKCµ are presented in Fig. 2, B and C. The V_max and K_m values determined for each of the peptides with each of the PKC isozymes are summarized in Table IV. It is clear from Fig. 2 and Table IV that different PKC family members selectively phosphorylate different subsets of the peptides. The optimal substrates for PKC, PKCI, PKC, and PKCµ (as judged by V_max/K_m ratios) were the -peptide, I-peptide, -peptide, and µ-peptide respectively, in agreement with the library predictions. Curiously, the -peptide was not the optimal peptide for PKC as judged by V_max/K_m ratio, although it was the lowest K_m substrate for PKC. In fact, the predicted optimal peptide substrate for each PKC was the lowest K_m substrate for that PKC, except in the case of PKC where the I and -peptides had slightly lower K_m values (2.7 µM and 2.8 µM, respectively) than the -peptide (3.8 µM). These results are in agreement with previous results with the peptide library approach that have indicated that this technique selects substrates on the basis of low K_m and/or high V_max/K_m ratios rather than on the basis of V_max alone (11, 12, 13).

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