154

Biochimica et Biophysica Acta, 1132 (1992) 154-160 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

BBAEXP 92412

Sequence and expression of human protein kinase C-e Patricia Basta, Mary Beth Strickland, William Holmes, Carson R. Loomis, Lawrence M. Ballas and David J. Burns Sphinx Pharmaceuticals Corporation, Molecular Biology Section, Durham, NC (USA) (Received 1 May 1992)

Key words: Protein kinase C-E; Phorbol ester; Gene expression; Catalytic domain; Cloning

Two human homologues of protein kinase C-• (El and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-• cDNAs from several species indicated that each of these human • clones contained cloning artifacts. Thus, a composite PKC-• (E3) clone was derived from clones E1 and E2. Human PKC-• (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human • sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-• clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct •-specific polyclonal antibodies.

Introduction

Materials and Methods

Protein kinase C (PKC), initially isolated by Nishizuka and colleagues is a major cellular regulatory enzyme [1]. It has been implicated in a variety of responses including secretion, modulation of ion conductance, gene expression, proliferation and tumor promotion [2,3]. It is defined as a phospholipiddependent, diacylglycerol or phorbol ester activated, serine/threonine protein kinase, and is ubiquitously expressed in a host of species and tissues [1-4]. Molecular cloning analyses of tissue from a number of mammalian sources have identified eight structurally related members of the protein kinase C family [2-4]. These eight members are derived from both multiple genes and from alternative splicing events [1-4]. In this manuscript, we report on the isolation, expression and characterization of two human protein kinase C-e clones.

Cloning of nucleic acid sequence encoding human PKC-e AZap human temporal cortex, and frontal cortex, cDNA libraries, the helper phage R408 and Escherichia coli strains BB4 and XL-1 blue were all purchased from Stratagene (La Jolla, CA) Oligonucleotides based on the V1 (PKC-e) and regions of the previously published rat PKC-e sequence [6] were purchased from Midland Certified Reagent Co. (Midland, TX) or prepared as needed using phosphoramidite chemistry on an Applied Biosystems (Foster City, CA) Model 381A DNA synthesizer.

Correspondence to: D. Burns, Sphinx Pharmaceuticals Corporation, Molecular Biology Section, P.O. Box 52330, Durham, NC 27717, USA. The human PKC-E sequence has been sent to the EMBL Data Library: accession number X65293 H. sapiens mRNA for protein kinase C-E. Abbreviations: PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; Ps, phosphatidylserine; DG, diacylglycerol.

Isolation of human PKC-e cDNA clones E1 and E2 A human temporal cortex cDNA library (Stragatene, La Jolla, CA) was screened as described below with the V 1 (PKC-E) oligonucleotide probe (5'CTGTGGGCTTCAAGCTCACGGCCTCGCAGATTTTGATCTTAAGAA-3'). Duplicate plaque lifts of a total of approx. 120000 plaques were made onto nitrocellulose circles which were then denatured in 1.5 M NaCI, 0.5 M NaOH, neutralized in 1.5 M NaCl, 0.5 M Tris and finally rinsed in 0.2 M Tris, 2 × SSC. The filters were then baked under vacuum at 80°C for 1 - 1 / 2 h. The filters were then prehybridized in 0.1 M NaCI, 0.1 M sodium citrate, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 0.2% ficoll, 0.02 M NaH2P Q , 0.5% SDS and 0.5 m g / m l sonicated salmon

155 sperm DNA at 42°C for 4 h. Hybridization was performed in 0.1 M NaC1, 0.1 M sodium citrate, 0.02 M NaHzH4, 0.4 mg/ml sonicated salmon sperm with 8 • 105 cpm/ml of the oligonucleotide at 42°C overnight. Filters were washed the following day in a solution containing 0.1 M NaC1, 0.1 sodium citrate, and 0.1% SDS. Washing was performed until only background counts remained. Each wash was for 15 min and the temperature of the washes started at 42°C. At each successive wash the temperature was increased by 3°C. The final wash temperature was 52°C. These filters were then placed between plastic wrap and exposed to film (Kodak XAR) with a lightening plus intensifying screen, at - 70°C for 3 days. Eleven potentially positive plaques were obtained from this primary screen. These plaques were then replated and rescreened as secondaries with the same oligonucleotide probe. Results from the secondary screen indicated that one out of the eleven initial primaries was positive. This clone was then designated as E2. Human clone E1 was obtained by screening a human brain frontal cortex library (Stratagene, La Jolla, CA) with the human E2 clone under the following hybridization conditions. Prehybridization and hybridization was performed in 50% formamide, 0.1% bovine serum albumin (BSA), 0.1% fieoll, 0.1% polyvinylpyrrolidonel 0.75 M NaC1, 0.025 M NaHzPO 4 + 7H20 , 0.005 M EDTA, 0.1% sodium dodecyl sulfate (SDS) and 100 ~ g / m l salmon sperm at 42°C, either in the presence or absence of 32p-labeled clone E2.

Analysis of human PKC-• clones After isolation of the A clones E2 and El, the E2 and E1 plasmids were excised out of the A vector in vivo and converted to a Bluescript plasmid (pBluescriptI, Stratagene, La Jolla, CA). The E. coli strain XL-1 blue (Stratagene, La Jolla, CA) was coinfected with both the appropriate phage lysate and R408 helper phage (Stratagene, La Jolla, CA). This rescued DNA was then used to make double-stranded DNA by transforming fresh XL-1 E. coli cells. A large scale plasmid preparation was prepared and the DNA obtained was used as template for sequencing and restriction enzyme analyses. Clone E1 contained an insert of 61 basepairs 6f non-PKC DNA beginning at nucleotide position 2082. Clone E2 contained two basepair differences between clone E1 and the previously characterized mouse, rat and rabbit PKC-• clones. Thus, a spliced construct was made to eliminate these cloning artifacts. The spliced product, designated clone E3, was created using the PKC-• clones E1 and E2 as templates. This was accomplished by ligating together the 1.9 kilobase N c o I / HindlII fragment of E1 to the 0.25 kilobase HindIII/EcoRI fragment of E2. An EcoRI/NheI adapter was then prepared to reincorporate the 44

basepairs of • sequence eliminated by the EcoRI digestion (see below).

Construction and isolation of recombinant PKC-E baculot~iruses The cDNA clones for human PKC-E (clones El, E2, and E3) were inserted into the NheI site of the BlueBac baculovirus transfer vector (Invitrogen, San Diego, CA). For the insertion of clones E2 and E3 into BlueBac, an NcoI-EcoRI fragment encoding 723 amino acids for both the E2 and E3 subspecies was isolated from the corresponding pBluescriptI plasmids previously described. NheI-NcoI adaptors (d(CTAGCCCGGGC))/(d(CATGGCCCGGG)) and Eco RI-Nhe I adaptors (d(AATTCAAAGGTTTCTCCTACTTTGGTGAAGACCTGATGCCCTGAGG))/(d(CTAGCTCAGGGCATCAGGTCTTCACCAAAGTAGGAGAAACCTTI'G)) coding for the additional PKC-E amino acids were used for insertion of the two PKC-E fragments into the single NheI site of the BlueBac vector. Separate transfer vectors were created for the E2 and E3 clones. An NcoI-EcoRI fragment was also isolated from clone El, and inserted into the BlueBac vector using the same adaptors. Transfer of genes coding for the various epsilon clones (El, E2, and E3) into Baculovirus genome was accomplished as previously described [16]. Expression and purification of recombinant PKC-E Protein from the various epsilon clones was produced in S. frugiperda cells (Sf9) infected with the appropriate recombinant baculoviruses. Sf9 cells were routinely harvested and characterized 60-72 h after infection. For purification, Sf9 cells containing recombinant PKC-E (E2 or E3 clones) were centrifuged, and the cell pellet was stored at -80°C prior to protein purification. The pellet was resuspended in 20 ml of homogenization buffer (20 mM Tris-HC1, pH 7.5, 0.25 M sucrose, 10 mM EGTA, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.02% leupeptin) and allowed to thaw on ice. The ceils were then homogenized in a glass homogenizer with a teflon pestle, and further disrupted by two 30 s bursts with a probe sonicator (Fisher) with a microtip used at 35% of maximum. The homogenate was then adjusted to pH 7.5 with 2 M Tris-HCl and centrifuged at 100000 × g for 60 min. The supernatant was applied to a DEAE anion exchange chromatography column (Millipore (MemSep chromatography cartridge), Bedford, MA) which had been previously equilibrated with Running Buffer (20 mM Tris-HC1, pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, 10 mM /3-mercaptoethanol). The chromatography column was run on a fast protein liquid chromatography (FPLC) system (Pharmacia LKB Biotechnology). The column was then washed with two column volumes of

156 Running Buffer, and eluted with an increasing salt gradient of 0 to 500 mM NaC1. Fractions that displayed protein kinase C activity (as determined by the protein kinase C assay described herein), or protein kinase C epsilon immunoreactivity were pooled together. The pooled fractions were then stabilized with a 0.05% Triton X-100/10% glycerol mixture and frozen at 80oc.

scribed [17]. The first rabbit injection utilized Freund's complete adjuvant, following by 3-5 bi-weekly booster injections using Freund's incomplete adjuvant.

Protein kinase C activily Protein kinase C activity was determined using the protein kinase C vesicle assay of Ogita et al. [18], with modifications as described herein. Briefly, a reaction mixture contained in a final volume of 250 /xl the following components (final concentrations): 40 ~ g / m l phosphatidylserine (Avanti Polar Lipids); 1.76 /zg/ml diacylglycerol (Avanti Polar Lipids); 47.5 /zM EGTA; 10 mM MgC12; 20 mM Hepes (pH 7.5); 200 ~zg/ml histone type HI or myelin basic protein (Worthington Biochemical and Sigma); 20/~M [3zp]ATP (New England Nuclear Research); and partially purified enzyme. This mixture was incubated for 10 min at 30°C, and filtered onto Whatman G F / C filters The filters were

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Antibody binding Two distinct PKC-e antibodies were prepared as follows: two peptides corresponding to the COOHterminus (NH~-CNQEEFKGFSYFGEDLMP-NHz) and the C1 region (Acetyl-SGEAPKDNEERC-NH2) of the human PKC-e were synthesized (Multiple Peptide Systems), coupled to Limulus polyphemus hemocyanin with m-maleimidobenzoic acid-N-hydroxysuccinimide ester (MBS), and injected into rabbits as de50

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Fig. 1. Complete nucleotide and amino acid sequence of human PKC-e. The complete nucleotide and amino acid sequences of the human protein kinase C-e E3 clone are shown. Amino acid residues number 475 and 532 are altered in human ~ clone E2 to a Tyr and His, respectively. The two nucleotide differences between the E3 and E2 clones are at positions 1439 (G (E3) -~ A (E2)) and 1609 (G (E3) ~ C (E2)).

157 washed with 10 ml 10% trichloroacetic acid and placed in glass scintillation vials containing 8 ml scintillation fluid. The vials were then counted in a scintillation counter (model 1209 liquid scintillation counter, LKB). For the no lipid control determinations, both phosphatidylserine and diacylglycerol were removed from the reaction mixture. A few modifications of the standard vesicle assay were required when a modified peptide ( N H 2E R M R P P K R Q G S V R R R V - C O O H ) was used as a substrate for PKC-E. The peptide is a modified peptide derived from the pseudosubstrate site of PKC-e. The peptide (200/~M final concentration) was added to the standard components of the protein kinase C vesicle assay, as described above, and incubated at 30°C for 10 min. A portion of the reaction mixture was spotted onto filters (P-81 filters, Whatman, Hillsboro, OR), and washed four times with 75 mM H3PO 4. The filters were then dried and counted as described above.

( E l ) contained an insert of non-PKC D N A (61 basepairs) between nucleotide positions 2082 and 2083 (Fig. 1). Translation of this clone would produce a truncated PKC-E protein that would be missing 44 amino acids from its carboxy-terminus. This was confirmed by expression of the E1 clone in Sf9 cells (data not shown). The second human E clone (E2) appeared to be a full-length clone based on sequence comparisons to the mouse, rat and rabbit PKC-E clones. A comparison of the sequences of both human PKC-e clones ( E l and E2) indicated only two basepair differences between the two clones (the 61 basepairs of non-PKC sequence f r o m clone E1 are not included in this comparison). These nucleotide differences are at positions 1439 (G ( E l ) ~ A (E2) and 1609 (G ( E l ) --* C (E2)) (Fig. 1) and translate into two amino acid differences at positions 475 (Cys ( E l ) ~ Tyr (E2)) and 532 (Asp ( E l ) ~ His (E2)). We believe that the two nucleotide changes present in the E2 clone represent cloning artifacts for

Phorbol binding assay Binding of [3H]phorbol 12,13-dibutyrate (PDBu) to PKC-E was measured according to the vesicle assay of Burns and Bell [16]. Briefly, for the vesicle assay a 100 /xl reaction mix containing 50 /zl of total insect cell homogenate containing recombinant PKC-E (E2 or E3 clones) and the following components (final concentration): 40 / z g / m l phosphatidylserine (Avanti Polar Lipids), 2 m M CaC12, 20 m M H e p e s (pH 7.5) and 100 nM [3H]PDBu (New England Nuclear Research) was incubated for 10 min at room temperature. 50/zl of the reaction mixture was spotted onto a W h a t m a n G F / C filter and washed with 5 ml of ice-cold wash buffer (wash buffer: 5 m M Hepes, p H 7.5 and 0.2 m M CaC12). The filter was then placed in a vial containing 10 ml scintillation fluid and counted on a scintillation counter. Nonspecific binding was determined in the presence of 2 0 / z M of unlabeled PDBu (Sigma).

E2

E3

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Results

Fig. 1 shows the nucleotide and amino acid sequence of the human PKC-E. This clone (E3) is a composite derived from two clones isolated from two distinct human brain c D N A libraries (see Materials and Methods). H u m a n PKC-E has an overall sequence identity (at the nucleotide level) of 9 0 - 9 2 % compared to the previously identified mouse, rat and rabbit clones [5-7]. At the amino acid level, the deduced human PKC-e sequence shows a 9 8 - 9 9 % identity with the mouse, rat and rabbit E sequences, respectively (Fig 1). Two human PKC-E clones were originally isolated from two independent c D N A libraries. Based on sequence comparisons to mouse, rat and rabbit PKC-c clones, it was apparent that both of these clones contained cloning artifacts. The first human epsilon clone

Fig. 2. Antibody characterization of the expressed PKC-E clones (E2 and E3). PKC-~ clones E2 and E3 were expressed in the insect cell-baculovirus expression system using established protocols [16]. Total extracts from E2 or E3 infected Sf9 cells or partially purified E2 and E3 protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting with Especific polyclonal antibodies. This is a representative Western blot using the E antibody derived from the COOH-terminus; a similar pattern was observed using the Cl-region PKC E antibody. EXTRACT E2 and EXTRACT E3, total extracts of Sf9 cells infected with either recombinant E2 or E3 baculoviruses. PEAK 1 AND PEAK 2, total extracts of Sf9 cells infected with either recombinant E2 or E3 baculoviruses separated into two distinct peaks by DEAE column chromatography (see Materials and Methods). MW STD, molecular weight standards (kDa).

158 the following reasons: (1) the mouse, rat, rabbit PKC-e clones, the E1 human clone, and most other PKC family members contain a cysteine residue at amino acid position 475 and an aspartic acid residue at position 532 [5-7]; (2) the E2 clone did not display functional protein kinase C activity when expressed in the baculovirus-insect cell expression system (see below). The in vivo significance of a catalytically inactive PKC family member is not readily apparent (see Discussion). Therefore, a complete human PKC-e clone (designated E3) was prepared by splicing together • clones, E1 and E2, to eliminate the cloning artifacts. Human PKC-• clones, E2 and E3 were both expressed in the baculovirus-insect cell expression system. When insect cells expressing the • clones were

40000

subjected to DEAE column chromatography, two distinct peaks of protein kinase C immunoreactivity were observed for both of the recombinant • proteins. Each of these peaks were recognized by two distinct e-specific antipeptide antibodies. Immunoblots of one such experiment are shown in Fig. 2. The observed • immunoreactive bands were approx. 96 and 93 kDa. These size differences most likely represent different phosphorylation states of PKC-e. Purified PKC-e from rat brain displayed two prominent bands at 96 and 93 kDa on silver stained SDS polyacrylamide gels [19]. Phosphatase treatment of this purified PKC-e resulted in the loss of the 96 and 93 kDa bands with the concomitant appearance of a 90 kDa band [19]. Thus, these experiments confirm that the two PKC-e bands proba-

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Fig. 3. Cofactor dependencies for PKC-E. The cofactor dependencies for human PKC-e were determined using the standard vesicle assay [18]. All components in the assay were kept constant except for the following: (A) the phosphatidylserine (PS) concentration was varied from 0-70 /~g/ml; (B) diacylglycerol (DO) concentrations ranged from 0 - 4 / z g / m l ; (C) [3zp]ATP concentrations were varied from 0-60 p.M; and (D) PKC activity was measured using increasing amounts of two separate substrates - histone ]]]-S (closed circles) or myelin basic protein (open circles).

159 TABLE I Lipid dependence of the recombinant proteins PKC E3 and PKC E2 in the absence or presence of lipid activators (phosphatidylserine and diacylglycerol) with three different substrates

E clone

cpm no lipid

cpm lipid

Fold increase

E3 substrate histone myelin basic protein peptide

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E2 substrate histone myelin basic protein peptide

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13000 11000 27000

0 0 1

bly represent different phosphorylation states of the enzyme. Extracts of insect cells expressing each of the human PKC epsilon proteins (E2 and E3) displayed [3H]PDBu binding activity (data not shown). However, only recombinant protein derived from epsilon clone E3 displayed functional protein kinase C activity (Table I). Experimentally, only peak fractions containing the 96 kDa (peak No. 1) form of immunoreactive PKC-E (see Fig. 2) displayed phosphatidylserine- and diacylglycerol-dependent protein kinase activity. D E A E fractions containing the 93 kDa form of PKC-E (Peak No. 2) displayed kinase activity that was independent of added lipids (data not shown). The reason for this discrepancy is unknown, but may be reflective of the different phosphorylation states of the • enzyme. Myelin basic protein was approx, a 5-fold better substrate for the 96 kDa form of PKC-• (E3) than either histone HI or a modified peptide derived from the pseudosubstrate site of PKC-E (Table I and Fig. 3D). Activation of • required both phosphatidylserine and diacylglycerol; this was true for all of the tested substrates (histone H1, myelin basic protein and the modified pseudosubstrate peptide). Maximal activation of enzyme occurred at approx. 20 / z g / m l of phosphatidylserine (Fig. 3A) and 2 / z g / m l of diacylglycerol when myelin basic protein was used as the substrate (Fig. 3B). Maximal ATP concentration was around 20 /xM (Fig. 3C). Calcium was not required for activation of the enzyme. In fact, calcium inhibited epsilon activity at concentrations higher than 1 mM (data not shown).

Discussion In this paper, we have demonstrated the cloning and expression of two human PKC-• clones, E2 and E3. We have demonstrated phorbol binding activity with

both clones, but kinase activity with only E3. The properties of E3 are comparable to those observed with the mouse, rat and rabbit PKC-E enzymes [5-7]. The only difference between the fully functional E3 and the phorbol binding competent E2 are two amino acid differences in the catalytic domain. PKC-e clone E3, as well as mouse, rat and rabbit • contain a cysteine residue at amino acid position 475 and an aspartic acid residue at position 532 [5-7]. The catalytically inactive PKC-e clone, E2 has a tyrosine residue at position 475, and a histidine residue at position 532. In addition to the mouse, rat and rabbit PKC-•, rat ,PKC-a, /3 and ~" all contain a cysteine residue at position 475. This cysteine residue is replaced by a threonine in the rat PKC-8 and y, and by a serine in the yeast and Drosophilia in protein kinase C homologues [15]. This residue is not conserved in a variety of other serine/threonine kinases [15]. However, the aspartic acid residue at position 532 is highly conserved in all of the known members protein kinase C family [15]. In fact, the aspartic acid residue is conserved in the catalytic domains of all identified s e r i n e / t h r e o n i n e and tyrosine kinases [8,10,15]. This is one of several critical aspartic acid residues present in the active site of protein kinases. The residue is apparently involved in the binding of Mg 2÷ to the active site of the cAMPdependent protein kinase [8-11]. Therefore, it is not surprising that a cloning artifact that converts this highly conserved aspartic acid residue into a positively charged histidine residue, could explain the lack of protein kinase C activity observed with the PKC-• (E2) clone. In fact, the mutation of a single, higly-conserved amino acid residue in the ATP-binding site of protein kinase C renders the enzyme catalytically inactive [1214]. The contribution of the tyrosine residue at position 475 in the PKC-E (E2) clone to the production of a non-functional protein kinase, however, cannot be completely ruled out at this time. A site-directed mutagenesis strategy could be used to determine whether the mutation of only the aspartic acid residue, or a mutation of both the aspartic acid and cysteine residues is required to produce a catalytically inactive PKC-E.

Acknowledgement We would like to thank Margaret Waiters for her technical support.

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