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Molecular and Cellular Biochemistry 233: 99–105, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Purification of a lipid-activated and Ca2+-independent protein kinase from the mantle tissueof Mytilus galloprovincialis Lmk.

Luis Mercado, Asunción Cao, Ramiro Barcia and Juan Ignacio Ramos-MartínezDepartamento de Bioquimica y Biología Molecular, Universidad de Santiago de Compostela, Facultad de Veterinaria,(Campus de Lugo), Lugo, Spain

Received 19 October 2001; accepted 18 January 2002

Abstract

A phospholipid-sensitive Ca2+-independent protein kinase (p105) was purified to homogeneity from mantle tissue of the mus-sel Mytilus galloprovincialis Lmk., employing consecutively DE-52 cellulose, Sephacryl S-200 and Biogel HTP chromatog-raphies. The purified enzyme appeared as a single band on 10% SDS-PAGE, and had a molecular weight of 105 kDa.

The positive Western blotting of the purified eluate for anti-human-PKCδ and PKCε suggests that the enzyme from musselmantle may be an ancestral nPKC isoform, with the kinetic properties of the enzyme very close to those of PKCε isoform ofvertebrates.

Western blotting of samples from different steps of purification using specific mouse anti-p105, showed two protein bandsin samples from the initial steps. However, only one band was detected in the Biogel-HTP eluate, the most purified fraction.The purification steps did not affect the presence of P-serine in p105. No P-tyrosine peptides were detected in any of the pu-rification steps. These results open a new field of work on the study of several molecular processes related to energetic me-tabolism and reproduction in molluscs, whose regulation is associated with the activation of protein kinases. (Mol Cell Biochem233: 99–105, 2002)

Key words: Mytilus, PKC, protein kinase, purification

Abbreviations: DAG – diacylglycerol; DE-52 – diethylaminoethyle-52; DTT – dithiotreitol; EDTA – ethylene diamine-tetraaceticacid; EGTA – ethylene glycol bis-β-aminoethyl ether; FPLC – fast performance liquid chromatography; HTP – hydroxylapatite;MBP – myelin basic protein; PDGF – platelet derived growth factor; PKA – cAMP-dependent protein kinase; PKC – proteinkinase C; PMA – 4β-phorbol 12-myristate 13-acetate; PMSF – phenyl methyl sulfonyl fluoride; PS – phosphatidyl serine

in the control of the relationship between the seasonal vari-ation of the energetic metabolism and the ripening stages ofthe sexual cells [2, 3]. Also, oocytes ripening process in somemarine invertebrates is at least in part regulated by the revers-ible phosphorylation of certain proteins [4].

The first of the processes described above has been stud-ied in our laboratory [2, 3] and the cAMP-dependent protein

Introduction

Reversible phosphorylation is a universal mechanism of meta-bolic regulation that is involved in many physiological func-tions [1].

In the sea mussel (Mytilus galloprovincialis Lmk.), thephosphorylation state of regulatory proteins is a key factor

Address for offprints: J.I. Ramos-Martínez, Departamento de Bioquimica y Biología Molecular, Facultad de Veterinaria, Universidad de Santiago deCompostela (Campus de Lugo), 27002 Lugo, Spain (E-mail: bniramos@lugo.usc.es)

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kinase (PKA) from various mussel tissues has been purifiedto homogeneity [5–7]. The tissue distribution of differentlipid-dependent protein kinases (PKC) was also studied us-ing anti-PKC from vertebrates antibodies. The α and δ iso-forms were detected in the cytosolic fraction in the adductorand retractor muscles of the M. galloprovincialis [8].

Protein kinase C (PKC) is a family of lipid-regulated ser-ine/threonine kinases that phosphorylates a great variety ofcellular proteins and plays an essential role in signal trans-duction mechanisms [9–11].

The purification of a DAG-, PS-activable and Ca2+-in-dependent protein kinase present in the mantle of Mytilusgalloprovincialis Lmk. is described in the present work. Itsidentification and its inclusion among the different PKC fami-lies are discussed.

Materials and methods

Materials

Chemicals were obtained from Merck (Darmstdt, Germany).[γ32P]ATP (30,000 Ci/mol), enhanced chemiluminescence(ECLR) kit, Sephacryl S-200 prepacked column and theVRKRTLRRL peptide substrate were supplied by AmershamPharmacia Biotech (Buckinghamshire, UK). ATP, Histone(Type HIIIS), protamine sulphate, myelin basic protein andsynthide-2 were from Sigma Chemical Co. (St. Louis, MO,USA). DE-52 cellulose and disc cellulose (P-81) were fromWhatman Scientific Ltd (Maistone, UK). All electrophore-sis products and Biogel HTP were purchased from Bio-Rad(Hercules, CA, USA). Polyclonal affinity purified antibod-ies against different human PKC isoenzymes were from Gibco-BRL (Inchinnan, UK). The (28–48) fragment of Neurogranin(AAKIGASFRGHMARKK), PKCδ and ε peptide substrateswere from Calbiochem (La Jolla, CA, USA). All other rea-gents were of highest available quality.

Tissue preparations and homogenisation

Sea mussels of the species Mytilus galloprovincialis Lmk.,were collected in winter from a sea farm located at the Riaof Betanzos (NW Spain). Molluscs were placed in tanks con-taining aerated sea water and transported to the laboratory.The mantles were then rapidly dissected and immediatelyfrozen at –80ºC until use.

Tissues were homogenised with a glass-teflon potter-Elvej-hem homogenizer (1:1 v/v) in ice-cold 1 mM EDTA, 1 mMEGTA, 10 mM MgCl

2, 5 mM 2-β-mercaptoetanol, 1 mM

PMSF, 5 mM benzamidine, 2 µg/ml leupeptine in 20 mMHCl-tris buffer (pH 8.0). The homogenate was centrifuged

at 100,000 × g for 80 min at 4°C in a Beckman (Fullerton,CA, USA) L7 Ultracentrifuge (70 Ti rotor), and the super-natant obtained was filtered through washed glass-wool andused as the crude cytosolic homogenate.

Protein kinase C assay

The enzymatic activity was determined measuring the trans-ference of the terminal radiolabeled phosphate from [γ32P]ATPto the protamine sulphate. The standard reaction mixture (55µl) contained 45 mM HCl-tris buffer (pH 7.5); 34 µg/ml phos-phatidyl serine; 3.4 mM DTT; 100 mM protamine sulphate;0.11 mM MgATP2–; [γ32P]ATP (3.5 µCi/ml) and 25 µl of sam-ple. 2.7 µg/ml of DAG or PMA were also included. After 15min incubation at 37°C, 10 µl of 300 mM orthophosphoric acidwere added. After 3 sec centrifugation at 7200 × g (Denver In-struments Co.), 35 µl of reaction mixture were transferred tobinding paper disc of phosphocellulose (P-81). The discs werewashed 3 times with 5 ml of 300 mM orthophosphoric acidand transferred to scintillation vials. 5 ml of β-scintillant coun-ter (Ready Safe, Beckman, Fullerton, CA, USA) were addedto each vial and counted for 1 min. A blank was run withoutphosphatidyl serine. The unit of enzymatic activity was definedas the amount of enzyme that phosphorylates 1 µmol of sub-strate per min at 37°C and pH 7.5.

Purification of a lipid-activated and Ca2+-independentprotein kinase (p105) from mussel mantle

All the chromatographic procedures were carried out at 4°Cusing a FPLC system (Amersham Pharmacia Biotech., Upp-sala, Sweden). The crude cytosolic extract obtained from 100g of tissue was applied to a DE-52 cellulose column (30 ×2.5 cm) equilibrated with 0.5 mM EGTA, 0.5 mM EDTA, 10mM 2-β-mercaptoetanol, 1 mM PMSF, 10% glycerol in 20mM HCl-tris buffer (pH 8.0) (Buffer A). The column was firstwashed with 150 ml of buffer A supplemented with 25 mMNaCl and 0.1% Triton X-100. The column was then washedwith 800 ml of Buffer A supplemented with 25 mM NaCl.Finally, the active fractions for protamine sulphate phospho-rylating Ca2+-independent activity were eluted with 300 mlof a linear gradient between 25 and 300 mM NaCl in bufferA. The active fractions were pooled and dialysed overnightat 4°C against (1:1000 v/v) of Buffer B made with, 0.5 mMEGTA, 0.5 mM EDTA, 10 mM 2-β-mercaptoetanol, 1% glyc-erol in 20 mM HCl-tris buffer (pH 7.5) (Buffer B). The dia-lysed material was concentrated to a final volume of 2 ml,using an Amicon PM-30 ultraconcentrator. The material wasapplied to a prepacked Sephacryl S-200 column (90 × 1.5 cm)and equilibrated with buffer B. The flow rate was 0.5 ml/minand 1 ml fractions were collected. Both the absorbance at 280

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nm and the enzymatic activity were monitored in all elutedfractions. The fractions with lipid-activated and Ca2+-inde-pendent protein kinase (p105) activity were pooled and con-centrated to 2 ml as above.

The material was then applied to a Biogel HTP column (9× 1 cm) previously equilibrated with 2 vol. of Buffer B. Theflow rate was 0.4 ml/min and 0.6 ml fractions were collected.After the injection of the sample, the column was washed with15 ml of 0.5 mM EGTA, 0.5 mM EDTA, 10 mM 2-β-mer-captoethanol and 10% glycerol. The fractions containing PS-activated and Ca2+-independent protein kinase (p105) werepooled and concentrated as above. Samples were stored at–80oC until use.

Protein assay

Protein was determined by the method of Bradford [12] us-ing bovine serum albumin as standard.

Western blotting

Following 10% SDS-PAGE, the separated proteins were trans-ferred to a PVDF membrane at 4°C using a Bio-Rad transferblot device (100 volts/90 min). Non specific sites were blockedby incubation of membranes with skimmed milk. Different pri-mary antibodies were used to identify the proteins. Horserad-ish peroxidase-conjugated sheep anti-mouse Ig was used assecondary antibody for detection by the commercial ECLR

detection system. The dilutions of the specific antibodies were1:1000 for mouse anti-p105, and anti-PKCε, 1:500 for humananti-PKCδ and finally 1:2000 for anti-P-ser and anti-P-Tyr. Thedilution of the second antibody was 1:2000 for rabbit-HRP anti-IgG or 1:20000 for mouse-HRP anti-IgG when used.

Purification of antibodies against the p105 protein

Mice from Swiss strain were immunised with 50 µg of emul-sionated antigen with complete Freund’s adjuvant (1:9 v/v)[13]. The pristane was used as inducer of ascitic tumour. TheIgGs were purified using consecutive ammonium sulphateprecipitation and protein G-Sepharose chromatography. Theidentification of heavy chain of Fc from mouse IgG and theimmunospecificity were tested by Western blotting.

Results

A protein kinase with DAG-dependent, PS-activated and Ca2+-independent activity (p105) was purified from mussel mantletissue. Table 1 shows the summary of a standard purification

process. It can be seen that, after 3 consecutive chromatog-raphies, the enzyme was purified 2000-fold with a yield of11% with respect to the initial activity.

The crude cytosolic extract containing the whole cytosolicactivity was loaded onto DE-52 cellulose chromatographiccolumn. As shown in Fig. 1A, the enzyme was eluted as asingle active peak by means of a NaCl gradient in the elu-tion buffer. The most active fractions were concentrated andsubsequently loaded onto a Sephacryl S-200 column (Fig.1B). All the activity was eluted as a single peak, which wasconcentrated to 2 ml and loaded onto a Biogel HTP column(Fig. 1C). The enzyme was eluted as a single peak at the endof the phosphate gradient (Fig. 1C). The fractions correspond-ing to this peak were analysed by means of 10% SDS-PAGEand stained with Coomassie blue. All the fractions containedexclusively a protein with a molecular weight of 105 kDa.The active peak was concentrated and analysed by 10% SDS-PAGE (Fig. 1D) and gel-filtration through Sephacryl S-200(results not shown). The homogeneous protein displayed amolecular weight of 105 kDa.

Kinetic characterisation of p105

p105 phosphorylating activity was tested on different sub-strates specific for both Ca2+-dependent and Ca2+-independ-ent PKCs (cPKC and nPKC, respectively).

Standard conditions for DAG and PS concentrations wereused in this assay as described in Materials and methods. Themaximum phosphorylating activity was obtained with pro-tamine sulphate as substrate, and its value considered as thereference. The activities obtained with other substrates andpeptides are shown as percentages (Table 2).

Lower rates were obtained with these other substrates. Twokey points should be noted from this Table. First, the simi-larity between the velocities obtained with the commercialpeptide VRKRTLRRL (Amersham Pharmacia Biotech) andthe PKCε-specific peptide. Second, the lack of activity de-tected for both the PKCδ-specific peptide and Synthide-2.

Table 1. Purification procedure of the Ca2+-independent and DAG and PS-activated protein kinase (p105) from the mantle tissue of Mytilus gallo-provincialis Lmk

Protein Activity Specific Purification Yieldactivity

(mg) (mUI) (mUI × mg–1) (fold) (%)

Crude extract 3150 650 0.20 1 100DE-52 76 180 2.30 11.5 27.6Sephacryl S-200 5.75 158 27.47 137.35 24.30Biogel HTP 0.18 72 400.0 2000.0 11.07

Specific activity is expressed as enzymatic activity (I.U.) per mg of pro-tein. The enzymatic activity was determined as described in methods (2.3.).The data are the means of three purification processes.

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Immunological characterisation of p105

Polyclonal anti-p105 mouse antibodies were elicited usingas antigen the enzyme in its most pure form from HTP elu-ate. As it can be seen in Fig. 2A, both the ascitic fluid andthe purified IgG identify the band of 105 kDa correspondingto the pure enzyme. When the immunodetection was car-ried out with ascitic liquid in less purified extracts (Fig. 2B),two protein bands were detected with very similar molecu-lar weights, close to 105 kDa.

It was also observed that from the same eluate (DE-52), im-munodetection with anti-PKCδ antibodies of human origin de-tected one band of approximately 105 kDa, equivalent to thatidentified with the anti-p105 specific antibody (result not shown).

Fig. 1. Purification of lipid-activated and Ca2+-independent protein kinase (p105) activity from mantle tissue of Mytilus galloprovincialis Lmk. (A) Ion-exchange chromatography on DE-52 cellulose. (B) Gel filtration through sephacryl S-200. (C) Adsorption chromatography on Biogel HTP. The figures arerepresentative of each step. The active fractions were analysed by 10% SDS-PAGE. The proteins were stained with Coomassie Blue. (D) 10% SDS-PAGEof the different steps of the purification process. Lane 1 = molecular weight markers; lane 2 = cytosolic crude extract; lane 3 = DE-52; lane 4 = sephacrylS-200; lane 5 = Biogel HTP.

Table 2. Relative protein kinase activities of p105 with various peptidesand protein substrates

Substrate Relative activity (%)

Protamine sulfate 100.00PKCδ Peptide substrate 0 . 0 0PKCε Peptide substrate 52 .50Synthide-2 0 . 0 0Neurogranine 7 . 8 4VRKRTLRR 47.16HIIIS 3 . 4 4MBP 18.17

Substrates were added at 50 µM (peptides) or 1 mg/ml (proteins) inthe standard (see Materials and methods) reaction mixture with 34 µg/ml of PS and 1 µM PMA. Measurements were performed in triplicateand the results presented as the means of the three values.

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As observed in Fig. 2B, the two bands detected by anti-p105 in crude cytosolic extract became a single band at theend of the purification process. This was the only band de-tected in the purified eluate when using anti-human-PKCε

(Fig. 3).

Mouse anti-p105 antibody recognised a single band (105kDa) in crude and purified samples that was also positive formouse anti-P-Serine antibodies in the p105 purification proc-esses. When anti-P-Tyrosine was used as antibody in the as-say, no protein band was detected (Fig. 3).

Discussion

In a previous study, the presence of a DAG-dependent, PS-activable and Ca2+-independent protein kinase activity wasdetected in the mantle tissue of the mussel Mytilus gallo-provincialis Lmk. [8]. Identification was done by means ofwestern blotting, using anti-PKCδ of human origin. Also, verylow Ca2+-dependent PKC activities found in the mantle tis-sue were identified as the PKCα isoform and the PKCβ cata-lytic domain [8].

In the present paper, a lipid-activated and Ca2+-independ-ent protein kinase was purified in a 3-step process. The firststep consisted of an ion-exchange chromatography on DE-52 cellulose. This exchanger has often been successfully usedfor purifying PKCs of diverse origins [14–16]. The yieldobtained after this chromatography step was about 30%, whichis somewhat lower than that obtained in the purification ofPKC from rat spleen, brain and heart [14–16]. The yield wasalso lower than that obtained with the DE-52 column in thepurification of the Ca2+-independent PKC or Apl II from nerveganglia of the mollusk Aplysia californica [17]. Apl II wasthe first PKC purified from mollusks starting from the origi-nal tissue, and that apparently plays an important role in thecontrol of the neuronal function of the animal [17].

In the following steps for the purification of the enzyme frommussel mantle, gel-filtration chromatographies through Sepha-cryl S-200 and adsorption chromatographies with hydroxyl-apatite (Biogel HTP) were successfully used. Purification

Fig. 2. The protein kinase p105 is recognised by mouse anti-p105. (A) Thesources of enzyme were two different concentrations of purified HTP elu-ate. The sources of anti-p105 were ascitic fluids and purified IgGs as de-scribed in Materials and methods. (B) Mouse anti-p105 recognised p105in the different steps of purification processes. Lane 1 = crude cytosolicextract; lane 2 = sephacryl S-200; lane 3 = Biogel HTP, samples. 10 µgprotein were charged in each lane.

Fig. 3. Phosphorylation of tyrosine or serine residues of p105. The p105 samples were from crude cytosolic extract (1) or Biogel HTP (2) eluate. The Westernblottings were carried out as described in methods using the corresponding antibody. Anti-p105 and anti P-Ser or anti P-Tyr were used as described in Materialsand methods.

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values and yields obtained in this way were better than thosenoted in the purification of Apl II and other enzymes fromvertebrates [14–17]. In particular, the enzyme from musselmantle showed better yield in the HTP column than that shownby Apl II with the same column [17].

Ten percent SDS-PAGE of the purified form of the DAG-dependent, PS-activated and Ca2+-independent protein ki-nase suggested that the enzyme appeared homogeneous,and the single band showed a molecular weight of 105 kDa(p105). This is higher than that of the Apl II, which wasfound to be 87 kDa, and also higher than those of other PKCisoenzymes belonging to any of the conventional or newfamilies of diverse origin [18]. The value of the molecularweight was confirmed by means of gel-filtration chromatog-raphy (result not shown).

In vertebrates, different isoenzyme forms of Ca2+-independ-ent or ‘new’ PKCs have been obtained by means of geneticexpression. δ, η and θ isoforms have been identified in thisway, showing lower molecular weights than the enzyme fromM. galloprovincialis mantle. Only PKCε has a molecularweight somewhat higher (97 kDa) than others of the family[19]. PKD enzymes also named PKCµ isoform of diverseorigins has a molecular weight of 104 kDa [20], but the im-munodetection with anti-human PKCµ proved negative.

The results obtained with the studies on kinetics indicatefunctional similarities between p105 from M. galloprovin-cialis and the PKCε form from vertebrates. The similar veloci-ties, assessed with the PKCε pseudosubstrate specific peptideand the artificial VRKRTLRRL peptide, suggest that p105could catalyse phosphorylation reactions similar to those ofPKCε in vertebrates [21]. The lack of phosphoryating activ-ity on the PKCδ specific peptide, together with the limitedactivity on histone are further data in support of the identifi-cation of p105 with PKCε [22, 23]. These results are similar tothose found in Apl II from brain ganglia of Aplysia californica[17, 23]. The positive immunodetection of p105 with bothanti-PKCδ and anti-PKCε could be in keeping with the con-servation of certain specific regions among members of thenPKC family [24]. Moreover, the high molecular weight de-tected would confirm a possible ancestral character for p105of M. galloprovincialis with regard to Apl II of A. californica[17, 23].

The use of anti-p105 in Western blotting permitted thedetection of two bands in crude cytosolic extract and a sin-gle band in the purified eluate from HTP. This result sug-gested a change in the phosphorylation state of the enzymealong the purification process. This event is very frequent inenzymes modulated by phosphorylation or having autophos-phorylating mechanisms [25, 26].

Regardless of the purification state of the enzyme (Fig. 3),a single band of 105 kDa was always obtained, positive forP-serine. This result suggested that, as indicated by Keranen

et al. [27], the purification state of the protein did not affectthe phosphorylation of a serine likely to be present in thecarboxyl end and that usually belongs to the sequence of thepseudosubstrate [27].

It was also observed that p105 did not have any phospho-rylated tyrosine in any of the purification steps in whichWestern blotting was performed. This result agreed with thewell-known absence of tyrosine kinase activity of the nPKCspresent in many kinds of cells [28], and suggested that p105was in an inactive state. The phosphorylation of tyrosines isa process that develops in vivo as a consequence of the ac-tion of either src-Tyr-K or Tyr-kinase receptors such as PDGFor insulin receptors [29, 30]. In some cases, the process isinduced in vitro by PMA in the presence of ATP [22].

p105 activation would supposedly require Tyr phosphor-ylation, which would facilitate its binding to PS and DAG.This process suggested by Keranen et al. [27] is currentlybeing studied in our laboratory.

Acknowledgements

This work was supported by the Grants XUGA 26100 PB96and PGIDTOOMAR26102PR from the autonomous Govern-ment of Galicia (Spain). We thank Maria Mosquera for ex-cellent technical assistance.

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