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Molecular and Cellular Biochemistry 250: 41–45, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Phorbol esters induce translocation of the nPKCp105 to membrane in mussel hemocytes

Luís Mercado, Asunción Cao, Ramiro Barcia andJuan Ignacio Ramos-MartinezDepartamento de Bioquímica y Biología Molecular, Universidad de Santiago de Compostela, Facultad de Veterinaria,Campus de Lugo, Lugo, Spain

Received 9 July 2002; accepted 18 December 2002

Abstract

Previous works revealed the presence of the nPKC enzyme p105 in hemocytes of M. galloprovincialis Lmk. Specific musselantibodies were obtained from mouse and used in confocal microscopy and Western blotting. These techniques allowed theobservation of p105 cytosol-to-membrane translocation induced by TPA for the first time in hemocytes of molluscs. The incu-bation of mussel immune cells with TPA for longer than 30 min also triggered a down-regulation process. Mussel hemocytesare an excellent model to study the molecular processes of innate immunity. (Mol Cell Biochem 250: 41–45, 2003)

Key words: hemocytes, immunity, Mytilus, mussel, PKC, translocation

Abbreviations: nPKC – new protein kinase C; cPKC – classical protein kinase C; TPA – tumor-promoting phorbol ester; DAG– diacyl glycerol; WB – Western blotting; PS – phosphatidyl serine; P-Tyr – phospho-tyrosine

Introduction

The hemocytes of the marine mussel Mytilis galloprovincialisLmk. are responsible for the innate immune system of thismollusc [1–7], since these animals lack a clonal-type immunesystem [3, 4]. As happens in macrophages from mammals,the activation of a specific receptor in the hemocytes triggersthe particular net of signal internalization. Some PKCs inter-vene in the process [6–9], and in this sense, several nPKC andcPKC isoenzymes were detected in tissues of Mytilus gallo-provincialis Lmk. [10].

Two PKC forms were detected in the nerve ganglia of themollusc Aplysia californica; Apl I is Ca2+-dependent, whereasApl II is Ca2+-independent [11, 12]. A Ca2+-independent PKCform present in mussel mantle was purified and called p105[13]. As Apl II, p105 is autophosphorylable, but no P-Tyro-sine residues were detected in it [13].

The presence of p105 was detected in mussel hemocytes[14]. Activation by TPA and translocation of the enzyme to

the membrane seems to be a general feature of the proteinkinases C from vertebrates [9]. However, such processes donot seem to take place in the same way in different PKC fami-lies, showing remarkable differences in the Apl isoenzymesfrom Aplysia californica [11, 12, 15, 16].

The tumor-promoting action of phorbol ester on p105 isstudied in the present work. The hemocytes of Mytilus gallo-provincialis prove an excellent model to study innate immu-nity, whose cell activation processes involve a protein kinase[3, 4, 17].

Materials and methods

Materials

Enhanced chemiluminescence (ECLR) kit, (γ32P)ATP (30,000Ci/mol), and VRKRTLRRL peptide substrate were suppliedby Amersham Pharmacia Biotech (Buckinghamshire, UK).

Address for offprints: J.I. Ramos-Martinez, Departamento de Bioquímica y Biología Molecular, Universidad de Santiago de Compostela, Facultad deVeterinaria, Campus de Lugo, E-27002 Lugo, Spain (E-mail: bniramos@lugo.usc.es)

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Disc cellulose (p-81) was from Whatman Scientific (Maidstone,UK). β-scintillant counter (Ready-safe) was from Beckman(Fullerton, CA, USA). All other reagents were from Merck(Darmdastd, Germany) or Sigma Chemicals (St. Louis, MO,USA). Mouse anti p105 was obtained as described previously[13]. Texas Red-conjugated mouse anti-IgG antibodies for im-munofluorescence were from Jackson Immunoresearch Labs(West Grove, PA, USA).

Biological material and hemocyte cultures

Marine mussels (Mytilus galloprovincialis Lmk.) were col-lected from a sea farm located at the Betanzos estuary (N.W.Spain). Molluscs were placed in tanks containing aeratedseawater and transported to the laboratory. Hemolymph andcells were prepared as described previously [17]. Hemolymphwas aspirated by a syringe from the adductor muscle in 0.1ml of ALS (Alseve) buffer [17]. Hemocytes were counted ina Neubauer hemocytometer. A total of 9 × 106 cells were ob-tained from 30 mussels. Cells were cultured for 3 days inmodified L-15 medium [17]. Cell viability was assayed withthe Trypan blue exclusion technique.

106 hemocytes were suspended in 200 µl of lysis buffer (20mM Tris-HCl buffer containing 1 mM EGTA, 1 mM EDTA,5 mM β-mercaptoethanol, 1 mM PMSF, 5 mM benzamidineand 2 µg/ml leupeptine at pH 8.0). Lysis was performed for30 min at 4ºC. Samples were then centrifuged at 100,000 ×g for 70 min at 4ºC. The precipitate was suspended in 200 µlof extracting buffer (20 mM MES buffer containing 10 mMEGTA, 2 mM EDTA, 250 mM sucrose, 10 mM β-mercapto-ethanol, 10% glycerol and 1% triton X-100, pH 6.5). It wasstirred for 1 h at 4ºC and centrifuged again, in the same con-ditions as before. This sample was called hemocytes mem-brane fraction.

Protein kinase C assay

The enzymatic activity was determined by measuring thetransference of the terminal radiolabeled phosphate from(γ32P)ATP to the commercial peptide VRKRTLRRL. The re-action mixture (55 µl) contained 45 mM HCl-Tris buffer (pH7.5); 34 µg/ml phosphatidyl serine (PS); 3.4 mM DTT; 100mM commercial peptide substrate; 0.31 mM MgCl

2, 0.11 mM

ATP2–; (γ32P)ATP (3.5 µCi/ml) and 25 µl of sample. 2.7 µg/ml were also included. After 15 min incubation at 37ºC, 10µl of 300 mM ortophosphoric acid were added. After 3 seccentrifugation, 35 µl of reaction mixture were transferred tophosphocellulose binding paper discs (P-81). The discs werewashed 3 times with 5 ml of 300 mM ortophosphoric acidand transferred to scintillation vials. 5 ml of β-scintillationcounter were added to each vial and counted for 1 min. A

blank was run without PS. The unit of enzymatic activity wasdefined as the amount of enzyme that phosphorylates 1 µmolof substrate per min at 37ºC and pH 7.5.

Protein assayProtein was determined by the method of Bradford [18], us-ing bovine serum as standard.

Effect of TPA on PKC activityAfter 3 days of culture [17], several samples containing 106

hemocytes each were incubated for different times in 1 mlof fresh L-15 supplemented medium respectively, in the pres-ence of 80 nM TPA. Controls without TPA were performed.The reaction was stopped by centrifugation at 13,000 × g for15 min at 4ºC.

Hemocytes were lysed as previously indicated and cy-tosol and membrane samples were used for protein kinaseenzymatic activity assay and Western blotting (WB). In WBassays, 25 µg of protein were loaded on each lane in 10%SDS-PAGE. The separated proteins were transferred to aPVDF membrane at 4ºC using a Bio-Rad transfer blot de-vice (100 volts/90 min). Non specific sites were blocked byincubation of membranes with skimmed milk. The dilutionof the specific mouse anti-mussel p105 was 1:1000. Horse-radish peroxidase conjugated sheep anti-mouse Ig was usedas secondary antibody for detection by the commercial ECLR

detection system.

Indirect immunofluorescenceSamples of hemocytes were incubated with 80 nM TPA for30 min at 18ºC. A blank without TPA was performed.

25 µl of a treated suspension of hemocytes (106 cells)were smeared on a slide, washed for 5 min with PBS (10mM Na

2HPO

4, 2 mM KH

2PO

4, 135 mM NaCl, 3 mM KCl,

pH 7.4) and blocked with horse serum (1:20) in PBS at 37ºC.The sample was incubated with the primary antibody (1:1000)for 30 min at 37ºC. After washing 3 times in PBS the slideswere incubated for 30 min with a 1:100 dilution of Texas-Red-conjugated donkey antibody to IgG (1:100) for 30 minin a humid chamber at 37ºC. Slides were fixed in acetone at–20°C for 10 min, air dried and rinsed in PBS. The samplewas eventually washed and mounted in glycerol 90% anddistilled water. Samples of hemocytes were observed in anepifluorescence microscope and the images were taken byconfocal microscopy.

Results and discussion

p105 is a nPKC or Ca2+-independent protein kinase C that canbe found in different tissues of Mytilus galloprovincialis Lmk.[14], but it is in hemocytes that its presence is most remark-

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Fig. 1. Translocation of p105 from cytosol to membrane induced by TPA. The cells were treated as described in Materials and methods. p105 was detectedwith Texas Red fluorochrome-conjugated mouse anti-IgG (1:100) for 30 min in humid chamber at 37°C. The samples were taken by confocal microscopy.(A) Hemocytes control; (B) hemocytes cultured with 80 nM TPA for 30 min at 18°C TPA.

Fig. 2. Down-regulation of p105 by the incubation for different times of M. galloprovincialis hemocytes with 80 nM TPA. (A) Western blotting and (B)p105 enzymatic activity were made as described previously. Cytosol and membrane fractions of cell incubated at different times with TPA were obtainedas described in Materials and methods. In the Western blotting 25 µg of protein were charged in each lane. For the measurements of enzymatic activity 1.5µg of protein were used in each assay. The assays were repeated 5 times and bars represents the standard deviation of the means.

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able. After 3 days in culture, mussel hemocytes were markedwith Texas Red-conjugated mouse anti-p105 and the cyto-solic distribution of the enzyme was then observed (Fig.1A). Western blotting of the cytosolic and membrane frac-tions of the hemocytes confirmed this datum (Fig. 2A). Thetwo well-known bands corresponding to different p105 phos-phorylation states detected in crude extracts [13, 14] wereobserved in the control assay, but only in cytosol. The factthat p105 appeared exclusively in cytosol proves the energeticsynchronicity and basal activity of the cultured hemocytes [17].On the contrary, other tissues of Mytilus galloprovincialisshowed these two bands in both cytosol and membrane [13,14]. This could mean different activity states in the tissues,reflected in the distribution of the enzyme in both phases.Figure 1B shows that incubation with TPA for 30 min pro-voked cytosol-to-membrane translocation of p105.

Western blotting showed a progressive translocation ofp105 from cytosol to membrane when hemocytes were in-cubated with TPA for different times (Fig. 2A). This trans-location coincided with an increase of p105 phosphorylatingactivity (Fig. 2B). After 30 min, almost all p105 was boundto the membrane (Figs 2A and 2B). When incubating forlonger than 60 min, there was an inversion and p105 ap-peared exclusively in the cytosolic fraction (Figs 2A and2B).

In both Aplysia californica and Mytilus galloprovincialisLmk., PKC forms with a Ca2+-independent phosphorylatingactivity were purified: Apl II and p105 respectively [12, 13].Although these enzymes are kinetically similar to the ε PKCisoform from brain of vertebrates, other properties make themdifferent [9, 19]. It is possible to notice the differences be-tween the enzymes of both molluscs with respect to TPA acti-vation. Apl II is not TPA-activated, and cytosol-to-membranetranslocation by effect of the phorbol has not been proved [15,16]. In Aplysia enzymes and cPKC from vertebrates, thisprocess is triggered by Ca2+ [20]. On the contrary, p105 wasinhibited by Ca2+ [14]; therefore, in this case this cation doesnot trigger the reaction.

No down-regulation by effect of TPA was proved on AplII [15], whereas prolonged incubation of mussel hemocyteswith TPA clearly triggered the down-regulation process.p105 forms bound to the membrane were observed to dis-appear while proteins were being formed in the cytosol (Fig.2B).

The ensemble of results suggest that p105 from Mytilusgalloprovincialis have the primary structural base to be con-sidered an ancestral PKC with some of the regulatory prop-erties of classic PKCs, even in spite of its independence ofCa2+ levels (nPKC). We also believe that mussel hemocytescan suit further studies on the innate immunity, given theireasy extraction and culture [17].

Acknowledgements

This work was supported by the grant PGIDT MAR26102PRfrom the Autonomous Government of Galicia (Spain). Theauthors wish to thank María Mosquera for excellent techni-cal assistance.

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