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Eukaryotic Cell, January 2006, p. 77-91, Vol. 5, No. 1
1535-9778/06/$08.00+0     doi:10.1128/EC.5.1.77-91.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Multigene Family Encoding 3',5'-Cyclic-GMP-Dependent Protein Kinases in Paramecium tetraurelia Cells

Roland Kissmehl,1* Tim P. Krüger,1 Tilman Treptau,1 Marine Froissard,2 and Helmut Plattner1

Department of Biology, University of Konstanz, 78457 Konstanz, Germany,1 Centre de Génétique Moléculaire, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France2

Received 9 June 2005/ Accepted 1 November 2005


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ABSTRACT
 
In the ciliate Paramecium tetraurelia, 3',5'-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca2+ stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation.


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INTRODUCTION
 
In eukaryotic cells, 3',5'-cyclic GMP (cGMP) is one of the second messengers participating in signal transduction pathways. Its significance has been only partially elucidated. Three effector activities are currently discussed (48, 77), namely, (i) cGMP-induced activation of ion channels, (ii) cGMP-sensitive phosphodiesterase (PD) activity, and (iii) Ser/Thr-specific cGMP-dependent protein kinase (PKG) activity. PKGs are found not only in metazoans of different phyla (48, 60) but also in flowering plants (73) and lower eukaryotes (16, 23). The metazoan PKGs can be divided into two types, a soluble type I form with several splice forms (58, 66, 83) and a single membrane-bound type II form (33, 76). Unlike mammalian and invertebrate PKGs, which are typically homodimers, the protozoan PKGs known so far are all monomeric proteins (16, 23, 55, 80). The multiple interactions of PKGs with other signaling systems (77) are particularly intriguing. Signaling via PKGs presupposes the occurrence of a guanylate cyclase (GC) and termination by PD activity.

Among potential signaling components, the following are known to occur in the ciliate Paramecium: a Ca2+-dependent GC (47, 67), PD (68), and a PKG (53). The broad spectrum of cellular activities governed or modulated by PKGs led us to identify the corresponding genes.

Our approach is based on the recent availability of an indexed genomic library (34), on an international Paramecium genome project (13, 72), and on sequencing data from either a 1-megabase chromosome (86) or from an ongoing genome project provided by Genoscope (France). We were surprised by the occurrence of an extensive family of PKG genes in Paramecium, in contrast to the global description of PKG activity in cilia and whole-cell bodies (53) without any further differentiation.

The large number of genes we found may reflect the broad range of activities observed with or related to cGMP formation in Paramecium. This includes depolarization-induced ciliary reversal (84), circadian rhythms (25), and exocytosis, particularly with regard to rephosphorylation of pp63/pf (parafusin), an exocytosis-sensitive phosphoprotein of 63 kDa (36, 43, 54). The recent suggestion of a potential role of PKG includes actin filament reorganization and/or intracellular trafficking in mammalian cells (32). For Paramecium, this may turn out to be important with regard to extensive membrane trafficking (62).

In summary, the broad spectrum of PKG genes we now find may facilitate further elucidation of PKGs in widely different functions of Paramecium cells. Moreover, a comparison with pathogenic relatives of Paramecium in the phylum Alveolata, i.e., Toxoplasma, Plasmodium, Eimeria, etc. (Apicomplexa), revealed interesting observations.


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MATERIALS AND METHODS
 
Cell cultures and cell fractionation. The wild-type strains of Paramecium tetraurelia used were stock strains 7S and d4-2, derived from stock strain 51S (71). Cells were cultivated either in a bacterized medium or, for subcellular fractionation, in an axenic medium as previously described (40). Whole-cell homogenates were prepared in phase buffer (20 mM Tris-maleate, 20 mM NaOH, 20 mM NaCl, 250 mM sucrose, pH 7.0) by ~100 hand strokes in a glass homogenizer equipped with a Teflon pestle. Soluble and particulate fractions were separated by centrifugation at 100,000 x g for 60 min at 4°C. Cell surface complexes (cortices) were prepared according to the method of Lumpert et al. (49), and cilia were purified by differential centrifugation after applying the Mn2+-shock method (57). A protease inhibitor cocktail containing 15 µM pepstatin A, 100 mU/ml aprotinin, 100 µM leupeptin, 0.26 mM N-p-tosyl-L-arginine methyl ester, 28 µM E64, and 0.2 mM Pefabloc SC was used throughout.

PCRs with genomic DNA. For PCR, total wild-type DNA was prepared from log-phase d4-2 cell cultures as described previously (28). For PKG1-1, the probe consisted of a 474-bp PCR amplification product made with the following primers (MWG-Biotech, Ebersberg, Germany): 5' oligonucleotide 1, 5'-CAAGTGACCAAACAAGGC-3'; and 3' oligonucleotide 2, 5'-GATTGATTTTTCGTTTTTGTTC-3'. For PKG2-1, the probe was a 514-bp PCR amplification product created with the following two primers: 5' oligonucleotide 3, 5'-TTCAATAATATTATGAGATGGAG-3'; and 3' oligonucleotide 4, 5'-ACCTTTTTTGTTTCTTTGTGC-3'. Each PCR mixture (50 µl) contained 20 ng of DNA, a 200 nM concentration of each primer, a 0.2 mM concentration of each deoxynucleoside triphosphate, and 1 µl of Advantage 2 DNA polymerase (Clontech Laboratories, Inc., Heidelberg, Germany). Reactions were carried out for 1 cycle of denaturation (1 min, 95°C) and 35 cycles of denaturation (30 s, 95°C), annealing (45 s, 46°C), and extension (60 s, 68°C), with a final extension step (5 min, 68°C).

Screening of a genomic {lambda}ZAPII library of EcoRI-digested macronucleic DNA from P. tetraurelia (5 x 106 primary plaques) was performed by using a PCR-based strategy (4) in which the universal T3 primer was combined with each of three nested PKG2-1-specific 3' oligonucleotides, oligonucleotides 5 to 7 (3' oligonucleotide 5, 5'-ACCTTTTTTGTTTCTTTGTGC-3'; 3' oligonucleotide 6, 5'-CTTTTGGAGTGAATCTTGAGG-3'; and 3' oligonucleotide 7, 5'-TCTACATCCTCTCCTCCTC-3'). Following one denaturation step (95°C, 1 min), 39 cycles of amplification (30 s at 95°C, 45 s at 56°C, and 60 s at 68°) and a final elongation step of 3 min at 68°C were carried out. For three consecutive PCRs, the resulting PCR product (~50 ng) was used as a new template for the subsequent reaction with the nested primer. PKG-specific PCR products were cloned into the plasmid pCR2.1 by using a TOPO-TA cloning kit (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. After transformation into Escherichia coli (DH5{alpha} or TOP10F' cells), positive clones were sequenced as described below.

PCRs with cDNAs. The open reading frames (ORFs) of PKG1-1 and PKG2-1 were amplified by reverse transcriptase PCR (RT-PCR), using total RNA prepared according to the protocol described by Haynes et al. (29). RT-PCR was performed in a programmable T3 thermocycler (Biometra, Göttingen, Germany), using oligonucleotide 8 and PowerScript reverse transcriptase (Clontech Labs) for first-strand cDNA synthesis (3' oligonucleotide 8, 5'-AACTGGAAGAATTCGCGGCCGCGGAATTTTTTTTTTTTTTT-3'). The subsequent PCR (50 µl) was performed with Advantage 2 cDNA polymerase mix (Clontech Labs), using either the PKG1-1-specific primers 9 and 10 (5' oligonucleotide 9, 5'-GAGATCAGATGATTCCTGGGT-3'; and 3' oligonucleotide 10, 5'-TTCAAAAGTCTTAATCCCAGTC-3') or the PKG2-1-specific primers 11 and 12 (5' oligonucleotide 11, 5'-ATCTTTGAAAAGTGCCAGTA-3'; and 3' oligonucleotide 12, 5'-GTTCTGTATTATATTCAAAAGTC-3'), with the last two containing artificial restriction sites added at their 5' ends (SpeI and XhoI, respectively). Amplifications were performed with 1 cycle of denaturation (95°C, 1 min) and 39 cycles of denaturation (95°C, 30 s), annealing (58°C, 45 s), and extension (68°C, 3 min), followed by a final extension step at 68°C for 5 min. PCR products were cloned and sequenced as described below.

Sequencing. Sequencing was done by the MWG Biotech (Ebersberg, Germany) custom sequencing service. DNA sequences were aligned with CLUSTAL W integrated into the DNASTAR Lasergene software package (Madison, WI).

Annotation of further PKG genes. In order to identify further paralogs of PKGs, the developing Paramecium database (http://paramecium.cgm.cnrs-gif.fr/ptblast/) was screened by using the nucleotide and amino acid sequences of PKG1-1 and PKG2-1. Positive hits were further analyzed by performing BLAST searches at the NCBI database (2). Conserved motif searching was performed with either PROSITE (6) or BLAST-RPS, using pfam entries of the corresponding CDD database (52).

Southern hybridization. Radioactive probes were synthesized by [{alpha}-32P]dATP incorporation using a random primer labeling system (Gibco-BRL, Cergy-Pontoise, France) according to the supplier's protocol. Nonradioactive probes were made using digoxigenin-labeled nucleotides (Roche, Indianapolis, Ind.). Hybridizations with an indexed library of Paramecium macronuclear DNAs were carried out as described by Keller and Cohen (34).

Expression of a Paramecium PKG1-1-specific peptide in E. coli. For heterologous expression of a PKG-specific peptide, we selected the amino acid sequence of PKG1-1 (accession number AJ550184 [GenBank] ). After changing all deviating Paramecium glutamine codons (TAA and TAG) to universal glutamine codons (CAA and CAG) by PCR methods (15), the coding region for F266 to Q445 of Paramecium PKG1-1 was cloned into the XhoI/BamHI restriction sites of the pET16b expression vector of the Novagen pET system (Madison, WI), which adds a 24-amino-acid peptide at the N terminus of the selected sequence, including a His10 tag for purification of the recombinant peptide.

Purification of a recombinant PKG1-1 peptide and preparation of polyclonal antibodies. The recombinant PKG1-1 peptide, PKG1-1F266-Q445, was purified by affinity chromatography on Ni2+-nitrilotriacetate-agarose under denaturing conditions, as recommended by the manufacturer (Novagen), and was used for immunization of rabbits. After several boosts, positive sera were taken and affinity purified by two subsequent chromatography steps, as described in detail by Kissmehl et al. (40).

SDS-polyacrylamide gel electrophoresis and immunoblotting. Protein samples were denatured by boiling for 3 min in a sample buffer (0.4 M Tris-HCl, 1% sodium dodecyl sulfate [SDS], 0.5% dithiothreitol, 20% glycerol, pH 8.0) without subsequent alkylation and then subjected to electrophoresis in 10% SDS-polyacrylamide gels using a discontinuous buffer system as described previously (37). The replicas were electroblotted onto nitrocellulose membranes, and immunobinding was carried out as described previously (40), using affinity-purified antibodies (Abs) against PKG1-1. Bound Abs were detected with a corresponding peroxidase-conjugated secondary Ab (anti-rabbit immunoglobulin G), using the Amersham enhanced chemiluminescence (ECL) detection system.

Isolation of PKGs and phosphorylation studies. The isolation of PKGs was performed according to a protocol described by Kissmehl et al. (36). Standard assays for PKG activity were performed with histone II-S, an established in vitro substrate for PKGs (36). Phosphorylation of recombinant pp63/pf (26) was accomplished in a reaction volume of 45 µl containing 20 mM triethanolamine-HCl, 240 mM NaCl, 10% glycerin in the presence of 5 mM MgCl2, 0.12 mM ATP, [{gamma}-32P]ATP (185 MBq/µmol), 3 µg of recombinant pp63/pf, with or without cGMP (0.15 µM), 8-chlorophenylthio (pCPT)-cGMP (0.15 µM), and the PKG fraction (1.275 µg). At the end of the incubation (20 min at 20°C), 25-µl aliquots were subjected to SDS-polyacrylamide gel electrophoresis and processed for autoradiography.

Immuno-LM and -EM analysis. For immuno-light microscopy (immuno-LM), cells were prepared basically as previously described (39), except that 1% Triton X-100 was replaced by 0.5% digitonin as a permeabilization agent. After exposure to affinity-purified anti-PKG1-1 Abs and fluorescein isothiocyanate-conjugated anti-rabbit Abs (Sigma-Aldrich, St. Louis, MO), both diluted 1:100 in phosphate-buffered saline plus 1% bovine serum albumin, fluorescence staining was analyzed either by conventional LM or by confocal laser scanning microscopy, as previously described (40). Images acquired with LSM 510 software were processed with Photoshop software (Adobe Systems, San Jose, CA). For immuno-electron microscopy (immuno-EM) analysis, samples were prepared as described previously (40). Primary anti-PKG Abs from rabbits were detected with protein A-gold conjugates with a 5-nm diameter (Au5 nm) on ultrathin sections which were "stained" with uranyl acetate.


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RESULTS
 
On the basis of three independently initiated Paramecium genome projects, we were able to identify a PKG multigene family with at least 35 members. These projects included a pilot sequencing project (13, 72), the sequencing of a macronucleic 1-megabase chromosome (86), and the current genome project initiated by the GDRE (Groupement de Recherches Européen), coordinated by Jean Cohen and Linda Sperling (CNRS, France) in collaboration with Genoscope (Evry, France). PKGs of Paramecium can be classified into 16 subfamilies with several members each, two of which, PKG1-1 and PKG2-1, were analyzed in more detail after their molecular cloning.

PKG 1 subfamily. First, a partial sequence of a PKG encoding protein was found in the pilot genome project of Paramecium (13, 72). Sequencing of the corresponding clone, 7i7, resulted in the identification of the 3' end of this gene. In order to obtain the missing 5' end, we took advantage of an indexed genomic library, which we analyzed in two subsequent hybridization steps (34) by using a specific probe designed from the 7i7 sequence (Fig. 1A, top). Besides clone 7i7, another three clones were then retrieved, namely, 8g1, 8n17, and 114d24. All three contain the missing 5' sequence information of this gene, called PKG1-1 (accession number AJ550184 [GenBank] ).



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  		FIG. 1. Characteristics of cGMP-dependent protein kinases PKG1-1 and PKG2-1 from Paramecium tetraurelia. (A and G) Schematic diagrams of PKG1-1 (A) and PKG2-1 (G) at the nucleotide (top) and amino acid (bottom) levels. (Top) Positions of introns are indicated (gray arrowheads). Orange areas are untranslated regions at the 5' and 3' ends of the coding sequence. The region used as a probe is labeled in green. The numbers indicated represent the nucleotide positions within the genomic versions of the two genes. (Bottom) Characteristic features such as the Ser/Thr kinase domain and the two cyclic nucleotide-binding domains are labeled with different shades of gray. The region of PKG1-1 used for raising Abs is shown in more detail in panel F. (B and H) Hydrophilicity plots according to the algorithms of Kyte and Doolittle (44). The positions and sequences of putative N-myristoylation sites (C and I) as well as of the two signatures within each cyclic nucleotide-binding domain (D and J) are shown. Deviations from the consensus are shown with lowercase letters. Magenta, highly conserved amino acids, e.g., for the maintenance of the structural integrity of the ß-barrel; yellow, binding sites for the ribose phosphate moieties; green, binding site for the guanine base. (E and K) Sequences important for catalysis include the ATP-binding regions with a consensus for ATP binding (gray), the loop with the catalytic aspartate at position 580 or 615 (yellow), the activation loops with a threonine for putative autophosphorylation at positions 614 and 650 (red), and the C-terminal part of the catalytic domains with some invariant residues (green). (F) The PKG1-1 peptide containing a His tag at the N terminus (underlined) consists of 203 amino acids (left). After heterologous expression in E. coli, it is visible predominantly as a 24-kDa band on a silver-stained SDS-polyacrylamide gel (right).

The PKG1-1 gene, consisting of 2,489 bp, encodes a protein of 770 amino acids with a calculated molecular mass of 89.3 kDa (Table 1). A comparison of the genomic sequence with the cDNA equivalent revealed that the gene is expressed and that the ORF is interrupted by seven short introns (Fig. 1A, top), which all display the characteristics of Paramecium introns (65, 72), i.e., bordering by 5'-GT and AG-3' and a size of 21 to 30 nucleotides.


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  		TABLE 1. Characteristics of PKG genes in Paramecium tetraurelia

The overall structure of the gene product resembles that of proteins in higher eukaryotes (60). PKG1-1 is composed of three putative functional domains, i.e., a catalytic domain which would catalyze the transfer of {gamma}-phosphate from ATP to a serine/threonine residue of a substrate protein, a regulatory domain with two tandem cyclic nucleotide-binding sites, and an amino-terminal domain (Fig. 1A, bottom). It does not contain a transmembrane domain (Fig. 1B), but there are several putative N-myristoylation sites (Fig. 1C), one of which occurs at the amino terminus, as is typical for type II PKGs (78). This would allow the enzyme to be localized not only in the cytosolic fraction but also in particulate fractions. In contrast to PKGs from higher eukaryotes, the amino terminus of PKG1-1 does not contain a hydrophobic leucine/isoleucine zipper motif, which is required for dimerization (5, 46), suggesting its occurrence as a monomeric enzyme in P. tetraurelia. There are also no autophosphorylation sites at the amino terminus, which are known to enhance the basal activity of the enzyme as well as to increase the affinity of type I PKGs for cAMP (45, 70). They normally are located in close proximity to a highly conserved region at the amino terminus. This region interacts with the catalytic center and inhibits enzyme activity in the absence of bound cGMP (35). However, in PKG1-1, such a pseudosubstrate sequence is either absent or not very well conserved.

The regulatory domain consists of two highly conserved cyclic nucleotide-binding domains of ~120 amino acids each (69, 82), both of which are present in PKG1-1 and display remarkable sequence identity (Fig. 1D). From the analysis of the prokaryotic CAP gene, it is known that such a domain is composed of a ß-barrel formed by eight antiparallel ß-sheets (ß1 to 8) and three flanking {alpha}-helices (81). There are several invariant amino acids, three of which are glycine residues considered essential for maintenance of the structural integrity of the beta-barrel. Two of them are located within ß2 and ß3 (signature 1), whereas the third conserved glycine occurs in the second signature (within ß6 and ß7) as part of the consensus sequence F-G-E, which forms a conserved binding pocket for the ribose phosphate moiety. This signature also occurs in PKG1-1 (Fig. 1D), where it contains another invariant amino acid, an arginine residue, which chelates the cyclic phosphate diester as part of the conserved sequence R-S/T/A-A. However, the two cGMP-binding domains also differ remarkably from each other and therefore may have different biochemical properties. This has been reported for mammalian PKGs, where only the domain located close to the amino terminus has been identified as having slow dissociation characteristics and a high binding affinity (64).

The boundary between the putative catalytic domain and the second cGMP-binding domain is located seven residues upstream from the first glycine of the highly conserved consensus motif (G-X-G-X-X-G-X-V/A) for ATP binding (Fig. 1E), where a hydrophobic residue (phenylalanine) is usually found (24). Another motif, termed the catalytic loop, contains an aspartate (D580) as the putative base accepting the proton from the attacking substrate hydroxyl group during the phosphate transfer reaction. This motif is also present in PKG1-1 (Fig. 1E). The lysine at position 582 of this loop may help to facilitate the phosphor transfer by neutralizing the negative charge of the {gamma}-phosphate during transfer. PKG1-1 also contains a putative activation loop close to the active center (Fig. 1E), in which a phosphothreonine would be required for a catalytically active enzyme (18). Beyond that, there are several highly conserved amino acids in the C-terminal part of the catalytic domain of PKG1-1, including those forming the motif H-X-W-F, which defines the boundary of the carboxyl-terminal domain (Fig. 1E). In mammalian PKGs, the latter domain is thought to contribute to substrate recognition (60).

The overall identities of PKG1-1 with type I or type II PKGs from higher eukaryotes range, on the amino acid level, from 20.0% to 22.7% (Table 2). However, the overall identity to PKGs from the more related Apicomplexan group is much higher (~33%), although the PKGs are not classified as one or the other of the two subtypes occurring in metazoans (16, 23).


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  		TABLE 2. Multiple alignment of PKGs from different speciesa

By BLAST searching with the complete sequence of PKG1-1 in the Paramecium database, another three members of this PKG 1 subfamily were identified, which have been grouped together not only due to their close relationship but also due to the same number and position of introns interrupting their ORFs (Table 1). Therefore, they were designated PKG1-2 to PKG1-4. They all encode proteins of 770 amino acids, with calculated molecular masses ranging from 88.8 to 89.5 kDa (Table 1). Their identities to PKG1-1 vary between ~74% and 83% at the nucleotide sequence level and between ~84 and 89% at the protein sequence level (Table 1). Sequence alignments with PKG proteins from other species revealed similar values for each of the new members of this Paramecium subfamily to those found for PKG1-1, showing highest similarities to PKGs from Apicomplexan organisms (Table 2).

PKG 2 subfamily. In the pilot genome project of Paramecium (13, 72), a second partial sequence was found resembling PKGs. In order to obtain the full gene information, we first completed sequencing of the corresponding clone, 22C23, by which we obtained the 3' end. For the missing 5' end, we performed Southern hybridizations in which the indexed genomic library (34) was probed with a PCR fragment located in the middle of the gene (Fig. 1G, top). However, none of the clones obtained contained the missing 5' sequence information for this PKG gene. We finally were successful by screening a genomic {lambda}ZAPII library of EcoRI-digested macronucleic DNA from P. tetraurelia with a nested PCR technique (4). The complete gene was called PKG2-1 (accession number AJ550185 [GenBank] ). With its 2,642 bp, it encodes a soluble enzyme of 819 amino acids with a calculated molecular mass of 94.0 kDa (Fig. 1G; Table 1). RT-PCR analysis revealed that PKG2-1 is expressed and that its ORF is interrupted by seven short introns (Fig. 1G). The overall domain structure closely resembles that of PKG1-1 (Fig. 1H to K). However, there are several insertions in the amino-terminal region as well as at the carboxy terminus of the enzyme. In metazoans, such regions are known to be involved in the dimerization of homologous subunits, autoinhibition of the catalytic domain in the absence of cGMP, and substrate recognition (60). Therefore, the variations we observed may entail different functional consequences to be analyzed in future studies.

By BLAST searching with the complete sequence of PKG2-1 in the Paramecium database, another member of this PKG 2 subfamily has been identified (Table 1). This gene, designated PKG2-2, encodes a protein of 819 amino acids which is >92% identical to PKG2-1 and >45% identical to members of the PKG 1 subfamily (Table 1). Sequence alignments with PKG proteins from other species show the highest similarity to PKGs from the Apicomplexan group, as observed also for the members of the PKG 1 subfamily (Table 2).

Further PKG subfamilies. BLAST results from the Paramecium database revealed the presence of further PKG-encoding genes in the macronucleic genome, which have been divided into subfamilies 3 to 16 according to their characteristic gene and protein structures (Table 1). Within a given subfamily, the members differ by only up to 26% at the nucleotide level and even less (≤15%) at the amino acid level. Their ORFs are interrupted by the same numbers of introns, all located at corresponding positions (Table 1). However, between the various subfamilies, the numbers and positions of introns are extremely variable, with their number ranging from 0 to 12 (Table 1). This heterogeneity is also reflected by a phylogenetic analysis which suggests that all Paramecium PKGs may be derived from a common ancestral intron-free gene (Fig. 2). The analysis also reveals that several duplication events might be the reason for the occurrence of the numerous PKG paralogs in P. tetraurelia, including a recent duplication of the whole genome (L. Sperling and J. Cohen, personal communication) that resulted in a second, closely related paralog within most of the subfamilies (Fig. 2; Table 1). Remarkably, similarity values between the subfamilies are still high, ranging from 35 to 45% on average when referred to PKG1-1 (Table 1). Compared with PKGs from other species, the sequence alignment data suggest for each of the Paramecium PKG isoforms a closer relationship to the Apicomplexan group than to the well-characterized type I and type II PKGs from higher eukaryotes (48, 60). For the latter, identities are only ~20% (Table 2). This is in contrast to the domain structures of the gene products, which much more closely resemble those from higher eukaryotes. These include a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis (Fig. 3). Their calculated molecular masses range from 77.1 kDa to 94.8 kDa (Table 1). This is due not only to a variable N-terminal domain but also to the number of putative cGMP-binding sites within the regulatory domains of individual PKG subfamilies (Fig. 3). The consequences of both must be elucidated in future work.



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  		FIG. 2. Evolutionary relationships of the Paramecium PKG superfamily. Predictions from multiple sequence alignments are shown in a rooted phylogenetic tree generated with the Clustal W program of the DNASTAR Lasergene software (Madison, WI). The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the numbers of substitution events. The scale below the tree indicates the numbers of "nucleotide substitutions" for both DNA and protein sequences. Note that most of the subfamilies contain two PKG genes (see the text).



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  		FIG. 3. Domain structures of individual members of the Paramecium PKG superfamily derived from conserved motif searching. Characteristic features are shown as follows: light gray, cyclic nucleotide-binding domains; dark gray, catalytic domain; and P, potential autophosphorylation sites. Note the variance in the number of cyclic GMP-binding domains within individual PKG subfamilies.

Immunolocalization of PKGs. In order to analyze the subcellular distribution of PKGs, we have chosen the region between F266 and Q445 of PKG1-1 (Fig. 1A) for heterologous expression in E. coli (after changing all deviating Paramecium glutamine codons to universal glutamine codons) and the subsequent production of polyclonal Abs (Fig. 1F). This region is rather conserved between some of the isoforms (Table 3), and therefore a certain degree of cross-reactivity can be expected. However, cross-reactivity is likely to decrease rapidly with decreasing peptide similarities, as observed with some other PKG isoforms. The Abs easily recognized the recombinant peptide used for immunization when tested by an enzyme-linked immunosorbent assay and Western blotting (data not shown). They were used in an affinity-purified form for several approaches, including Western blotting and LM and EM localization studies.


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  		TABLE 3. Comparison of PKG regions used for raising polyclonal Abs

In Western blots under semireducing conditions, whole-cell homogenates displayed several cross-reactive bands with apparent molecular masses ranging between ~75 and 150 kDa, which may represent not only monomeric but also homo- or heterodimeric forms of PKGs (Fig. 4). The latter is supported by the observation that some of the PKGs may contain putative leucine/isoleucine zipper motifs, which are, however, not highly conserved (<80%). Moreover, it cannot be entirely excluded that the polyclonal Ab may also recognize proteins other than PKG if they contain similar epitopes. However, this can be excluded, at least for a cAMP-dependent protein kinase of Paramecium, a closely related member of the same superfamily, as no cross-reactive bands of ~44 kDa (10) were observed in the corresponding lanes (Fig. 4, lanes 6 to 10). Some of the bands occurred not only in the soluble fraction but also in particulate fractions, suggesting the existence of both soluble and membrane-associated isoforms. The same is true of the isolated cilium fraction, which preferentially contains PKG bands of ~150 kDa, i.e., presumably dimeric forms, whereas a monomeric 83-kDa PKG species seems to be enriched selectively in the Paramecium cortex. None of the bands were visible when Western blots were produced with the corresponding preimmune serum (Fig. 4, lanes 1 to 5) or in controls with the secondary Ab only (data not shown). Nevertheless, it cannot be entirely excluded that the polyclonal Ab may also recognize other proteins besides PKG when they also contain similar epitopes.



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  		FIG. 4. Western blot analysis of the subcellular distribution of PKGs, using Abs against a cross-reactive region of PKG1-1. Aliquots (50 µg) of whole-cell homogenates (lanes 1 and 6), supernatants from centrifugation at 100,000 x g (lanes 2 and 7), pellets from centrifugation at 100,000 x g (lanes 3 and 8), isolated cell cortices (lanes 4 and 9), and isolated cilia (lanes 5 and 10) were processed for immunobinding using either polyclonal affinity-purified Abs against the recombinant PKG1-1 peptide shown in Fig. 1E (lanes 6 to 10) or the corresponding preimmune serum (lanes 1 to 5). Several cross-reactive bands between ~70 kDa and 150 kDa were observed, not only in the 100,000 x g supernatant fraction (lane 7) but also in particulate fractions (lanes 8 and 9), suggesting the existence of both soluble and membrane-associated isoforms as well as of monomeric (*) and homo- or heterodimeric (**) forms of PKGs. The same was true for isolated cilia (lane 10), although cilia may preferentially contain dimeric forms of PKGs, an interesting aspect to be analyzed in future work. This is in contrast to the cortex fraction (lane 9), which was devoid of cilia and contained a strong band of 83 kDa. None of the bands were detected by the corresponding preimmune serum (lanes 1 to 5).

The occurrence of both soluble and structure-bound forms of PKGs within a Paramecium cell is also compatible with data obtained by confocal immunofluorescence analyses (Fig. 5). Besides some general cytosolic labeling, several subcellular organelles, such as cilia, the oral cavity, numerous vesicle-like structures, and some cortical structures, were distinctly labeled.



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  		FIG. 5. Immunofluorescence localization of PKGs by CLSM. Cells permeabilized under conditions preserving cilia were labeled either by affinity-purified Abs against a cross-reactive region of PKG1-1 (B and C) or with the corresponding preimmune serum (A). Besides slight cytosolic labeling, several subcellular organelles were much more intensely labeled, including cilia (ci), the oral cavity (oc), numerous vesicle-like structures (arrowhead, B), and various structures occurring at the cell cortex (arrow, C). No signal was observed with the preimmune serum (A). Bars = 10 µm.

For most of the results achieved by confocal laser scanning microscopy (CLSM) analysis, we could identify equivalents in immunogold EM analyses (Fig. 6 and 7). Particularly consistent labeling occurred along the cell boundary formed by the intimately connected complex of the cell membrane and the outer part of the alveolar sac compartment, whose "inner" part (facing the cell center) was also labeled (Fig. 6A). Other "hot spots" in the cell cortex are the surroundings of trichocyst tips (Fig. 6A and B) and of ciliary basal bodies (Fig. 6A and 7A). The most consistently labeled structures were the membranes of cilia (Fig. 6C and D). Less densely labeled areas were the surface membranes and basal bodies along the oral cavity (Fig. 7B) and cytoplasmic domains around vesicles presumably involved in trafficking. In the cortex, this includes regions with medium-sized vesicles and the endoplasmic reticulum (Fig. 7A). Deeper in the cytoplasm, domains with "discoidal vesicles" (engaged in recycling from the phagocytic vacuoles and the cytoproct to the oral cavity [1]) and the surfaces of larger vesicles attributable to the phagolysosomal apparatus (Fig. 7C) were labeled.



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  		FIG. 6. Immunogold EM localization of PKGs. (A) The structures most heavily labeled with Au5 nm particles (encircled) are complexes formed by the cell membrane and the tightly apposed outer part of the alveolar sac (as) membrane (left side). Additional labeling occurs on the inner part of the alveolar sac membrane, around a trichocyst tip (tt), and around a ciliary basal body (bb). Note the absence of the label from "irrelevant" structures, such as the outside medium, lumens of alveolar sacs, kinedesmal fibers (kf), and a mitochondrion (m). (B) Enrichment of Au5 nm along the flanks of a trichocyst tip, in contrast to the trichocyst body (tb), shown below. (C and D) Labeling was predominantly enriched along the membranes of cilia, as shown in longitudinal (C) and cross (D) sections. Bars = 0.1 µm.



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  		FIG. 7. Immunogold EM localization. (A) Cortical labeling (Au5 nm [encircled]) of "hot spots" on the cell surface (cell membrane-alveolar sac complex, top right), around a ciliary basal body (bb), and in association with the endoplasmic reticulum and vesicles (v) of different sizes. (B) Oral cavity with label on the cell membrane near basal bodies (bb). (C) Labeling of discoidal vesicles (dv) and of larger, round to oblong vesicles (v), probably members of the lysosomal apparatus. Bars = 0.1 µm.

In total, the results obtained by immuno-EM analysis are fully compatible with those from immunofluorescence analysis (Fig. 5), including labeling of the oral cavity, cilia, vesicle-type structures, and elements of the cell cortex.

pp63/pf as putative substrate for PKG. Since PKGs seem to be enriched in cortical structures, we were interested in whether pp63/pf, an exocytosis-sensitive phosphoprotein of 63 kDa which is rapidly (within 0.1 s) dephosphorylated upon stimulation and slowly rephosphorylated during ≤1 min (31, 87), would be a putative candidate for an endogenous substrate, inasmuch as it is also a soluble protein that occurs in the cortex (38). In order to establish pp63/pf as a putative PKG substrate, we used a chromatographically enriched PKG fraction for in vitro phosphorylation studies with recombinant pp63/pf. As shown in Fig. 8, recombinant pp63/pf was intensely phosphorylated under conditions when PKGs were fully stimulated (lanes 5 and 6). Under conditions of autophosphorylation (without recombinant pp63/pf), we were also able to identify several phosphorylation bands between 75 and 105 kDa (Fig. 8, lane 3). Similar bands have also been observed in Western blots and therefore may represent autophosphorylated forms of PKGs rather than copurified substrate proteins. The specificity of these phosphorylation studies is also supported by the finding that enzyme activity is stimulated by cGMP (compare Fig. 8, lane 2, with lanes 5 and 6) but not by cAMP (data not shown).



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  		FIG. 8. Phosphorylation of recombinant pp63/pf by endogenous PKG in vitro. Phosphorylation of recombinant pp63/pf was observed when 3 µg of recombinant pp63/pf (lanes 1, 2, 4, 5, and 6) was assayed with ~1.25 µg of a PKG-containing fraction (lanes 2, 3, 5, and 6) either in the absence (lane 2) or in the presence of 0.15 µM cGMP (lane 5) or 0.15 µM 8-pCPT-cGMP (lane 6). The other two bands, at ~75 and 100 kDa, were also observed under autophosphorylation conditions in the PKG fraction (without pp63/pf, lane 3). Note the stimulatory effect of cGMP on the phosphorylation of pp63/pf (compare lane 2 with lanes 5 and 6). In contrast, recombinant pp63-1/pf was not phosphorylated when assayed under similar conditions in the absence of PKG (lanes 1 and 4).


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DISCUSSION
 
General aspects. Unlike higher eukaryotic cells, Paramecium is not known to perform alternative splicing of mRNAs. Diversification of PKGs may therefore be achieved by increasing gene numbers, probably facilitated by recent whole-genome duplication (L. Sperling and J. Cohen, personal communication). In mammals, splice variants of PKG occur (19, 48) which are either differentially located, just like PKG isoforms in Drosophila Malpighian tubule cells (50), or regulate the expression of another isoform (21). One may speculate that some of the paralogs we found in Paramecium may potentially exert such a regulatory function. Moreover, the plethora of PKG gene products may contribute to the regulation of the elaborated vesicle trafficking system and to the elaborated surface patterning of a Paramecium cell with their clear-cut functional domains.

Gene structure and domains of PKG molecules. The gene structures of the PKG 1 and PKG 2 subfamilies, shown in Fig. 1, fulfill a variety of criteria typical of PKGs, e.g., of mammalian species (19, 48). (i) They represent single molecules with typical molecular masses, thus fitting with PKG fractions isolated from Paramecium cells (53) or from other lower eukaryotic cells (55, 80), including Apicomplexan parasites (16, 23). This is in contrast to cAMP-dependent protein kinases, including those from Paramecium (9; R. Kissmehl and H. Plattner, unpublished observation), which possess separate catalytic and regulatory subunits. (ii) Most of the Paramecium PKGs possess a molecular organization characteristic of mammalian PKGs (19, 48), including a regulatory part with two cGMP-binding domains in the N-terminal half and a catalytic (kinase) domain in the C-terminal half, with considerable similarities in the deduced amino acid sequences (Table 2). (iii) They display most of the relevant motifs typical of PKGs (19, 48), with the exception that the amino-terminal domains of PKG1-1 and PKG2-1 seem to have neither a dimerization site (no conserved leucine/isoleucine heptad repeat) nor an autoinhibitory region. The latter is also true for the other PKG paralogs in Paramecium compared with the consensus sequence K/R-K/R-X-G/A-S-E-P/S described previously (60).

An interesting aspect also comprises the variable numbers of putative cGMP-binding domains within the regulatory domains of individual PKG subfamilies (Fig. 3). Although their individual properties still have to be elucidated in future work, e.g., binding affinities, dissociation kinetics, etc., the relevance and consequences of this heterogeneity, together with the large number of PKG paralogs, may reflect the responsiveness of Paramecium cells to widely varying environmental conditions. It would also be interesting to know whether and how PKG paralogs with nonconserved cGMP-binding domains, such as PKG16-1, may be able to contribute as effectors for cGMP.

Comparison with other Alveolata. Parasitic relatives, such as Plasmodium falciparum, have larger PKG molecules deduced from larger genes, with three active and a degenerate fourth cGMP-binding domain (11, 12). PKGs from Eimeria tenella have three cGMP-binding domains, and two isoforms are formed from a single-copy gene by alternative translation initiation (23). Interestingly, the overall domain structure of most of the PKG genes of Paramecium therefore resembles much more those from less related species, such as mammals or plants, than those from their closest parasitic relatives. However, the similarity is much closer when the domains of PKGs are compared at the amino acid level (Table 2).

Localization and potential functions of PKGs. For Paramecium, no data are available on cGMP-sensitive ion channels or cGMP-activated PD activity, while the occurrence of PKG activity is well established (53). Previous attempts to localize PKGs in Paramecium (41) are difficult to interpret, in contrast to the case of GC. The latter has been clearly localized to ciliary membranes, to the complex formed by the outer alveolar sac membrane and the cell membrane, and to the lower (inner) part of the alveolar sac envelope (47). In conceivable agreement with this is the immunofluorescence localization of PKG in cilia and some cell surface compartments (Fig. 6), which in part is reminiscent of green fluorescent protein-labeled alveolar sacs (27). This would suggest a rapid and well-localized interaction between cGMP, after formation by GC, and PKG. The question becomes, then, what may be the functional implications of this interaction?

Relevance of PKGs for ciliary activity. Ciliary beat activity in Paramecium is rapidly reversed by depolarization-dependent Ca2+ influx (17), and this process seems to be modulated by a [cGMP] increase (51, 59). cGMP, in turn, is produced by Ca2+-dependent GC activation. Concomitantly, Ca2+ has been established as the primary signal, since pressure injection of cGMP under voltage clamp conditions does not change the ciliary beat direction (56). Furthermore, when cGMP is applied in vitro to permeabilized Paramecium models, it increases the beat frequency without changing its direction, with only a minor change in the beating pattern (8). Surprisingly, the overall reaction is not much different from the effects of cAMP. An increase in cAMP, as it occurs upon hyperpolarization, is accompanied by an increased ciliary beat frequency without any change in swimming direction (84). Thus, the [cGMP] increase seen during depolarization is at odds with the absence of a reversal effect of cGMP application in vitro. How can this be reconciled?

One reason could be the lack of signal transduction in the absence of PKGs, assuming extraction during cell permeabilization. In fact, at least some of the PKGs are soluble (see Results), e.g., the enzyme extracted from cilia under previously described conditions (53). If so, the involvement of PKGs would be plausible despite the restricted effects of cGMP in vitro. Concomitantly, the application of PKGs to permeabilized Paramecium cells induces ciliary reversal in atalanta A mutants, which normally do not show this reaction (3). Although a ciliary protein of 59 kDa can be selectively phosphorylated in vitro in a cGMP-dependent manner (3), less is known about PKGs and the molecular machinery regulating ciliary beat direction in Paramecium. However, the involvement of PKGs appears to be very likely due to the three forms that have been extracted in previous biochemical work (53).

Potential relevance for exo-endocytosis. In metazoan cells, the global response generally achieved by PKG activation is lowering the intracellular [Ca2+] and therefore reestablishing [Ca2+] homeostasis (30, 48, 77). This may also hold for Paramecium because of the following observations. (i) GC is associated with alveolar sac membranes (47), which are established cortical Ca2+ stores activated upon exocytosis stimulation (61). (ii) [cGMP] rises during exocytosis stimulation (42), although this lags behind the Ca2+ signal determined by fast fluorochrome analyses or by analytical EM methods, i.e., quenched-flow/X-ray microanalysis (= "EDX") (61). Both of these methods provide considerable spatial resolution, while the [cGMP] changes could be measured, although quickly, only globally. (iii) During exocytosis, a 63-kDa phosphoprotein, pp63/pf, is rapidly dephosphorylated within 0.1 s and slowly rephosphorylated during ≤1 min (31, 75, 87). It is localized in the cell cortex (38), as is PKG (this paper). The gene has been cloned, and the recombinant expression product showed phosphoglucomutase activity (26). The actual Ser/Thr phosphorylation sites have been determined by matrix-assisted laser desorption ionization analysis and prognosticated as sites for casein kinase 2 and PKG activities (43). Based on structure analysis by X-ray diffraction, phosphorylation sites could be positioned on the surface of the pp63/pf molecule so that they are accessible to the respective kinases (54). The predicted phosphorylation of pp63/pf has been verified previously for casein kinase 2 (37, 79). In the present paper, we have achieved this for PKGs, although the precise PKG form(s) contributing remains to be identified. A putative role of this phosphorylation has already been discussed recently (63), including regulating contact with substrate proteins (7).

In summary, considering exo-endocytosis-regulated processes, the potential role of PKG involvement is not less intriguing than, for instance, the level of discussion concerning neuronal (14, 85) and a variety of other systems (20, 22).

Relevance for other activities in Paramecium. Other Ca2+-based signaling mechanisms in Paramecium include spontaneous circadian rhythms involving Ca2+ and cGMP/cAMP oscillations (25), unfortunately with unknown signal transduction pathways. In this regard, periodic cGMP changes and the involvement of PKGs have been established for some mammalian neurons (74).

PKG-mediated phosphorylation processes have recently been extended to Rac-type proteins (32), with an involvement of cytoskeleton structuring and vesicle trafficking. The latter is highly diversified in Paramecium, where it awaits an in-depth molecular analysis (62).

Conclusions. We could establish in P. tetraurelia cells the occurrence of an unexpectedly large family of PKG genes. The large number is not surprising if one considers the large number of sequences altogether coding for protein kinases, as found already at an early stage of the Paramecium genome project (72). The different PKG isoforms may serve specific functions depending on their positions in the cell. In cilia, PKGs can phosphorylate proteins to be more clearly defined. Their potential role as a switch for changing ciliary beat direction has yet to be established in detail. In the cell soma of Paramecium, PKGs will perform independent activities, e.g., in the context of rephosphorylation of the exocytosis-sensitive phosphoprotein pp63/pf. The data presented here will allow one to address these questions in much more detail beyond the biochemical data previously published.


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ACKNOWLEDGMENTS
 
We gratefully thank Jean Cohen for access to the library screening, J. E. Schultz (University of Tübingen) for providing us with the genomic Paramecium library, Claudia Stuermer for access to the CLSM, Ivonne Sehring for excellent technical help in the CLSM analysis, and Doris Bliestle for electronic image processing.

This work was supported by Deutsche Forschungsgemeinschaft grants to H.P.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, University of Konstanz, P.O. Box 5560, 78457 Konstanz, Germany. Phone: 49-7531-88-3712. Fax: 49-7531-88-2245. E-mail: roland.kissmehl{at}uni-konstanz.de. Back


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Eukaryotic Cell, January 2006, p. 77-91, Vol. 5, No. 1
1535-9778/06/$08.00+0     doi:10.1128/EC.5.1.77-91.2006
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