- Email: [email protected]
Identification of Possible Phosducins in the Ciliate Blepharisma japonicum
Protist
Protist, Vol. 155, 181–192, June 2004 http://www.elsevier.de/protist Published online 14 June 2004
ORIGINAL PAPER
Identification of Possible Phosducins in the Ciliate Blepharisma japonicum Hanna Fabczak, Katarzyna Sobierajska, and Stanislaw Fabczak1 Polish Academy of Sciences, M. Nencki Institute of Experimental Biology, Department of Cell Biology, 3 Pasteur St., PL-02 093 Warsaw, Poland Submitted July 16, 2003; Accepted December 22, 2003 Monitoring Editor: Janine Beisson
Examination of ciliate Blepharisma japonicum whole cell lysates with an antibody against phosphoserine and in vivo labeling of cells with radioactive phosphate revealed that the photophobic response in the ciliate is accompanied by a rapid dephosphorylation of a 28 kDa protein and an enhanced phosphorylation of a 46 kDa protein. Analysis with antibodies raised against rat phosducin or human phosducin-like proteins, identified one major protein of a molecular weight of 28 kDa, and two protein bands of 40 kDa and 93 kDa. While the identified ciliate phosducin is phosphorylated in a light-dependent manner, both phosducin-like proteins exhibit no detectable dependence of phosphorylation upon illumination. An immunoprecipitation assay also showed that the ciliate phosducin is indeed phosphorylated on a serine residue and exists in a phosphorylated form in darkness and that its dephosphorylation occurs in light. Immunocytochemical experiments showed that protozoan phosducin and phosducin-like proteins are localized almost uniformly within the cytoplasm of cells adapted to darkness. Cell exposure to light caused a pronounced displacement of the cell phosducin to the vicinity of the plasma membrane; however, no translocation of phosducin-like proteins was observed upon cell illumination. The obtained results are the first demonstration of the presence and morphological localization of a possible phosducin and phosducin-like proteins in ciliate protists. Phosducin and phosducin-like proteins were found to bind and sequester the β γ -subunits of G-proteins with implications for regulation of G-protein-mediated signaling pathways in various eukaryotic cells. The findings presented in this study suggest that the identified phosphoproteins in photosensitive Blepharisma japonicum may also participate in the regulation of the efficiency of sensory transduction, resulting in the motile photophobic response in this cell.
Introduction The ciliate Blepharisma japonicum possesses an endogenous photoreceptor system that renders the cells capable of avoiding light (Fabczak 2000a; Giese 1973; Maeda et al. 1997; Matsuoka et al. 2000b; Tao et al. 1994). This cell behavior results 1
Corresponding author; fax 48 22 822 5342 e-mail [email protected]
from its motile photophobic response to increasing light intensity (Fabczak et al. 1993a; Kraml and Marwan 1983). The cell photophobic response consists of a delayed cessation of forward swimming, a period of backward movement (ciliary reversal) followed by restoration of forward swimming mostly in an altered direction (Fabczak et al. 1993a). During prolonged intense illumination, an increase in forward movement velocity (positive photokinesis) and 1434-4610/04/155/02-181 $ 30.00/0
182
H. Fabczak et al.
a marked elongation of the cell body is also observed (Fabczak et al. 1993a; Ishida et al. 1989; Kraml and Marwan 1983). The ciliary reversal during the photophobic response is mediated by a membrane action potential, which can be elicited by an early light-induced receptor potential (Fabczak et al. 1993a). The ciliate photoreceptor system seems to be coupled to membrane potentials and photomotil-
ity changes, as in the case of photoreceptor cells of higher organisms (Rayer et al. 1990), via a Gprotein-mediated sensory transduction pathway (Fabczak 2000a, b; Fabczak et al. 1993b; 1998; 1999). It has been shown that phosphorylated proteins play an important role in regulating various sensory transduction pathways (Bünemann and Hosey 1999; Cohen 1982; Dickman and Yarden 1999; Graves and Krebs 1999; Greengard 1978). There is evidence that in photoreceptor cells of the vertebrate retina, phosphorylation and dephosphorylation of phosducin and phosducin-like proteins participate in the regulation of the phototransduction cascade (Lee et al. 1984; Polans et al. 1979; Schultz 2001). These phosphoproteins were found to bind with a high affinity to βγ-subunits of the specific Gprotein, transducin, with a significant implication for the regulation of the availability of transducin for light activation via rhodopsin (Bauer et al. 1992; Lee et al. 1992). Phosducin was originally discovered about 15 years ago in photoreceptor cells of bovine retina, and more recent reports show that phosducin as well as phosducin-like proteins have been found in many other cells (Blaauw et al. 2003; Danner and Lohse 1996; Flanary et al. 2000; Kasahara et al. 2000; Lee et al. 1987; Reig et al. 1990). Since phosducin and phosducin-like proteins are so ubiquitous, it is of great interest to know from an evolutionary point of view whether these functionally important phosphoproteins also exist in photosensitive lower eukaryotes, such as the ciliate Blepharisma japonicum. Therefore, the purpose of this study was the detection of proteins exhibiting light-
Figure 1. Detection of light-dependent protein phosphorylation in whole cell homogenates from Blepharisma japonicum. (A) Western blot with monoclonal antibody PSER-4A9. (B) Test for equal loading and quantification, performed with control antibody against βtubulin. (C) Quantification of protein phosphorylation under different experimental conditions. Lane 1: cells adapted to darkness (control); lane 2: cells exposed to light for 2 s; lanes 3 and 4: cells exposed to light for 2 s and then adapted to darkness for 3 min and 5 min, respectively; lane 5: cells treated with 4 mM potassium ions, lane 6: cells adapted to darkness in control medium then treated with 4 mM potassium ions and finally exposed to light for 2 s. The arrowheads indicate two main protein bands, one of 28 kDa and a second of 46 kDa, which displayed distinct alterations in the level of phosphorylation upon cell illumination. The protein phosphorylation level in control cells is defined as a 100% value. The polyacrylamide gel was calibrated with SDS-PAGE protein molecular weight markers.
Phosducins in Blepharisma japonicum
Figure 2. In vivo phosphorylation of proteins in Blepharisma japonicum. (A) SDS-PAGE of cell lysate stained by Coomasie Brilliant Blue. (B) Corresponding autoradiogram. Lane 1: cells adapted to darkness (control); lane 2: ciliates exposed to light for 2 s; lane 3: dark-adapted cells incubated in control solution with addition of 4 mM potassium ions; lane 4: cells adapted to darkness in control medium supplemented with 4 mM potassium ions and then exposed to light for 2 s. Arrowheads mark the same 28 kDa and 46 kDa phosphoproteins shown in Figure 1. Arrows show proteins of about 70 kDa and 80 kDa, which display phosphorylation increase upon light exposure and double arrowheads indicate proteins of about 40 and 54 kDa whose phosphorylation level is lower after illumination. Other details as in Figure 1.
dependent changes in the extent of their phosphorylation, their identification and morphological localization in ciliate Blepharisma japonicum with in vivo phosphorylation assays and immunocytochemical methods.
Results Light-Regulated Protein Phosphorylation Western blot analysis of whole cell lysates from Blepharisma japonicum cells demonstrated that the antibody raised against phosphorylated serine epitopes PSER-4A9, recognizes several protein bands (Fig. 1). The phosphorylation level of most of the labeled proteins was unaffected in illuminated cells, except for two prominent protein bands, one of molecular weight of 28 kDa and another of 46 kDa (Fig. 1A and C, lanes 1 and 2). The 28 kDa protein was most extensively phosphorylated in dark-
183
Figure 3. Immunoblotting for identification of phosducin and phosducin-like protein isoforms in whole cell lysate from Blepharisma japonicum cells and homogenates from bovine rod outer segments. (A) SDSPAGE stained by Coomassie Brilliant Blue. Lane 1: molecular weight markers; lane 2: lysate from ciliates; lane 3: bovine rod outer segment homogenate. (B) Corresponding immunoblot, which shows that antibody raised against phosducin recognizes one protein band with an apparent molecular weight of 28 kDa in the lysate from ciliates (lane 1) and of 33 kDa in the homogenate from bovine rod outer segments (lane 2). (C) Western blot indicating an interaction of antibody prepared against phosducin-like protein with protein bands of molecular weights of 40 kDa and 93 kDa in lysate from ciliates (lane 1) and of 33 kDa in bovine rod outer segment homogenate (lane 2). The polyacrylamide gel was calibrated with SDS-PAGE protein markers.
adapted cells (Fig. 1A and C, lane 1) and its phosphorylation level was significantly reduced in cells exposed to light (Fig. 1A and C, lane 2). In contrast, the 46 kDa protein displayed a low phosphorylation level during darkness (Fig. 1A and C, lane 1) and an enhanced phosphorylation in illuminated cells (Fig. 1A and C, lane 2). The light-induced phosphorylation changes of both proteins were entirely reversible, since progressive rephosphorylation of the 28 kDa protein and dephosphorylation of the 46 kDa protein were observed when cells, previously exposed to light, adapted to darkness (Fig. 1A and C, lane 3). About 5 min dark adaptation is sufficient to
184
H. Fabczak et al.
Figure 4. Immunoprecipitation of an endogenous phosducin protein isoform in Blepharisma japonicum with antibody raised against phosducin. The precipitated proteins in lysates from both dark-adapted (lane 1) and illuminated (lane 2) ciliates were analyzed by anti-phosphoserine antibody. The blot is the representive image of three independent experiments performed for detection of phosphoserine residues. Other details as in Figure 3.
evoke protein phosphorylation of light-treated cells that reaches the phosphorylation levels measured in the control organisms (Fig. 1A and C, lane 4). To test whether the observed changes in phosphorylation levels of the 28 kDa and 46 kDa proteins were specifically induced by light or resulted simply from cell membrane depolarization, the effect of ionic stimulation was examined. These experiments showed that treatment of dark-adapted cells with high concentrations of external potassium ions did not elicit dephosphorylation of the 28 kDa protein and phosphorylation of the 46 kDa protein (Fig. 1A and C, lane 5) as observed in cells exposed to light (Fig. 1A and C, lane 2). Levels of phosphorylation of both phosphoproteins in homogenates from darkadapted ciliates, which were first treated with high potassium ions concentrations and subsequently exposed to light (Fig. 1A and C, lane 6), were similar to those obtained for cells that were only illuminated (Fig. 1A and C, lane 2). As an additional control of protein phosphorylation in Blepharisma japonicum, an in vivo analysis was performed using radioactive phosphate [32P]. These studies revealed that most of the labeled phosphoproteins in the cell lysate had molecular
weights higher than 80 kDa. Their phosphorylation was not appreciably affected by illumination (Fig. 2). However, two proteins of about 70 kDa and 80 kDa showed higher levels of phosphorylation upon cell illumination (Fig. 2B, arrows) and proteins of about 40 kDa and 55 kDa had lower levels of phosphorylation under the same light conditions (Fig. 2B, double arrowheads). In the dark, the most extensively labeled phosphoprotein among the lower molecular weight proteins had an apparent molecular weight of 28 kDa (Fig. 2B, lane 1). The phosphorylation level of this protein was significantly lowered by illumination (Fig. 2B, lane 2). The protein phosphorylation of 28 kDa in the homogenate from dark-adapted cells treated with high concentrations of potassium ions and then illuminated was similar to that of control cells (Fig. 2B, lanes 2 and 4). A major phosphorylated protein band of 46 kDa could be observed in cells exposed to light (Fig. 2B, lanes 2 and 4) whereas in dark-adapted ciliates the level of phosphorylation of this protein was much lower (Fig. 2B, lanes 1 and 3).
Identification of Possible Phosducin and Phosducin-Like Proteins An immunoblotting assay with an antibody against phosducin showed clear immunoreactivity only with one protein band of 28 kDa in the whole cell lysate from ciliates (Fig. 3B, lane 1) and with a 33 kDa band in bovine rod outer segment homogenate (Fig. 3B, lane 2), indicating sufficient selectivity of the applied antibody. The 28 kDa protein detected in this assay evidently resembles the 28 kDa protein band shown in Figures 1 and 2, exhibiting light-dependent phosphorylation (Fig. 1A and C, lanes 1 and 2; Fig. 2B). An antibody raised against a phosducin-like protein revealed a distinct immunoreactivity and recognized two protein bands with molecular weights of 40 kDa and 93 kDa in the cell homogenate (Fig. 3C, lane 1), as well as a 33 kDa protein band in bovine rod outer segment homogenate (Fig. 3C, lane 2). None of these detected phosphoproteins was identified in the phosphorylation pattern presented in Figure 1A as a protein which exhibited phosphorylation dependence upon illumination. However, the changes in the phosphorylation levels of the 40 kDa protein were established in the in vivo experiments using [32P] (Fig. 2B). In control blots with identical protein load and incubated with the secondary antibody only, no immunolabeling was found, confirming a high specificity of labeling (not shown). To further examine whether phosphorylation changes of the detected ciliate phosducin were induced by light, the cell lysates from control and illu-
Phosducins in Blepharisma japonicum
185
Figure 5. Morphological immunolocalization of phosducin and phosducin-like proteins in Blepharisma japonicum. (A) Control cells incubated with TBS containing 1% BSA in place of the primary antibody. Labeling of cells with antibody against phosducin (B, C, E, F) and phosducin-like proteins (G, H, I) indicates the localization of these phosphoproteins within the cell cytoplasm. (B, E) Translocation of phosducin protein from the cytoplasm to the vicinity of plasma membrane occurring in organisms exposed to light for 30 s. (H, I) Picture indicating a lack of redistribution of phosducin-like protein upon cell exposure to light. (D) Cell cortical structures immunolabeled with mAb12G9 antibody. Ciliary rows (cr) and the basal bodies, adoral membranelles (AZM) of oral apparatus with the metachronal waves (arrowheads) and paroral membrane (PM) are marked.
minated ciliates were immunoprecipitated with the antibody against phosducin. Next, the precipitated proteins were analyzed by the PSER-4A9 antibody. In both dark-kept and illuminated cell samples, the anti-phosducin antibody precipitated exclusively one protein phosphorylated on a serine residue, which had a molecular weight of 28 kDa (Fig. 4). In light-treated cells, phosphorylation of the 28 kDa protein decreased significantly (Fig. 4, lane 2), whilst in the case of untreated cells, the phosphorylation level of this protein was higher (Fig. 4, lane 1). These
experimental results further prove that in Blepharisma japonicum there is a 28 kDa phosducin protein that displays enhanced phosphorylation in darkness and that becomes markedly dephosphorylated in light.
Localization of Ciliate Phosducin and Phosducin-Like Proteins The localization of identified phosducin and phosducin-like proteins in the ciliate was investigated by
186
H. Fabczak et al.
immunocytochemical analysis using the antibodies against these phosphoproteins. The examinations revealed prominent fluorescence in cells, indicating the occurrence of phosducin and phosducin-like protein isoforms in the ciliate tested (Fig. 5). The dark-adapted cells shown in Figure 5C, F and G exhibit fluorescence which is almost evenly distributed in their cytoplasm. After exposure of cells to light for 30 s, displacement of ciliate phosducin protein from the cytosol to the close vicinity of the plasma membrane is observed (Fig. 5B and E). In Figure 5E, phosducin translocation is indicated during the ongoing presence of light stimulation as a bright fluorescence, which concentrates at the cell margin along with a simultaneous slight reduction of the fluorescence intensity in the central part of the cell. While the phosducin protein clearly translocates upon light stimulation, no redistribution of phosducin-like proteins was observed within the cell under the same light conditions (Fig. 5H and I). No immunolabeling was observed in control cells treated with secondary antibodies only (Fig. 5A). To verify that the cell fixation and permeabilization procedures were performed correctly, a mAb12G9 antibody, known to label specifically some cell cortical structures in ciliates (Strzyzewska-Jówko ˙ et al. 2003), was used. The experiments showed that the antibody used did indeed decorate some cortical structures of Blepharisma japonicum cells as in other ciliates. Figure 5D shows marked immunolabeling of ciliary rows (cr) and basal bodies at the anterior part of the cell, adoral membranelles (AZM) with metachronal waves (indicated by arrowheads) and paroral membrane (PM) within the cell oral apparatus (Aescht and Foissner 1998). These data proved sufficient permeation of antibodies used to localize phosducin and phosducin-like proteins in the protozoan cells tested.
Discussion Protein phosphorylation is recognized as a highly important mechanism by which internal events in cells are controlled by external physiological stimuli (Bünemann and Hosey 1999; Dickman and Yarden 1999; Cohen 1982; Graves and Krebs 1999; Greengard 1978). In visual systems, it has been shown that the process of protein phosphorylation and dephosphorylation is involved in the inactivation of the active form of rhodopsin and in its restoration (Bownds et al. 1972; Frank et al. 1973; Kühn 1974). Phosducin and phosducin-like proteins, major cytosolic phosphoproteins in mammalian photoreceptor cells, have been postulated to participate in the
downregulation of phototransduction systems (Schulz 2001). It has also been shown that phosducins are phosphorylated in darkness and that this phosphorylation may be catalyzed by protein kinase A, Ca2+-calmodulin kinase II or G-protein-coupled receptor kinase 2 (Lee et al. 1990; Ruiz-Gómez et al. 2000; Thulin et al. 2001; Wilkins et al 1996; Willardson et al. 1996; Yoshida et al. 1994). Dephosphorylation of these phosphoproteins may occur by activation of type 1 or 2A phosphatases upon cell exposure to light (Lee et al. 1984; Pagh-Roehl et al. 1995). The most important characteristic of phosducin appears to be its high affinity sequestration of the βγ-subunit of heterotrimeric G-proteins (Gβγ) (Gaudet et al. 1996; 1999; Schulz 2001; Yoshida et al. 1994). Competition between phosducin and the α-subunit of retinal specific G-protein (transducin) for available Gβγ diminishes signal amplification through interference with the reassembly of the Gprotein complex that is essential for G-protein recycling with activated rhodopsin. In general, similar mechanisms have been postulated for phosducinlike proteins (Schulz 2001). While phosducin was originally discovered in vertebrate retinas and developmentally related pineal glands (Kuo et. al. 1989; Lee et al. 1990; Reig et al. 1990), more recently, the existence of many phosducin-related proteins has been reported in numerous other tissues, including olfactory epithelium, liver, brain and spleen (Schulz 2001). Phosducin isoforms have also been lately found in some lower eukaryotic organisms. In the yeast Saccharomyces cerevisiae, two genes were identified, encoding phosducin-like proteins, Plp1 and Plp2, which bind Gβγ (Flanary et al. 2000). The Cryphonectria parasitica gene, designated bdm-1, encodes a phosducin-like protein involved in Gβγ function and Gα accumulation (Kasahara et al. 2000). Similarly, in Dictyostelium discoideum three genes, phlp-1, phlp-2 and phlp-3, each encoding a phosducin-like protein of a different group, were discovered and it was established that one of them, named phosducin-I, also plays a role in the control of G-protein pathway signaling (Blaauw et al. 2003). In this study, we have explored the possibility that light stimulation followed by motile photophobic response may also alter the phosphorylation level of proteins in Blepharisma japonicum. The photoreceptor system in the ciliate is thought to be coupled to membrane potential and motility alterations via G-protein and also possibly different second messenger pathways, this stems from observations that the photophobic response and another light-evoked physiological phenomenon, cell body elongation, were significantly affected by modulation of internal cGMP levels pointing to cyclic nucleotide as a pos-
Phosducins in Blepharisma japonicum
sible signal transmitter in both behavioral processes (Fabczak et al. 1993b, Ishida et al. 1989). However, there is a line of evidence that phosphoinositides may also contribute to photophobic behavior in Blepharisma japonicum, i.e. cell treatment with drugs interfering effectively with the phosphoinositide signaling pathway, strongly inhibits photophobic responses and prolongs response latency significantly (Fabczak et al. 1996). The reported immunoblotting and immunocytochemical examination also show that within the ciliate cortex an inositol trisphosphate receptor-like protein is present (Fabczak et al. 1998; Matsuoka et al. 2000a). And finally, recent radioimmunological studies allowed the demonstration of fluctuations in the internal inositol trisphosphate level of cells upon exposure to light, which seemed to result from modulation of the activity of cellular phospholipases (Fabczak 2000a; Fabczak et al. 1999). Based on the above-mentioned data, it is obvious that the detailed molecular mechanism of light signal transduction in Blepharisma japonicum is still elusive at present and its complete clarification awaits further investigations. Our immunoblotting investigations of Blepharisma japonicum revealed the existence of two proteins exhibiting light-dependent alterations in their phosphorylation levels (Figs 1A, C and 2B). These phosphoproteins have apparent molecular weights of 28 kDa and 46 kDa and display reversible dephosphorylation and phosphorylation upon illumination and darkness, respectively. The changes in phosphorylation level were specifically initiated by light, since no significant differences in the level of phosphorylation were detected in cells treated with high external concentration of potassium ions (Fig. 1A and C, lane 5). As shown in Figure 1A and C, lane 6, and also in Figure 2, only subsequent illumination of potassium-treated ciliates results in dephosphorylation of the 28 kDa protein and increased phosphorylation of the 46 kDa protein. Potassium ions at higher concentrations are known to effectively depolarize the cell membrane and may trigger an action potential followed by ciliary reversal, as in the case of depolarizing photoreceptor potential in the ciliate tested (Fabczak et al. 1993a). The immunoblotting assays show that only the 28 kDa protein is recognized by the applied antibody against phosducin (Fig. 3A) and it correlates well with the 28 kDa protein found in the protein phosphorylation pattern that displays enhanced phosphorylation in the dark and is dephosphorylated upon exposure to light (Fig. 1A and C, lanes 1 and 2; Fig. 2B, lanes 1 and 2 and also Fig. 4, lanes 1 and 2). Similar light-evoked changes in the phosphorylation level of both the 28 kDa and 46 kDa proteins
187
were observed in Blepharisma japonicum in in vivo phosphorylation analysis using radioactive phosphate (Fig. 2B). Moreover, the experiments revealed four other proteins of molecular weights of 40, 55, 70 and 80 kDa, which seem to undergo a light-dependent phosphorylation as well. The diversity in phosphorylation patterns shown in Figures 1 and 2, possibly results from the labeling by [32P] not only serine but also tyrosine or threonine residues. This may be caused by low specificity of the applied Pser-4A9 antibody, which has been shown to depend greatly on the surrounding amino acid sequence. In addition, the immunoprecipitation assay indicated that the ciliate phosducin is phosphorylated on serine residues, and indeed it exists in highly phosphorylated and dephosphorylated forms in darkness (Fig. 4, lane 1) and in light (Fig. 4, lane 2), respectively The apparent molecular weight of 28 kDa of the ciliate phosducin is similar to that of 26 kDa and 32 kDa phosducin isoforms found in yeast (Flanary et al. 2000) or in mammalian phosducins whose molecular weight was estimated from the protein amino acid sequence to be 28 kDa, although they migrate on gels as 33 kDa proteins (Lee et al. 1984; Schulz 2001). The cell homogenates probed with the antibody against phosducin-like protein revealed two proteins of 40 kDa and 93 kDa. Although none of these phosphoproteins corresponds to any proteins exhibiting light-dependent phosphorylations as shown in the immunoblotting experiments (Fig. 1A), in the in vivo phosphorylation experiments, the 40 kDa protein exhibits phosphorylation alterations upon light stimulation (Fig. 2B). There is a possibility that this protein resembles the 40 kDa protein detected with the antibody against phosducin-like protein (Fig. 3C). However, immunoprecipitation of the [32P] labeled phosphoproteins with the antibody against phosducin-like proteins has not proved this (data not shown). In the photoreceptor cells of the visual systems, it has been reported that photoreceptor activation by light is followed by phosducin dephosphorylation that results in its translocation between inner and outer rod segments (Kuo and Miki 1989; Lee et al. 1992) or redistribution towards the membrane, where it binds Gβγ subunits of G-proteins (Schulz et al. 1998). The cellular components that translocate in response to light, besides phosducin, also include transducin, arrestin and 2A phosphatase (Brown et al. 2002; McGinnis et al. 2002; Sokolov et al. 2002). These translocations have been shown to affect photoreceptor adaptation to background illumination (Hardie 2002). However, in the study by Nakano et al. (2001), little if any light-induced movement of phosducin was found.
188
H. Fabczak et al.
Nevertheless, immunocytochemical examination of section images of Blepharisma japonicum showed that light exposure promotes significant concentration of the ciliate phosducin at the cell margin (Fig. 5B and E), while the distribution of both phosducin-like proteins was unchanged under the applied light conditions (Fig. 5H and I). It appears from these observations that phosducin and phosducin-like proteins identified in Blepharisma japonicum may play an important physiological role, similar to that proposed in the vertebrate retina (Thulin et al. 1999). These data support our suggestion that the identified cytoplasmic 28 kDa protein (Figs 1 and 2) may represent a ciliate phosphoprotein exhibiting properties similar to that of phosducin (Figs 3 and 4), i.e. light-dephosphorylated phosducin may translocate towards the cell membrane and thereby binds βγ-subunits of G-proteins and influences the cell phototransduction cascade. Phosducin binding to Gβγ may control the kinetics of second messenger signaling in ciliates by inhibition of Gβγ-mediated phosphoinositide hydrolysis, as is the case in other cells (Hawes et al. 1994; McLaughlin et al. 2002). This supposition is quite likely, since the presence of a protein homologous to the α-subunit of bovine Gproteins in Blepharisma japonicum has been established recently (Fabczak 2000a, b). Among the ciliate protozoans, the participation of G-proteins in signaling pathways has already been postulated for Paramecium bursaria (Shinozawa et al. 1996) and Stentor coeruleus, in which the α-subunits of G-proteins have been partially cloned (Fabczak et al. 1993c; Marino et al. 2001). The results obtained in this study demonstrate for the first time that the ciliate protist Blepharisma japonicum possesses phosducin and phosducinlike proteins, which have been shown to be present in various tissues of higher eukaryotes (Schulz 2001), and more recently in lower eukaryotic cells (Blaauw et al. 2003; Flanary et al. 2000; Kasahara et al. 2000). These phosphoproteins are characterized as the cytosolic regulators of G-protein function. These data also have important evolutionary implications since they suggest that light transduction in the ciliate protist seems to be under control of similar processes of phosducin phosphorylation and dephosphorylation as in the evolutionarily distant photoreceptor cells of higher organisms. Beyond this suggestion, little is currently known about the detailed mechanism by which the discovered ciliate phosducin is involved in the transduction of light signals in Blepharisma japonicum cells. The knowledge of phosducin function in vivo in the ciliate photoperception system may support in vitro studies on the role of phosducin in other G-protein-
mediated signaling pathways. Sequencing of these proteins and detailed characterization of their function in the cell’s photobehavior is the topic of future investigations.
Methods Cell culture: Stock cultures of ciliate Blepharisma japonicum were grown in 300 ml glass dishes in Pringsheim solution at pH 7 at room temperature under semi dark conditions (Fabczak 2000b). The cells were fed twice a week with the small ciliate Tetrahymena pyriformis, axenically grown at room temperature in a medium composed of 1.0% proteose peptone (Difco, USA) and 0.1% yeast extract (Difco, USA). The cultures of Tetrahymena pyriformis were centrifuged and washed in Pringsheim solution before feeding Blepharisma japonicum. Prior to each experiment, the Blepharisma japonicum cells were starved for at least 24 h, then gently collected by a low speed centrifugation, and subsequently washed in an excess of fresh culture medium lacking nutritional components. Finally, the chosen cell samples were used for biochemical assays after incubation in darkness in fresh culture medium, referred to as a control medium, or in test solutions of designed compositions. Cell stimulation: Before an experiment, tested samples of dark-adapted cells were first left at rest for about 10 min to avoid any mechanical disturbances, and then exposed to light or ionic stimulations. Cell illumination (for 2 s) was provided by a 150 W fiber optic light source (MLW, Germany), equipped with an electromagnetic programmable shutter (model l22-841, Ealing Electro-Optics, England). Ionic stimulation was performed by incubation of cells in an external 4 mM potassium solution prepared by addition of a proper amount of 0.1 M KCl to the control medium. Electrophoresis and Western blotting: For immunoblotting analysis, cell samples were mixed with sample buffer (2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 1.0 mM EDTA and 62.5 mM Tris at pH 6.8) (Laemmli 1970), supplemented with protease and phosphatase inhibitors (50 mM NaF, 2 mM PMSF, 10 µM okadaic acid, 10 µg/ml aprotinin, 100 µg/ml leupeptin) to terminate reactions and then boiled for 5 min. Equal amounts of protein (30 µg) from solubilized cells were separated by 10% SDSPAGE with a Hoefer System (Amersham, USA) and transferred to nitrocellulose membranes (Bio-Rad, USA) during 60 min at 100 V in a transfer buffer (192 mM glycine, 20% methanol and 25 mM Tris at pH 8.3) as described elsewhere (Towbin et al. 1979).
Phosducins in Blepharisma japonicum
The membranes were then blocked for 2 h at room temperature by incubation in TBS buffer (150 mM NaCl, l0 mM Tris, pH 7.5) with 2% bovine serum albumin (BSA) and 0.2% Tween-20 (TBS-BSA-Tween blocking solution). For detection of specified cell phosphoproteins, the blots were incubated with monoclonal anti-phosphoserine antibody, clone PSER-4A9 (Alexis, Switzerland) at a concentration of 0.1 µg/ml in TBS-BSA-Tween solution or with a 5,000-fold dilution of the polyclonal antibodies raised against rat phosducin and human phosducinlike proteins (kindly provided by Professor Craig Thulin from Brigham Young College in Provo, USA; Thulin et al. 1999) in TBS-BSA-Tween solution overnight at 4 ºC. After several washes in TBS with 0.1% Tween-20, blots were incubated for 60 min at room temperature with secondary antibodies, antimouse or anti-rabbit IgG-horseradish peroxidase conjugates (Calbiochem, Germany) at a 1:10,000 dilution in TBS-BSA-Tween solution. Finally, membranes were washed in TBS-Tween buffer and developed with an Amersham ECL detection system (Amersham, Sweden). The intensities of immunoreactive protein bands were quantified using a laser densitometry and ImageQuant software (Bio-Rad). Molecular weights of proteins were determined based on their relative electrophoretic mobility with molecular weight markers (Bio-Rad). In the control sets of experiments, incubations with primary antibodies were omitted. Protein concentration in individual cell samples was estimated with a method reported elsewhere using BSA as a standard (Bradford 1976). Tests for equal loading and protein quantity were done throughout experiments with a control antibody, anti-β-tubulin (Sigma, USA). In vivo phosphorylation assay: The method used for labeling of cells in vivo was a slight modification of that described by Haystead and Garrison (1999). Samples of cells were incubated in darkness in phosphate-free Pringsheim solution supplemented with 0.5 mCi of [32P]-phosphate (Polatom ´ Radioisotope Center, Otwock-Swierk, Poland) for 2 h at room temperature to allow incorporation of 32P into endogenous cell phosphoproteins. After incubation, cell samples were transferred into Pringsheim solution without the radioactive isotope and then exposed to light or potassium ions as described above. Subsequently, control and treated cell samples were solubilized in Triton buffer (1% Triton X-100, 150 mM NaCl, 1.0 mM EDTA, 20 mM Tris, pH 7.4) supplemented with protease and phosphatase inhibitors (50 mM NaF, 1 mM phenylmethylsulfonylfluoride (PMSF), 10 µM okadaic acid, 5 µg/ml aprotinin, 10 µg/ml leupeptin) and centrifuged for 15 min at 12,000 rpm at 4 ºC. The obtained su-
189
pernatant was then collected and protein concentration in each cell sample was estimated (Bradford 1976). Finally, the protein samples were denatured by boiling for 5 min in sample buffer (Laemmli 1970) and subjected to 10% SDS-PAGE as described in the foregoing section. Autoradiography of the 32P-labeled phosphoproteins was done by exposing the dried polyacrylamide gels to X-ray film (Amersham, Sweden) in X-ray cassettes lined with high-speed and intensifying screens (DuPont) at –70 ºC. Immunoprecipitation: Samples of dark-adapted cells (control) or cells exposed to light were solubilized in Triton buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1mM EDTA) and then centrifuged for 15 min at 12,000 rpm at 4 ºC. The resultant supernatants after supplementing with protease and phosphatase inhibitors (10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 100 µM okadaic acid, 5 mM NaF) were used for the immunoprecipitation assay. The cell samples were subjected to preclearance for 1 h with 30% (v/v) protein A-Sepharose CL-4B (Amersham, Sweden) at 4 ºC. After this, the mixture was centrifuged for 1 min at 12,000 rpm at 4 ºC. The supernatant fraction was incubated for 1.5 h at 4 ºC with serum-containing antibody against phosducin and then for 1.5 h with a fresh portion of protein-A-Sepharose. The resin was washed three times in washing buffer containing 20 mM Tris-HCl, 150 mM NaCl at pH 7.4, transferred to new tubes for the third wash, then resuspended in sample buffer, boiled for 5 min and analyzed on SDS-PAGE according to the method of Laemmli (1970). The protein phosphorylation assay was carried out using the Western blot technique with monoclonal antibody against phosphorylated serine residues, clone PSER-4A9 (Alexis, Switzerland) as described above. Immunocytochemistry: The ciliates suspended in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgCl2, pH 6.9, and protease inhibitors of 1 mM PMSF, 1 µM leupeptin, 1 µg/ml aprotinin) were fixed for 10 min at –70 °C in 70% ethanol solution. After the fixing procedure, cells were washed twice in PHEM buffer and permeabilized for 10 min at 0 °C in PHEM buffer containing 0.05% Triton X-100. The cell samples were then washed twice with PHEM buffer and incubated for 1 h at room temperature in PHEM buffer with 2% BSA to block nonspecific binding. Subsequently, the cell preparations were exposed overnight at 4 °C to polyclonal antibodies against phosducin or phosducin-like proteins at 1:500 dilution in TBS solution with 1% BSA (TBS-BSA-Tween). The cell samples were also exposed to monoclonal mAbs 12G9 antibody (kindly provided by Professor Maria Jerka-Dzi-
190
H. Fabczak et al.
adosz from the Nencki Institute of Experimental Biology) at 1: 20 dilution in TBS-BSA. This antibody was shown to label specifically some ciliate cortical structures (Strzy˙zewska-Jówko et al. 2003). Following primary incubation and extensive washings in TBS-Tween, cell samples were finally exposed for 60 min at room temperature to a goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (Calbiochem) at 1:300 dilution in TBS-BSA-Tween or anti-mouse IgG conjugated with fluorescein isothiocyanate (Calbiochem) at 1: 200 dilution in TBS-BSATween. After incubation, cells were washed again in TBS solution and examined with a laser scanning confocal microscope (Leica, Germany). Nonspecific fluorescence was determined in cell samples suspended in TBS-BSA buffer and devoid of primary antibody.
Acknowledgements We wish to express our gratitude to Professor C. D. Thulin (Brigham Young University, Provo, Utah, USA) for generously supplying samples of polyclonal antibodies against phosducin and phosducin-like proteins. We would also like to thank Professor Maria Jerka-Dziadosz for the generous gift of mAb12G9 monoclonal antibody and helpful instructions concerning cell fixation and immunolabeling. This work was supported in part by grant No 6P04C-057-18 from the Polish Committee for Scientific Research to SF and statutory funding of the Nencki Institute of Experimental Biology in Warsaw.
References Aescht E, Foissner W (1998) Divisional morphogenesis in Blepharisma americanum, B. undulans, and B. hyalinum (Ciliophora: Heterotrichida). Acta Protozool 37: 71–92 Bauer PH, Muller S, Puzicha M, Pippig S, Obermaier B, Helmreich EJ, Lohse MJ (1992) Phosducin is a protein kinase A-regulated G-protein regulator. Nature 358: 73–75 Blaauw M, Knol JC, Kortholt A, Roelofs J, Ruchira, Posma M, Visser AJWG, Van Haaster PJM (2003) Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins. EMBO J 22: 5047–5057 Bownds MD, Bawes J, Miller J, Stahlman M (1972) Phosphorylation of frog photoreceptor membranes induced by light. Nature New Biol 237: 125–127 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein uti-
lizing the principle of protein-dye binding. Anal Biochem 72: 248–254 Brown BM, Carlson BL, Zhu X, Lolley RN, Craft CM (2002) Light-driven translocation of the protein phosphatase 2A complex regulates light/dark dephosphorylation of phosducin and rhodopsin. Biochemistry 41: 13526–13538 Bünemann M, Hosey MM (1999) G-protein coupled receptor kinases as modulators of G-protein signaling. J Physiol 517: 5–23 Cohen P (1982) The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature 296: 613–620 Danner S, Lohse MJ (1996) Phosducin is a ubiquitous G-protein regulator. Proc Natl Acad Sci USA 93: 10145–10150 Dickman MB, Yarden O (1999) Serine/threonine protein kinases and phosphatases in filamentious fungi. Fungal Gen Biol 26: 99–117 Fabczak H (2000a) Protozoa as model system for studies of sensory light transduction: photophobic responses in ciliate Stentor and Blepharisma. Acta Protozool 39: 171–181 Fabczak H (2000b) Contribution of the phosphoinositide-dependent signal pathway to photomotility in Blepharisma. J Photochem Photobiol B 55: 120–127 Fabczak H, Walerczyk M, Fabczak S (1998) Identification of protein homologous to inositol trisphosphate receptor in ciliate Blepharisma. Acta Protozool 37: 209–213 Fabczak H, Park B-P, Fabczak S, Song P-S (1993c) Photosensory transduction in ciliates. II. Possible role of G-protein and cGMP in Stentor coeruleus. Photochem Photobiol 57: 702–706 Fabczak H, Tao N, Fabczak S, Song P-S (1993b) Photosensory transduction in ciliates. IV. Modulation of the photomovement response of Blepharisma japonicum by cGMP. Photochem Photobiol 57: 889–892 Fabczak H, Walerczyk M, Fabczak S, Groszynska B (1996) InsP3-modulated photophobic responses in Blepharisma. Acta Protozool 35: 251–255 Fabczak H, Walerczyk M, Groszynska B, Fabczak S (1999) Light induces inositol trisphosphate elevation in Blepharisma japonicum. Photochem Photobiol 69: 254–258 Fabczak S, Fabczak H, Song P-S (1993a) Photosensory transduction in ciliates. III. The temporal relation between membrane potentials and photomototile responses in Blepharisma japonicum. Photochem Photobiol 57: 872–876 Flanary PL, DiBello PR, Estrada P, Dohlman HG (2000) Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast. J Biol Chem 275: 18462–18469
Phosducins in Blepharisma japonicum
191
Frank RN, Cavanagh HD, Kenyon KR (1973) Lightstimulated phosphorylation of bovine visual pigments by adenosine triphosphate. J Biol Chem 248: 596–609
Lee RH, Brown BM, Lolley RN (1990) Protein kinase A phosphorylates retinal phosducin on serine 73 in situ. J Biol Chem 265: 15860–15866
Gaudet R, Bohm A, Sigler PB (1996) Crystal structure at 2.4 Å resolution of the complex of transducin βγ and its regulators, phosducin subunits of G-proteins. Cell 87: 577–588
Lee RH, Liebermann BS, Lolley RN (1987) A novel complex from bovine visual cells of a 33 kDa phosphoprotein with β- and γ-transducin: purification and subunit structure. Biochemistry 26: 3983–3990
Gaudet R, Savager JR, McLaughlin JN, Willardson BM, Sigler PB (1999) A molecular mechanism for the phosphorylation-dependent regulation of heteromeric G proteins by phosducin. Mol Cell 3: 649–660
Lee RH, Ting TD, Lieberman BS, Tobias DE, Lolley RN, Ho YK (1992) Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin. J Biol Chem 267: 25104–25112
Giese AC (1973) Blepharisma. The Biology of a Lightsensitive Protozoan. In Stanford University Press, Stanford, USA Graves JD, Krebs EG (1999) Protein phosphorylation and signal transduction. Pharmacol Ther 82: 111–121 Greengard P (1978) Phosphorylated proteins as physiological effectors. Science 199: 146–152 Hardie R (2002) Adaptation through translocation. Neuron 34: 3–5 Hawes BE, Touhara K, Kurose H, Lefkowitz RJ, Inglese J (1994) Determination of the Gβγ-binding domain of phosducin. J Biol Chem 269: 29825–29830 Haystead TAJ, Garrison JC (1999) Study of Protein Phosphorylation in Intact Cells. In Hardie DG (ed) Protein Phosphorylation. Oxford University Press, Oxford, USA, pp 1–31 Ishida M, Shigenaka Y, Taneda K (1989) Studies on the mechanism of cell elongation in Blepharisma japonicum. I. A physiological mechanism how light stimulation evokes cell elongation. Europ J Protistol 25: 182–186 Kasahara S, Wang P, Nuss DL (2000) Identification of bdm-1, a gene involved in G protein β-subunit function and α-subunit accumulation. Proc Natl Acad Sci USA 97: 412–417 Kraml M, Marwan W (1983) Photomovement responses of the heterotrichous ciliate Blepharisma japonicum. Photochem Photobiol 37: 313–319 Kühn H (1974) Light-dependent phosphorylation of rhodopsin in living frogs. Nature 250: 588–590 Kuo CH, Miki N (1989) Translocation of photoreceptor specific MEKA protein by light. Neurosci Lett 103: 8–10
Maeda M, Haoki H, Matsuoka T, Kato Y, Kotsuki H, Utsumi K, Tanaka T (1997) Blepharismin 1-5, novel photoreceptor from the unicellular organism Blepharisma japonicum. Tetrahedron Lett 38: 7411–7414 Marino MJ, Sherman TG, Wood DC (2001) Partial cloning of putative G-proteins modulating mechanotransduction in ciliate Stentor. J Eukaryot Microbiol 48: 527–536 Matsuoka T, Moriyama N, Kida A, Okuda K, Suzuki T, Kotsuki H (2000a) Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma japonicum. J Photochem Photobiol B 54: 131–135 Matsuoka T, Tokumori D, Kotsuki H, Ishida M, Matsushita M, Kimura S, Itoh T, Checcucci G (2000b) Analyses of structure of photoreceptor organelle and blepharismin-associated protein in unicellular eukaryote Blepharisma. Photochem Photobiol 72: 709–713 McGinnis JF, Matsumoto B, Whelan JP, Cao W (2002) Cytoskeleton participation in subcellular trafficking of signal transduction proteins in rod photoreceptor cells. J Neurosci Res 67: 290–297 McLaughlin JN, Thulin CD, Bray SM, Martin MM, Elton TS, Willardson BM (2002) Regulation of angiotensin II-induced G protein signaling by phosducinlike protein. J Biol Chem 277: 34885–34895 Nakano K, Chen J, Tarr GE, Yoshida T, Flyn JM, Bitensky MW (2001) Rethinking the role of phosducin: Light-regulated binding of phosducin to 140303 in rod inner segments. Proc Natl Acad Sci USA 98: 4693–4698
Kuo CH, Akiyama M, Miki N (1989) Isolation of a novel retina-specific clone (MEKA cDNA) encoding a photoreceptor soluble protein. Mol Brain Res 6: 1–10
Pagh-Roehl K, Lin D, Burnside B (1995) Phosducin and PP33 are in vivo targets of PKA and type 1 or 2A phosphatases, regulators of cell elongation in teleost rod rod inner-outer segments. J Neurosci 15: 6475–6488
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680–685
Polans AS, Hermolin J, Bownds MD (1979) Light-induced dephosphorylation of two proteins in frog rod outer segments. J Gen Physiol 74: 595–613
Lee RH, Brown BM, Lolley RN (1984) Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina. Biochemistry 23: 1972–1977
Rayer B, Naynert M, Stieve H (1990) Phototransduction. Different mechanisms in vertebrates and invertebrates. J Photochem Photobiol B 7: 107–148
192
H. Fabczak et al.
Reig JA, Yu L, Klein DC (1990) Pineal transduction. Adrenergic-cyclic AMP-dependent phosphorylation of cytoplasmic 33-kDa protein (MEKA) which binds betagamma-complex of transducin. J Biol Chem 265: 5816–5824 Ruiz-Gómez A, Humrich J, Murga AC, Quitterer U, Lohse MJ, Major FJR (2000) Phosphorylation of phosducin and phosducin-like protein by G proteincoupled receptor kinase 2 (GRK2). J Biol Chem 275: 29724–29730 Schulz R (2001) The pharmacology of phosducin. Pharmacol Res 43: 1–10 Schulz R, Schulz K, Wehmeyer A, Murphy J (1998) Translocation of phosducin in living neuroblastoma X glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein. Brain Res 790: 347–356 Shinozawa T, Hashimoto H, Fujita J, Nakaoka Y (1996) Participation of GTP-binding protein in the photo-transduction of Paramecium bursaria. Cell Struct Funct 21: 469–474 Sokolov M, Lyubarsy AL, Strissel KJ, Savchenko AB, Govardovskii VI, Pugh ENJR, Arshavsky VY (2002) Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation. Neuron 34: 95–106 Strzyzewska-Jówko ˙ I, Jerka-Dziadosz M, Frankel J (2003) Effect of alteration in the global body plan on the deployment of morphogenesis-related protein epitopes labeled by monoclonal antibody 12G9 in Tetrahymena thermophila. Protist 154: 71–90
Tao N, Diforce L, Romanowski M, Meza-Keuthen S, Song P-S, Furuya M (1994) Stentor and Blepharisma photoreceptors: structure and function. Acta Protozool 39: 171–181 Thulin CD, Howes K, Driscoll CD, Savage JR, Rand TA, Baehr W, Willardson BM (1999) The immunolocalization and divergent roles of phosducin and phosducin-like protein in the retina. Mol Vision 5: 40–49 Thulin CD, Savager JR, McLaughlin JN, Truscott SM, Old WM, Ahn NG, Resing KA, Hamm HE, Bitensky MW, Willardson BM (2001) Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding. J Biol Chem 276: 23805–23815 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: Procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 Wilkins JF, Bitensky MW, Willardson BM (1996) Regulation of the kinetics of phosducin phosphorylation in retinal rods. J Biol Chem 271: 19232–19237 Willardson BM, Wilkins JF, Yoshida T, Bitensky MW (1996) Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-depenednt adenyl cyclase. Proc Natl Acad Sci USA 93: 1475–1479 Yoshida T, Willardson BM, Wilkins JF, Jensen GJ, Thornton BD, Bitensky MW (1994) The phosphorylation state of phosducin determines its ability to block transducin subunit interaction and inhibits transducin binding to activated rhodopsin. J Biol Chem 269: 24050–24057
Protist, Vol. 155, 181–192, June 2004 http://www.elsevier.de/protist Published online 14 June 2004
ORIGINAL PAPER
Identification of Possible Phosducins in the Ciliate Blepharisma japonicum Hanna Fabczak, Katarzyna Sobierajska, and Stanislaw Fabczak1 Polish Academy of Sciences, M. Nencki Institute of Experimental Biology, Department of Cell Biology, 3 Pasteur St., PL-02 093 Warsaw, Poland Submitted July 16, 2003; Accepted December 22, 2003 Monitoring Editor: Janine Beisson
Examination of ciliate Blepharisma japonicum whole cell lysates with an antibody against phosphoserine and in vivo labeling of cells with radioactive phosphate revealed that the photophobic response in the ciliate is accompanied by a rapid dephosphorylation of a 28 kDa protein and an enhanced phosphorylation of a 46 kDa protein. Analysis with antibodies raised against rat phosducin or human phosducin-like proteins, identified one major protein of a molecular weight of 28 kDa, and two protein bands of 40 kDa and 93 kDa. While the identified ciliate phosducin is phosphorylated in a light-dependent manner, both phosducin-like proteins exhibit no detectable dependence of phosphorylation upon illumination. An immunoprecipitation assay also showed that the ciliate phosducin is indeed phosphorylated on a serine residue and exists in a phosphorylated form in darkness and that its dephosphorylation occurs in light. Immunocytochemical experiments showed that protozoan phosducin and phosducin-like proteins are localized almost uniformly within the cytoplasm of cells adapted to darkness. Cell exposure to light caused a pronounced displacement of the cell phosducin to the vicinity of the plasma membrane; however, no translocation of phosducin-like proteins was observed upon cell illumination. The obtained results are the first demonstration of the presence and morphological localization of a possible phosducin and phosducin-like proteins in ciliate protists. Phosducin and phosducin-like proteins were found to bind and sequester the β γ -subunits of G-proteins with implications for regulation of G-protein-mediated signaling pathways in various eukaryotic cells. The findings presented in this study suggest that the identified phosphoproteins in photosensitive Blepharisma japonicum may also participate in the regulation of the efficiency of sensory transduction, resulting in the motile photophobic response in this cell.
Introduction The ciliate Blepharisma japonicum possesses an endogenous photoreceptor system that renders the cells capable of avoiding light (Fabczak 2000a; Giese 1973; Maeda et al. 1997; Matsuoka et al. 2000b; Tao et al. 1994). This cell behavior results 1
Corresponding author; fax 48 22 822 5342 e-mail [email protected]
from its motile photophobic response to increasing light intensity (Fabczak et al. 1993a; Kraml and Marwan 1983). The cell photophobic response consists of a delayed cessation of forward swimming, a period of backward movement (ciliary reversal) followed by restoration of forward swimming mostly in an altered direction (Fabczak et al. 1993a). During prolonged intense illumination, an increase in forward movement velocity (positive photokinesis) and 1434-4610/04/155/02-181 $ 30.00/0
182
H. Fabczak et al.
a marked elongation of the cell body is also observed (Fabczak et al. 1993a; Ishida et al. 1989; Kraml and Marwan 1983). The ciliary reversal during the photophobic response is mediated by a membrane action potential, which can be elicited by an early light-induced receptor potential (Fabczak et al. 1993a). The ciliate photoreceptor system seems to be coupled to membrane potentials and photomotil-
ity changes, as in the case of photoreceptor cells of higher organisms (Rayer et al. 1990), via a Gprotein-mediated sensory transduction pathway (Fabczak 2000a, b; Fabczak et al. 1993b; 1998; 1999). It has been shown that phosphorylated proteins play an important role in regulating various sensory transduction pathways (Bünemann and Hosey 1999; Cohen 1982; Dickman and Yarden 1999; Graves and Krebs 1999; Greengard 1978). There is evidence that in photoreceptor cells of the vertebrate retina, phosphorylation and dephosphorylation of phosducin and phosducin-like proteins participate in the regulation of the phototransduction cascade (Lee et al. 1984; Polans et al. 1979; Schultz 2001). These phosphoproteins were found to bind with a high affinity to βγ-subunits of the specific Gprotein, transducin, with a significant implication for the regulation of the availability of transducin for light activation via rhodopsin (Bauer et al. 1992; Lee et al. 1992). Phosducin was originally discovered about 15 years ago in photoreceptor cells of bovine retina, and more recent reports show that phosducin as well as phosducin-like proteins have been found in many other cells (Blaauw et al. 2003; Danner and Lohse 1996; Flanary et al. 2000; Kasahara et al. 2000; Lee et al. 1987; Reig et al. 1990). Since phosducin and phosducin-like proteins are so ubiquitous, it is of great interest to know from an evolutionary point of view whether these functionally important phosphoproteins also exist in photosensitive lower eukaryotes, such as the ciliate Blepharisma japonicum. Therefore, the purpose of this study was the detection of proteins exhibiting light-
Figure 1. Detection of light-dependent protein phosphorylation in whole cell homogenates from Blepharisma japonicum. (A) Western blot with monoclonal antibody PSER-4A9. (B) Test for equal loading and quantification, performed with control antibody against βtubulin. (C) Quantification of protein phosphorylation under different experimental conditions. Lane 1: cells adapted to darkness (control); lane 2: cells exposed to light for 2 s; lanes 3 and 4: cells exposed to light for 2 s and then adapted to darkness for 3 min and 5 min, respectively; lane 5: cells treated with 4 mM potassium ions, lane 6: cells adapted to darkness in control medium then treated with 4 mM potassium ions and finally exposed to light for 2 s. The arrowheads indicate two main protein bands, one of 28 kDa and a second of 46 kDa, which displayed distinct alterations in the level of phosphorylation upon cell illumination. The protein phosphorylation level in control cells is defined as a 100% value. The polyacrylamide gel was calibrated with SDS-PAGE protein molecular weight markers.
Phosducins in Blepharisma japonicum
Figure 2. In vivo phosphorylation of proteins in Blepharisma japonicum. (A) SDS-PAGE of cell lysate stained by Coomasie Brilliant Blue. (B) Corresponding autoradiogram. Lane 1: cells adapted to darkness (control); lane 2: ciliates exposed to light for 2 s; lane 3: dark-adapted cells incubated in control solution with addition of 4 mM potassium ions; lane 4: cells adapted to darkness in control medium supplemented with 4 mM potassium ions and then exposed to light for 2 s. Arrowheads mark the same 28 kDa and 46 kDa phosphoproteins shown in Figure 1. Arrows show proteins of about 70 kDa and 80 kDa, which display phosphorylation increase upon light exposure and double arrowheads indicate proteins of about 40 and 54 kDa whose phosphorylation level is lower after illumination. Other details as in Figure 1.
dependent changes in the extent of their phosphorylation, their identification and morphological localization in ciliate Blepharisma japonicum with in vivo phosphorylation assays and immunocytochemical methods.
Results Light-Regulated Protein Phosphorylation Western blot analysis of whole cell lysates from Blepharisma japonicum cells demonstrated that the antibody raised against phosphorylated serine epitopes PSER-4A9, recognizes several protein bands (Fig. 1). The phosphorylation level of most of the labeled proteins was unaffected in illuminated cells, except for two prominent protein bands, one of molecular weight of 28 kDa and another of 46 kDa (Fig. 1A and C, lanes 1 and 2). The 28 kDa protein was most extensively phosphorylated in dark-
183
Figure 3. Immunoblotting for identification of phosducin and phosducin-like protein isoforms in whole cell lysate from Blepharisma japonicum cells and homogenates from bovine rod outer segments. (A) SDSPAGE stained by Coomassie Brilliant Blue. Lane 1: molecular weight markers; lane 2: lysate from ciliates; lane 3: bovine rod outer segment homogenate. (B) Corresponding immunoblot, which shows that antibody raised against phosducin recognizes one protein band with an apparent molecular weight of 28 kDa in the lysate from ciliates (lane 1) and of 33 kDa in the homogenate from bovine rod outer segments (lane 2). (C) Western blot indicating an interaction of antibody prepared against phosducin-like protein with protein bands of molecular weights of 40 kDa and 93 kDa in lysate from ciliates (lane 1) and of 33 kDa in bovine rod outer segment homogenate (lane 2). The polyacrylamide gel was calibrated with SDS-PAGE protein markers.
adapted cells (Fig. 1A and C, lane 1) and its phosphorylation level was significantly reduced in cells exposed to light (Fig. 1A and C, lane 2). In contrast, the 46 kDa protein displayed a low phosphorylation level during darkness (Fig. 1A and C, lane 1) and an enhanced phosphorylation in illuminated cells (Fig. 1A and C, lane 2). The light-induced phosphorylation changes of both proteins were entirely reversible, since progressive rephosphorylation of the 28 kDa protein and dephosphorylation of the 46 kDa protein were observed when cells, previously exposed to light, adapted to darkness (Fig. 1A and C, lane 3). About 5 min dark adaptation is sufficient to
184
H. Fabczak et al.
Figure 4. Immunoprecipitation of an endogenous phosducin protein isoform in Blepharisma japonicum with antibody raised against phosducin. The precipitated proteins in lysates from both dark-adapted (lane 1) and illuminated (lane 2) ciliates were analyzed by anti-phosphoserine antibody. The blot is the representive image of three independent experiments performed for detection of phosphoserine residues. Other details as in Figure 3.
evoke protein phosphorylation of light-treated cells that reaches the phosphorylation levels measured in the control organisms (Fig. 1A and C, lane 4). To test whether the observed changes in phosphorylation levels of the 28 kDa and 46 kDa proteins were specifically induced by light or resulted simply from cell membrane depolarization, the effect of ionic stimulation was examined. These experiments showed that treatment of dark-adapted cells with high concentrations of external potassium ions did not elicit dephosphorylation of the 28 kDa protein and phosphorylation of the 46 kDa protein (Fig. 1A and C, lane 5) as observed in cells exposed to light (Fig. 1A and C, lane 2). Levels of phosphorylation of both phosphoproteins in homogenates from darkadapted ciliates, which were first treated with high potassium ions concentrations and subsequently exposed to light (Fig. 1A and C, lane 6), were similar to those obtained for cells that were only illuminated (Fig. 1A and C, lane 2). As an additional control of protein phosphorylation in Blepharisma japonicum, an in vivo analysis was performed using radioactive phosphate [32P]. These studies revealed that most of the labeled phosphoproteins in the cell lysate had molecular
weights higher than 80 kDa. Their phosphorylation was not appreciably affected by illumination (Fig. 2). However, two proteins of about 70 kDa and 80 kDa showed higher levels of phosphorylation upon cell illumination (Fig. 2B, arrows) and proteins of about 40 kDa and 55 kDa had lower levels of phosphorylation under the same light conditions (Fig. 2B, double arrowheads). In the dark, the most extensively labeled phosphoprotein among the lower molecular weight proteins had an apparent molecular weight of 28 kDa (Fig. 2B, lane 1). The phosphorylation level of this protein was significantly lowered by illumination (Fig. 2B, lane 2). The protein phosphorylation of 28 kDa in the homogenate from dark-adapted cells treated with high concentrations of potassium ions and then illuminated was similar to that of control cells (Fig. 2B, lanes 2 and 4). A major phosphorylated protein band of 46 kDa could be observed in cells exposed to light (Fig. 2B, lanes 2 and 4) whereas in dark-adapted ciliates the level of phosphorylation of this protein was much lower (Fig. 2B, lanes 1 and 3).
Identification of Possible Phosducin and Phosducin-Like Proteins An immunoblotting assay with an antibody against phosducin showed clear immunoreactivity only with one protein band of 28 kDa in the whole cell lysate from ciliates (Fig. 3B, lane 1) and with a 33 kDa band in bovine rod outer segment homogenate (Fig. 3B, lane 2), indicating sufficient selectivity of the applied antibody. The 28 kDa protein detected in this assay evidently resembles the 28 kDa protein band shown in Figures 1 and 2, exhibiting light-dependent phosphorylation (Fig. 1A and C, lanes 1 and 2; Fig. 2B). An antibody raised against a phosducin-like protein revealed a distinct immunoreactivity and recognized two protein bands with molecular weights of 40 kDa and 93 kDa in the cell homogenate (Fig. 3C, lane 1), as well as a 33 kDa protein band in bovine rod outer segment homogenate (Fig. 3C, lane 2). None of these detected phosphoproteins was identified in the phosphorylation pattern presented in Figure 1A as a protein which exhibited phosphorylation dependence upon illumination. However, the changes in the phosphorylation levels of the 40 kDa protein were established in the in vivo experiments using [32P] (Fig. 2B). In control blots with identical protein load and incubated with the secondary antibody only, no immunolabeling was found, confirming a high specificity of labeling (not shown). To further examine whether phosphorylation changes of the detected ciliate phosducin were induced by light, the cell lysates from control and illu-
Phosducins in Blepharisma japonicum
185
Figure 5. Morphological immunolocalization of phosducin and phosducin-like proteins in Blepharisma japonicum. (A) Control cells incubated with TBS containing 1% BSA in place of the primary antibody. Labeling of cells with antibody against phosducin (B, C, E, F) and phosducin-like proteins (G, H, I) indicates the localization of these phosphoproteins within the cell cytoplasm. (B, E) Translocation of phosducin protein from the cytoplasm to the vicinity of plasma membrane occurring in organisms exposed to light for 30 s. (H, I) Picture indicating a lack of redistribution of phosducin-like protein upon cell exposure to light. (D) Cell cortical structures immunolabeled with mAb12G9 antibody. Ciliary rows (cr) and the basal bodies, adoral membranelles (AZM) of oral apparatus with the metachronal waves (arrowheads) and paroral membrane (PM) are marked.
minated ciliates were immunoprecipitated with the antibody against phosducin. Next, the precipitated proteins were analyzed by the PSER-4A9 antibody. In both dark-kept and illuminated cell samples, the anti-phosducin antibody precipitated exclusively one protein phosphorylated on a serine residue, which had a molecular weight of 28 kDa (Fig. 4). In light-treated cells, phosphorylation of the 28 kDa protein decreased significantly (Fig. 4, lane 2), whilst in the case of untreated cells, the phosphorylation level of this protein was higher (Fig. 4, lane 1). These
experimental results further prove that in Blepharisma japonicum there is a 28 kDa phosducin protein that displays enhanced phosphorylation in darkness and that becomes markedly dephosphorylated in light.
Localization of Ciliate Phosducin and Phosducin-Like Proteins The localization of identified phosducin and phosducin-like proteins in the ciliate was investigated by
186
H. Fabczak et al.
immunocytochemical analysis using the antibodies against these phosphoproteins. The examinations revealed prominent fluorescence in cells, indicating the occurrence of phosducin and phosducin-like protein isoforms in the ciliate tested (Fig. 5). The dark-adapted cells shown in Figure 5C, F and G exhibit fluorescence which is almost evenly distributed in their cytoplasm. After exposure of cells to light for 30 s, displacement of ciliate phosducin protein from the cytosol to the close vicinity of the plasma membrane is observed (Fig. 5B and E). In Figure 5E, phosducin translocation is indicated during the ongoing presence of light stimulation as a bright fluorescence, which concentrates at the cell margin along with a simultaneous slight reduction of the fluorescence intensity in the central part of the cell. While the phosducin protein clearly translocates upon light stimulation, no redistribution of phosducin-like proteins was observed within the cell under the same light conditions (Fig. 5H and I). No immunolabeling was observed in control cells treated with secondary antibodies only (Fig. 5A). To verify that the cell fixation and permeabilization procedures were performed correctly, a mAb12G9 antibody, known to label specifically some cell cortical structures in ciliates (Strzyzewska-Jówko ˙ et al. 2003), was used. The experiments showed that the antibody used did indeed decorate some cortical structures of Blepharisma japonicum cells as in other ciliates. Figure 5D shows marked immunolabeling of ciliary rows (cr) and basal bodies at the anterior part of the cell, adoral membranelles (AZM) with metachronal waves (indicated by arrowheads) and paroral membrane (PM) within the cell oral apparatus (Aescht and Foissner 1998). These data proved sufficient permeation of antibodies used to localize phosducin and phosducin-like proteins in the protozoan cells tested.
Discussion Protein phosphorylation is recognized as a highly important mechanism by which internal events in cells are controlled by external physiological stimuli (Bünemann and Hosey 1999; Dickman and Yarden 1999; Cohen 1982; Graves and Krebs 1999; Greengard 1978). In visual systems, it has been shown that the process of protein phosphorylation and dephosphorylation is involved in the inactivation of the active form of rhodopsin and in its restoration (Bownds et al. 1972; Frank et al. 1973; Kühn 1974). Phosducin and phosducin-like proteins, major cytosolic phosphoproteins in mammalian photoreceptor cells, have been postulated to participate in the
downregulation of phototransduction systems (Schulz 2001). It has also been shown that phosducins are phosphorylated in darkness and that this phosphorylation may be catalyzed by protein kinase A, Ca2+-calmodulin kinase II or G-protein-coupled receptor kinase 2 (Lee et al. 1990; Ruiz-Gómez et al. 2000; Thulin et al. 2001; Wilkins et al 1996; Willardson et al. 1996; Yoshida et al. 1994). Dephosphorylation of these phosphoproteins may occur by activation of type 1 or 2A phosphatases upon cell exposure to light (Lee et al. 1984; Pagh-Roehl et al. 1995). The most important characteristic of phosducin appears to be its high affinity sequestration of the βγ-subunit of heterotrimeric G-proteins (Gβγ) (Gaudet et al. 1996; 1999; Schulz 2001; Yoshida et al. 1994). Competition between phosducin and the α-subunit of retinal specific G-protein (transducin) for available Gβγ diminishes signal amplification through interference with the reassembly of the Gprotein complex that is essential for G-protein recycling with activated rhodopsin. In general, similar mechanisms have been postulated for phosducinlike proteins (Schulz 2001). While phosducin was originally discovered in vertebrate retinas and developmentally related pineal glands (Kuo et. al. 1989; Lee et al. 1990; Reig et al. 1990), more recently, the existence of many phosducin-related proteins has been reported in numerous other tissues, including olfactory epithelium, liver, brain and spleen (Schulz 2001). Phosducin isoforms have also been lately found in some lower eukaryotic organisms. In the yeast Saccharomyces cerevisiae, two genes were identified, encoding phosducin-like proteins, Plp1 and Plp2, which bind Gβγ (Flanary et al. 2000). The Cryphonectria parasitica gene, designated bdm-1, encodes a phosducin-like protein involved in Gβγ function and Gα accumulation (Kasahara et al. 2000). Similarly, in Dictyostelium discoideum three genes, phlp-1, phlp-2 and phlp-3, each encoding a phosducin-like protein of a different group, were discovered and it was established that one of them, named phosducin-I, also plays a role in the control of G-protein pathway signaling (Blaauw et al. 2003). In this study, we have explored the possibility that light stimulation followed by motile photophobic response may also alter the phosphorylation level of proteins in Blepharisma japonicum. The photoreceptor system in the ciliate is thought to be coupled to membrane potential and motility alterations via G-protein and also possibly different second messenger pathways, this stems from observations that the photophobic response and another light-evoked physiological phenomenon, cell body elongation, were significantly affected by modulation of internal cGMP levels pointing to cyclic nucleotide as a pos-
Phosducins in Blepharisma japonicum
sible signal transmitter in both behavioral processes (Fabczak et al. 1993b, Ishida et al. 1989). However, there is a line of evidence that phosphoinositides may also contribute to photophobic behavior in Blepharisma japonicum, i.e. cell treatment with drugs interfering effectively with the phosphoinositide signaling pathway, strongly inhibits photophobic responses and prolongs response latency significantly (Fabczak et al. 1996). The reported immunoblotting and immunocytochemical examination also show that within the ciliate cortex an inositol trisphosphate receptor-like protein is present (Fabczak et al. 1998; Matsuoka et al. 2000a). And finally, recent radioimmunological studies allowed the demonstration of fluctuations in the internal inositol trisphosphate level of cells upon exposure to light, which seemed to result from modulation of the activity of cellular phospholipases (Fabczak 2000a; Fabczak et al. 1999). Based on the above-mentioned data, it is obvious that the detailed molecular mechanism of light signal transduction in Blepharisma japonicum is still elusive at present and its complete clarification awaits further investigations. Our immunoblotting investigations of Blepharisma japonicum revealed the existence of two proteins exhibiting light-dependent alterations in their phosphorylation levels (Figs 1A, C and 2B). These phosphoproteins have apparent molecular weights of 28 kDa and 46 kDa and display reversible dephosphorylation and phosphorylation upon illumination and darkness, respectively. The changes in phosphorylation level were specifically initiated by light, since no significant differences in the level of phosphorylation were detected in cells treated with high external concentration of potassium ions (Fig. 1A and C, lane 5). As shown in Figure 1A and C, lane 6, and also in Figure 2, only subsequent illumination of potassium-treated ciliates results in dephosphorylation of the 28 kDa protein and increased phosphorylation of the 46 kDa protein. Potassium ions at higher concentrations are known to effectively depolarize the cell membrane and may trigger an action potential followed by ciliary reversal, as in the case of depolarizing photoreceptor potential in the ciliate tested (Fabczak et al. 1993a). The immunoblotting assays show that only the 28 kDa protein is recognized by the applied antibody against phosducin (Fig. 3A) and it correlates well with the 28 kDa protein found in the protein phosphorylation pattern that displays enhanced phosphorylation in the dark and is dephosphorylated upon exposure to light (Fig. 1A and C, lanes 1 and 2; Fig. 2B, lanes 1 and 2 and also Fig. 4, lanes 1 and 2). Similar light-evoked changes in the phosphorylation level of both the 28 kDa and 46 kDa proteins
187
were observed in Blepharisma japonicum in in vivo phosphorylation analysis using radioactive phosphate (Fig. 2B). Moreover, the experiments revealed four other proteins of molecular weights of 40, 55, 70 and 80 kDa, which seem to undergo a light-dependent phosphorylation as well. The diversity in phosphorylation patterns shown in Figures 1 and 2, possibly results from the labeling by [32P] not only serine but also tyrosine or threonine residues. This may be caused by low specificity of the applied Pser-4A9 antibody, which has been shown to depend greatly on the surrounding amino acid sequence. In addition, the immunoprecipitation assay indicated that the ciliate phosducin is phosphorylated on serine residues, and indeed it exists in highly phosphorylated and dephosphorylated forms in darkness (Fig. 4, lane 1) and in light (Fig. 4, lane 2), respectively The apparent molecular weight of 28 kDa of the ciliate phosducin is similar to that of 26 kDa and 32 kDa phosducin isoforms found in yeast (Flanary et al. 2000) or in mammalian phosducins whose molecular weight was estimated from the protein amino acid sequence to be 28 kDa, although they migrate on gels as 33 kDa proteins (Lee et al. 1984; Schulz 2001). The cell homogenates probed with the antibody against phosducin-like protein revealed two proteins of 40 kDa and 93 kDa. Although none of these phosphoproteins corresponds to any proteins exhibiting light-dependent phosphorylations as shown in the immunoblotting experiments (Fig. 1A), in the in vivo phosphorylation experiments, the 40 kDa protein exhibits phosphorylation alterations upon light stimulation (Fig. 2B). There is a possibility that this protein resembles the 40 kDa protein detected with the antibody against phosducin-like protein (Fig. 3C). However, immunoprecipitation of the [32P] labeled phosphoproteins with the antibody against phosducin-like proteins has not proved this (data not shown). In the photoreceptor cells of the visual systems, it has been reported that photoreceptor activation by light is followed by phosducin dephosphorylation that results in its translocation between inner and outer rod segments (Kuo and Miki 1989; Lee et al. 1992) or redistribution towards the membrane, where it binds Gβγ subunits of G-proteins (Schulz et al. 1998). The cellular components that translocate in response to light, besides phosducin, also include transducin, arrestin and 2A phosphatase (Brown et al. 2002; McGinnis et al. 2002; Sokolov et al. 2002). These translocations have been shown to affect photoreceptor adaptation to background illumination (Hardie 2002). However, in the study by Nakano et al. (2001), little if any light-induced movement of phosducin was found.
188
H. Fabczak et al.
Nevertheless, immunocytochemical examination of section images of Blepharisma japonicum showed that light exposure promotes significant concentration of the ciliate phosducin at the cell margin (Fig. 5B and E), while the distribution of both phosducin-like proteins was unchanged under the applied light conditions (Fig. 5H and I). It appears from these observations that phosducin and phosducin-like proteins identified in Blepharisma japonicum may play an important physiological role, similar to that proposed in the vertebrate retina (Thulin et al. 1999). These data support our suggestion that the identified cytoplasmic 28 kDa protein (Figs 1 and 2) may represent a ciliate phosphoprotein exhibiting properties similar to that of phosducin (Figs 3 and 4), i.e. light-dephosphorylated phosducin may translocate towards the cell membrane and thereby binds βγ-subunits of G-proteins and influences the cell phototransduction cascade. Phosducin binding to Gβγ may control the kinetics of second messenger signaling in ciliates by inhibition of Gβγ-mediated phosphoinositide hydrolysis, as is the case in other cells (Hawes et al. 1994; McLaughlin et al. 2002). This supposition is quite likely, since the presence of a protein homologous to the α-subunit of bovine Gproteins in Blepharisma japonicum has been established recently (Fabczak 2000a, b). Among the ciliate protozoans, the participation of G-proteins in signaling pathways has already been postulated for Paramecium bursaria (Shinozawa et al. 1996) and Stentor coeruleus, in which the α-subunits of G-proteins have been partially cloned (Fabczak et al. 1993c; Marino et al. 2001). The results obtained in this study demonstrate for the first time that the ciliate protist Blepharisma japonicum possesses phosducin and phosducinlike proteins, which have been shown to be present in various tissues of higher eukaryotes (Schulz 2001), and more recently in lower eukaryotic cells (Blaauw et al. 2003; Flanary et al. 2000; Kasahara et al. 2000). These phosphoproteins are characterized as the cytosolic regulators of G-protein function. These data also have important evolutionary implications since they suggest that light transduction in the ciliate protist seems to be under control of similar processes of phosducin phosphorylation and dephosphorylation as in the evolutionarily distant photoreceptor cells of higher organisms. Beyond this suggestion, little is currently known about the detailed mechanism by which the discovered ciliate phosducin is involved in the transduction of light signals in Blepharisma japonicum cells. The knowledge of phosducin function in vivo in the ciliate photoperception system may support in vitro studies on the role of phosducin in other G-protein-
mediated signaling pathways. Sequencing of these proteins and detailed characterization of their function in the cell’s photobehavior is the topic of future investigations.
Methods Cell culture: Stock cultures of ciliate Blepharisma japonicum were grown in 300 ml glass dishes in Pringsheim solution at pH 7 at room temperature under semi dark conditions (Fabczak 2000b). The cells were fed twice a week with the small ciliate Tetrahymena pyriformis, axenically grown at room temperature in a medium composed of 1.0% proteose peptone (Difco, USA) and 0.1% yeast extract (Difco, USA). The cultures of Tetrahymena pyriformis were centrifuged and washed in Pringsheim solution before feeding Blepharisma japonicum. Prior to each experiment, the Blepharisma japonicum cells were starved for at least 24 h, then gently collected by a low speed centrifugation, and subsequently washed in an excess of fresh culture medium lacking nutritional components. Finally, the chosen cell samples were used for biochemical assays after incubation in darkness in fresh culture medium, referred to as a control medium, or in test solutions of designed compositions. Cell stimulation: Before an experiment, tested samples of dark-adapted cells were first left at rest for about 10 min to avoid any mechanical disturbances, and then exposed to light or ionic stimulations. Cell illumination (for 2 s) was provided by a 150 W fiber optic light source (MLW, Germany), equipped with an electromagnetic programmable shutter (model l22-841, Ealing Electro-Optics, England). Ionic stimulation was performed by incubation of cells in an external 4 mM potassium solution prepared by addition of a proper amount of 0.1 M KCl to the control medium. Electrophoresis and Western blotting: For immunoblotting analysis, cell samples were mixed with sample buffer (2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 1.0 mM EDTA and 62.5 mM Tris at pH 6.8) (Laemmli 1970), supplemented with protease and phosphatase inhibitors (50 mM NaF, 2 mM PMSF, 10 µM okadaic acid, 10 µg/ml aprotinin, 100 µg/ml leupeptin) to terminate reactions and then boiled for 5 min. Equal amounts of protein (30 µg) from solubilized cells were separated by 10% SDSPAGE with a Hoefer System (Amersham, USA) and transferred to nitrocellulose membranes (Bio-Rad, USA) during 60 min at 100 V in a transfer buffer (192 mM glycine, 20% methanol and 25 mM Tris at pH 8.3) as described elsewhere (Towbin et al. 1979).
Phosducins in Blepharisma japonicum
The membranes were then blocked for 2 h at room temperature by incubation in TBS buffer (150 mM NaCl, l0 mM Tris, pH 7.5) with 2% bovine serum albumin (BSA) and 0.2% Tween-20 (TBS-BSA-Tween blocking solution). For detection of specified cell phosphoproteins, the blots were incubated with monoclonal anti-phosphoserine antibody, clone PSER-4A9 (Alexis, Switzerland) at a concentration of 0.1 µg/ml in TBS-BSA-Tween solution or with a 5,000-fold dilution of the polyclonal antibodies raised against rat phosducin and human phosducinlike proteins (kindly provided by Professor Craig Thulin from Brigham Young College in Provo, USA; Thulin et al. 1999) in TBS-BSA-Tween solution overnight at 4 ºC. After several washes in TBS with 0.1% Tween-20, blots were incubated for 60 min at room temperature with secondary antibodies, antimouse or anti-rabbit IgG-horseradish peroxidase conjugates (Calbiochem, Germany) at a 1:10,000 dilution in TBS-BSA-Tween solution. Finally, membranes were washed in TBS-Tween buffer and developed with an Amersham ECL detection system (Amersham, Sweden). The intensities of immunoreactive protein bands were quantified using a laser densitometry and ImageQuant software (Bio-Rad). Molecular weights of proteins were determined based on their relative electrophoretic mobility with molecular weight markers (Bio-Rad). In the control sets of experiments, incubations with primary antibodies were omitted. Protein concentration in individual cell samples was estimated with a method reported elsewhere using BSA as a standard (Bradford 1976). Tests for equal loading and protein quantity were done throughout experiments with a control antibody, anti-β-tubulin (Sigma, USA). In vivo phosphorylation assay: The method used for labeling of cells in vivo was a slight modification of that described by Haystead and Garrison (1999). Samples of cells were incubated in darkness in phosphate-free Pringsheim solution supplemented with 0.5 mCi of [32P]-phosphate (Polatom ´ Radioisotope Center, Otwock-Swierk, Poland) for 2 h at room temperature to allow incorporation of 32P into endogenous cell phosphoproteins. After incubation, cell samples were transferred into Pringsheim solution without the radioactive isotope and then exposed to light or potassium ions as described above. Subsequently, control and treated cell samples were solubilized in Triton buffer (1% Triton X-100, 150 mM NaCl, 1.0 mM EDTA, 20 mM Tris, pH 7.4) supplemented with protease and phosphatase inhibitors (50 mM NaF, 1 mM phenylmethylsulfonylfluoride (PMSF), 10 µM okadaic acid, 5 µg/ml aprotinin, 10 µg/ml leupeptin) and centrifuged for 15 min at 12,000 rpm at 4 ºC. The obtained su-
189
pernatant was then collected and protein concentration in each cell sample was estimated (Bradford 1976). Finally, the protein samples were denatured by boiling for 5 min in sample buffer (Laemmli 1970) and subjected to 10% SDS-PAGE as described in the foregoing section. Autoradiography of the 32P-labeled phosphoproteins was done by exposing the dried polyacrylamide gels to X-ray film (Amersham, Sweden) in X-ray cassettes lined with high-speed and intensifying screens (DuPont) at –70 ºC. Immunoprecipitation: Samples of dark-adapted cells (control) or cells exposed to light were solubilized in Triton buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1mM EDTA) and then centrifuged for 15 min at 12,000 rpm at 4 ºC. The resultant supernatants after supplementing with protease and phosphatase inhibitors (10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM PMSF, 100 µM okadaic acid, 5 mM NaF) were used for the immunoprecipitation assay. The cell samples were subjected to preclearance for 1 h with 30% (v/v) protein A-Sepharose CL-4B (Amersham, Sweden) at 4 ºC. After this, the mixture was centrifuged for 1 min at 12,000 rpm at 4 ºC. The supernatant fraction was incubated for 1.5 h at 4 ºC with serum-containing antibody against phosducin and then for 1.5 h with a fresh portion of protein-A-Sepharose. The resin was washed three times in washing buffer containing 20 mM Tris-HCl, 150 mM NaCl at pH 7.4, transferred to new tubes for the third wash, then resuspended in sample buffer, boiled for 5 min and analyzed on SDS-PAGE according to the method of Laemmli (1970). The protein phosphorylation assay was carried out using the Western blot technique with monoclonal antibody against phosphorylated serine residues, clone PSER-4A9 (Alexis, Switzerland) as described above. Immunocytochemistry: The ciliates suspended in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgCl2, pH 6.9, and protease inhibitors of 1 mM PMSF, 1 µM leupeptin, 1 µg/ml aprotinin) were fixed for 10 min at –70 °C in 70% ethanol solution. After the fixing procedure, cells were washed twice in PHEM buffer and permeabilized for 10 min at 0 °C in PHEM buffer containing 0.05% Triton X-100. The cell samples were then washed twice with PHEM buffer and incubated for 1 h at room temperature in PHEM buffer with 2% BSA to block nonspecific binding. Subsequently, the cell preparations were exposed overnight at 4 °C to polyclonal antibodies against phosducin or phosducin-like proteins at 1:500 dilution in TBS solution with 1% BSA (TBS-BSA-Tween). The cell samples were also exposed to monoclonal mAbs 12G9 antibody (kindly provided by Professor Maria Jerka-Dzi-
190
H. Fabczak et al.
adosz from the Nencki Institute of Experimental Biology) at 1: 20 dilution in TBS-BSA. This antibody was shown to label specifically some ciliate cortical structures (Strzy˙zewska-Jówko et al. 2003). Following primary incubation and extensive washings in TBS-Tween, cell samples were finally exposed for 60 min at room temperature to a goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (Calbiochem) at 1:300 dilution in TBS-BSA-Tween or anti-mouse IgG conjugated with fluorescein isothiocyanate (Calbiochem) at 1: 200 dilution in TBS-BSATween. After incubation, cells were washed again in TBS solution and examined with a laser scanning confocal microscope (Leica, Germany). Nonspecific fluorescence was determined in cell samples suspended in TBS-BSA buffer and devoid of primary antibody.
Acknowledgements We wish to express our gratitude to Professor C. D. Thulin (Brigham Young University, Provo, Utah, USA) for generously supplying samples of polyclonal antibodies against phosducin and phosducin-like proteins. We would also like to thank Professor Maria Jerka-Dziadosz for the generous gift of mAb12G9 monoclonal antibody and helpful instructions concerning cell fixation and immunolabeling. This work was supported in part by grant No 6P04C-057-18 from the Polish Committee for Scientific Research to SF and statutory funding of the Nencki Institute of Experimental Biology in Warsaw.
References Aescht E, Foissner W (1998) Divisional morphogenesis in Blepharisma americanum, B. undulans, and B. hyalinum (Ciliophora: Heterotrichida). Acta Protozool 37: 71–92 Bauer PH, Muller S, Puzicha M, Pippig S, Obermaier B, Helmreich EJ, Lohse MJ (1992) Phosducin is a protein kinase A-regulated G-protein regulator. Nature 358: 73–75 Blaauw M, Knol JC, Kortholt A, Roelofs J, Ruchira, Posma M, Visser AJWG, Van Haaster PJM (2003) Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins. EMBO J 22: 5047–5057 Bownds MD, Bawes J, Miller J, Stahlman M (1972) Phosphorylation of frog photoreceptor membranes induced by light. Nature New Biol 237: 125–127 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein uti-
lizing the principle of protein-dye binding. Anal Biochem 72: 248–254 Brown BM, Carlson BL, Zhu X, Lolley RN, Craft CM (2002) Light-driven translocation of the protein phosphatase 2A complex regulates light/dark dephosphorylation of phosducin and rhodopsin. Biochemistry 41: 13526–13538 Bünemann M, Hosey MM (1999) G-protein coupled receptor kinases as modulators of G-protein signaling. J Physiol 517: 5–23 Cohen P (1982) The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature 296: 613–620 Danner S, Lohse MJ (1996) Phosducin is a ubiquitous G-protein regulator. Proc Natl Acad Sci USA 93: 10145–10150 Dickman MB, Yarden O (1999) Serine/threonine protein kinases and phosphatases in filamentious fungi. Fungal Gen Biol 26: 99–117 Fabczak H (2000a) Protozoa as model system for studies of sensory light transduction: photophobic responses in ciliate Stentor and Blepharisma. Acta Protozool 39: 171–181 Fabczak H (2000b) Contribution of the phosphoinositide-dependent signal pathway to photomotility in Blepharisma. J Photochem Photobiol B 55: 120–127 Fabczak H, Walerczyk M, Fabczak S (1998) Identification of protein homologous to inositol trisphosphate receptor in ciliate Blepharisma. Acta Protozool 37: 209–213 Fabczak H, Park B-P, Fabczak S, Song P-S (1993c) Photosensory transduction in ciliates. II. Possible role of G-protein and cGMP in Stentor coeruleus. Photochem Photobiol 57: 702–706 Fabczak H, Tao N, Fabczak S, Song P-S (1993b) Photosensory transduction in ciliates. IV. Modulation of the photomovement response of Blepharisma japonicum by cGMP. Photochem Photobiol 57: 889–892 Fabczak H, Walerczyk M, Fabczak S, Groszynska B (1996) InsP3-modulated photophobic responses in Blepharisma. Acta Protozool 35: 251–255 Fabczak H, Walerczyk M, Groszynska B, Fabczak S (1999) Light induces inositol trisphosphate elevation in Blepharisma japonicum. Photochem Photobiol 69: 254–258 Fabczak S, Fabczak H, Song P-S (1993a) Photosensory transduction in ciliates. III. The temporal relation between membrane potentials and photomototile responses in Blepharisma japonicum. Photochem Photobiol 57: 872–876 Flanary PL, DiBello PR, Estrada P, Dohlman HG (2000) Functional analysis of Plp1 and Plp2, two homologues of phosducin in yeast. J Biol Chem 275: 18462–18469
Phosducins in Blepharisma japonicum
191
Frank RN, Cavanagh HD, Kenyon KR (1973) Lightstimulated phosphorylation of bovine visual pigments by adenosine triphosphate. J Biol Chem 248: 596–609
Lee RH, Brown BM, Lolley RN (1990) Protein kinase A phosphorylates retinal phosducin on serine 73 in situ. J Biol Chem 265: 15860–15866
Gaudet R, Bohm A, Sigler PB (1996) Crystal structure at 2.4 Å resolution of the complex of transducin βγ and its regulators, phosducin subunits of G-proteins. Cell 87: 577–588
Lee RH, Liebermann BS, Lolley RN (1987) A novel complex from bovine visual cells of a 33 kDa phosphoprotein with β- and γ-transducin: purification and subunit structure. Biochemistry 26: 3983–3990
Gaudet R, Savager JR, McLaughlin JN, Willardson BM, Sigler PB (1999) A molecular mechanism for the phosphorylation-dependent regulation of heteromeric G proteins by phosducin. Mol Cell 3: 649–660
Lee RH, Ting TD, Lieberman BS, Tobias DE, Lolley RN, Ho YK (1992) Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin. J Biol Chem 267: 25104–25112
Giese AC (1973) Blepharisma. The Biology of a Lightsensitive Protozoan. In Stanford University Press, Stanford, USA Graves JD, Krebs EG (1999) Protein phosphorylation and signal transduction. Pharmacol Ther 82: 111–121 Greengard P (1978) Phosphorylated proteins as physiological effectors. Science 199: 146–152 Hardie R (2002) Adaptation through translocation. Neuron 34: 3–5 Hawes BE, Touhara K, Kurose H, Lefkowitz RJ, Inglese J (1994) Determination of the Gβγ-binding domain of phosducin. J Biol Chem 269: 29825–29830 Haystead TAJ, Garrison JC (1999) Study of Protein Phosphorylation in Intact Cells. In Hardie DG (ed) Protein Phosphorylation. Oxford University Press, Oxford, USA, pp 1–31 Ishida M, Shigenaka Y, Taneda K (1989) Studies on the mechanism of cell elongation in Blepharisma japonicum. I. A physiological mechanism how light stimulation evokes cell elongation. Europ J Protistol 25: 182–186 Kasahara S, Wang P, Nuss DL (2000) Identification of bdm-1, a gene involved in G protein β-subunit function and α-subunit accumulation. Proc Natl Acad Sci USA 97: 412–417 Kraml M, Marwan W (1983) Photomovement responses of the heterotrichous ciliate Blepharisma japonicum. Photochem Photobiol 37: 313–319 Kühn H (1974) Light-dependent phosphorylation of rhodopsin in living frogs. Nature 250: 588–590 Kuo CH, Miki N (1989) Translocation of photoreceptor specific MEKA protein by light. Neurosci Lett 103: 8–10
Maeda M, Haoki H, Matsuoka T, Kato Y, Kotsuki H, Utsumi K, Tanaka T (1997) Blepharismin 1-5, novel photoreceptor from the unicellular organism Blepharisma japonicum. Tetrahedron Lett 38: 7411–7414 Marino MJ, Sherman TG, Wood DC (2001) Partial cloning of putative G-proteins modulating mechanotransduction in ciliate Stentor. J Eukaryot Microbiol 48: 527–536 Matsuoka T, Moriyama N, Kida A, Okuda K, Suzuki T, Kotsuki H (2000a) Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma japonicum. J Photochem Photobiol B 54: 131–135 Matsuoka T, Tokumori D, Kotsuki H, Ishida M, Matsushita M, Kimura S, Itoh T, Checcucci G (2000b) Analyses of structure of photoreceptor organelle and blepharismin-associated protein in unicellular eukaryote Blepharisma. Photochem Photobiol 72: 709–713 McGinnis JF, Matsumoto B, Whelan JP, Cao W (2002) Cytoskeleton participation in subcellular trafficking of signal transduction proteins in rod photoreceptor cells. J Neurosci Res 67: 290–297 McLaughlin JN, Thulin CD, Bray SM, Martin MM, Elton TS, Willardson BM (2002) Regulation of angiotensin II-induced G protein signaling by phosducinlike protein. J Biol Chem 277: 34885–34895 Nakano K, Chen J, Tarr GE, Yoshida T, Flyn JM, Bitensky MW (2001) Rethinking the role of phosducin: Light-regulated binding of phosducin to 140303 in rod inner segments. Proc Natl Acad Sci USA 98: 4693–4698
Kuo CH, Akiyama M, Miki N (1989) Isolation of a novel retina-specific clone (MEKA cDNA) encoding a photoreceptor soluble protein. Mol Brain Res 6: 1–10
Pagh-Roehl K, Lin D, Burnside B (1995) Phosducin and PP33 are in vivo targets of PKA and type 1 or 2A phosphatases, regulators of cell elongation in teleost rod rod inner-outer segments. J Neurosci 15: 6475–6488
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680–685
Polans AS, Hermolin J, Bownds MD (1979) Light-induced dephosphorylation of two proteins in frog rod outer segments. J Gen Physiol 74: 595–613
Lee RH, Brown BM, Lolley RN (1984) Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina. Biochemistry 23: 1972–1977
Rayer B, Naynert M, Stieve H (1990) Phototransduction. Different mechanisms in vertebrates and invertebrates. J Photochem Photobiol B 7: 107–148
192
H. Fabczak et al.
Reig JA, Yu L, Klein DC (1990) Pineal transduction. Adrenergic-cyclic AMP-dependent phosphorylation of cytoplasmic 33-kDa protein (MEKA) which binds betagamma-complex of transducin. J Biol Chem 265: 5816–5824 Ruiz-Gómez A, Humrich J, Murga AC, Quitterer U, Lohse MJ, Major FJR (2000) Phosphorylation of phosducin and phosducin-like protein by G proteincoupled receptor kinase 2 (GRK2). J Biol Chem 275: 29724–29730 Schulz R (2001) The pharmacology of phosducin. Pharmacol Res 43: 1–10 Schulz R, Schulz K, Wehmeyer A, Murphy J (1998) Translocation of phosducin in living neuroblastoma X glioma hybrid cells (NG 108-15) monitored by red-shifted green fluorescent protein. Brain Res 790: 347–356 Shinozawa T, Hashimoto H, Fujita J, Nakaoka Y (1996) Participation of GTP-binding protein in the photo-transduction of Paramecium bursaria. Cell Struct Funct 21: 469–474 Sokolov M, Lyubarsy AL, Strissel KJ, Savchenko AB, Govardovskii VI, Pugh ENJR, Arshavsky VY (2002) Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation. Neuron 34: 95–106 Strzyzewska-Jówko ˙ I, Jerka-Dziadosz M, Frankel J (2003) Effect of alteration in the global body plan on the deployment of morphogenesis-related protein epitopes labeled by monoclonal antibody 12G9 in Tetrahymena thermophila. Protist 154: 71–90
Tao N, Diforce L, Romanowski M, Meza-Keuthen S, Song P-S, Furuya M (1994) Stentor and Blepharisma photoreceptors: structure and function. Acta Protozool 39: 171–181 Thulin CD, Howes K, Driscoll CD, Savage JR, Rand TA, Baehr W, Willardson BM (1999) The immunolocalization and divergent roles of phosducin and phosducin-like protein in the retina. Mol Vision 5: 40–49 Thulin CD, Savager JR, McLaughlin JN, Truscott SM, Old WM, Ahn NG, Resing KA, Hamm HE, Bitensky MW, Willardson BM (2001) Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding. J Biol Chem 276: 23805–23815 Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: Procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354 Wilkins JF, Bitensky MW, Willardson BM (1996) Regulation of the kinetics of phosducin phosphorylation in retinal rods. J Biol Chem 271: 19232–19237 Willardson BM, Wilkins JF, Yoshida T, Bitensky MW (1996) Regulation of phosducin phosphorylation in retinal rods by Ca2+/calmodulin-depenednt adenyl cyclase. Proc Natl Acad Sci USA 93: 1475–1479 Yoshida T, Willardson BM, Wilkins JF, Jensen GJ, Thornton BD, Bitensky MW (1994) The phosphorylation state of phosducin determines its ability to block transducin subunit interaction and inhibits transducin binding to activated rhodopsin. J Biol Chem 269: 24050–24057