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Regulation of the Circadian Oscillator in Xenopus Retinal Photoreceptors by Protein Kinases Sensitive to the Stress-activated Protein Kinase Inhibitor, SB 203580

2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês

10.1074/jbc.m401389200

1083-351X

Minoru Hasegawa, Gregory M. Cahill,

Light effects on plants

Circadian rhythms are generated by transcriptional and translational feedback loops. Stress-activated protein kinases (SAPKs) are known to regulate transcription factors in response to a variety of extracellular stimuli. In the present study, we examined whether the SAPKs play a role in the circadian system in cultured Xenopus retinal photoreceptor layers. A 6-h pulse of SB 203580, an inhibitor of SAPKs, reset the circadian rhythm of melatonin in a phase-dependent manner similar to dark pulses. This phase-shifting effect was dose-dependent over the range of 1-100 μm. Treatment with SB 203580 also affected light-induced phase shifts, and light had no effect on the circadian oscillator in the presence of 100 μm SB 203580. In-gel kinase assays showed that SB 203580 selectively inhibited a small group of protein kinases in the photoreceptor cells. These SB 203580-sensitive kinases correspond to two isoforms of phosphorylated p38 MAPK and three isoforms of c-Jun N-terminal kinase (JNK). Further in vitro study demonstrated that SB 203580 also inhibited casein kinase Iϵ (CKIϵ), which has been shown to regulate circadian rhythms in several organisms. However, a pharmacological inhibition of CKI reset the circadian oscillator in a phase-dependent manner distinct from that of SB 203580. This argues against a primary role of CKI in the phase-shifting effects of SB 203580. These results suggest that SB 203580 affects the circadian system by inhibiting p38 MAPKs or JNKs and that these protein kinases are candidate cellular signals in the regulation of the circadian oscillator in the Xenopus retinal photoreceptors. Circadian rhythms are generated by transcriptional and translational feedback loops. Stress-activated protein kinases (SAPKs) are known to regulate transcription factors in response to a variety of extracellular stimuli. In the present study, we examined whether the SAPKs play a role in the circadian system in cultured Xenopus retinal photoreceptor layers. A 6-h pulse of SB 203580, an inhibitor of SAPKs, reset the circadian rhythm of melatonin in a phase-dependent manner similar to dark pulses. This phase-shifting effect was dose-dependent over the range of 1-100 μm. Treatment with SB 203580 also affected light-induced phase shifts, and light had no effect on the circadian oscillator in the presence of 100 μm SB 203580. In-gel kinase assays showed that SB 203580 selectively inhibited a small group of protein kinases in the photoreceptor cells. These SB 203580-sensitive kinases correspond to two isoforms of phosphorylated p38 MAPK and three isoforms of c-Jun N-terminal kinase (JNK). Further in vitro study demonstrated that SB 203580 also inhibited casein kinase Iϵ (CKIϵ), which has been shown to regulate circadian rhythms in several organisms. However, a pharmacological inhibition of CKI reset the circadian oscillator in a phase-dependent manner distinct from that of SB 203580. This argues against a primary role of CKI in the phase-shifting effects of SB 203580. These results suggest that SB 203580 affects the circadian system by inhibiting p38 MAPKs or JNKs and that these protein kinases are candidate cellular signals in the regulation of the circadian oscillator in the Xenopus retinal photoreceptors. The central mechanisms of circadian oscillators are transcriptional, and translational autoregulatory feedback loops consisting of several clock gene products (1Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2369) Google Scholar, 2Shearman L.P. Sriram S. Weaver D.R. Maywood E.S. Chaves I. Zheng B. Kume K. Lee C.C. van der Horst G.T.J. Hastings M.H. Reppert S.M. Science. 2000; 288: 1013-1019Crossref PubMed Scopus (1125) Google Scholar, 3Lee C. Etchegaray J.P. Cagampang F.R. Loudon A.S. Reppert S.M. Cell. 2001; 107: 855-867Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar). Many of the clock proteins are phosphorylated, and some of the relevant protein kinases have been identified. It has been shown in hamster and Drosophila that mutations of casein kinase Iϵ (CKIϵ), 1The abbreviations used are: CKIϵ, casein kinase I epsilon; ANOVA, analysis of variance; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; JNKI1, JNK inhibitor 1; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PRC, phase response curve; PTC, phase transition curve; SAPK, stress-activated protein kinase; SCN, suprachiasmatic nucleus; ZT, Zeitgeber time; LD, light/dark sycle. and double-time, a Drosophila homologue gene of CKIϵ, changed the periods of the circadian oscillators (4Kloss B. Price J.L. Saez L. Blau J. Rothenfluh A. Wesley C.S. Young M.W. Cell. 1998; 94: 97-107Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 5Price J.L. Blau J. Rothenfluh A. Abodeely M. Kloss B. Young M.W. Cell. 1998; 94: 83-95Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar, 6Lowrey P.L. Shimomura K. Antoch M.P. Yamazaki S. Zemenides P.D. Ralph M.R. Menaker M. Takahashi J.S. Science. 2000; 288: 483-491Crossref PubMed Scopus (694) Google Scholar). Phosphorylation of FRQ protein, a central component of the Neurospora circadian system, has also been shown to affect the period length of the circadian oscillator (7Liu Y. Loros J. Dunlap J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 234-239Crossref PubMed Scopus (168) Google Scholar). These results indicate that protein kinases play important roles in regulation of circadian feedback loops. The stress-activated protein kinases (SAPKs) are candidates for involvement in circadian system regulation. The SAPKs are members of the mitogen-activated protein kinase (MAPK) family and are activated by a variety of extracellular stimuli such as UV light, heat shock, osmotic stress, or inflammatory cytokines. The activated SAPKs regulate several transcription factors to control gene expression (reviewed in Refs. 8Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar and 9Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (357) Google Scholar). The SAPKs are classified into at least two groups based on the amino acid sequences of their dual-phosphorylation sites. One group is c-Jun N-terminal kinases (JNKs, also known as SAPK1), and the other group is p38 MAPKs (also known as SAPK2, -3, and -4). Each SAPK is activated by a different set of upstream kinase and has a different substrate preference (8Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (515) Google Scholar, 9Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (357) Google Scholar, 10Young P.R. McLaughlin M.M. Kumar S. Kassis S. Doyle M.L. McNulty D. Gallagher T.F. Fisher S. McDonnell P.C. Carr S.A. Huddleston M.J. Seibel G. Porter T.G. Livi G.P. Adams J.L. Lee J.C. J. Biol. Chem. 1997; 272: 12116-12121Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). Because some of the stimuli that activate SAPKs are known to reset the circadian oscillator (11Zatz M. Wang H.-M. Am. J. Physiol. 1991; 260: R769-R776PubMed Google Scholar, 12Zatz M. Wang H.-M. Am. J. Physiol. 1991; 261: R1424-R1430PubMed Google Scholar, 13Provencio I. Foster R.G. Brain Res. 1995; 694: 183-190Crossref PubMed Scopus (156) Google Scholar), the SAPKs may play a role in regulation of the circadian oscillator feedback loops. In the present study, we investigated whether the SAPKs are involved in the regulation of the circadian oscillator in Xenopus retinal photoreceptors. We used SB 203580, an inhibitor of SAPKs, to pharmacologically manipulate SAPK pathways. SB 203580 was originally described as an exceptionally specific inhibitor of some of the p38 MAPKs (9Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (357) Google Scholar, 14Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar, 15Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar, 16Kumar S. McDonnell P.C. Gum R.J. Hand A.T. Lee J.C. Young P.R. Biochem. Biophys. Res. Commun. 1997; 235: 533-538Crossref PubMed Scopus (450) Google Scholar, 17Eyers P.A. Craxton M. Morrice N. Cohen P. Goedert M. Chem. Biol. 1998; 5: 321-328Abstract Full Text PDF PubMed Scopus (280) Google Scholar). More recently it has also been shown to inhibit JNKs at higher concentrations (18Clerk A. Sugden P.H. FEBS Lett. 1998; 426: 93-96Crossref PubMed Scopus (206) Google Scholar, 19Whitmarsh A.J. Yang S.-H. Su M.S.-S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (438) Google Scholar). We found that SB 203580 reset the photoreceptor circadian oscillator and prevented light-induced phase shifts. We also showed that the inhibitory effect of SB 203580 is relatively specific to a small group of protein kinases in the photoreceptor cells and that these SB 203580-sensitive kinases include at least JNKs and p38 MAPKs. We also found that SB 203580 suppresses not only SAPKs but also CKIϵ in an in vitro kinase assay. Animals and Photoreceptor Layer Preparation—Adult, male Xenopus laevis (length, 5-6.5 cm) were purchased from Nasco (Fort Atkinson, WI) and exposed to 12-h light:12-h dark (LD 12:12) cycles for at least 3 weeks before use. Photoreceptor layers were prepared as described previously (20Hasegawa M. Cahill G.M. J. Neurochem. 1998; 70: 1523-1531Crossref PubMed Scopus (24) Google Scholar). Briefly, eyecups were prepared, and the inside of each eyecup was sequentially washed with 0.3% Triton X-100, distilled water, and culture medium to lyse the inner nuclear layer. Then the damaged inner retina was peeled out. The photoreceptor layers and attached pigment epithelium were isolated and incubated overnight in modified Wolf and Quimby amphibian tissue culture medium (Invitrogen, Grand Island, NY) (20Hasegawa M. Cahill G.M. J. Neurochem. 1998; 70: 1523-1531Crossref PubMed Scopus (24) Google Scholar). An incubator maintained the LD cycle, constant temperature (21.0 ± 0.2 °C), and 5% CO2 conditions. The pigment epithelium was removed from photoreceptor layer on the next day to complete the preparation. The experimental protocols meet the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Superfusion Culture—Superfusion culture was carried out for 5 days in constant darkness as described previously (20Hasegawa M. Cahill G.M. J. Neurochem. 1998; 70: 1523-1531Crossref PubMed Scopus (24) Google Scholar). Briefly, photoreceptor layers were cultured individually in flowthrough culture chambers, and the chambers were placed in light-tight water jackets. Temperature was maintained at 21 ± 0.1 °C by a circulating water bath. To maintain the pH of the culture medium, the inflow tubing inside the water jackets was gas-permeable silicone, and circulating water was bubbled with 95% O2/5% CO2. Culture medium was a mixture of 80% defined balanced salts and amino acids (21Cahill G.M. Besharse J.C. J. Neurosci. 1991; 11: 2959-2971Crossref PubMed Google Scholar) and 20% Wolf & Quimby amphibian tissue culture medium, with 0.1 mm ascorbic acid, 100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 100 μm 5-hydroxy-l-tryptophan added. Culture medium was delivered by a syringe pump at a rate of 0.4 ml/h. Experimental photoreceptor layers were exposed to drug pulses by changing syringes and/or to pulses of light at times specified relative to the animals' previous LD cycles (Zeitgeber time (ZT), dark onset = ZT 12). White light was delivered by fiber optic cables from an illuminator (Cole Parmer, Vernon Hills, IL) with a 150-watt quartz-halogen lamp. Control photoreceptor layers were exposed to the control medium containing Me2SO and kept in constant darkness throughout the experiments with syringe changes at the times of drug pulses. Melatonin in superfusate samples was measured by radioimmunoassay, using an 125I-melatonin analogue (Covance, Vienna, VA) and the antiserum produced by Rollag and Niswender (22Rollag M.D. Niswender G.D. Endocrinology. 1976; 98: 482-489Crossref PubMed Scopus (559) Google Scholar). Quantitative measurement of phase shifts of melatonin rhythms was performed as described previously (23Hasegawa M. Cahill G.M. J. Neurochem. 1999; 72: 1812-1820Crossref PubMed Scopus (31) Google Scholar). First, the melatonin release record from each photoreceptor layer was smoothed by a three-point moving average, and long-term trends were removed by subtracting the average of values 12 h before and after each point (using the Chrono computer program, written by Till Roenneberg, Universität München). The times of half-rise and half-fall of the third, fourth, and sometimes fifth circadian peaks were measured as phase reference points for each record. For each photoreceptor layer, the time of each phase reference point was compared with the corresponding mean time in the control group, and the magnitude of the overall phase shift for each photoreceptor layer was taken as the mean of the phase differences measured at those phase reference points. Tissue Collection for Protein Kinase Assays—For each sample, four photoreceptor layers were cultured together in a 24-well tissue culture dish, starting at the dark onset time on the day after dissection. Culture medium was the same as that used for the superfusion culture. The culture dishes were placed in the CO2 incubator and kept in constant darkness. Tissue collections were performed at ZT 6 as follows. First, the photoreceptor layers were rinsed four times with ice-cold phosphate-buffered saline (pH 7.4) then lysed in 100 or 400 μl of ice-cold cell lysis buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Triton X-100, 5mm EGTA, 5 mm EDTA, 50 mm β-glycerophosphate, 20 mm NaF, 1 mm Na3VO4, 2 μm microcystin-LR, 2.5 mm benzamidine, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride) for 10 min on ice. Tissues were homogenized and centrifuged (16,000 × g) for 10 min at 4 °C. The supernatant fractions were kept at -80 °C until immunoprecipitation or in-gel kinase assay. Immunoprecipitation—The protein samples were incubated overnight with phospho-JNK antibody (1:50), phospho-p38 MAPK antibody (1:50) (New England Biolabs, Beverly, MA), or CKIϵ antibody (1:19; BD Transduction Laboratories, Lexington, KY) at 4 °C. On the next day, the samples were incubated for 2-3 h with either 10 μl of protein A-Sepharose beads (Amersham Biosciences, Piscataway, NJ) or mixture of protein A-Sepharose beads and protein G-agarose beads (Invitrogen) at 4 °C. For in-gel kinase assay, the samples were centrifuged (16,000 × g) at 4 °C, and precipitates were washed three times with ice-cold cell lysis buffer. The immunoprecipitated samples were solubilized in 10 μl of 2× SDS gel loading buffer (125 mm Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mm dithiothreitol (DTT)) then heated at 95-100 °C for 5 min. After centrifugation, supernatant fractions were kept at -80 °C until in-gel kinase assay. For kinase assay, the samples incubated with the beads were centrifuged at 4 °C, and precipitates were washed two times with ice-cold cell lysis buffer and one time with 1.1× kinase buffer (1× kinase buffer contained 25 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 2 mm DTT, 2 μm microcystin-LR, 0.1 mm Na3VO4, 5 mm β-glycerophosphate). The immunoprecipitated samples were resuspended into 20 μl of 1.1× kinase buffer and used for the kinase assay. In-gel Kinase Assay—In-gel kinase assays were performed as described (24Kameshita I. Fujisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (434) Google Scholar) with slight modifications. Briefly, equal amounts of total protein samples or immunoprecipitated samples solubilized in 2× SDS gel loading buffer were resolved on 10% SDS-polyacrylamide gel containing 0.5 mg/ml myelin basic protein (MBP). After electrophoresis, the gel was washed with 20% 2-propanol in 50 mm Tris-HCl (pH 8.0) for 1 h and then with buffer A (50 mm Tris-HCl (pH 8.0), 5 mm 2-mercaptoethanol) for 1 h to remove SDS. The proteins on the gel were denatured by 6 m guanidine-HCl in buffer A for 1 h, then renatured by 0.04% Tween 40 in buffer A for 19 h at 4 °C. After renaturation, the gel was preincubated in in-gel kinase assay buffer (40 mm Hepes-NaOH (pH 8.0), 2 mm DTT, 0.1 mm EGTA, 5 mm MgCl2) for 30 min, then the kinase reaction was carried out in in-gel kinase assay buffer containing [γ-32P]ATP (25 μCi/ml buffer) for 1 h at room temperature, in the absence or presence of SB 203580 (1, 10, or 100 μm). The in-gel kinase assay buffer for the control group contained 0.13% Me2SO. After the kinase reaction, the gel was washed with 5% trichloroacetic acid containing 1% sodium pyrophosphate then dried on paper. Phosphorylated proteins were detected by autoradiography. For quantitative analysis, each gel was exposed for multiple different periods of time, and each protein kinase band was analyzed by the appropriately exposed film using the National Institutes of Health Image software (crude extract samples) or the MacBass (immunoprecipitated samples). In both programs, the levels of phosphorylation were obtained by subtracting the background level from the signal intensity of each band. The value of each band in the experimental groups was compared with that of the corresponding band in the control group on the same film. Kinase Assay—Equal amounts of the immunoprecipitated samples were incubated in kinase assay buffer containing 1 μg of phosvitin as a substrate and [γ-32P]ATP (5 μCi) for 30 min at 30 °C, in the presence or absence of SB 203580 (1, 10, or 100 μm). The kinase assay buffer for the control group contained 0.13% Me2SO. The kinase reaction was terminated by addition of 2× SDS gel loading buffer. The samples were heated at 95-100 °C for 5 min and then centrifuged. The supernatant fractions were resolved on 12% SDS-polyacrylamide gel. The phosphorylated substrates were detected by autoradiography and analyzed using MacBass as described above. Materials—Culture medium salts were obtained from Sigma (St. Louis, MO), and all other medium components were from Invitrogen. SB 203580, PD 98059, and JNK inhibitor I were purchased from Calbiochem (La Jolla, CA). SP600125 was a gift from Signal Pharmaceuticals Inc. (San Diego, CA). For the melatonin radioimmunoassay, the 125I-melatonin analogue was from Covance (Vienna, VA), and the antiserum was donated by Dr. M. D. Rollag (Uniformed Services University of the Health Sciences). For the in-gel kinase assay and kinase assay, [γ-32P]ATP was from ICN (Costa Mesa, CA) and PerkinElmer Life Sciences (Boston, MA). SB 203580 Resets the Photoreceptor Circadian Oscillator—A 6-h pulse of SB 203580 (30 μm) reset the phase of the melatonin release rhythm. The magnitude and direction of the phase shift depended on the timing of the pulse (Fig. 1). A pulse of SB 203580 beginning at ZT 3 shifted the phase of the melatonin rhythm ∼12 h (Fig. 1A), a pulse beginning at ZT 12 caused a 7-h phase advance (Fig. 1B), and a pulse at ZT 15 had no effect on the phase of the melatonin rhythm (Fig. 1C). The new phase was attained rapidly and persisted through the end of the experiment. In contrast to its strong effect on the phase of the circadian oscillator, SB 203580 did not cause an apparent acute change in melatonin levels (Fig. 1). This is an interesting observation, because all of the entrainment stimuli reported previously such as light, dopamine, and cyclic AMP acutely affect melatonin level in the retinal photoreceptors (20Hasegawa M. Cahill G.M. J. Neurochem. 1998; 70: 1523-1531Crossref PubMed Scopus (24) Google Scholar, 23Hasegawa M. Cahill G.M. J. Neurochem. 1999; 72: 1812-1820Crossref PubMed Scopus (31) Google Scholar), indicating that those signals act on both the entrainment pathway and the melatonin output pathway. On the other hand, the effect of SB 203580 occurs distinctively on the entrainment pathway (or on the oscillator), and therefore, the SB 203580-sensitive cellular signal does not play a significant role in oscillator output for melatonin release. We produced a phase-response curve (PRC) for SB 203580 by starting pulses at eight different phases throughout the circadian cycle (Fig. 2A). Pulses beginning in the late subjective night to the early subjective day caused phase delays, and pulses beginning in the late subjective day to the early subjective night caused phase advances, whereas a pulse centered at midnight did not change the phase of the melatonin rhythms, resulting in a dark-pulse type PRC (Fig. 2A). When the data are replotted as a phase-transition curve, they indicate type-0 resetting; a pulse given at any time of the day always moved the oscillator phase to the night time (Fig. 2B). We tested whether inhibition of extracellular signal regulated kinases (ERKs, other members of MAPK superfamily) reset the circadian rhythm of melatonin by using PD 98059, an inhibitor of ERK kinase. ERK has been suggested to regulate the circadian system in mouse suprachiasmatic nucleus (SCN) (25Obrietan K. Impey S. Storm D.R. Nat. Neurosci. 1998; 1: 693-700Crossref PubMed Scopus (319) Google Scholar), chick pineal gland (26Sanada K. Hayashi Y. Harada Y. Okano T. Fukada Y. J. Neurosci. 2000; 20: 986-991Crossref PubMed Google Scholar, but also see 27Yadav G. Straume M. Heath 3rd, J. Zatz M. J. Neurosci. 2003; 23: 10021-10031Crossref PubMed Google Scholar), and Bullfrog retina (28Harada Y. Sanada K. Fukada Y. J. Biol. Chem. 2000; 275: 37078-37085Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) as well as several cell lines (29Akashi M. Nishida E. Genes Dev. 2000; 14: 645-649PubMed Google Scholar, 30Cermakian N. Pando M.P. Thompson C.L. Pinchak A.B. Selby C.P. Gutierrez L. Wells D.E. Cahill G.M. Sancar A. Sassone-Corsi P. Curr. Biol. 2002; 12: 844-848Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). However, a 6-h pulse of PD 98059 (30 μm) given either during the midday or during the midnight had no effect on the phase of melatonin rhythms (Fig. 2A). This argues against a role for ERK signaling in regulation of the circadian oscillator in Xenopus retinal photoreceptors. A Dose-dependent Effect of SB 203580 on the Circadian Oscillator Phase—We determined the dose-response relationship for SB 203580 effects on the circadian oscillator by starting pulses at ZT 6, where the oscillator is the most sensitive to the drug. A 6-h pulse of the drug induced phase shift of the circadian oscillator in a dose-dependent manner (Fig. 3). A pulse of 1 μm SB 203580 did not affect oscillator phase (Fig. 3A), whereas a pulse of 10 μm caused a significant phase shift (Fig. 3B). A larger phase shift was induced when the photoreceptor layers were given a pulse at a concentration of 100 μm (Fig. 3C). This result showed that the phase-shifting effects of SB 203580 are dose-dependent and that a threshold concentration for resetting the circadian oscillator is somewhere in the range of 1-10 μm in the photoreceptor cells (Fig. 3D). Effects of SB 203580 on Light-induced Phase Shifts—We also investigated effects of SB 203580 on photic entrainment. When photoreceptor layers were exposed to a 6-h light pulse (∼600 lux) beginning at ZT 15, the expected phase delays of the melatonin rhythms were induced (Figs. 4A and 5A). At 10 or 30 μm SB 203580, a 6-h pulse of the drug alone did not cause a significant phase shift at this time (Fig. 5A). When the light treatments were applied together with either 10 μm or 30 μm SB 203580, significant phase advances were induced (Figs. 4B and 5A). These phase advances were not caused by either light or the drug treatment alone, indicating a complex interaction in the effects of the two treatments on the oscillator. On the other hand, treatment with a higher concentration of SB 203580 (100 μm) together with light caused a phase delay that is indistinguishable from that caused by SB 203580 (100 μm) alone, but significantly different from that caused by the light alone (Fig. 5A). This indicates that light had no effect on oscillator phase in the presence of 100 μm SB 203580. We also performed similar experiments at ZT 21 to confirm the SB 203580 effects on the light-induced phase shift. When the photoreceptor layers were exposed to a 6-h light pulse beginning at ZT 21, the expected phase advances were induced (Fig. 5B). On the other hand, a 6-h pulse of SB 203580 caused phase delays of melatonin rhythms, and the effect was dose-dependent (Fig. 5B). When the light was applied together with either 10 or 30 μm SB 203580, the phase shift that is significantly different from that produced by either light or the drug treatment alone was induced (Fig. 5B). However, simultaneous treatment with light and 100 μm SB 203580 together caused a phase delay that is indistinguishable from that caused by SB 203580 (100 μm) alone. Thus, SB 203580 consistently affected both phase advances and phase delays induced by light, and light had no effect on the oscillator phase in the presence of 100 μm SB 203580.Fig. 5Quantitative summary of circadian phase shifts induced by light (∼∼600 lux), SB 203580, and simultaneous treatment with light and SB 203580. The 6-h pulses were started at ZT 15 (A) or at ZT 21 (B). Three light-induced phase shifts in each graph were obtained from three separate experiments to test effects of three different concentrations of SB 203580 on the light-induced phase shift. Phase advances and delays are represented as positive and negative values, respectively. In both ZTs, the phase shift caused by light and SB 203580 (100 μm) together was indistinguishable from that caused by SB 203580 (100 μm) alone but was significantly different from that caused by light alone. Group means ± S.E. (n = 5 in each group) of magnitude of phase shifts are represented. *, p < 0.05, significant phase shift compared with control groups (ANOVA, Dunnett two-tailed test).View Large Image Figure ViewerDownload (PPT) In contrast, a 6-h pulse of PD 98059 (30 μm) did not affect the phase shifts caused by a light pulse (∼600 lux) at ZT 15 (Fig. 4, C and D). This result argues against a role for ERK signaling in regulation of the circadian oscillator in Xenopus retinal photoreceptors by light. It has been reported that inhibition of protein synthesis resets the circadian oscillator in a phase-dependent manner similar to SB 203580 and blocks light-induced phase shifts (31Dunlap J.C. Feldman J.F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1096-1100Crossref PubMed Scopus (54) Google Scholar, 32Takahashi J.S. Murakami N. Nikaido S.S. Pratt B.L. Robertson L.M. Recent Prog. Horm. Res. 1989; 45: 279-352PubMed Google Scholar, 33Johnson C.H. Nakashima H. J. Biol. Rhythms. 1990; 5: 159-167Crossref PubMed Scopus (30) Google Scholar). This raised the possibility that the effects of SB 203580 on the circadian system are due to nonspecific inhibition of protein synthesis. We therefore examined whether SB 203580 inhibited protein synthesis in our system. The photoreceptor layers were incubated under constant darkness in the culture medium in which leucine was replaced by [3H]leucine (40 μCi/ml; ICN, Costa Mesa, CA) for 6 h in the presence or absence of SB 203580, and the [3H]leucine incorporated into newly synthesized protein was measured by the trichloroacetic acid precipitation method (34Eskin A. Yeung S.J. Klass M.R. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7637-7641Crossref PubMed Scopus (42) Google Scholar). We found that a 6-h pulse of SB 203580 did not inhibit total protein synthesis (data not shown). It is unlikely, therefore, that the effects of SB 203580 are caused by nonspecific inhibition of protein synthesis. Effects of SB 203580 on Photoreceptor Protein Kinases—We assessed the overall specificity of SB 203580 for protein kinase activities in the photoreceptors by in-gel kinase assays of total protein extracts. This technique detects both substrate-dependent phosphorylation and autophosphorylation of kinases separated by molecular mass. Using MBP as a substrate, we detected various protein kinases in the photoreceptor cells (Fig. 6). The signals represent specific incorporation of radiolabeled phosphates into the substrate protein, because no signal was detected when [γ-32P]ATP was replaced by [α-32P]ATP (data not shown). The apparent molecular masses of the detected protein kinases were distributed approximately between 30 and 110 kDa. For quantitative analysis, those kinases were divided into 10 groups (peaks 1-10) based on the differences in the molecular mass (Fig. 6). When the kinase reaction was carried out in the presence of SB 203580 (1, 10, or 100 μm), only two groups of protein kinases were significantly inhibited in a dose-dependent manner (Fig. 6 and Table I). The apparent molecular masses of those SB 203580-sensitive protein kinase groups were ∼55 kDa (peak 5) and 43-45 kDa (peak 6). This result suggests that the inhibitory effect of SB 203580 is relatively specific to a small group of protein kinases.Table IEffect of SB 203580 on protein kinases in Xenopus retinal photoreceptor cellsPeak (~kDa)32P incorporation1 μm10 μm100 μm% of controlPeak 1 (80-105)99.3 ± 7.5102.0 ± 10.886.1 ± 6.6Peak 2 (74)102.9 ± 15.1106.7 ± 10.0129.4 ± 16.6Peak 3 (68)88.2 ± 16.687.8 ± 20.7119.3 ± 17.4Peak 4 (61)85.4 ± 5.889.2 ± 6.470.3 ± 9.0Peak 5 (55)84.1 ± 7.052.7 ± 1.3ap < 0.05, significantly different from the group treated with 1 μm SB 203580 (ANOVA, Dun