Abstract
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 microm. Treatment with SB 203580 also affected light-induced phase shifts, and light had no effect on the circadian oscillator in the presence of 100 microm 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 Iepsilon (CKIepsilon), 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.
Highlights
CKI⑀, changed the periods of the circadian oscillators (4 – 6)
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 mitogen-activated protein kinase (MAPK) or Jun N-terminal kinase (JNK) and that these protein kinases are candidate cellular signals in the regulation of the circadian oscillator in the Xenopus retinal photoreceptors
We 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
Summary
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. 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. For the in-gel kinase assay and kinase assay, [␥-32P]ATP was from ICN (Costa Mesa, CA) and PerkinElmer Life Sciences (Boston, MA)
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