The vertebrate retina retains a circadian oscillator, and its oscillation is self-sustained with a period close to 24 h under constant environmental conditions. Here we show that bullfrog retinal mitogen-activated protein kinase (MAPK) exhibits an in vivo circadian rhythm in phosphorylation with a peak at night in a light/dark cycle. The phosphorylation rhythm of MAPK persists in constant darkness with a peak at subjective night, and this self-sustained rhythm is also observed in cultured

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... s, indicating its close interaction with the retinal oscillator. The rhythmically phosphorylated MAPK is detected only in a discrete subset of amacrine cells despite ubiquitous distribution of MAPK throughout the retinal layers. Treatment of the cultured retinas with MAPK kinase (MEK) inhibitor PD98059 suppresses MAPK phosphorylation during the subjective night, and this pulse perturbation of MEK activity induces a significant phase delay (4 -8 h) of the retinal circadian rhythm in MAPK and MEK phosphorylation. These observations strongly suggest that the site-specific and time-of-dayspecific activation of MAPK contributes to the circadian time-keeping mechanism of the retinal clock system. The physiology and behavior of living organisms from bacteria to humans show daily fluctuations with a period of approximately 24 h, and the oscillations controlled by autonomous circadian oscillators are termed circadian rhythms (1, 2). These rhythms can be synchronized (entrained) to environmental time cues such as light or temperature but are sustained under constant environmental conditions in the absence of time cues (1). A lot of studies on the Drosophila melanogaster clock system have demonstrated that a transcription/translation-based feedback loop is involved in generating the circadian rhythmicity (2, 3). In Drosophila, a complex of two transcription factors, dCLOCK and CYCLE (dBMAL1), positively regulates both period (per) and timeless (tim) transcription through CACGTG E-box elements found in their promoters (4 -6), leading to an increase in protein levels of both PER and TIM. PER and TIM proteins form a heterodimer and translocate to the nucleus where PER/TIM negatively regulate dCLOCK/CYCLE-induced transcription of their own promoters (5, 7-11). Thus, these positive and negative elements constitute an autoregulatory feedback loop. In vertebrates, autonomous circadian oscillators are located in several neuronal tissues such as the retina, pineal gland, and suprachiasmatic nucleus (SCN) 1 (1, 12-17) . Homologs of per, clock, and bmal1 have been identified in vertebrates, and they are expressed in SCN of mammals (reviewed in Refs. 2 and 18). It is now postulated that the molecular framework of the circadian oscillator is conserved from flies to mammals, although each clock component appears to function in a slightly different manner (reviewed in Refs. 19 and 20). In addition to the transcriptional and translational regulation, post-translational modifications of clock gene products play important roles in the clock system. Drosophila PER, TIM, and CLOCK proteins are phosphorylated in a daily/circadian manner (21-23), and another clock gene product, DOUBLE-TIME, closely related to human casein kinase I⑀, has been shown to reduce the stability of PER by phosphorylation (24, 25) . Also, Drosophila TIM is degraded upon receiving light, probably in a tyrosine kinase-and proteasome-dependent manner (10, 22, 26) . Although these posttranslational events significantly contribute to the circadian timing mechanism in Drosophila, little is known about the molecular nature of protein kinases, phosphatases, and proteases in vertebrate clock systems. Recently, we have demonstrated circadian phosphorylation of mitogen-activated protein kinase (MAPK) in the chick pineal gland (27). Importantly, circadian-phosphorylated MAPK was localized in follicular pinealocytes (27) , where the pineal circadian oscillator is located (28), suggesting an intracellular linkage between the circadian activation of MAPK and the oscillator. This is also true of the mouse SCN (29), although the phase of the circadian activation of MAPK in the mouse SCN is opposite to that of pineal MAPK (i.e. daytime-versus nighttime-specific activation; Refs. 27 and 29). Here we investigated the MAPK activation cycle and its contribution to the oscillator in the retina, which is another well defined clock-containing tissue of vertebrates. It was clearly demonstrated that bullfrog (Rana catesbeiana) retinal MAPK exhibits in vivo and in vitro circadian rhythm in activation peaked at nighttime, and this rhythmicity was found in a discrete subset of amacrine cells, which could be a candidate for clock location in the retina. Noticeably, the retinal circadian rhythms in MAPK and MAPK kinase (MEK) phosphorylation were phase-delayed by a pulse perturbation of MEK activity, suggesting that the MEK-MAPK pathway plays an important role in maintenance of the circadian time-keeping mechanism basically common to vertebrate clock systems.