Circadian activation of bullfrog retinal mitogen-activated protein kinase associates with oscillator function

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 retinas, 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-day-specific activation of MAPK contributes to the circadian time-keeping mechanism of the retinal clock system.