Quantitative Two-Dimensional Gel Electrophoretic Analysis of Clock-Controlled Proteins in Cultured Chick Pineal Cells: Circadian Regulation of Tryptophan Hydroxylase

Abstract

The progression of the circadian oscillator through its cycle and the circadian rhythm of melatonin production in dissociated chick pineal cultures both require daily de novo protein synthesis during defined circadian phases. To identify specific proteins involved in these two processes, we have performed a quantitative two-dimensional polyacrylamide gel electrophoretic screen of proteins that are synthesized at different times of the day in chick pineal cell cultures. Out of ∼700 proteins analyzed, we have identified several proteins whose levels of ³⁵S incorporation oscillate in a light/dark cycle. One protein of 56 kDa, pI 6 (p56) undergoes a diurnal oscillation that parallels the melatonin rhythm, reaching a peak early in the night and falling to minimal levels during the day. A second protein of 22 kDa, pI 4.5 (p22) also expresses a diurnal rhythm in ³⁵S incorporation; however, it peaks at the end of the night. The oscillations of both proteins persist, with a reduced amplitude, in constant darkness. Furthermore, the phases of the p56 and p22 rhythms are regulated by the light/dark cycle. Both p56 and p22 appear to be under direct control of the chick pineal circadian oscillator, and therefore can be described as "clock-controlled proteins." We have identified p56 as tryptophan hydroxylase by microsequencing and western blotting. Chick pineal tryptophan hydroxylase also expresses a 24-h oscillation in abundance both in vitro and in vivo. The rhythm in tryptophan hydroxylase expression represents a newly discovered level of regulation of the melatonin synthesis pathway by the circadian clock in chick pineal cells.