Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock

Abstract

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock.

Figure 1

6-DMAP blocks FRQ phosphorylation in vivo. At two different circadian time points, DD14 (CT2, Top) and DD24 (CT13, Bottom), half of the cultures were treated with 5 mM 6-DMAP. Cultures were harvested at the indicated times after the treatment. Western blot analysis shows the phosphorylation profiles of FRQ protein. The asterisks indicate missing LFRQ phosphorylation bands in the 6-DMAP treated sample.

Figure 2

6-DMAP slows down FRQ degradation and lengthens the period of the Neurospora clock in a dose-dependent manner. (A) Western blot analysis showing the reduction of FRQ degradation after an LD transition in the presence of 5 mM 6-DMAP. Cultures were first grown in LL for 1 day before being transferred into DD. Just before the LD transition, 5 mM 6-DMAP was added to half of the cultures, and cultures were harvested at the indicated times in DD. (B) Densitometric analysis of the Western blot shown in A. (C) Race tube data showing that 6-DMAP lengthens the period of the clock in a dose-dependent manner. (Left) The concentrations of the drug. Two replicate race tubes for each concentration are shown.

Figure 3

Identification of three FRQ phosphorylation sites. (A) Schematic diagram of SFRQ deletion constructs. The dashed box represents the entire SFRQ ORF (aa 100–989). The black bars below indicate the locations of the deleted regions in different sFRQ constructs. “4” denotes sFRQ4; 4A-D represent subsections of “4.” (B) FRQ phosphorylation profiles in various sFRQ deletion strains. Cultures were grown in LL for at least 24 hr before harvesting. * marks the strains in which SFRQ is less phosphorylated than the wild-type SFRQ. The various FRQ phosphorylation forms were separated by using a longer than normal electrophoresis time to emphasize the mobility differences caused by phosphorylation. (C) The sequence of the 20-aa region deleted in sFRQ4C. The three potential phosphorylation sites conserved among different fungal frq homologs are indicated by *. The point mutations introduced are described in parentheses. (D) SFRQ phosphorylation profiles of the mutants containing point mutations at the three potential sites. The arrow indicates the phosphorylation band missing in the three mutants.

Figure 4

The effects of deletion and mutation of the FRQ phosphorylation sites on FRQ degradation and the circadian clock. (A) Western blot analysis showing that deletion of region 4C (aa 500–519) and mutation of S513 slow down the degradation of SFRQ. After the cultures were grown in LL for a day, 10 μg/ml of CHX was added at time 0. The appearance of more compact FRQ signals (compared with FRQ signals in Fig. 3 B and D) was the result of a shorter electrophoresis time. (B) The densitometric analysis of the race tube data showing that S513R mutations dramatically increase the period of the clock in DD. In contrast to the strains used in A, these strains are derived from a wild-type frq gene and produce both LFRQ and SFRQ forms. (C and D) Western blot and densitometric analyses showing that S513R mutations slow down FRQ degradation after an LD transition.

Figure 5

Two different mutations at S513 result in differential responses of FRQ degradation. (A) Western blot analysis showing that the S513I and S513D mutations lead to the loss of the top SFRQ phosphorylation band. The arrow indicates the missing band in the mutants. (B) Western blot analysis showing that the S513I mutation dramatically slows down the degradation of SFRQ. Cultures were grown in LL for a day before CHX was added at time 0. (C) Grouped data showing densitometric analysis of Western blots from three independent experiments as in B. Bars = SD.

Figure 6

The S513I mutation results in significantly slower FRQ degradation and in a very long circadian period. (A) Western blot analysis of FRQ degradation after an LD transition in the wild-type and the two S513 mutant strains. The numbers above the blot indicate the number of hours after the LD transition. (B) Densitometric analysis of Western blots from three independent experiments as in A. Bars = SD. (C) Race tube data of the wild-type, KAJ120, and S513 mutant strains. The first black bar on each race tube indicates the time of the LD transfer. The subsequent black bars indicate the growth fronts of the cultures every 24 hr. The periods of the strains are shown on the right. ARR, arrhythmic.