Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events

Abstract

Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora, we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events.

Fig. 1.

Map of FRQ. FRQ ORF with the identified phosphorylation sites and known FRQ domains are indicated. Amino acids in red are identified in vivo FRQ phosphorylation sites. The asterisks above the residues indicate in vitro FRQ phosphorylation sites by CK-1a, CKA, or CK-1a and CKA combined. Short and long blue lines indicate sites and phosphopeptides preferentially phosphorylated in hyperphosphorylated FRQ samples detected by quantitative MS experiments, respectively. Underlined sequences (M1–19) indicate mutated phosphorylation sites.

Fig. 2.

Mapping phosphorylation sites and peptides preferentially phosphorylated in hyperphosphorylated FRQ by ¹⁵N metabolic labeling and quantitative MS. (A) MS spectrum of one FRQ phosphopeptide (with both ¹⁴N and ¹⁵N forms) showing the increase of ¹⁵N-labeled peptide peaks in the purified FRQ sample (DD6 vs. DD18). The m/z of the ¹⁵N/¹⁴N phosphopeptide pair and their mass difference (Δmass) are indicated. Hyperphosphorylated FRQ was ¹⁵N labeled. (B) A table of identified phosphopeptides with significant enrichment of ¹⁵N peaks compared with unphosphorylated peptide controls. (C) MS spectrum of one representative unphosphorylated peptide showing the disappearance of its ¹⁵N form in the purified FRQ. (D) Table showing the identified outliers of unphosphorylated peptides that have low ¹⁵N/¹⁴N ratios.

Fig. 3.

Mutational analyses of FRQ phosphorylation sites. (A) Western blot analysis showing the FRQ phosphorylation profiles of the indicated strains' cultures in LL. Arrows indicate the extensively phosphorylated FRQ forms in the KAJ120 strain. (B) Race tube assays showing the circadian conidiation rhythms of the phosphorylation mutants in DD. (C) Summary diagram showing the period differences between the KAJ120 strain and the phosphorylation mutants. Error bars indicate standard deviation of the mean.

Fig. 4.

Regulation of FRQ stability by multiple FRQ phosphorylation events. (A) Western blot analysis showing the circadian expression of FRQ in the wild-type and indicated mutant strains in DD. (B) Western blot and densitometric analyses showing the pairwise comparisons of FRQ degradation kinetics between the KAJ120 strain and indicated mutants. Each comparison was independently performed at least 3 times in LL. Error bars indicate standard deviations.

Fig. 5.

Both period-lengthening and period-shortening mutations contribute to period and FRQ stability. (A) Race tube analyses showing the circadian conidiation rhythms of the indicated strains. (B) Western blot analysis showing the comparison of FRQ degradation kinetics between the M9 and M9+18 mutants.

Fig. 6.

A model explaining how multiple FRQ phosphorylation events control FRQ stability by regulating the interaction between FRQ and SCF^FWD-1. Lollipops on FRQ symbolize phosphorylation events. Multiple phosphorylation events in the middle of FRQ promote its degradation via SCF^FWD-1-dependent ubiquitination and degradation whereas the phosphorylation events near the C terminus stabilize FRQ.