FRQ-CK1 Interaction Underlies Temperature Compensation of the Neurospora Circadian Clock

Abstract

Temperature compensation is a fundamental property of all circadian clocks; temperature compensation results in a relatively constant period length at different physiological temperatures, but its mechanism is unclear. Formation of a stable complex between clock proteins and casein kinase 1 (CK1) is a conserved feature in eukaryotic circadian mechanisms. Here, we show that the FRQ-CK1 interaction and CK1-mediated FRQ phosphorylation, not FRQ stability, are main mechanisms responsible for the circadian temperature compensation phenotypes in Neurospora. Inhibition of CK1 kinase activity impaired the temperature compensation profile. Importantly, both the loss of temperature compensation and temperature overcompensation phenotypes of the wild-type and different clock mutant strains can be explained by temperature-dependent alterations of the FRQ-CK1 interaction. Furthermore, mutations that were designed to specifically affect the FRQ-CK1 interaction resulted in impaired temperature compensation of the clock. Together, these results reveal the temperature-compensated FRQ-CK1 interaction, which results in temperature-compensated CK1-mediated FRQ and WC phosphorylation, as a main biochemical process that underlies the mechanism of circadian temperature compensation in Neurospora. IMPORTANCE Temperature compensation allows clocks to adapt to all seasons by having a relatively constant period length at different physiological temperatures, but the mechanism of temperature compensation is unclear. Stability of clock proteins was previously proposed to be a major factor that regulated temperature compensation. In this study, we showed that the interaction between CK1 and FRQ, but not FRQ stability, explains the circadian temperature compensation phenotypes in Neurospora. This study uncovered the key biochemical mechanism responsible for temperature compensation of the circadian clock and further established the mechanism for period length determination in Neurospora. Because the regulation of circadian clock proteins by CK1 and the formation of a stable clock complex with CK1 are highly conserved in eukaryotic clocks, a similar mechanism may also exist in animal clocks.

Keywords: Neurospora; casein kinase 1; circadian clock; protein phosphorylation; temperature compensation.

FIG 1

Altered FRQ stability is not responsible for temperature compensation. (A, left) Representative photographs of race tubes used to evaluate circadian conidiation rhythm of the wild-type strain at indicated temperatures. Periods are given to the right. (Right) Plot of period versus temperature. Error bars are standard errors of means (n = 5). (B, left) Image of Western blot for FRQ from the wild-type strain grown in constant light at the indicated temperatures for the indicated number of hours after CHX addition. (Right) Plot of relative FRQ level as a function of time after CHX addition over a range of temperatures. Error bars are standard deviations (n = 3). The levels of large and small forms of FRQ due to alternative splicing and their various phosphorylated species were used for quantification. (C, left) Image of Western blot for FRQ from the wild-type strain grown in constant light for one day, transferred into constant darkness, and harvested at the indicated time. (Right) Plot of relative FRQ level as a function of time after light/dark (LD) transition over a range of temperatures. Error bars are standard deviations (n = 3). (D, left) Representative photographs of race tubes used to evaluate circadian conidiation rhythms of the wild-type (WT) and frq⁷ strains at the indicated temperatures. Periods are given to the right. (Right) Plot of period versus temperature. Error bars are standard errors of means (n = 5). (E, left) Image of Western blot for FRQ from wild-type and frq⁷ strains grown in constant light at 21 and 29°C for the indicated number of hours after CHX addition. (Right) Plot of relative FRQ quantity as a function of time after CHX addition over a range of temperatures. Error bars are standard deviations (n = 3). (F, left) Image of Western blot for FRQ from wild-type and frq⁷ strains grown at 21 and 29°C for the indicated number of hours after LD transition. (Right) Plot of relative FRQ quantity as a function of time after LD transition over a range of temperatures. Error bars are standard deviations (n = 3). *, P < 0.05; **, P < 0.01; Student’s t test.

FIG 2

Casein kinase 1 is involved in temperature compensation of the Neurospora clock. (A, left) Representative photos of race tubes used to evaluate effects of 6-DMAP treatment on conidiation rhythms in the wild-type strain over a range of temperatures. (Right) Plot of period versus temperature at indicated 6-DMAP concentrations. Error bars are standard errors of means (n = 3). (B, left) Representative photos of race tubes used to evaluate effects of PF670462 treatment on conidiation rhythms of the wild-type strain. (Right) Plot of period versus temperature at indicated PF670462 concentrations. Error bars are standard errors of means (n = 3). (C) Diagram depicting the strategy for creation of the homokaryotic ck1a^H123Y strain by homologous recombination (see Materials and Methods for details). (D) Western blot of FRQ and CK1a protein levels in the wild-type and ck1a^H123Y strains. (E, left) Representative photos of race tubes used to evaluate conidiation rhythms of the WT (ck1a^WT) and ck1a^H123Y mutant. (Right) Plot of period versus temperature for wild-type and mutant strains. Error bars indicate standard errors of means (n = 3).

FIG 3

Temperature sensitivity of the FRQ-CK1a interaction explains the temperature compensation profiles of the wild-type and frq⁷ strains. (A, left) Western blot analysis for CK1a and stained membrane (MEM) (loading control) in extracts of the wild-type strain grown at the indicated temperatures. (Right) Plot of the relative CK1a levels as a function of temperature. Error bars are standard deviations (n = 5). (B) In vitro kinase assay showing the phosphorylation of GST-FRQ (534-562) and GST-FRQ (425-683) by recombinant His-CK1a at different temperatures. Error bars are standard deviations (n = 3). (C, left) Western blot analysis for FRQ precipitated with Myc-CK1a from extracts of Neurospora cultures grown in constant light at the indicated temperatures in the presence of quinic acid (QA). (Right) Plot of the relative amount of FRQ-CK1a complex as a function of temperature. Quantification of relative FRQ-CK1a interaction levels is based on the ratio of IP to input and normalized to CK1a level. Error bars are standard deviations (n = 3). ns, not significant; *, P < 0.05; **, P < 0.01; Student’s t test. (D, left) Western blot analysis of WC-2 immunoprecipitates from the wild-type strain grown at the indicated temperatures and analyzed for FRQ and WC-2. (Right) Relative amounts of FRQ precipitated with WC-2 as a function of growth temperature (n = 6). Quantification of relative FRQ-WC-2 interaction levels is based on the ratio of IP to input and normalized with WC-2 level. (E, left) Western blot analysis for FRQ precipitated with endogenous CK1a from extracts of the wild-type and frq⁷ strains grown in constant light at 21 and 29°C. (Right) Relative amount of FRQ-CK1a complex at 21 and 29°C. Quantification of relative FRQ-CK1a interaction levels is based on the ratio of IP to input and normalized with CK1a level. Error bars are standard deviations (n = 5). ns, not significant; *, P < 0.05; Student’s t test. (F) Western blot analysis of FRQ from extracts of the wild-type strain grown in constant light at the indicated temperatures and subjected to partial trypsin digestion. Error bars are standard deviations (n = 4). **, P < 0.01; Student’s t test.

FIG 4

Temperature sensitivity of the FRQ-CK1a interaction results in the temperature overcompensation phenotype of the ckb^RIP strain and CK2 phosphorylation sites of FRQ mutants. (A, left) Representative photos of race tubes used to evaluate conidiation rhythms of the ckb^RIP strain. (Right) Plot of period versus temperature for wild-type and ckb^RIP strains. Error bars are standard errors of means (n = 5). (B, left) Western blot analysis for FRQ precipitated with Myc-CK1a from extracts of the wild-type strain and the ckb^RIP strain that expresses Myc-CK1a grown in constant light at the indicated temperatures in the presence of quinic acid. (Right) Plot of the relative amount of FRQ-CK1a complex as a function of temperature. Quantification of relative FRQ-CK1a interaction levels is based on the ratio of IP to input and normalized with CK1a level. Error bars are standard deviations (n = 4). **, P < 0.01; Student’s t test. (C, left) Representative photos of race tubes used to evaluate conidiation rhythms of the wild-type (frq complementation strain) and M19 strains. (Right) Plot of period versus temperature for wild-type and M19 strains. Error bars are standard errors of means (n = 4). (D) Myc-CK1a immunoprecipitation assays showing that the FRQ-CK1a interaction is temperature overcompensated in the M19 strain that expresses Myc-CK1a grown in constant light at the indicated temperatures. Quantification of relative FRQ-CK1a interaction levels is based on the ratio of IP to input and normalized with CK1a level. Error bars are standard deviations (n = 3). *, P < 0.05; **, P < 0.01; Student’s t test.

FIG 5

Mutations of the FCD domains of FRQ cause temperature-sensitive FRQ-CK1a association and partial loss of temperature compensation. (A to C, left) Representative photos of race tubes used to evaluate conidiation rhythms of the wild-type (frq complementation strain) and FCD mutant strains. (Right) Plot of period versus temperature for wild-type and FCD mutant strains. Error bars are standard errors of means (n = 5 to 9). (D, left) Western blot analyses of FRQ immunoprecipitated with anti-CK1a antibody from extracts of the wild-type and Q494N strains grown in constant light at 21 and 29°C. (Right) Plot of relative amount of FRQ in wild-type and Q494N strains. Quantification of relative FRQ-CK1a interaction levels is based on the ratio of IP to input and normalized to CK1a level. Error bars are standard deviations (n = 3). ns, not significant; *, P < 0.05; Student’s t test. (E, left) Western blot analyses of FRQ in the wild-type and Q494N strains grown in constant light at 21 or 29°C for the indicated number of hours after CHX addition. (Right) Plot of relative FRQ levels at the indicated hours after CHX treatment. Error bars are standard deviations (n = 3). *, P < 0.05; **, P < 0.01; Student’s t test.

FIG 6

Mutations of CK1a phosphorylation sites on FRQ cause temperature-sensitive FRQ-CK1a interaction and partial loss of temperature compensation. (A, left) Representative photos of race tubes used to evaluate conidiation rhythms of wild-type (frq complementation strain) and M10 and M9 mutant strains at the indicated temperatures. (Right) Plot of period versus temperature for wild-type and mutant strains. Error bars indicate standard errors of means (n = 5). (B, left) Western blot analyses of FRQ immunoprecipitated with anti-CK1a antibody from extracts of the wild-type, M9, and M10 mutant strains grown in constant light at 21 and 29°C. (Right) Plot of relative amount of FRQ in wild-type, M9, and M10 mutant strains. Quantification of relative FRQ-CK1a interaction levels is based on the ratio of IP to input and normalized to CK1a level. Error bars are standard deviations (n = 3). ns, not significant; *, P < 0.05; Student’s t test. (C, left) Western blot analyses of FRQ in the wild-type, M9, and M10 mutant strains grown in constant light at 21 or 29°C for the indicated number of hours after CHX addition. (Right) Plot of relative FRQ levels at the indicated hours after CHX treatment. Error bars are standard deviations (n = 3). *, P < 0.05; **, P < 0.01; Student’s t test. (D) FRQ-CK1a interaction is important for the maintenance of circadian period of the wild-type strain within the physiological temperature range (20 to 30°C). When temperature is above 34°C, the FRQ-CK1a interaction is enhanced, resulting in period shortening and impaired temperature compensation.