Phosphorylation of FREQUENCY protein by casein kinase II is necessary for the function of the Neurospora circadian clock

Abstract

FREQUENCY (FRQ), a key component of the Neurospora circadian clock, is progressively phosphorylated after its synthesis. Previously, we identified casein kinase II (CKII) as a kinase that phosphorylates FRQ. Disruption of the catalytic subunit of CKII abolishes the clock function; it also causes severe defects in growth and development. To further establish the role of CKII in clock function, one of the CKII regulatory subunit genes, ckb1, was disrupted in Neurospora. In the ckb1 mutant strain, FRQ proteins are hypophosphorylated and more stable than in the wild-type strain, and circadian rhythms of conidiation and FRQ protein oscillation were observed to have long periods but low amplitudes. These data suggest that phosphorylation of FRQ by CKII regulates FRQ stability and the function of the circadian feedback loop. In addition, mutations of several putative CKII phosphorylation sites of FRQ led to hypophosphorylation of FRQ and long-period rhythms. Both CKA and CKB1 proteins are found in the cytoplasm and in the nucleus, but their expressions and localization are not controlled by the clock. Finally, disruption of a Neurospora casein kinase I (CKI) gene, ck-1b, showed that it is not required for clock function despite its important role in growth and developmental processes. Together, these data indicate that CKII is an important component of the Neurospora circadian clock.

FIG. 1.

Disruption of the ckb1 gene in Neurospora resulting in circadian conidiation rhythms of low amplitude and long period. (A) Western blot analysis showing that expression of CKB1 protein was abolished in two independent ckb1^RIP strains. The asterisk designates a nonspecific band recognized by our CKB1 antiserum. (B) Race tube assay showing the conidiation rhythms in DD. Black lines indicate the growth front every 24 h. Severe independent ckb1^RIP strains were examined by multiple sets of race tubes, and the representative race tube results are shown. (C) Densitometric analysis of the results of the race tube assay shown in panel B.

FIG. 2.

FRQ expression in the ckb1^RIP strain. (A) Western blot analysis showing FRQ phosphorylation patterns in the wild-type, ckb1^RIP, and cka^RIP strains. Cultures were harvested in LL. Arrows indicate the hyperphosphorylated or hypophosphorylated FRQ species. (B) Western blot analysis showing FRQ degradation in the wild-type and ckb1^RI strains after an LD transition. (C) Densitometric analysis of the results from three independent experiments. Error bars are standard deviations. (D) Rhythmic expression of FRQ in the wild-type and ckb1^RI strains in DD. The experiment was repeated multiple times, and similar results were obtained. (E) Densitometric analysis of the results shown in panel D.

FIG. 3.

Rhythmic expression of frq, ccg-1, and ccg-2 in the ckb1^RIP strain. (A) Rhythmic expression of frq and ccg-1 in the wild-type and ckb1^RI strains in DD. The experiments were repeated multiple times, and similar results were obtained. (B and C) Densitometric analysis of the results shown in panel A. (D) Expression of ccg-2 in the wild-type and ckb1^RI strains in DD.

FIG. 4.

Mutations of putative CKII phosphorylation sites of FRQ resulting in the disappearance of phosphorylated FRQ species and long-period circadian rhythms. (A) FRQ phosphorylation patterns in frq mutant strains. The arrow indicates the hyperphosphorylated FRQ species in the wild-type strain. Mutations of FRQ in the mutant strains are indicated below. (B) Race tube assays showing the long-period conidiation rhythms in the mutant. The periods ([plusmn] standard deviation) of the mutants are indicated. (C) Rhythmic expression of FRQ in the wild-type and m5 strains in DD. (D) Densitometric analysis of the results shown in panel C.

FIG. 5.

Expression and cellular localization of CKA and CKB1 proteins. (A) Western blot analysis showing the expression of CKA and CKB1 in the wild-type strain in DD. Representative results from three independent experiments are shown. The slightly high levels of CKB at DD4 and DD16 were not consistently seen in other experiments. (B) (Top) Western blot analysis showing levels of CKA and CKB1 in the cytoplasm and in the nucleus in DD at different time points. (Bottom) Densitometric analysis of the results from three independent experiments showing the levels of CKA and CKB in the nucleus. Error bars are standard deviations.

FIG. 6.

Normal FRQ expression and oscillation in the ck-1b^RIP strain despite its severe defects in growth and development. (A) Race tube assay showing the slow growth rate of the ck-1b^RIP strain. Black lines mark the growth front every 24 h. (B) Western blot analysis showing the FRQ phosphorylation patterns in the wild-type and ck-1b^RIP strains. Cultures were harvested in LL. (C) Rhythmic expression of FRQ in the wild-type and ck-1b^RIP strains in DD.