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

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.