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Abstract

The White Collar Complex (WCC) is a transcription factor and light receptor formed by two subunits. Only White Collar-1 (WC 1) has a light receptor domain but both WC-1 and White Collar-2 (WC-2) have zinc finger DNA binding domains. In the negative feedback loop of the circadian clock in Neurospora crassa, WCC is the positive element that drives the clock and synchronizes it with the external cycle of day and night. According to these two functions, there are two groups of target genes, the clock-controlled genes (ccgs) and the light-inducible genes. The latter subset is activated by binding of the light-activated WCC (L-WCC). FREQUENCY (FRQ) is a target of WCC and is the negative element in the feedback loop of the circadian clock in N. crassa. FRQ recruits Casein Kinase 1a (CK1a) to inactivate WCC by phosphorylation. This circadian phosphorylation also stabilizes WCC, so the feedback of FRQ on WCC is both negative and positive. The light-induced activity of L-WCC is regulated by phosphorylation as well, the mechanism of this light-induced phosphorylation is the subject of this study.

L-WCC was proven to consist of two molecules WC-1 and two molecules WC-2. In this work, 34 phosphorylation sites of WC-1 (27 new) and 23 sites of WC-2 (22 new) have been determined. Neither phosphorylation of a protein domain, nor light- or dark-specific phosphorylation were found. There seems to be a pool of poorly and differently phosphorylated WCC molecules in the dark, and light increases the phosphorylation of each molecule. The search for kinase(s) revealed that FRQ also mediates the light-induced phosphorylation of WCC by CK1a and the activity of other kinases is presumed. Phosphorylation site mutants of WC 2 revealed a gradual reduction of the transcriptional activity of WCC, a regulatory compensation of WC-1 and WC-2 and suggested identity of circadian and light-induced phosphorylation. Phosphorylation sites followed by proline are overrepresented on WCC and were shown to be a trigger for FRQ-mediated phosphorylation. Other triggers of phosphorylation of WCC are DNA-binding and most likely light-induced dimerization. DNA-binding brings WCC close to the proline-directed kinases of the transcriptional machinery (TM). It was hypothesized that the TM feeds back on the active WCC to mark it for subsequent, activity-attenuating, FRQ-mediated phosphorylation. A recently published study of the circadian phosphorylation of WCC largely confirms these results but does not touch the topic of proline-directed phosphorylation although these sites are overrepresented in that study as well. Proline-directed phosphorylation is also overrepresented on CLOCK, the WCC ortholog in Drosophila melanogaster, further supporting the hypothesis of a feedback of the TM on WCC.