FIGURE 4.

Phosphorylation of CRY1 Ser-588 is rhythmic in liver and is increased when DNA-PK is absent or depleted. A, alignment of CRY1 sequence surrounding the SQ site at 588–589 with epitope for antibody that recognizes Ser(P)-33 of RPA32. This information comes from consensus site analysis of other proteins recognized by this antibody (43). Red denotes required amino acids SQ at the phosphorylation site, blue denotes amino acids that are highly preferred at these positions, and green denotes amino acids that are also frequently found in protein sequences recognized by this antibody. B, P-CRY1(S588) antibody recognizes immunoprecipitated (IP) WT CRY1 but not S588A mutant CRY1. C, P-CRY1(S588) antibody recognizes immunoprecipitated WT CRY1, but not if lysate is treated with phosphatase. Signal is restored if phosphatase inhibitor sodium vanadate (Na₃VO₄) is added along with the phosphatase. D, CRY1 Ser-588 phosphorylation is rhythmic in mouse liver nuclei. Nuclear fractions were isolated from livers collected every 4 h from mice housed in constant darkness, and an aliquot (500 μg) of each was subjected to immunoprecipitation with P-CRY1(S588) antibody. These immunoprecipitates (top panel) as well as whole nuclear lysates (bottom panels) were then Western blotted (WB) with the antibodies shown. An asterisk denotes a nonspecific band. CT denotes circadian time, where CT12 is defined as time of activity onset. E, Ser-588 phosphorylation increases in the absence of DNA-PKcs. Endogenous CRY1 was immunoprecipitated from WT and DNA-PKcs KO MEF and then analyzed by Western blot using the P-CRY1(S588) antibody (top panel) and CRY1 antibody (bottom panel). Cells were treated with MG132 (10 μm) and okadaic acid (30 nm) for 5 h before harvesting. F, HEK293 cells were transfected with WT or S588A mutant Myc-CRY1 and with either control siRNAs or siRNAs to GAPDH or DNA-PKcs as labeled. Myc-CRY1 was immunoprecipitated and analyzed by Western blot as in E. Western blots showing the levels of DNA-PK, Myc-CRY1, and tubulin in the input cell lysates are also shown.