FIGURE 5.

The S588D phosphomimetic mutation causes increased CRY1 stability by antagonizing FBXL3 function. A, repression assays showing the level of CLOCK/BMAL1 activation of Per1-luc reporter in the presence of different doses of WT or mutant CRY1. HEK293 cells were transfected with Clock and Bmal1 expression plasmids and WT or mutant Cry1 expression plasmids as shown. The raw data were normalized such that the average level of activation of cells transfected with Clock and Bmal1, and the reporter control was equal to 100%. Each data point is averaged from the results of three replicates, with the error bars representing the S.E. B, triple phosphomimetic mutant (S551D,S564D,S588D) has a longer half-life than WT or the triple alanine mutant. Cycloheximide (CHX) was added to HEK293 cells expressing WT, S551A,S564A,S588A, or S551D,S564D,S588D mutant CRY1; cells were collected after 0, 2, 4, 6, or 8 h; and levels of CRY1 were detected by Western blot (WB). C, the single mutation S588D is sufficient to stabilize the mutant CRY1 compared with WT CRY1. Cycloheximide was added to HEK293 cells expressing WT or S588D mutant CRY1; cells were collected after 0, 2, 4, 6, or 8 h; and levels of CRY1 were detected by Western blot using the LiCor system for quantitation. D, the means of three replicates of experiment shown in C. The error bars represent S.E. E, WT CRY1 levels are reduced when FBXL3 is co-expressed in HEK293 cells, but this is not the case when the S588D mutation is present. Shown are Western blots for Myc-CRY1 (top panel) and FLAG-FBXL3 (bottom panel).