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. 2016 Sep 23:7:159.
doi: 10.3389/fneur.2016.00159. eCollection 2016.

Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period

Na Liu  1 , Eric Erquan Zhang  2
Affiliations

Phosphorylation Regulating the Ratio of Intracellular CRY1 Protein Determines the Circadian Period

Na Liu et al. Front Neurol. .

Abstract

The core circadian oscillator in mammals is composed of transcription/translation feedback loop, in which cryptochrome (CRY) proteins play critical roles as repressors of their own gene expression. Although post-translational modifications, such as phosphorylation of CRY1, are crucial for circadian rhythm, little is known about how phosphorylated CRY1 contributes to the molecular clockwork. To address this, we created a series of CRY1 mutants with single amino acid substitutions at potential phosphorylation sites and performed a cell-based, phenotype-rescuing screen to identify mutants with aberrant rhythmicity in CRY-deficient cells. We report 10 mutants with an abnormal circadian period length, including long period (S280D and S588D), short period (S158D, S247D, T249D, Y266D, Y273D, and Y432D), and arrhythmicity (S71D and S404D). When expressing mutated CRY1 in HEK293 cells, we show that most of the mutants (S71D, S247D, T249D, Y266D, Y273D, and Y432D) exhibited reduction in repression activity compared with wild-type (WT) CRY1, whereas other mutants had no obvious change. Correspondingly, these mutants also showed differences in protein stability and cellular localization. We show that most of mutants are more stable than WT, except S158D, T249D, and S280D. Although the characteristics of the 10 mutants are various, they all impair the ratio balance of intracellular CRY1 protein. Thus, we conclude that the mutations caused distinct phenotypes most likely through the ratio of functional CRY1 protein in cells.

Keywords: cryptochrome; period length; phosphorylation; protein stability; subcellular localization.

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Figures

Figure 1
Figure 1
A cell-based screen to identify critical phosphorylation residues on mCRY1 through rescuing rhythmicity in Cry1−/−:Cry2−/− fibroblasts (DKO cells). (A) Dosage-dependent rescue of circadian rhythms in DKO cells by mCRY1. The mCRY1 expression vector was cotransfected into cells with the P(Per2)-dLuc reporter vector. Three days after transfection, the cells were synchronized by dexamethasone treatment and then moved to luciferin-containing medium for 5–6 days of bioluminescence recording. (B–F) The P(Per2)-dLuc reporter rhythms (baseline subtracted) from DKO cells transfected with WT or mutant mCRY1, as noted in the legend. Experiments were performed as in (A). (G) Quantitation of the period length from WT and mutant mCRY1 transfected cells that showed a distinct period phenotype as noted in the legend. Error bars represent SEM (n ≥ 5, **p < 0.01; ***p < 0.001; ****p < 0.0001, ANOVA).
Figure 2
Figure 2
Effects of mutant mCRY1 on BMAL1: CLOCK-induced transcriptional activation. (A) HEK293 cells were cotransfected with BMAL1 (10 ng plasmid), CLOCK (15 ng) expression plasmid, and Per2-dluc (10 ng) reporter with WT or mutant mCRY1 (5 ng) as noted. Twenty-four hours after transfection, the cells were changed to luciferin-containing medium for end-point bioluminescence recording. The raw data were normalized such that the reporter control without mCRY1 (WT or mutant) transfection was equal to 100%. Compared with WT mCRY1, the mutants exhibited two profiles: strong repression with similar repression activity to WT and weak repression with a significant reduction in repression activity. Mean and error bars (SEM) of three independent transfections are shown (****p < 0.0001, ANOVA). Two additional experiments gave similar results. (B) DKO cells were cotransfected with Per2-dluc (10 ng) reporter and WT or mutant mCRY1 (50 ng) as noted. Experiments and data analysis were done as in (A). Compared with WT, the mutants exhibited two profiles: strong repression with similar repression activity as WT and weak repression with a significant difference. Mean and error bars (SEM) of three independent transfections are shown (**p < 0.01, ***p < 0.001, ****p < 0.0001, ANOVA). Two additional experiments gave similar results.
Figure 3
Figure 3
Effects of phosphomimetic mutation on mCRY1 protein stability and interactions with FBXL3 and PER2. (A) Global protein stability (GPS) reporter system. The DsRed–IRES–EGFP target element was cloned into a lentiviral vector, and the fluorescent reporter proteins were co-expressed from a single mRNA via an internal ribosomal entry site (IRES). (B) HEK293 cells were infected with lentivirus of pLv–DsRed–IRES–EGFP–mCRY1 (WT or mutant) for 24 h, and then the fluorescent protein signals were analyzed by flow cytometry. The EGFP/DsRed ratio acts as a reporter for stability of the expressed WT or mutant mCRY1. The d1EGFP and d4EGFP are markers for 1- and 4-h half-lives, respectively. (C) Luciferase complementation assay. mCRY1 (WT or mutant) and FBXL3 (or PER2) were co-expressed as fusion proteins with luciferase fragments in HEK293 cells. Experiments were done as in Figure 2A and the data presented relative to mCRY1 (WT)-mFBXL3 (or mPER2). Mean and error bars (SEM) of three independent transfections are shown (****p < 0.0001, ANOVA). Two additional experiments gave similar results.
Figure 4
Figure 4
Effects of phosphomimetic mutation on subcellular localization of mCRY1 protein. GFP-tagged mCRY1 (WT or mutant) proteins were transiently overexpressed in HEK293 cells, and the subcellular distribution pattern of mCRY1 protein was analyzed. (A) Representative images of GFP-mCRY1 (WT or mutant) were detected by GFP fluorescence (green), and the nuclei were stained with Hoechst (blue). (B) Percentage of cells showing nuclear (N), nuclear–cytoplasmic (N + C), and cytoplasmic (C) staining as indicated in the plots. The ratio of cells with subcellular localization to the total transfected cells was analyzed by counting 100 cells three times in each experiment. (C) Percentage of colocalization of GFP-mCRY1 (WT or mutant) with nuclei. The 50 GFP-mCRY1 (WT or mutant)-expressing cells were analyzed by Image J software (Version 1.37c, NIH, USA). Mean and error bars (SEM) are shown (n = 3 for each experiment). Two additional experiments gave similar results (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, ANOVA). (D) Location of the important phosphorylated sites on the liner protein functional regions of mCRY1. The FAD binding domain, which contains phosphate binding loop, protrusion loop, C-terminal lid (Lid), and nuclear localization signal (NLS), has been indicated in this schematic diagram. The diagram was constructed using Illustrator for Biological Sequences (IBS) software (24).
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