Competing E3 Ubiquitin Ligases Determine Circadian Period by Regulated Degradation of CRY in Nucleus and Cytoplasm

The Psttm Mutation Causes Period Shortening

To confirm that Fbxl21 is the causative gene, we generated transgenic mice expressing the Psttm allele using the tetracycline-transactivator (tTA) system (Hong et al., 2007). Three independent tetO::Psttm transgenic lines were crossed to Scg2::tTA mice in which the Secretogranin-2 promoter drives tTA expression in the SCN and other brain areas to evaluate whether the Psttm allele affects circadian behavior. Western blotting analysis using cerebellum extracts confirmed that only double transgenic mice showed mutant protein expression (Figure S2A). To determine whether Scg2::tTA-driven PSTTM expression in the brain also shortens free-running period as seen in Psttm/+ mice (Figure S2B), we measured their circadian activity rhythms in constant darkness. Double transgenic mice displayed short circadian periods compared with single transgenic lines (Figure 2A, S2C). The period shortening was conditional because doxycycline (Dox) treatment, which represses tTA transgene expression, restored the period to wild-type (WT) values (Figures 2A, S2C). Removal of Dox from the drinking water after 4 wks of treatment caused the double transgenic mice to return to their previous, short free-running periods, confirming that inducible PSTTM expression was the cause of the short free-running periods (Figures 2B, S2D). In contrast, overexpression of WT FBXL21 under the control of the Scg2::tTA driver did not cause period changes in Scg2::tTA/tetO::Fbxl21 #38 mice (Figure S2, E, F and G). Together, these observations show that conditional expression of the Psttm allele in the SCN and brain shortens circadian period in vivo, thus, verifying that Fbxl21 is the causative gene. Because transgenic expression of Psttm in a WT background shortens period and because the Psttm allele is semidominant, this suggests that Psttm can act as an antimorphic mutation.

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Figure 2

The Psttm mutation shortens period and antagonizes the period lengthening effect of the Ovtm mutation in FBXL3

A. Representative actogram of tetO::Psttm #11 single transgenic mouse (left). Representative actogram of tetO::Psttm #11 transgenic mouse with the Scg2::tTA driver (right). Arrowheads indicate LD to DD transition. Doxycycline-containing (10 μg/ml) water was administered during the interval indicated by yellow shading on the actogram.

B. Free-running periods of single and double transgenic mice during constant darkness with water (black), Dox (yellow), and water after Dox treatment (grey). Error bars show SEM (tetO::Psttm #11 n = 3, Scg2::tTA/tetO::Psttm #11 n=5; *p < 0.05, Bonferroni corrected pair-wise comparison).

C. Representative PER2::LUC bioluminescence recording of SCN and pituitary explants from WT and homozygous Psttm mice. Blue and red traces represent PER2 rhythm from WT and Psttm mice, respectively. WT SCN mean circadian period: 24.76 hr (n=6), Psttm SCN mean circadian period: 23.36 hr (n=6). t test: *p-value 0.0027. WT pituitary mean circadian period: 23.2hr, N=6; Psttm pituitary mean circadian period: 22.61hr, N=6. t test: **p-value 0.0004.

D. Representative actograms of WT, Ovtm/Ovtm, Psttm/Psttm and Ovtm/Ovtm Psttm/Psttm mice subjected to the same experimental schedule as in Fig. 1B.

E. Period distribution. The four panels from top to bottom represent WT, Ovtm/Ovtm, Psstm/Psstm and Ovtm/Ovtm Psttm/Psttm mice, respectively. ANOVA of the period was performed among the genotype groups. Results: DF = 3; F= 388.507; p = 2.295 × 10^−51. See also Figure S2 and Table S1.

To investigate the effects of Psttm at the cellular and molecular level, we crossed the Psttm mice with mice carrying the Per2::luc reporter (Yoo et al., 2004), and examined the circadian rhythmicity in tissue explants from WT and Psttm homozygous mice (Figure 2C). SCN organotypic cultures from Psttm mice showed robust rhythms, yet with significantly shorter periods compared with those of WT SCNs (Figure 2C, lower right). Similarly, pituitary explants from Psttm mice also showed shorter periods than WT explants (Figure 2C, lower). Thus, the primary effect of the Psttm mutation on the circadian clock is period shortening at both behavioral and molecular levels.

The FBXL3 I364T mutation in Overtime (Ovtm) mutant mice lengthens period by about 2.5 hrs (Siepka et al., 2007), providing evidence that CRY stability is important for setting the pace of the clock (Busino et al., 2007; Godinho et al., 2007; Maywood et al., 2011). Given that both Ovtm and Psttm correspond to mutations in F-box genes related to CRY degradation, we created Psttm/Psttm Ovtm/Ovtm double-mutant mice to assess the genetic interaction of these mutant alleles (Figure 2, D and E). While WT mice showed a mean period of 23.34 hr (SD=0.30, N=32), Ovtm mice displayed a long period of 26.00 hr (SD=0.46, N=13). In contrast, Psttm mice exhibited a short period of 22.77 hr. Interestingly, the Ovtm/Psttm double-homozygous mutant mice displayed a free-running period of 23.16 hr (SD=0.27, N=29), similar to that of WT mice, and different from the predicted value of 24.4 hr for an additive effect (Table S1). Thus, the Psttm and Ovtm alleles appear to act in an antagonistic rather than additive manner.

Effects of the Psttm Mutation on Core Clock Gene Expression

To explore the effects of the Psttm mutation on clock gene expression, we collected liver and cerebellum samples from WT and Psttm mice maintained in constant darkness and measured cycling clock gene mRNA expression patterns. The Psttm mutation caused significant elevation of Per1, Per2, Cry1 and Cry2 transcripts in cerebellum and a moderate elevation of clock gene transcript levels in liver (Figure 3A, S3A), perhaps due to lower Fbxl21 expression in liver relative to cerebellum (mean Ct value of WT cerebellum and liver: 27.1 and 31.2, respectively; Figure S3D shows different levels of endogenous FBXL21 expression in various mouse tissues). Interestingly, the levels of several clock-controlled genes, including Rev-erbα, Dec2 and Dbp, were significantly increased in liver (Figure S3A).

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Figure 3

Psttm alters circadian clock gene expression in mice

A. Real-time RT-PCR analysis of clock gene expression in WT and Psttm mice. Blue and red circles represent WT and Psttm mice, respectively. Error bars represent SEM for each time point from four independent replicates. 2-way ANOVA shows significant statistical differences between WT and Psttm mutants for Per1 (p < 0.001) and Per2 (p < 0.0001). Cry1 expression shows statistically significant difference at CT12 (p < 0.01) between WT and Psttm mice. Clock protein oscillation in cerebellum. (B) and liver (C). Western blotting was performed using total protein extracts with the indicated antibodies. Representative blots from 3 independent experiments are shown. Quantification of 3 independent experiments are shown to the right of each representative blot. Blue and red circles represent values from WT and Psttm mice. Error bars represent SEM for each time point from 3 independent repeats. In B, 2-way ANOVA shows significant statistical differences between WT and Psttm for PER2 (p < 0.0001), CRY2 (p < 0.0001), and FBXL21 (p < 0.0001). In C, 2-way ANOVA shows significant statistical differences between WT and Psttm for FBXL21 (p < 0.0001).

D. Immunohistochemical staining of CRY1 in SCN sections from WT and Psttm mice. Representative images from ZT12 are shown.

E. Number of CRY1-positive nuclei in the SCN sections collected from WT and Psttm mice at ZT0, 8, and 12. Student t-test shows statistically significant difference between WT and Psttm mice at ZT0 and ZT12 (*** p < 0.001) and ZT8 (* p < 0.01). See also Figure S3.

Next we examined the effects of the Psttm mutation on core clock protein expression in cerebellum and liver. PER1 and PER2 levels were elevated in the cerebellum of Psttm mice (Figure 3B), consistent with the observed increase in transcript levels. Furthermore, Psttm also appeared to advance the phase of the PER protein rhythm. In contrast, CRY1 protein levels were not elevated, and CRY2 levels were significantly reduced (Figure 3B). Consistent with the previously reported rhythmic Fbxl21 mRNA accumulation (Dardente et al., 2008), we observed a circadian oscillation of FBXL21 protein in the cerebellum of WT mice. In Psttm mutants, FBXL21 protein levels in cerebellum were significantly reduced throughout the circadian cycle compared with WT (Figure 3B), suggesting the Psttm mutation impaired FBXL21 protein stability. In addition, in the liver of Psttm mutants, the phase of PER1/2 and CRY1/2 accumulation was advanced relative to WT (Figure 3C). FBXL21 protein accumulation was also both reduced and phase-advanced in Psttm liver. The reduction in CRY, despite enhanced transcript levels, suggests that the Psttm mutation leads to the destabilization of CRY protein. In addition, because the expression of FBXL21 is rhythmic and in phase with that of CRY1/2 (Figure 3B), it is possible that FBXL21 could exert phase-dependent effects.

To determine whether the Psttm mutation affects CRY expression in the SCN, we used immunocytochemical quantitation of SCN sections from WT and Psttm mice. As shown in Figure 3D, the number of CRY1 positive nuclei was significantly reduced in SCN from Psttm mice at ZT0, 8, 12 compared with WT controls (Figure 3E, S3B,C). Taken together, these experiments show that the Psttm mutation leads to a general increase in the RNA expression of CLOCK:BMAL1 target genes, while at the same time altering the expression of CRY1 and CRY2 proteins and substantially reducing FBXL21 protein levels. The reduction in CRY, despite enhanced transcript levels, suggests that the Psttm mutation leads to destabilization of CRY protein.

FBXL21 Stabilizes CRY by Competing Against FBXL3

The concomitant reduction of FBXL21 and CRY proteins caused by the Psttm mutation prompted us to investigate the biochemical interaction between CRY and FBXL21. Co-immunoprecipitation assays showed that among the 6 core clock proteins in the primary loop (CRY1/2, PER1/2, CLOCK, BMAL1), only CRY1 and CRY2 were able to interact with both FBXL21 and PSTTM (Figure S4A). All CRY1 mutants with C-terminal domain truncation were co-immunoprecipitated with both FBXL3 and FBXL21, indicating that FBXL binds to CRY in the photolyase homology domain of CRY1 (amino acids 1–530) (Figure S4). Next we expressed the F-box proteins in NIH3T3 cells and the cell lysates were immunoprecipitated with anti-FLAG (FBXL3, FBXL21, PSTTM) or anti-V5 (β-TRCP1) antibodies. All the F-box proteins including PSTTM were found to interact strongly with native CULLIN1 and SKP1 (the other components of the SCF complex), indicating FBXL21 and PSTTM can form SCF E3 ligase complexes (Figure 4A). Only low levels of CRY1 co-immunoprecipitated with ectopically expressed FBXL3, likely due to robust CRY1 degradation. To our surprise, FBXL21 or PSTTM co-immunoprecipitated much higher levels of CRY1 compared with FBXL3. The stable binding between CRY1 and FBXL21/PSTTM differs from the transient E3 ligase-substrate interaction commonly associated with rapid degradation (e.g., FBXL3-CRY interaction). In addition, ectopically expressed PSTTM and FBXL21 both showed strong affinity toward CRY, indicating that the mutation does not significantly affect CRY binding.

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Figure 4

FBXL21 forms an SCF E3 ligase complex that slowly ubiquitinates CRY1 and antagonizes the activity of SCF^Fbxl3

A. Interaction of FBXL proteins with Cullin1 and Skp1. NIH3T3 cells were transfected with Flag-Fbxl3, Flag-Fbxl21, Flag-Psttm and V5-βTrcp1 expression constructs. Co-immunoprecipitated proteins were analyzed by western blotting with anti-CRY1, anti-CUL1, and anti-SKP1 antibodies. Representative blots from 3 independent experiments are shown.

B and C. FBXL21 interacts with CRY1 (B) and CRY2 (C) in native extracts in a circadian manner. Liver extracts from CT0 to CT24 were immunoprecipitated (IP) with an FBXL21 antibody, and Western blotting was performed with CRY1 and CRY2 antibodies. Lower: quantification of co-immunoprecipitated CRY1 and CRY2 amounts. Data are taken from 2 independent experiments. Error bars show ± range (n = 2). 2-way ANOVA shows statistically significant differences between WT and Psttm for the amount of CRY1 co-precipitated with FBXL21 throughout the circadian cycle (p < 0.0001).

D. Differential effects of FBXL21 and PSTTM on CRY1 stability. 293A cells were co-transfected with indicated constructs. 32 hrs after transfection, cells were treated with 20 μg/ml cycloheximide and incubated for the indicated time before harvest. Western blotting was performed to monitor CRY1 levels using an anti-HA antibody. Lower: Quantification of the effects of FBXL3, FBXL21, PSTTM on CRY1 stability. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3). Half life was determined by using nonlinear, one phase exponential decay analysis (the half-life parameter, K, is significantly different in all four conditions: p<0.0001)

E. The Psttm mutation destabilizes FBXL21. 293A cells were transfected with Fbxl21-Flag or Psttm-Flag constructs. Cycloheximide treatment was performed as in D. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3). The half-life parameter, K, is significantly different: p=0.0436.

F. Competition between FBXL3 and FBXL21/PSTTM modulates CRY1 degradation. Cycloheximide treatment and CRY1 western blotting were performed as in D. Data for quantification represent 3 independent experiments (Figure S4F). The half-life parameter, K, is significantly different: p<0.0001. Data are taken from 3 independent experiments. Error bars show ± SEM (n = 3).

Lower: Competition between Ovtm and Fbxl21/Psttm in CRY1 degradation. Cycloheximide treatment and CRY1 western blotting were performed as in D. Data for quantification represent 3 independent experiments (representative western blots are shown in Figure S4G). The half-life parameter, K, is significantly different: p<0.0001. Error bars show ± SEM (n = 3).

G. In vitro ubiquitination assay. Sf9 cells were infected with the indicated baculovirus constructs. Samples were analyzed by western blotting with an anti-Myc antibody. Upper shows a short exposure image, and the bracket to the right marks polyubiquitinated CRY1. Lower shows a long exposure image, and the bracket to the right marks highly polyubiquitinated CRY1. Results are representative of more than 3 replicates.

H. In vitro ubiquitination was performed as indicated in G. Sf9 cells were infected with the indicated baculovirus. Co-infection of Fbxl baculovirus (Fbxl3+Fbxl21, Fbxl3+Psttm) attenuated the E3 ligase activity of FBXL3. Results are representative of 3 replicates.

I. FBXL3/FBXL21 mediated CRY1 ubiquitination requires Ubc5 as an E2 ligase. Top: Ubc5-mediated robust ubiquitination by FBXL3-SCF complexes and multi ubiquitination by FBXL21/PSTTM SCF complex. Lower: lack of CRY1 ubiquitination in the presence of Ubc13/Uev1A as the E2 ligase. Results are representative of 3 replicates.

J. 293A cells were co-transfected with Cry1-HA, ubiquitin (hUb-HA) and the indicated F-box constructs. Cells were treated with MG132 (10μg/ml) 6 hr before harvest. Whole-cell lysates were analyzed by western blotting with an anti-CRY1 antibody. Results are representative of 3 replicates. See also Figure S4.

To verify the interactions between FBXL21 and CRY1/2 in vivo, we assessed native protein interactions over the circadian cycle by immunoprecipitating endogenous FBXL21 proteins from WT and Psttm liver tissue lysates collected at different circadian times (Figure 4, B and C). Levels of co-immunoprecipitated CRY1/2 showed a circadian rhythm correlated with their periodic accumulation (Figure 3C) in WT tissue; on the other hand, lower levels of CRY1/2 were pulled down in Psttm liver, presumably due to reduced FBXL21 expression (Figure 3C). Next we compared the potential E3 ligase activity of FBXL3 with that of FBXL21 and PSTTM in CRY-degradation assays in 293A cells (Figure 4D, and Figure S4, B and C). In the absence of ectopic FBXL3 expression, transfected CRY1 had a half-life of 2.1 hr (Figure 4D and Figure S4C for CRY2). This rapid degradation was reversed when Cry1 was co-transfected with hFbxl3si RNA, indicating that endogenous hFBXL3 regulates CRY1-HA degradation in 293A cells (Figure S4D). As expected, co-transfection of FBXL3 accelerated CRY1 degradation (0.75 hr). In contrast, co-transfection of FBXL21 significantly decelerated CRY1 degradation (12.3 hr). PSTTM also appeared to slow down CRY1 degradation, but to a lesser extent than WT FBXL21 (3.7 hr). Because endogenous hFBXL3 is present, exogenously expressed FBXL21 likely antagonizes endogenous hFBXL3 to reduce CRY1 degradation. Since FBXL21 is a less efficient E3 ligase for CRY1 than FBXL3 (Fig. 4D), FBXL21 can act antagonistically as a partial agonist in a manner analogous to classic pharmacological experiments in which a partial agonist (FBXL21) can act as a competitive antagonist in the presence of a full agonist (FBXL3). In addition, because PSTTM itself is less stable than WT FBXL21 (Figure 4E), the unstable PSTTM (4 hr) would be expected to antagonize SCF^Fbxl3 less efficiently than WT FBXL21 (8.6 hr). Next we introduced the corresponding Psttm mutation into Fbxl3 (G143E) and measured the effect of the mutation on CRY1 degradation and FBXL3 stability (Figure S4B, lower). The G143E mutation in Fbxl3 did not alter protein stability or E3 ligase activity of FBXL3. Importantly, we rule out the possibility of FBXL21 or PSTTM as an E3 ligase for FBXL3: co-expression of FBXL21 or PSTTM showed no effects on FBXL3 stability independent of CRY, indicating that FBXL21 does not decelerate CRY degradation by serving as an E3 ligase for FBXL3 (Figure S4E).

To test the hypothesis that FBXL21 antagonizes FBXL3, we performed competition assays for CRY1 degradation in 293A cells (Figures 4F and S4F). In support of an antagonistic relationship between FBXL3 and FBXL21, co-expression of FBXL3 and FBXL21 resulted in an intermediate CRY1 half-life (4.4 hr) relative to expression of CRY1 with either FBXL3 (1.9 hr) or FBXL21 alone (11 hr). Interestingly, in the presence of FBXL3, PSTTM was less effective in decelerating CRY1 degradation (3 hr) than FBXL21 (4.4 hr). This supports the conclusion that PSSTM is a less effective antagonist of FBXL3-mediated CRY degradation than WT FBXL21. Next, we carried out similar assays using the hypomorphic Ovtm mutant of Fbxl3 to investigate whether the WT-like circadian periodicity in Ovtm/Psttm compound homozygous mice is associated with restored CRY degradation rate (Figure 2, D and E). Specifically, 293A cells were co-transfected with Cry1 and F-box pairs, Ovtm+Fbxl21, and Ovtm+Psttm. CRY1 degradation was attenuated in the presence of OVTM (4.9 hr; Figures 4F, lower, and S4G), whereas PSTTM expression in place of FBXL21 restored CRY1 degradation in the presence of OVTM (1.6 hr). These changes in CRY1 degradation rate are directly correlated with the period shortening or lengthening in the Psttm and Ovtm mouse mutants (Figure 2E), suggesting that the WT periods of Ovtm/Psttm mice resulted from an antagonistic interaction of the two mutations on CRY degradation in which impaired CRY degradation in OVTM was rescued (or increased) in the presence of PSTTM.

To verify definitively that FBXL21 forms an SCF E3 ligase complex, we performed in vitro ubiquitination assays to examine the ubiquitination activity of SCF^Fbxl3, SCF^Fbxl21 and SCF^Psttm. CRY1 was efficiently polyubiquitinated in the presence of SCF^Fbxl3 complex. Both FBXL21 and PSTTM were able to carry out CRY1 polyubiquitination, but were less active in generating highly polyubiquitinated forms of CRY compared with FBXL3 (Figure 4G, lower: long exposure). The observed differences in CRY ubiquitination processivity by FBXLs paralleled their effects on CRY degradation (Figure 4D). CRY1 ubiquitination by SCF^Fbxl3 was significantly reduced in the presence of SCF^Fbxl21 or SCF^Psttm (Figure 4H), suggesting that attenuated CRY1 degradation in the presence of both FBXL3 and FBXL21 (Figure 4F upper) was due to direct competition between FBXL3 and FBXL21 as CRY1 E3 ligases (Figure 4H). As shown in the end-point competition assay (Figure S4H), FBXL21 and PSTTM (as a lesser extent than WT) were able to protect CRY1 from FBXL3 mediated degradation in a dose-dependent manner, suggesting direct competition between FBXL3 and FBXL21.

No activity was detected from in vitro ubiquitination assays using the UBC13/Uev1A E2 ligase required for K63 ubiquitination (Figure 4I), indicating that FBXL21 is not involved in non-proteolytic K63 ubiquitination, and that CRY stabilization (Figure 4D) does not involve K63 ubiquitination by FBXL21. To confirm these results in intact cells, we performed ubiquitination assays by expressing CRY1, the F-box proteins, and ubiquitin (hUb) in 293A cells in the presence of the proteasome inhibitor MG132 (Figure 4J). Consistent with the in vitro assay results, FBXL21 and PSTTM showed significantly attenuated polyubiquitination activity relative to FBXL3.

Psttm and Fbxl21 Knockdown Differentially Alter Nuclear and Cytoplasmic CRY Degradation

To determine the allelic nature of the Psttm mutant, we compared the effects of the Psttm mutation with loss-of-function of Fbxl21 using shRNA knockdown of Fbxl21 (shFbxl21) on mPER2::LUC rhythms in mouse embryonic fibroblasts (MEFs). Psttm MEFs showed advanced phases with significantly shorter periods relative to WT MEFs (Figure 5, A and D). PSTTM was less abundant than WT FBXL21 in MEFs from the mPER2::LUC genetic background (Figure 5C left), consistent with what we observed in the tissue samples (Figure 3B,C), and knockdown efficiently depleted FBXL21 protein (Figure 5C right). Importantly, Fbxl21 knockdown also elicited phase advance and period shortening phenotypes compared with shRNA control MEFs (Figure 5, B and D), indicating that the Psttm allele behaves as either a loss-of-function hypomorphic allele or a dominant negative antimorphic allele. This is consistent with the in vitro ubiquitination assays that show that SCF^Psttm also does not show a significant gain-of-function in ubiquitination activity relative to SCF^Fbxl21.

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Figure 5

The Psttm mutation is mimicked by Fbxl21 knockdown and that promotes accelerated CRY1 degradation in the nucleus and stabilization in the cytoplasm

A. Psttm shortens circadian period in MEFs. Representative PER2::LUC bioluminescence recording from WT (blue trace) and Psttm (red trace) MEFs.

B. Fbxl21 knockdown shortens circadian period in MEFs. Representative PER2::LUC bioluminescence recording from Lenti-GFP infected (green trace) and Lenti-Fbxl21sh infected (orange trace) PER2::LUC MEFs.

C. Western blotting of WT, Psttm, Lenti-GFP and Lenti-Fbxl21sh cell lysates showing that FBXL21 is low in Psttm MEFs and depleted in Fbxl21 knockdown MEFs. Filled and open arrows indicate FBXL21 and a nonspecific band respectively.

D. Average period values of the experimental groups in A and B. Statistically significant difference in the mean period was detected between WT (24.4h) and Psttm (23.1hr) MEFs (t test: * p < 0.001,). Likewise, statistically significant difference in the mean period was detected between Lenti-GFP (24.06 h) and Lenti-Fbxl21si (22.68 hr) MEFs (t test: * p < 0.001).

E. The Psttm mutation decreases nuclear CRY1 levels and oscillation amplitude. Western blotting for CRY1 was performed using nuclear and cytosolic fractions. Right: Quantification from triplicates including examples shown here. CRY1 levels from Psttm nuclei (red) were significantly reduced (upper, 2-way ANOVA, p < 0.001) relative to CRY1 from WT nuclei (blue). In contrast, cytoplasmic CRY1 levels from Psttm (red) were significantly elevated throughout the circadian cycle (lower, 2-way ANOVA, p < 0.001). Error bars show ± SEM (n = 3).

F. Fbxl21 depletion reveals that Psttm behaves as a loss-of-function mutation relative to WT Fbxl21. Lenti-GFP and Lenti-Fbxl21sh infected mPER2::LUC MEFs were treated the same as in E. Right: Quantification from triplicates including example shown in F. Error bars show ± SEM (n = 3). CRY1 levels in Lenti-Fbxl21sh nuclei were significantly reduced (orange, upper, p < 0.001), whereas cytoplasmic CRY1 levels were significantly increased throughout the circadian cycle (orange, lower, p < 0.001).

G. Accelerated nuclear CRY degradation and decelerated cytoplasmic CRY degradation in Psttm and Fbxl21 knockdown MEFs. Western blotting was performed using anti-CRY1, -CRY2, -FBXL21 antibodies for nuclear and cytoplasmic fractions. Representative blots from 3 independent experiments are shown.

H. Quantification of nuclear and cytoplasmic CRY1 and CRY2 degradation in Psttm and Fbxl21sh MEFs from triplicate experiments including the representative blots shown in G. Error bars show ± SEM (n = 3). See also Figure S5.

To investigate the cellular localization of Fbxl21 and Psttm action on CRY abundance, we prepared nuclear and cytoplasmic fractions from samples collected over the circadian cycle and assessed CRY levels (Figures 5E, 5F, S5A, and S5B). In Psttm MEFs (red circles), nuclear CRY1 and CRY2 levels were significantly lower than in WT (blue circles) (Figures 5E, right upper, and CRY2 results are shown in Figure S5A). In contrast, cytoplasmic CRY levels were significantly elevated in Psttm MEFs (red circles) throughout the cycle (Figure 5E, right lower, and Figure S5A). In a similar manner, Fbxl21 knockdown (orange circles) reduced nuclear CRY1/2 (Figure 5F, right upper, and Figure S5B) compared to GFP control (green circles) yet enhanced cytoplasmic CRY1/2 levels (Figure 5F, right lower, and Figure S5B). To understand the mechanism underlying the differential effects of FBXL21/PSTTM on nuclear and cytoplamic CRY levels, we compared the rate of CRY degradation in nuclear and cytoplasmic fractions from WT vs. Psttm MEFs and shControl vs. shFbxl21 cell lines (Figure 5, G and H). Both the Psttm mutation and Fbxl21 knockdown accelerated nuclear CRY1/2 degradation compared with controls (Figure 5, G and H), with opposite effects on cytoplasmic CRY degradation. Endogenous FBXL21 was detected in both nuclear and cytoplasmic fractions, and the Psttm mutation impaired FBXL21 accumulation in both sub-cellular compartments (Figure 5G). Neither the Psttm mutation nor depletion of endogenous FBXL21 had any effect on nuclear FBXL3 protein levels (Figure S5C). Together, these findings demonstrate that reduced levels of FBXL21 elicited opposing effects on CRY degradation in the nucleus and the cytoplasm, leading to CRY accumulation in the cytoplasm but accelerated turnover in the nucleus.

FBXL21 Forms SCF Complexes in the Cytoplasm

To determine the subcellular localization of the FBXL proteins, we generated fluorescent protein fusions of the FBXL proteins. Consistent with previous results (Cenciarelli et al., 1999), Venus-FBXL3 localized primarily to the nucleus (Figure 6A). On the other hand, Venus-FBXL21 was found in both the nucleus and the cytoplasm (Figure 6A), in accordance with MEF fractionation experiments (Figure 5G). Venus-PSTTM showed a subcellular distribution similar to WT FBXL21, and Venus-CRY1 localized primarily in the nucleus as reported previously (Yagita et al., 2002). Next we used bimolecular fluorescence complementation (BiFC) to investigate the subcellular localization of FBXL-CRY1 complexes. Specifically, CRY1-VenC was co-expressed with the indicated VenN-FBXLs (Figure 6B), and protein interaction was measured at the Venus emission wavelength (528 nm). The CRY1-FBXL3 complex was observed primarily in the nucleus (~90%), whereas CRY1-FBXL21 or CRY1-PSTTM complexes were found in both the nucleus and cytoplasm. Furthermore, in a 3-way competitive BiFC assay (Kerppola, 2006), in which C-terminal Cerulean fused to CRY1 (CRY1-CerC) was allowed to interact with FBXL proteins fused with either Ven-N (VenN-FBXLs) or Cer-N (CerN-FBXLs), demonstrated that FBXL21, and to a lesser degree PSTTM, interact with CRY1 more strongly than FBXL3 (Figure 6C, lower: quantification). The stronger interaction of FBXL21 with CRY1 relative to FBXL3 likely accounts for how FBXL21 is capable of antagonizing FBXL3 ubiquitination in the nucleus to balance CRY stabilization in the WT situation.

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Figure 6

Differential localization of FBXL3 and FBXL21 SCF complexes in the nucleus and cytoplasm

A. Differential subcellular localization of FBXL3, FBXL21 and PSTTM. Full-length Venus tagged FBXL3, FBXL21, PSTTM, and CRY1 were expressed in 293A cells, and percentages of cells with fluorescence signals in nuclei only, cytoplasm only, or both were measured. Whereas FBXL3 (96%) and CRY1 (92%) were predominantly localized to the nucleus, FBXL21 and PATTM were found in the both nucleus and cytoplasm with 51% and 54% of cells in the cytoplasm, respectively. Smaller percentages of cells showed nuclei only localization (FBXL21: 18%, PSTTM: 12%) or both (FBXL21: 30%, PSTTM: 36%). Green: Venus; Blue: DAPI. Bar graphs show the mean ± SD of 3 replicate experiments. 2-way ANOVA shows the localization is significantly different, p < 0.0001.

B. Differential subcellular localization of CRY-FBXL complexes. VenN tagged FBXL3, FBXL21 and PSTTM formed BiFC complexes with VenC tagged CRY1 in 293A cells. FBXL3-CRY1 complexes were primarily localized to the nuclei (88%); however, FBXL21-CRY1 (62%) and PSTTM-CRY1 (70%) were distributed in both nuclei and cytoplasm. Green: Venus; Blue: DAPI. Bar graphs show the mean ± SD of 3 replicate experiments. 2-way ANOVA shows the localization is significantly different, p < 0.0001.

C. Reciprocal 2-color, 3-way BiFC competition assays in 293A cells using CRY1-CerC complementation with CerN-FBXL3 + VenN-FBXL21 or VenN-PSTTM (upper), VenN-FBXL3 + CerN-FBXL21 or CerN-PSTTM (lower). FBXL21, and to a lesser degree PSTTM, interact with CRY1 more strongly than FBXL3 (Bottom: quantification). Green: Venus; Blue: Cerulean. Bar graphs show the mean ± SEM of 3 replicate experiments; * p < 0.01, ** p < 0.001, *** p < 0.0001.

D. FBXL3 and SKP1 form complexes predominantly in the nuclei of 293A cells, whereas FBXL21 and PSTTM interact with SKP1 in the cytoplasm. Bar graphs show the mean ± SD of 3 replicate experiments. 2-way ANOVA shows the localization is significantly different, p < 0.0001.

E. FBXL3 and CULLIN form complexes mainly in the nuclei of U2OS cells (63%), whereas FBXL21 (75%) and FBXL21^Psttm (74%) bind to CULLIN mainly in the cytoplasm. All scale bars are 30 μm. Bar graphs show the mean ± SD of 3 replicate experiments. 2-way ANOVA shows the localization is significantly different, p < 0.0001.

To investigate the cause of the differential effects of FBXL21 depletion on subcellular CRY degradation, we examined SCF complex formation involving FBXL3, FBXL21 and PSTTM by using BiFC assays. Ven-C and Ven-N were fused to the N-termini of Cullin1 or SKP1 and the N-termini of FBXL3, FBXL21 or PSTTM, respectively. SKP1-FBXL3 and Cullin1-FBXL3 complexes were observed primarily in the nucleus (Figure 6D). In contrast, SKP1-FBXL21, SKP1-PSTTM, Cullin1-FBXL21, and Cullin1-PSTTM complexes were abundant in the cytoplasm (Figure 6D, E). Together, these results demonstrate that FBXL21 is primarily involved in CRY degradation in the cytoplasm, and the unstable Psttm allele or Fbxl21 knockdown lead to cytoplasmic CRY accumulation. On the other hand, reduced nuclear FBXL21 levels in Psttm or Fbxl21 knockdown MEFs reduce the antagonistic effects of FBXL21 against FBXL3, leading to accelerated CRY degradation in the nucleus.

We thank Choogon Lee for generously providing the anti-PER1 antibody, Homin Kim for LRR3 modeling, Junghea Park and Weimin Song for excellent technical support, and Yi Liu for editorial comments on the paper. Research was supported by NIH grants (U01 MH61915, P50 MH074924, and R01 MH078024) to J.S.T., (P50 MH074924 and R01 GM090247) to C.B.G., (F32 DA024556) to V.K., the Howard Hughes Medical Institute to J.S.T. and Z.J.C., and the American Heart Association (11SDG7600045) to Z.C. J.S.T. and Z.J.C. are Investigators, J.A.M. is an Associate, I.K. is a Laboratory Manager, and S.-H.Y., S.M.S., H.-K.H., V.K. were Associates in the Howard Hughes Medical Institute.