In order to gain insight into the role of CRYs in the molecular circadian clock, we sought to identify and further define functional domains in the CRY proteins of Xenopus laevis
. We have previously shown that the C-terminal tails of both xCRY proteins are necessary for each protein's nuclear localization in vitro (24
). To determine whether these regions are sufficient for nuclear localization, we fused these portions of the C-terminal tail of each xCRY protein in frame with eGFP and NOC, a cytoplasmic protein, and assayed these constructs for subcellular localization (Fig. ). While eGFP-NOC was localized in the cytoplasm as expected, both eGFP-xCRY1Cterm-NOC and eGFP-xCRY2Cterm-NOC were found predominantly in the nucleus (Fig. ). Therefore, residues E550 to R596 of xCRY1 and residues G515 to R556 of xCRY2 are not only necessary but also sufficient for nuclear localization in COS7 cells.
FIG. 1. The C termini of xCRY1 and xCRY2 are sufficient for nuclear localization of the CRY protein when expressed in COS7 cells. (A) Visualization of NOC, when cloned in frame with the C terminus of either xCRY1 (residues 550 to 596) or xCRY2 (residues 515 (more ...)
Next, we set out to define which residues within these domains are important for nuclear localization. In the C-terminal tail of xCRY1, residues 595 to 612 comprise a sequence that weakly resembles a bipartite NLS. This sequence is highly conserved in mCRY1 and has been shown to be functional in that protein. To determine if this sequence is sufficient for localization of xCRY1, we cloned it in frame with eGFP and NOC and assayed for subcellular localization. We found that there was only a small increase of this NOC fusion protein in the nucleus or in both the nucleus and the cytoplasm compared to eGFP-NOC without the putative NLS (37% versus 22%, respectively). It is for this reason that we did not include this putative NLS in our eGFP-xCry1Cterm-NOC constructs.
Since we found that the putative NLS in the C-terminal portion of xCRY1 was not sufficient for nuclear localization, we then investigated the area comprised of residues 550 to 596 into three regions, i.e., residues 550 to 564, residues 565 to 574, and residues 575 to 596, and then made various deletions in the xCRY1 C-terminal portion of eGFP-xCRY1Cterm-NOC. Any region or combination of regions that we deleted within the xCRY1 C terminus in the eGFP-xCRY1Cterm-NOC fusion protein led to partial or complete loss of nuclear localization (Fig. ). Deletion of region 1 from the xCRY1 C-terminal region had the most severe effect on NOC localization, but when it alone was fused to NOC, it was not sufficient to bring NOC into the nucleus. While we have shown that the xCRY1 C-terminal tail, residues 550 to 596, is sufficient for nuclear localization in these cells, it seems that this function is not confined to a single small domain.
In contrast, the C-terminal tail of xCRY2 contains a putative canonical nuclear NLS, which consists of six positively charged residues in a K(X9
)K(X)R pattern (15
). In order to test the functionality of this sequence, we replaced each cluster of basic residues with noncharged residues in the xCRY2Cterm-NOC fusion protein. When tested for subcellular localization, all of the mutated fusion proteins exhibited decreased nuclear localization; in fact, mutation of KRK541-543 or KVR554-556 resulted in complete loss of nuclear localization (Fig. ). Mutation of these same residues in full-length xCRY2 also resulted in complete loss of nuclear localization (data not shown). Therefore, xCRY2 has a functional bipartite NLS in its C-terminal tail.
Despite the fact that the C-terminal tails of xCRYs seem to be necessary and sufficient for the proteins' nuclear localization, the PHR of xCRY1 and xCRY2 is sufficient for repression of CLOCK-BMAL1-mediated activation when it is localized to the nucleus by a heterologous NLS (24
). Although the primary amino acid sequences of xCRY1, xCRY2, and xPHOTO are 85% similar in this region (Fig. ), the PHRs of xCRYs can repress CLOCK-BMAL1-mediated activation, but xPHOTO cannot (24
). The fact that these proteins share a high degree of sequence similarity yet have distinct functions allowed us to use a chimeric protein approach to characterize the function of the PHR of xCRY in the repression mechanism of CRYs and also to define functional domains within this region, while preserving the protein's structural integrity.
FIG. 2. xCRY/PHOTO chimeras show reduced repression of CLOCK-BMAL1-mediated activation. (A) Protein sequence alignment of the PHRs of xCRY1, xCRY2, and xPHOTO. The xCRY sequences are very similar to those of xPHOTO in the PHR, which includes all of the protein (more ...)
xCRY/xPHOTO chimeras which had the first half of either xCRY1 or xCRY2 and the second half of PHOTO were generated (Fig. ). Since xCRYs and xPHOTO are so similar, this was equivalent to making 46 amino substitutions in the second half of the PHR of xCRY. These chimeras were then tested for their ability to repress CLOCK-BMAL1-mediated transcription in a luciferase repression assay in COS7 cells. Both chimeras were still able to repress CLOCK-BMAL1, but they exhibited a significant decrease in repression compared to wild-type xCRY (P < 0.01) (Fig. ).
The decreased repression shown by the chimeras in the luciferase assay could be for many reasons; some examples include decreased protein stability, improper subcellular localization, or decreased interaction with CLOCK-BMAL1. To see if the chimeras' decreased repression is due to decreased protein expression or stability, the protein levels of the chimeras versus wild-type xCRYs were examined. These experiments demonstrated that the chimeric proteins were present at somewhat reduced levels compared to their wild-type xCRY counterparts: xCRY1/xPHOTO expression was reduced approximately twofold, and xCRY2/xPHOTO expression was reduced approximately fourfold (Fig. ). When the luciferase repression assay was repeated using doses of plasmid adjusted to yield comparable protein expression levels, the chimeras' abilities to repress CLOCK-BMAL1 improved slightly but still were significantly lower than that of wild-type xCRY (P < 0.01) (Fig. ). Therefore, the decrease in repression shown by the chimeras cannot be fully explained by decreased protein expression or stability.
FIG. 3. Decreased repression exhibited by chimeras is not due to decreased protein expression or improper subcellular localization. (A) Protein expression levels of xCRY and chimera proteins. COS7 cells were transfected with the indicated plasmids. A DNA dose (more ...)
Next, the subcellular localization of each chimera was investigated to see if the decrease in repression is due to mislocalized protein. While wild-type xCRY1, xCRY2, and xCRY2/xPHOTO were found predominantly in the nucleus, xCRY1/xPHOTO was localized mostly in the cytoplasm (Fig. ). Coexpression of CLOCK and BMAL1 with the xCRY1 chimera did not alter its localization (data not shown). To further elucidate whether the decrease in repression exhibited by xCRY1/PHOTO was due to its cytoplasmic localization, both Xenopus chimeras were cloned in frame with a heterologous NLS. Although both NLS-xCRY/xPHOTO proteins were localized to the nucleus (Fig. ), neither protein's repressive ability improved significantly (Fig. ), suggesting that improper localization of the chimera cannot explain its decreased repression.
Previous studies have shown that CRY binds to the CLOCK-BMAL1 heterodimer (6
) and that this binding is required for repression (19
). In light of these data, we hypothesized that that the disruption of repression seen with the xCRY chimeras may be due to an inability to bind the CLOCK-BMAL1 heterodimer. To test this hypothesis, we transfected COS7 cells with xCLOCK, xBMAL1-V5, and either FLAG-xCRY1, FLAG-xCRY2, FLAG-xCRY1/PHOTO, or FLAG-xCRY2/PHOTO. We then examined whether xBMAL1 immunoprecipitated with each CRY protein. While wild-type xCRY1 and xCRY2 exhibit a strong interaction with xBMAL1, neither chimera showed detectable interaction with xBMAL1 (Fig. ). This suggests that reduced interaction with theBMAL1 explains the decrease in repression by the chimeras.
FIG. 4. Chimeras do not interact with xBMAL1, unlike wild-type CRY proteins. IP, immunoprecipitation. COS7 cells were transfected with xCLOCK, xBMAL1-V5, and FLAG-xCRY constructs as indicated. CRY and chimera proteins were pulled down, using anti-FLAG-conjugated (more ...)
We could not determine whether CLOCK can bind to the chimeras since CLOCK was present at very low levels in our immunoprecipitation lysates and did not coimmunoprecipitate even with wild-type xCRY1 (data not shown). This is not surprising, since it has been previously shown that when CLOCK and BMAL are coexpressed in cell culture, CLOCK is rapidly degraded (8
The characterization of the xCRY/xPHOTO chimeras indicates that the second half of the xCRY PHR is required for full repression of CLOCK-BMAL1. To further define functionally important regions of this second half, we replaced portions of the xPHOTO moiety of the xCRY1/xPHOTO chimera with the equivalent portions of xCRY1 to see if restoration of xCRY1 residues would rescue repression of CLOCK-BMAL1. These substitutions corresponded to clusters of residues that are conserved in repressive-type vertebrate CRYs but not in xPHOTO (Fig. and ). When tested in the luciferase repression assay at a subsaturating dose (150 ng), these chimeras were expressed at levels comparable to those of wild-type xCRY1 and higher than those of the original xCRY1/xPHOTO chimera (Fig. ). However, the substitutions in these chimeras did not improve their repression of CLOCK-BMAL transcriptional activity, and in fact, the chimeras with further substitutions were less effective (Fig. ). From these data, we can conclude that restoration of any single cluster of xPHOTO residues to xCRY1 residues in the second half of the PHR was insufficient for full repression.
FIG. 5. Replacing portions of the xPHOTO portion of xCRY1/xPHOTO with xCRY1 residues does not rescue repression. (A) Schematic diagram of the primary structure of the xCRY1/xPHOTO chimera (xCRY1 moiety, blue; xPHOTO moiety, gray), highlighting portions of the (more ...)