All members of the photolyase/cryptochrome protein family share a 500-amino-acid core domain (containing two chromophore binding sites) but differ in the presence of N- or C-terminal extensions (39
). The large variation in length and amino acid composition of the C-terminal extension of CRY proteins suggests that this region constitutes the major structural determinant in shaping its function as either circadian core oscillator protein and/or (circadian) photoreceptor protein. In the present study, we have performed a detailed structure/function analysis of the C terminus of the mammalian circadian core oscillator protein CRY1.
By expressing a set of mutant CRY1 proteins in COS7 cells, we successfully identified domains that control the subcellular localization of the protein. We first uncovered a functional bipartite NLSc of the mouse CRY1 protein, which resembles the previously identified N-terminal monopartite NLSn (10
) in that it is evolutionary conserved in all vertebrate CRY homologs (48
). However, combined inactivation of the NLSc and the NLSn (by in vitro mutagenesis) could not prevent mCRY1 from entering the nucleus, suggesting the presence of an additional nuclear import mechanism, such as nuclear cotranslocation via interaction with another protein (piggyback transport). In the search for potential protein binding domains, we identified a putative CC domain at the beginning of the C-terminal extension of CRY1. Deletion of this CC in combination with inactivation of the NLSc resulted in complete cytoplasmic localization of mCRY1 (Fig. ). Proof of piggyback transport was obtained by our observation that mPER2 and BMAL1 can competitively bind to the CC of mCRY1 and facilitate nuclear localization of mCRY1mutNLSc (Fig. ). However, as HA-CRY1mutNLSn+c can still enter the nucleus in the absence of other overexpressed proteins, and endogenous mPER proteins and the CLOCK/BMAL1 heterodimer are expressed at very low levels in COS7 cells (data not shown), the endogenous factor mediating the CC-dependent nuclear import of mCRY1 remains to be identified. We consider TIMELESS an excellent candidate, as it is expressed robustly in these cells, and overexpressed TIM also associates with mCRY1, causing nuclear translocation of the latter protein (F. Tamanini, unpublished data). In conclusion, the C-terminal extension of mCRY1 is involved in a bimodal mechanism for nuclear import; (i) the binding of nuclear import factors to NLSc serves classical nuclear translocation, and (ii) the association of other (core clock) proteins to the CC can mediate nuclear cotranslocation. Yet, as cotranslocation has only been visualized with mutant mCRY1 proteins in a cellular transfection assay, the physiological relevance of the latter mechanism remains to be resolved.
Previously, we have shown that mPER2 shuttles between the nucleus and cytoplasm and accumulates in the nucleus after association with mCRY1, which led us to propose that signal-mediated regulation of subcellular localization of mCRY/mPER complexes controls their stability (and therefore activity) and as such can contribute to the phase delay in mRNA and protein rhythms (15
). In the present study, we have shown that mPER2 (and mPER1) binds to the CC of mCRY1 to form mCRY/mPER complexes and that the NLSc of mCRY1 is necessary to shift the subcellular equilibrium of the mCRY1/mPER2 complex from the cytoplasm to the nucleus. Oppositely, Miyazaki and coworkers provided evidence that the NLS of mPER2 is required for nuclear translocation of mCRY1 (20
). We have shown that the NLSc of mCRY1 and NLS of mPER2 are both required for nuclear accumulation of the complex (Fig. ). Importantly, inactivation of mPER2 NES domains or treatment of cells with the nuclear export inhibitor LMB promotes nuclear accumulation of mCRY1 (as visualized using mutant proteins with a defective NLSc). These findings suggest that mCRY and mPER shuttle as a complex (requiring the concerted action of NLS and NES sequences in the complex) and are in agreement with a recent report in which microinjected mCRY1 was shown to leave the Xenopus laevis
oocyte nucleus via association with mPER1 (17
). In conclusion, our data provide a molecular basis for the synchronous nuclear accumulation of mCRY and mPER proteins observed in vivo (15
) and define the C terminus of mCRY1 as a central domain in this process.
Despite the lack of amino acid sequence homology between the C-terminal extensions of mCRY1 and mCRY2, the latter protein also contains a putative CC and a bipartite NLSc (32
). Interestingly, serine 557 (underlined), which is in close proximity to the first basic cluster of the NLS (S
PKRK) of mCRY2, can be phosphorylated by mitogen-activated protein kinase (33
). Phosphorylation has been identified as a posttranslational event for the regulation of the activity of NLS domains, including that of mPER1 (12
). The opposite circadian phenotype of mCry1−/−
mice, carrying fast- and slow-ticking circadian oscillators, respectively (40
), suggests that nuclear accumulation of mCRY2/mPER complexes runs slightly ahead of mCRY1/mPER complexes. A difference in kinetics could be achieved by phosphorylation-mediated control over the relative strength of NLS (and NES) sequences that together determine the equilibrium between nuclear and cytoplasmically localized mCRY/mPER complexes.
The prime function of mCRY proteins is to inhibit CLOCK/BMAL1-mediated transcription activation of E-box genes in the negative limb of the mammalian circadian core oscillator. Neither the CC, nor the tail, nor the NLSc of mCRY1, when deleted individually, affects the CLOCK/BMAL1-inhibitory capacity of mCRY1, which suggests that these domains are dispensable. Xenopus
CRY1 and CRY2 (xCRY) resemble mammalian CRY proteins in carrying a CC and NLS-containing tail in their C-terminal extension (48
). Any truncation of the tail of xCRY caused CLOCK/BMAL1 inhibition in the transcription assay. However, this was only achieved after compensation with an exogenous NLS, which implies that tail-deficient xCRY proteins, unlike their mammalian counterparts, do not reach the nucleus by cotranslocation with CLOCK and/or BMAL1. Although a comparison between the Xenopus
and mammalian systems is difficult, taken together, these observations suggest differences between mCRY1 and xCRY1 in their cross talk with the CLOCK/BMAL1 heterodimer. Furthermore, we have established the first mutant CRYPTOCHROME with a deficiency in CLOCK/BMAL1-inhibitory capacity; HA-CRY1ΔCCtail (nuclear as a result of cotranslocation with BMAL1 and CLOCK) could no longer inhibit E-box gene expression. Thus, the presence of at least part of the C-terminal extension (CC or tail) of mCRY1 is mandatory for CLOCK/BMAL1 inhibition activity.
It has been suggested that the inhibition of CLOCK/BMAL-mediated transcription requires the interaction of mCRY proteins with either CLOCK alone or both CLOCK and BMAL1 (37
). We provide mechanistic evidence in support of the first hypothesis by showing that the association of mCRY1 with only CLOCK is necessary (e.g., HA-CRY1ΔCC), yet not sufficient (e.g., HA-CRY1ΔCCtail), to mediate this function. Given the slightly reduced CLOCK/BMAL1-inhibitory capacity of HA-CRY1ΔCC, CRY1/BMAL1 interactions could serve to stabilize the binding of mCRY1 to the promoter complex during the negative phase of the circadian loop.
Finally, we identified amino acids 371 to 470 from the core domain as being very important for the transcription-inhibitory activity of mCRY1. An extended C terminus (aa 371 to 606), but not the C-terminus itself (aa 471 to 606), when fused to Arabidopsis thaliana
(6-4PP)-specific photolyase, is able to confer CLOCK/BMAL1 transcription-inhibitory activity to the resulting chimeric protein. However, the extended C terminus requires the remainder of the core region (either mCRY1 or photolyase derived) to fully exert its function, possibly by mediating the association with CLOCK. These observations suggest that mCRY1 requires a complex network of interactions and intrinsic structural requirements for proper transcription inhibition, which involve the core domain and the C terminus. This concept is further illustrated by our observation that from a panel of full-length mCRY1 proteins with random insertions of 5 amino acids, mutant proteins that are not able to repress CLOCK/BMAL1-mediated transcription are most likely unfolded (F. Tamanini, unpublished data). Interestingly, it has recently been shown that whereas the C termini of animal and plant CRY proteins are intrinsically unstructured, they acquire a stable tertiary structure upon intermolecular interaction with the core domain (25
). Given the critical roles of amino acids 371 to 470 and amino acids 471 to 606, it is tempting to speculate that these two regions could be involved in the proposed interaction. This intermolecular binding can cause a structural change within the mCRY1 molecule, which in turn would activate the C terminus. While CLOCK and BMAL1 keep mCRY1 in the proximity of the E-box promoter, the activated C-terminal domains may recruit multiple transcriptional corepressor complexes (histone deacetylases and Sin3A) to silence CLOCK/BMAL1-driven transcription. This model (Fig. ) takes into account the fact that transcriptional corepressors have been recently shown to associate and possibly regulate the function of mCRY1 (21
) and that the binding of CLOCK/BMAL1 at the promoter is not affected by the coexpression of mCRY proteins (11
). In addition, the C terminus would be engaged in posttranslational processes (e.g., stability of mPER proteins, competitive association with clock factors, and nuclear import) that are essential for the robustness of the circadian mechanism.
FIG. 5. Model for the mechanism of action of mCRY1 within the mammalian core oscillator. The C terminus of mCRY1 is involved in association with mPER1 and mPER2 proteins and therefore regulates the stability and cellular localization of the latter proteins. The (more ...)
Functional cross talk between the photolyase-like domain and the C-terminal extension of CRY proteins is not unprecedented. Homodimerization of the Arabidopsis
CRY photoreceptor through the photolyase-like domain has been shown to be essential for light-mediated activation of the C terminus (35
). In the case of Drosophila
CRY, the photolyase-like domain is essential and sufficient for light detection, while the C terminus is involved in the degradation of Drosophila
TIM in response to light (2
). Our study on mammalian CRY1 suggests that communication between the core domain and the C-terminal extension may be a common feature of cryptochromes. Yet we propose that, in terms of protein function, the acquirement of different species-specific C-terminal extensions during evolution not only functionally separated cryptochromes from photolyases but also caused diversity within the cryptochrome protein family, making cryptochromes act like photoreceptors (as in Arabidopsis
), both photoreceptors and core oscillators proteins (as in Drosophila
and zebra fish), or pure core oscillator proteins (as in Xenopus