As in other organisms, a negative feedback loop constitutes the backbone of the molecular circadian clock in mammals (18
). The mPER-mCRY complex plays an essential role in the negative feedback loop by exerting inhibitory activity on CLOCK-BMAL1-driven transcription in a time-dependent manner (14
). Although mCRYs play a major role in the inhibition, the timing of this inhibition seems to be regulated by mPERs (15
). The present study suggests that the CKBD of an mPER protein dictates its clock-relevant functions.
has been placed outside of the core feedback loop based on previous genetic studies (3
), we show here that mPER3 is in fact part of the transcriptional inhibitory complex and is regulated in a manner similar to the regulation of mPER1 and mPER2. Like mPER1 and mPER2, mPER3 is rhythmically phosphorylated and translocated into the nucleus, and it interacts rhythmically with other clock proteins. Among clock proteins, mPER3 is most strongly associated with mPER1. In addition, in the absence of mPER1 (mPer1ldc
double-mutant mice) mPER3 phosphorylation is abolished. Thus, mPER3 is regulated by mPER1 at a posttranslational level. Consistent with these observations, phosphorylation of mPER3 by CKI
is enhanced in the presence of mPER1 in NIH 3T3 cells (Fig. ), and the nuclear translocation of mPER3 is promoted by mPER1 in NIH 3T3 cells (14
). The presence of mPER3 in circadian clock protein complexes is likely mediated by interaction with mPER1.
mPER1 and mPER2 in mPer2/3 and mPer1/3 double-mutant mice, respectively, show apparently normal phosphorylation, interaction with other clock proteins, and nuclear translocation, suggesting that the single remaining mPER can support the molecular clock. The levels of the remaining mPER protein in the mutant mice were comparable to those of the mPER proteins in wild-type mice, suggesting that circadian changes of mPER abundance in liver were not significantly altered by disruption of mPer1/3 or mPer2/3 genes.
Unlike mPER1 or mPER2, mPER3 alone cannot support the molecular clock even for one cycle. mPER3 in mPer1/2
double mutant mice is apparently not phosphorylated and interacts very weakly with other clock proteins. In addition, mPER3 in the double-mutant mice is always cytoplasmic. Our data suggest that the lack of physical association of mPER3 with CKI
might explain the functional inadequacy of mPER3 in terms of supporting the circadian clock. In fact, CKI
has been implicated in the nuclear translocation of the mPER proteins (1
Our results suggest that stable interactions between CKI
and mPERs are important for effective phosphorylation and likely lie upstream of nuclear translocation of mPERs. If mPER3 alone cannot support the circadian clock due to the lack of stable interaction with CKI
, then replacement of CKBD of mPER3 with that of mPER2 should rescue the inability of mPER3 to support the clock. Our study indeed shows that chPER3 physically interacts with CKI
and is efficiently phosphorylated in vitro. Taken together, our data strongly suggest that the CKBD of mPERs is essential for stable interaction with CKI
, effective phosphorylation by CKI
, and possibly other clock-relevant functions, such as nuclear translocation. The importance of the CKBD for a functioning circadian clock is supported by a human genetic study showing that a mutation in the CKBD of hPER2 is associated with a severe sleeping disorder known as familial advanced sleep phase syndrome (23