SUMMARY Period determination in the mammalian circadian clock involves the turnover rate of the repressors, CRY and PER. Here we show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate (G149E) missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss-of-function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm, but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.

The inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are widely localized in both the heterochromatin and euchromatin regions. We found recently the presence of nucleoplasmic complexes that are composed of phospholipids, IP(3)R/Ca(2+) channels, and Ca(2+) storage protein chromogranin B (CGB). Close examination and 3D image reconstruction of these complexes revealed numerous vesicular structures with an average diameter of approximately 50 nm that are primarily interspersed between the heterochromatins. IP(3) rapidly released Ca(2+) from these structures, but other inositol phosphates, inositol 1,4-bisphosphate, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate, failed to release Ca(2+). Addition of heparin or IP(3)R antibody blocked the IP(3)-induced Ca(2+) releases, indicating the release of Ca(2+) through the IP(3)R/Ca(2+) channels. Given the presence of the IP(3)R/Ca(2+) channels and Ca(2+) storage protein CGB in these vesicular structures, we postulate that these vesicles are the IP(3)-sensitive nucleoplasmic Ca(2+) stores. Abundance of the vesicular Ca(2+) stores between the heterochromatins appeared to imply critical roles these vesicular Ca(2+) stores play in controlling the Ca(2+) concentrations of the chromosomes.

Although the inositol 1,4,5-trisphosphate (IP(3)) induced nuclear Ca(2+) releases have been shown to play key roles in nuclear functions, the presence and operation of the IP(3)-dependent Ca(2+) control mechanism in the nucleoplasm have not been shown. Recently, we found the presence of a high-capacity, low-affinity Ca(2+)-storage protein chromogranin B (CGB) and all three IP(3) receptor (IP(3)R) isoforms in the nucleoplasm, localizing widely in both the heterochromatin and euchromatin regions. In view of the essential role of CGB-IP(3)R coupling in IP(3)-dependent Ca(2+) release in the endoplasmic reticulum, the potential coupling between CGB and the IP(3)Rs in the nucleoplasm was investigated. Hence, we found in the present study the presence of a nucleoplasmic complex, which is composed of the IP(3)R, CGB, and phospholipids, with an estimated molecular mass of approximately 2-3 x 10(7) Da, suggesting the possibility of the presence of an IP(3)-sensitive Ca(2+) store in the nucleoplasm. Moreover, double-labeling immunogold electron microscope studies showed the colocalization of all three IP(3)R isoforms with CGB to the extent that the majority of each IP(3)R isoform-labeling gold particles found in the nucleoplasm was literally next to the CGB-labeling gold particles. In line with the potential existence of an IP(3)-dependent vesicular nucleoplasmic Ca(2+) store, our preliminary results indeed showed a sudden release of Ca(2+) from a putative nucleoplasmic Ca(2+) store in response specifically to IP(3) but not to inositol 1,4-bisphosphate or inositol 1,3,4,5-tetrakisphosphate.

Recently, secretory granule Ca(2+) storage protein chromogranin B (CGB) was shown to be present in the nucleoplasm proper in a complex structure that consists of the inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels and the phospholipids. Further, the amounts of IP(3)Rs present in the nucleus of bovine chromaffin cells were shown to be comparable to that of the endoplasmic reticulum. Therefore, we investigated here the potential contribution of nuclear CGB on the IP(3)-dependent Ca(2+) mobilization in the nucleus, using both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells. Chromogranin A (CGA) expression in the NIH3T3 cells, which do not contain intrinsic chromogranins, increased the IP(3)-induced Ca(2+) releases in the nucleus by 45%, while CGB expression in the same cells increased the IP(3)-induced Ca(2+) releases in the nucleus by 80%. Microinjection of IP(3) into the nucleus of CGB-expressing NIH3T3 cells increased the IP(3)-dependent nuclear Ca(2+) mobilization approximately 3-fold, whereas in CGA-expressing cells it remained the same as that of control cells. In contrast, inhibition of CGA expression in PC12 cells by siRNA treatment decreased the IP(3)-induced Ca(2+) releases in the nucleus by 17%, while inhibition of CGB expression decreased the IP(3)-induced Ca(2+) releases in the nucleus by 55%. Microinjection of IP(3) into the nucleus of siCGB-treated PC12 cells decreased the IP(3)-dependent nuclear Ca(2+) mobilization by approximately 75%, whereas in siCGA-treated cells it remained the same as that of control cells. Given the presence of CGB in the nucleus, these results further highlight the critical contribution of nuclear CGB in the IP(3)-induced Ca(2+) release in the nucleus.