Figure 3.

The diurnal nucleus/cytoplasm shuttling of clock factors is mediated by phosphorylation. During the day (ZT0-ZT12), Clock is phosphorylated by CDK5 at specific sites Threonine 451 and 461, while BMAL1 is phosphorylated by CK2 at Serine 90. Somehow these events can promote heterodimerization and shuttle from the cytoplasm to the nucleus. Here, CLOCK is phosphorylated at Serine 440-441-446, and this post-translational modification promotes the transactivation mediated by CLOCK: BMAL1. During the early night (ZT12-16), CDK5 mediated phosphorylation of PER2 at Serine 394. This event promotes heterodimerization with CRY1 and probably, the assembling of the cytoplasmic macromolecular negative complex consisting also of PER1, CRY1, CK2β, and CKI ε/δ. This macromolecular complex goes into the nucleus and it complexes with the CLOCK: BMAL1 heterodimer. This event is promoted by phosphorylation of CRY1 at Serine 247 and 588, CRY2 at Serine 265. CLOCK is phosphorylated at Serine 38 and 42 which leads to the transactivation shut down. During the late-night (ZT16,-20), the nuclear macromolecular complex is disassembled. CLOCK undergoes subsequent phosphorylation at Serine 431 and 427, the second one mediated by GSK3β, which also phosphorylates BMAL1 at Serine 17 and 21. This even triggers CLOCK: BMAL1 shuttling to the cytoplasm followed by proteasomal degradation. CKI can phosphorylate both PER2 and PER1, respectively at Serine 477 (PER2) and Serine 902/916 (PER1). PER2 is additionally phosphorylated by CK2 at serine 53. All these events promote PER1/PER2 degradation in the cytoplasm mediated by β-TRCP1/2. CRY1 is translocated into the nucleus after being dephosphorylated at Serine 588. Here it is degraded by the proteasomal complex formed by FBXL3 and FBXL21. On the other hand, CRY2 is phosphorylated at Threonine 300, and this modification drives its degradation mediated by FBXW7-SCF. All these events between ZT16 and ZT24 close the 24 h cycle.