PMID- 17115977 OWN - NLM STAT- MEDLINE DCOM- 20070319 LR - 20191210 IS - 1470-8728 (Electronic) IS - 0264-6021 (Print) IS - 0264-6021 (Linking) VI - 402 IP - 3 DP - 2007 Mar 15 TI - Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression. PG - 525-36 AB - CLOCK and BMAL1 [brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator)-like protein 1] are central components of the molecular clock in mammals and belong to the bHLH (basic helix-loop-helix)/PAS [PER (Period)/ARNT/SIM (single-minded)] family. Features of their dimerization have never been investigated. Here, we demonstrate that PAS domain function requires regions extending over the short PAS core repeats. Strikingly, while deleting PAS core repeats does not overtly affect dimerization, it abolishes the transcriptional activity of the heterodimer. Interestingly, these deletions also abolish co-dependent phosphorylation of CLOCK and BMAL1, suggesting a link between the phosphorylation status of the heterodimer and its transactivation potential. We demonstrate that NPAS2 (neuronal PAS domain protein 2) and BMAL2 also undergo similar posttranslational modifications, thereby establishing the mechanism proposed for CLOCK-BMAL1 as a common feature of transcriptional activators in the circadian clock. The discovery of two novel splice variants of BMAL2 confirms the crucial role of the PAS domain and further strengthens the view that co-dependent phosphorylation is of functional significance. In agreement with this, we demonstrate that CRY1-2 (cryptochromes 1-2) affect transactivation and phosphorylation of transcriptional activators of the clock. Furthermore, CRY proteins stabilize the unphosphorylated forms of BMAL1(BMAL2) thereby shifting the phosphorylated/unphosphorylated ratio towards a predominantly unphosphorylated (transcriptionally inactive) form. In contrast, PER proteins, which are weak repressors, are without effect. From these results, we propose a general mechanism for the inhibition of CLOCK(NPAS2)-BMAL1(BMAL2) circadian transcriptional activation by CRY1-2. FAU - Dardente, Hugues AU - Dardente H AD - Laboratory of Molecular Chronobiology, Douglas Hospital Research Centre, McGill University, Montreal, QC, Canada H4H 1R3. FAU - Fortier, Erin E AU - Fortier EE FAU - Martineau, Vincent AU - Martineau V FAU - Cermakian, Nicolas AU - Cermakian N LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PL - England TA - Biochem J JT - The Biochemical journal JID - 2984726R RN - 0 (ARNTL Transcription Factors) RN - 0 (Basic Helix-Loop-Helix Transcription Factors) RN - 0 (Cryptochromes) RN - 0 (Flavoproteins) RN - 0 (Trans-Activators) RN - 9007-49-2 (DNA) RN - EC 2.3.1.48 (CLOCK Proteins) SB - IM MH - ARNTL Transcription Factors MH - Amino Acid Motifs MH - Animals MH - Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism MH - *Biological Clocks MH - CLOCK Proteins MH - COS Cells MH - Chlorocebus aethiops MH - Cryptochromes MH - DNA/metabolism MH - Dimerization MH - Flavoproteins/genetics/*metabolism MH - Phosphorylation MH - Protein Binding MH - Protein Processing, Post-Translational MH - Trans-Activators/genetics/*metabolism MH - Transcription, Genetic/genetics MH - Two-Hybrid System Techniques PMC - PMC1863574 EDAT- 2006/11/23 09:00 MHDA- 2007/03/21 09:00 PMCR- 2007/09/15 CRDT- 2006/11/23 09:00 PHST- 2006/11/23 09:00 [pubmed] PHST- 2007/03/21 09:00 [medline] PHST- 2006/11/23 09:00 [entrez] PHST- 2007/09/15 00:00 [pmc-release] AID - BJ20060827 [pii] AID - bj4020525 [pii] AID - 10.1042/BJ20060827 [doi] PST - ppublish SO - Biochem J. 2007 Mar 15;402(3):525-36. doi: 10.1042/BJ20060827.