Second messenger and Ras/MAPK signalling pathways regulate CLOCK/CYCLE‐dependent transcription

Abstract

The heterodimeric complex of the transcription factors CLOCK (CLK) and CYCLE (CYC) constitutes the positive element of the circadian clock in Drosophila and mammals. Phosphorylation of clock proteins represents an essential mechanism for promotion and control of the molecular oscillator. However, the kinases and signalling pathways that regulate CLK/CYC function remain largely elusive. In the present study we performed a chemical screen of kinase inhibitors in a cell culture reporter assay to identify functional regulators of CLK/CYC-dependent gene expression. These studies and analysis of constitutively active forms of kinases revealed that cyclic nucleotide/protein kinase A (PKA), calcium/calmodulin-dependent kinase (CaMK) II and Ras/mitogen-activated protein kinase (MAPK) regulate CLK/CYC activity. In vitro phosphorylation analysis showed a direct phosphorylation of CLK by CaMK II and p42 MAPK [extracellular signal-regulated kinase (ERK) 2], suggesting that these kinases regulate CLK/CYC-dependent transcription by direct phosphorylation of CLK.

Sequential and Compartment-specific Phosphorylation Controls the Life Cycle of the Circadian CLOCK Protein

“…5 ϫ 10 5 cells were transfected with 1 or 2 g of plasmid DNA and 10 l of Lipofectin, as described previously (16,22,26). Cells were cultured in poly-L-lysine (Sigma)-coated cover slide chambers (Nalgene, Rochester, NY).…”

“…Transcription Activity Assays-Transcriptional activation assays in Drosophila S2 and S2Rϩ cells were performed as described previously (16,22,26). In brief, cells from 500 l of culture (10 6 cells/ml) were transfected after 12-h incubation at 25°C with 200 l of serum-free Schneider's insect medium (Sigma) containing 1.5% Lipofectin (Invitrogen), 25 ng of pRLcopia, 10 ng of pGL3-(4-per-E-box)hs::lucϩ, 200 ng of pHT control vector, and 0.25 ng of pAc-Clk construct.…”

“…Expression Constructs-CLK proteins were expressed from a pAc5.1/V5-HisA vector (Invitrogen) either without any tag (pAc-Clk) for Western blot analysis and transcription activation assays as described in a previous study (26,27), or as fusion proteins with a N-terminal DsRed1 red fluorescent protein tag (pDsRed-Clk) for fluorescence microscopy (30). Mutations and deletions were inserted into these constructs with the QuikChange II XL system (Stratagene, La Jolla, CA) according to the instructions by the manufacturer and using the following primers: CLK NLS (substitution of Pro-484 to Leu, Lys-485 to Glu, and Lys-487 to Glu; fwd, CCA CAG GAA TAT CGC TCG AGG CCG AAC GAA AGT GC; rev, GCA CTT TCG TTC GGC CTC GAG CGA TAT TCC TGT GG), CLK ⌬NES (deletion of 25 amino acids from Gln-836 to Leu-860; fwd, GGC TCG CAA TCC ACC ATT AAT C; rev, TTG TTG CAG CTG CAA CTG TTG CTG).…”

“…PER-mediated inhibition of CLK/CYC (22,23) involves the recruitment of the casein kinase I⑀ homolog DOUBLE-TIME, which contributes to phosphorylation-induced inhibition and degradation of the CLK protein (24,25). Activation of CLK/CYC is affected by calcium/calmodulin-dependent kinase II and mitogen-activated protein kinase, which both phosphorylate CLK in vitro (26). Phosphorylation of CLK may also control recruitment of the transcription co-activator CREB-binding protein to the CLK/CYC complex, which is essential for trans-activation by CLK/CYC (27,28).…”

Circadian rhythmicity mediated by temporal regulation of the activity of p38 MAPK

“…Additionally, the oscillator components BMAL1 (in mice) and CLK (in Drosophila) can be directly phosphorylated by MAPKs (46,47). Although our work demonstrates circadian clock control of activity of a homolog of the p38 family of MAPKs involved in stress responses, circadian rhythms in the activity of the ERK family of MAPKs involved in growth control are also known in several systems (44,(47)(48)(49)(50)(51)(52)(53)(54)(55). These data suggest that control of MAPK pathways by the circadian clock may be a conserved feature of the output pathways.…”