Second messenger and Ras/MAPK signalling pathways regulate CLOCK/CYCLE-dependent transcription.
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.
Weber
Frank
F
Biochemie-Zentrum Heidelberg, Ruprecht-Karls Universität Heidelberg, Heidelberg, Germany.
Hung
Hsiu-Cheng
HC
Maurer
Christian
C
Kay
Steve A
SA
D056930:ARNTL Transcription Factors; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000502:physiology*; D056926:CLOCK Proteins; D020013:Calcium Signaling / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000502:physiology; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D008156:Luciferases; D014157:Transcription Factors; C120526:Clk protein, Drosophila; C112805:cyc protein, Drosophila