Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

Apiruck Watthanasurorot,Pikul Jiravanichpaisal,Sittiruk Roytrakul,Netnapa Saelee,Kenneth Söderhäll,Amornrat Phongdara,Irene Söderhäll

doi:10.1371/journal.pgen.1003361

Abstract

Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.

Highlights

The physiology and behavior of most organisms are regulated according to daily environmental changes in a circadian manner
Melatonin is a darkness hormone produced in the brain of most animals during the night, and here we show that melatonin controls the formation of a protein named AST2 in crayfish
We demonstrate that AST2 is induced by melatonin at night and functions in the internal biological clock by preventing BMAL1 and CLK to form a complex

Summary

Introduction

The physiology and behavior of most organisms are regulated according to daily environmental changes in a circadian manner. The homologues of the CLK and PER proteins have the same name, while the homologues of TIM and CYC are called cryptochrome (CRY) and brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1 (BMAL1) respectively [5]. The PERTIM heterodimer functions as an autoregulatory negative feedback loop that causes a decrease in transcriptional activation by CLK and CYC [6]. The CLK-CYC heterodimer directly interacts with the upstream E-boxes (CACGTG) of the PER and TIM genes to activate their transcription [7]. These clock genes are detected in several neural and non-neural tissues, suggesting that these feedback loops are not restricted to neurons [8]. RACK1 was recently identified as an inhibitor of mammalian CLK-BMAL1 activity by recruiting PKCa during the negative feedback phase of the cycle [9]

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Conclusion