Abstract
All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential “clock kinase” phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.
Highlights
Circadian clocks provide a key advantage to organism allowing them to prepare in advance for daily environmental changes
Circadian clocks depend on species-specific clock genes and proteins that interact in complex feedback loops to rhythmically control gene transcription
These temporal changes in PER phosphorylation are crucial for a functioning clock, since they modulate the stability of PER as well as the time of its nuclear entry, and in this way determine the pace of the clock [11,12,13]
Summary
Circadian clocks provide a key advantage to organism allowing them to prepare in advance for daily environmental changes. Circadian clocks depend on species-specific clock genes and proteins that interact in complex feedback loops to rhythmically control gene transcription (reviewed in [1,2]). Recent findings indicate that PER proteins in animals possess up to 25–30 phosphorylation sites [5,11] many of which undergo daily changes in phosphorylation These temporal changes in PER phosphorylation are crucial for a functioning clock, since they modulate the stability of PER as well as the time of its nuclear entry, and in this way determine the pace of the clock [11,12,13]. In Drosophila just a few kinases have been identified that interact with PER: DBT [15,16,17], SGG [12], CK2 [18,19,20] and proline-directed kinases as
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