A research team led by Joseph S. Takahashi, professor of neurobiology and physiology at Northwestern University, has discovered the first enzyme to play a role in the circadian clock of mammals. The team's findings are detailed in the April 21 issue of Science.
This discovery contributes another known piece to the circadian clock puzzle and should help increase researchers' understanding of circadian rhythm-related problems in humans, including jet lag, sleep disorders and affective disorders, such as depression and bipolar disease. Also, the enzyme could make an ideal target for the development of drugs to treat such problems.
"Our research provides the first evidence that a gene called casein kinase I epsilon is a critical component of the mammalian circadian clock," said Takahashi, who also is a Howard Hughes Medical Institute investigator. "This is the most exciting development to come out of my laboratory since we cloned the mammalian circadian gene, Clock, in 1997."
While casein kinase I epsilon, or CKIe, is the ninth gene to be identified with circadian rhythms in mammals, it is the first gene that acts as an enzyme.
Circadian (Latin for "about a day") clocks are known to rely on a simple and elegant feedback loop of gene activation and inhibition. When that loop is working right, normal circadian rhythms of sleep and wakefulness adhere to a 24-hour cycle. When something is awry, the sleep-wake cycle is thrown off its normal pattern.
In mammals, the clock's feedback loop begins when a pair of proteins in the cell's nucleus, activation proteins called CLOCK-BMAL1, bind to and turn on a genetic switch. This switch regulates genes that produce various negative feedback proteins, including PERIOD proteins. The proteins bind to each other and build up in the cell's cytoplasm until they reach a peak level, at which time they return to the nucleus and "shut off" the switch by inhibiting the CLOCK-BMAL1 proteins. Production of the negative feedback proteins ceases, and their numbers decline over time. Without the proteins to inhibit them, the CLOCK-BMAL1 team is free to again "turn on" the genetic switch and the cycle begins anew. A complete cycle takes 24 hours.
In identifying CKIe's role in clocks, Takahashi's research team focused on a known circadian gene mutation called tau in a strain of Syrian hamsters. The researchers bred a normal strain of hamsters with the tau hamsters, and then tested the offspring's circadian behavior. The animals broke down in to three groups. Normal hamsters had a 24-hour sleep-wake cycle; hamsters with one mutant tau gene (heterozygous) had a 22-hour cycle; and hamsters with two copies of the mutant gene (homozygous) had a 20-hour cycle.
By genetically analyzing normal hamsters and homozygous tau hamsters, using a novel approach called genetically directed representational difference analysis, the researchers were able to locate the tau mutation to a position on the hamster chromosome that coincides with the CKIe gene. Their analysis proved that tau represents a mutant allele, or an alternate form, of the hamster CKIe gene. The genetic analysis further showed that the region on the hamster chromosome with the CKIe gene is similar to regions on human chromosome 22 and mouse chromosome 15.
"This is a remarkable finding," said Takahashi, "because in the fruit fly Drosophila the circadian mutation, called double-time, also is encoded by casein kinase I, in a form similar to that found in mammals."
Takahashi and his team next looked to link CKIe to circadian function. In order to do this, they needed to determine the effects of the tau mutation on the biochemistry of CKIe. An extensive functional analysis of the enzyme showed that CKIe interacted with PERIOD proteins, the negative feedback proteins that make the circadian clock tick.
The next question was that if CKIe interacts with PERIOD, what effect does the tau mutation have on that interaction? Analysis showed that the mutant CKIe enzyme does not phosphorylate, or degrade, the PERIOD proteins in the cell's cytoplasm as efficiently as the normal enzyme. The researchers propose, therefore, that the PERIOD proteins build up earlier and return to the cell's nucleus ahead of schedule to inhibit CLOCK-BMAL1. In other words, the genetic switch gets turned off earlier than normal and the cycle starts again. If two mutant CKIe genes are present, the switch is turned off even earlier than when there is only one mutant gene.
"Casein kinase I epsilon basically is a regulator of the clock's loop or cycle," said Takahashi. "We believe that normal enzyme activity causes a delay -- a desirable delay -- in the negative feedback signal of the mammalian circadian clock, keeping it on a 24-hour cycle. But if the enzyme has a mutation, the CLOCK-BMAL1 mechanism while be inhibited earlier than desired, resulting in a shorter than normal cycle."
This study shows that CKIe is involved in the circadian rhythms of hamsters, but Takahashi is already thinking ahead to what these findings might mean for the human system. His team currently is testing whether or not the CKIe mutation affects circadian timing in humans.
Other authors on the paper are Phillip L. Lowrey, Peter D. Zemenides, Kazuhiro Shimomura and Marina P. Antoch from Northwestern University, Shin Yamazaki and Michael Menaker from the University of Virginia and Martin R. Ralph from the University of Toronto.
The research was supported by the Howard Hughes Medical Institute, the National Institute of Mental Health, National Science Foundation Center for Biological Timing and The Bristol-Myers Squibb Foundation.
Journal
Science