The Quiet Truth of Circadian Rhythm: Light and Phosphorylation
Jay is a college student at UC Davis studying Biology, but like most college students, he lacks a consistent sleep schedule due to his late-night bouts of studying on his computer. There are many factors that impact his sleep, such as caffeine intake, room temperature, noisy roommates... and the list continues. But surprisingly, light generates the largest impact on our sleep. And from light, phosphorylation occurs, driving cell signaling within the body. Light drives circadian rhythm as it causes phosphate groups to activate many of the necessary proteins within the sleep mechanism.
The 24-hour cycle that our bodies run on, with roughly eight hours dedicated to sleep and 16 hours awake, can serve as the simple definition of circadian rhythm. The mechanism for circadian rhythm is a relay race, with three runners in their positions, waiting for the baton to arrive and dormant until that point. In reality, three different protein complexes, CREB, BMAL1-CLOCK, and PER-CRY, await the arrival of a phosphate group.
When phosphorylated, CREB allows BMAL1-CLOCK to regulate the expression of PER-CRY, our main clock effector proteins. The levels of PER-CRY rise throughout the day and fall during the night, creating our circadian rhythm. But how do we arrive at this point?
When Jay opens up his window in the morning, the light from the sun hitting his eyes begins the circadian rhythm mechanism, like a starting pistol in a relay race. The responsible pathway takes light from the eyes and turns it into a signal, which will travel through the optic nerve to the suprachiasmatic nucleus (SCN), the internal clock in our brain. The tissues from the SCN are well suited to the role of an internal clock because even when placed in a petri dish, the tissues still operate on a ~24-hour cycle. [1]
The mechanism begins with our eyes taking in light during the day and transforming it into a message to the brain via neurotransmitters. These chemical messengers carry information specific to their chemical structure that will either excite or inhibit their appropriate receptor. The two messengers, L-glutamate and PACAP, bind to their receptors along the membrane of the SCN, which ultimately results in the activation of two different kinases.
Kinases are the runners in the race within the cell delivering the baton, phosphate, to allow the next runner to continue. Within the SCN cell, Protein Kinase A (PKA) and Calcium-Calmodulin-Dependent Kinase 2 (CaMKII) carry out phosphorylation, the delivery of a phosphate group. These two kinases act on the cAMP Response Element Binding protein (CREB), the first point to receive the baton. CREB lays on top of DNA strands within the nucleus of the SCN, acting as the on-off switch for DNA transcription. Under normal conditions, phosphorylated CREB allows for the activation of the BMAL1-CLOCK complex, which in turn enables the production of clock effector proteins, PER-CRY. But what occurs under suboptimal conditions?
Jay has been vigorously studying since 11:00 pm and checks his phone to see that it's 1:00 am and has a reminder to take his friend to the airport at 6:00 am. He goes to sleep, takes his friend to the airport, and then goes about his day normally. However, he notices more difficulty sleeping the following couple of nights. What is going on inside his SCN cells?
In a relay race, if the runner cramped up halfway through, knocking them out of the race, the next runner would not receive the baton. In biology, to prove the efficacy of a particular compound, researchers will remove that compound and observe the results. A group of scientists found that the phosphorylation status of sleep-promoting kinases, such as CaMKII, is altered by sleep deprivation. The altered status of these sleep-promoting kinases negatively impacts sleep regulation [2]. Furthermore, the study demonstrated that a significant decrease in sleep duration per day results from the embryonic knockout of CaMKIIa and CaMKIIb [2]. “Embryonic knockout” removes a specific gene or protein within the embryo of an organism, in this case, the removal of CaMKIIa and CaMKIIb. The embryonic knockout and altered status of CaMKII displays the integral role of kinases and phosphorylation in circadian rhythms.
Jay reportedly feels fine after only sleeping 5 hours but his body tells a much different, sleep-deprived story. Sleep deprivation leads to a decrease in phosphorylation activity among sleep-promoting kinases while low levels of CaMKII lead to a decrease in sleep duration per day, demonstrating how fragile our circadian rhythm is to arrhythmic activity. Furthermore, this relationship between sleep-promoting kinases and sleep deprivation enlightens us on how strongly phosphate impacts our circadian rhythm. Without CaMKII delivering phosphate to CREB, the body will endure less sleep which causes a decrease in the CaMKII activity, and the cycle repeats. However, with CREB activated via phosphorylation, the BMAL1-CLOCK protein complex can now carry out its function.
Mice are a model organism in studying neurobiological processes, specifically the circadian rhythm and the BMAL1-CLOCK protein complex. When studying the circadian rhythm and activity of mice, scientists found that when Ser-90, the 90th amino acid in BMAL, lacks a phosphate group, the mice fibroblasts of the SCN demonstrated arrhythmic activity as well as decreased circadian gene expression [3]. Another study stated that the activation of the BMAL1-CLOCK protein depends upon the phosphorylation status of BMAL1, meaning that kinases modulate its activity [4]. The phosphorylation status of BMAL1-CLOCK is utterly important to the functioning of our circadian clock, allowing for the production of PER-CRY proteins.
The production of these PER-CRY proteins partially concludes the mechanism that occurs in response to Jay opening his computer. PER and CRY, clock effector proteins, control their own modulation through a process called transcription-translation feedback loop, or TTFL. This process includes translating mRNA into a protein and that modified protein then acts as a modulator to the levels of translation for that mRNA. When the levels of PER-CRY rise too high in the SCN cell cytoplasm, they join together to inhibit BMAL-CRY, which controls PER-CRY transcription. Levels of PER and CRY slowly rise throughout the day with the body's response to light and then dip down around night time as they execute the TTFL, in accordance with a ~24 hour cycle. Jay opening up his computer at night stimulates the production of PER-CRY during a time when their levels decrease, demonstrating how late-night screen time impacts our sleep. Like the aforementioned proteins, does PER-CRY depend upon phosphorylation as well?
The PER-CRY complex receives the last baton to finish out the race of our circadian rhythm. When PER-CRY levels increase in the cytoplasm, there are two possible courses of action: re-enter the nucleus or be degraded by various enzymes. The joining of PER-CRY, known as dimerization, allows them to come back into the nucleus and engage in the TTFL. Interestingly, experiments demonstrated that the phosphorylation of PER favors the dimerization of PER binding to CRY [3]. A different experiment demonstrated that when PER lacked a phosphate group, it remained in the cytoplasm and was eventually degraded by enzyme activity [5]. This demonstrates that from start to finish, our circadian rhythm depends on the essential activity of phosphorylation.
Relay race? Phosphorylation? Mice? What does all of this mean? Light starts the mechanism of circadian rhythm and ultimately produces PER-CRY, regardless of time or place. The natural cycle for circadian rhythms consists of higher levels of PER-CRY during the day and lower levels at night. Exposing ourselves to light at night, via computer or phone, disrupts the natural circadian rhythm by the creation of PER-CRY. Additionally, the normalization of sleeping less than recommended, specifically among college students, directly causes chaos in the process of phosphorylation [6]. Sleep deprivation disrupts phosphorylation which, in turn, leads to improper function of our circadian rhythm. Overall, light exposure and sleep deprivation alter phosphorylation status which controls our circadian rhythms. So put down the phone and go to sleep.
About the Author: Callahan Kincaid
Callahan Kincaid is a class of 2025 Neurobiology, Physiology and Behavior major with an interest in memory forming, sleep and Alzheimers. Being obsessed with the mystery of the brain from a young age, Callahan naturally chose Neurobiology to uncover all the secrets that exist inside the brain. Understanding the cellular and molecular processes of basic human actions, such as sleep, provides a deeper understanding and appreciation of the human body. Hopefully readers will gain an appreciation for the complexities of the human body, as well as a solid foundation of how sleep and circadian rhythms actually work. These processes are often misunderstood due to the influx of sleep hack gurus, so this paper shall serve as a step towards the truth of sleep and circadian rhythms. Callahan is aiming to attend graduate school to further his studies of the brain.
Author's Note
I wrote this paper as a requirement for my UWP course, with the intended audience being my peers and professors. I chose the topic of phosphorylation and circadian rhythm because I have always been fascinated by circadian rhythms and was finally introduced to the mechanism for one of my major courses. I wanted to know more about the process at a molecular level, so I decided to research the impact of phosphorylation on our circadian rhythms. I want the readers to understand more about what a circadian rhythm is, how it works on a molecular level and how light impacts it by phosphorylation.
References
- Honma, Sato, and Ken-ichi Honma. “Single Cell Neuronal Circadian Clocks.” Encyclopedia of Neuroscience, Squire , pp. 843–847.
- Ode, Koji L., and Hiroki R. Ueda. “Phosphorylation hypothesis of sleep.” Frontiers in Psychology, vol. 11, 2 Oct. 2020, pp. 4. https://doi.org/10.3389/fpsyg.2020.575328. Print.
- Brenna, Andrea, and Urs Albrecht. “Phosphorylation and circadian molecular timing.” Frontiers in Physiology, vol. 11, 26 Nov. 2020, pp. 6. https://doi.org/10.3389/fphys.2020.612510. Print.
- Schibler, Ueli. “BMAL1 dephosphorylation determines the pace of the circadian clock.” Genes & Development, vol. 35, no. 15–16, 1 Aug. 2021, pp. 2. 1076–1078, https://doi.org/10.1101/gad.348801.121. Print.
- Cheng, Arthur H., and Hai-Ying Mary Cheng. “Genesis of the master circadian pacemaker in mice.”Frontiers in Neuroscience, vol. 15, 23 Mar. 2021, pp. 5. https://doi.org/10.3389/fnins.2021.659974.
- Lund, Hannah G., et al. “Sleep patterns and predictors of disturbed sleep in a large population of college students.” Journal of Adolescent Health, vol. 46, no. 2, Feb. 2010, pp. 124–132, https://doi.org/10.1016/j.jadohealth.2009.06.016