Now whilst it’s our yearly fashion to celebrate the start of the New Year, to a circadian rhythm, New Year’s Eve is just like any other day. The circadian clock is a cellular internal oscillator that operates through feedback loops coordinating multiple physiological processes. One such activity known to oscillate is of the protein kinase complex, mTORC1. Through an elegant and expansive array of pull downs and Western blots, a recent paper published in Cell Metabolism1, by Wu et. al, provides clear evidence of circadian regulation of mTORC1 by Per2.
The cell with clocks not in its walls
The most interesting aspect of the circadian clock is its ability to self-perpetuate. The molecular basis behind this self-perpetuation is of transcriptional-translational feedback loops. Key protein players of the circadian clock are, you guessed it, CLOCK, and the less guessable, BMAL1. CLOCK and BMAL1 promote the expression of thousands of genes, including Period (Per) and Cryptochrome (Cry). Per and Cry then inhibit the action of CLOCK and BMAL1 (Figure 1). This mechanism works then based on a negative feedback with delay. (Check out this recent review –> (2) if you’re interested in more!).
One complex caught up in this circadian cycle is mTORC1.
What are you (m)TORC-ing about?
The key catalytic subunit of mTORC1 is mTOR – the mammalian target of rapamycin. mTOR possesses kinase activity, phosphorylating numerous proteins promoting cell growth and metabolism. Along with mTOR, mTORC1 also contains the proteins RAPTOR and mLST8. Specifically, mTORC1 phosphorylates two proteins, 4E-BP1 and S6K to promote translation and another protein, ULK1, to prevent autophagy (the cells own degradation and recycling system).
Due to its control over critical cellular processes, it is not surprising that mTORC1 activity is regulated. Upstream of mTORC1 activation is the tuberous sclerosis complex (TSC). The current understanding is that this complex inhibits mTORC1 by inactivating an activator of mTORC1, Rheb (Figure 2).
A Per-spective on how this circadian regulation is achieved
Whilst the coordination of the mammalian circadian clock factors in transcriptional regulation (which takes place in the nucleus) has been well studied, it is becoming apparent that the factors may also perform roles in the cytoplasm. It is in the cytoplasm where mTORC1 functions.
One way you can study a protein’s function is by examining other proteins it interacts with. Wu’s team did just this with one clock factor, period 2 (Per2) and found that RAPTOR strongly interacted. Follow-up studies showed Per2 to interact with other mTORC1 components including mTOR and mLST8. The strongest interactions were made with the C-terminus of Per2.
But what is the purpose of this interaction?
When Per2 was overexpressed, there was a decrease in mTORC1 activity. The same response occurred when just the C-terminus of Per2 was overexpressed. Conversely, decreasing Per2 protein levels increased mTORC1 activity.
So, looks like Per2 is preventing mTORC1 activity (Figure 3). Corroborating this, a lack of Per2 greatly reduced the interaction between mTOR and Tsc1 (the upstream inhibitor of mTORC1).
However, the authors showed this reduction in mTORC1 activity to be Rheb-independent.
So, if TSC1 isn’t inhibiting Rheb to prevent mTORC1 activity, what is going on?
Could it be that inhibition of mTORC1 activity is due to blocking its substrates or activators from binding? These are key questions the team are keen to be answered.
Per-sonally my favourite part?
To conclude their study, the team analysed the regulation of Per2 and how that feeds into the roles and regulation of mTORC1. Interestingly, they found that glucagon signalling (occurs during fasting), induces Per2 expression which then inhibits mTORC1 activity.
Understanding the circadian clock and its influence over cellular processes is important since disruptions have been associated with cancer. A key hallmark of cancer is uncontrolled cell growth. With Per2’s ability to reduce mTORC1 activity in the cell, further insight into these molecular interactions could provide new therapeutic targets.
1. Wu et al., The Circadian Protein Period2 Suppresses mTORC1 Activity via Recruiting Tsc1 to mTORC1 Complex, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.11.006
2. Takahashi, J. S. Transcriptional architecture of the mammalian circadian clock. Nature Reviews Genetics 18, 164–179 (2017)