Cycling Cells - The circadian rhythm

Circadian rhythm is the cyclic control of cellular processes over a period of roughly twenty-four hours. There are many processes within the body that are held under circadian control; the need to eat and sleep, blood pressure and some hormone production to name a few. Circadian control is an important development in evolution, as it allows behaviour to adapt to appropriate times in the day. Humans have not adapted to function particularly well at night, so using that time for sleeping means they can be more alert during the day and in the hour or so before you wake up (on weekdays at least), the body gets busy increasing the blood-pressure, preparing you to need the bathroom and, slightly bizarrely, increasing testosterone levels.

I wasn't planning on doing a post on circadian rhythms, but Alejandro mentioned it in one of the comments, so it was in the back of my head when I was looking for a paper to review this week. And it's about time I started getting back into more general molecular biochemistry (less than five months left till exams!) rather than concentrating exclusively on bacteria.

From a cellular point of view circadian rhythms are controlled by careful feedback loops between interacting proteins. In this paper, the main positively regulating proteins they were looking at were proteins named CLOCK and BMAL1, which bind to promoter sequences of circadian rhythm genes and switch them on. They also turn on the negative regulators PER and CRY which, when they get to high enough levels, bind to CLOCK and BMAL1 and stop them from functioning, turning the circadian rhythm genes off again.

Many clock proteins undergo post-translational modifications in order to give a further level of control. In particular they can be phosphorylated (a phosphate group is added onto the protein) by proteins known as protein kinases. The main work of the paper I decided to look at (reference below) was identifying GSK3β, a protein kinase which phosphorylates BMAL1. Like most kinases, GSK3β is not just involved in circadian rhythm control, but takes part in many other cellular functions such as control of glucose homeostasis, cell fate determination, and cell survival. It's not surprising, therefore, that is can be involved in a number of pathological conditions, including diabetes, Alzheimer's disease, cancer and bipolar disorder.

The first study done for the paper was to show that GSK3β will bind to BMAL1 and phosphorylate it. This was done by adding pure samples of the proteins together, along with radiolabelled ATP as a source of phosphate. The result is shown below (all diagrams taken from the reference at the bottom) wiith GST used as a control to check what the GSK3β does when there's nothing for it to phosphorylate (it phosphorylates itself):


As only radiolabelled material shows up on the autoradiogram, this shows that the GSK3β has transferred the radiolabelled phosphate onto the BMAL1.

For further proof that the GSK3β was carrying out the phosphorylation under physiologically relevant conditions, cell lines were used which contained no GSK3β (-/- mutants). Comparing the levels of BMAL1 in these cells with wild type cells showed higher levels of BMAL1 in the mutant strain. This is expected as the addition of the phosphate is thought to lead to the addition of another group, ubiquitin. Ubiquitin is (as far as I am aware) an almost universal signal for 'Degrade This Protein'.

The next stage was to look for the actual sites of phosphorylation; the places on the BMAL1 where the GSK3β sticks the additional phosphate group. As with most kinases, GSK3β recognises a specific pattern of protein residues (T/SXXXS/T for anyone who's interested) and fifteen of these sequences were found in BMAL1. The exact residues were found by point mutation; certain amino-acids were changed and the resulting change in phosphorylation measured:
The wild type protein is shown on the left and the mutant, with the significant T residue converted to an alanine(A) on the left (the change is notated as T21A; the T in position 21 has been changed to an A). This change in one amino acid has decreased the phosphorylation by 40%, although it would be nice to see some actual values rather than relative ones.

The final test to confirm that GSK3β is involved in circadian rhythm control of BMAL1 was to show some actual cyclic behaviour of the protein. For this two cell lines were used, the wild type and the GSK3β -/- mutant. α-tubulin was used as a control; this protein is expressed at constant levels over time and therefore shows that the decreasing and increasing levels of BMAL1 is actually due to changing levels in the cell, rather than just a smaller cell sample containing fewer proteins overall:
That's quite a beautiful gel; in the wild type cells the BMAL1 cycles nicely over the time period (they don't run the experiment for terribly long, but BMAL1 cycling has been proved adequately elsewhere, and the wild type cells are more of a control than the actual experiment). When you knock out the GSK3β, however, the cycling pretty much stops. The paper is careful to point out that is doesn't completely stop, some evidence of differing levels is still seen, but this is to be expected. It's very rare that important cellular processes in mammals are placed entirely under the control of one protein, and there are likely to be other pathways involved in the circadian control of BMAL1.

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Sahar, S., Zocchi, L., Kinoshita, C., Borrelli, E., & Sassone-Corsi, P. (2010). Regulation of BMAL1 Protein Stability and Circadian Function by GSK3β-Mediated Phosphorylation PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008561

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