Adding to the growing body of evidence for how the protein Cyclin-Dependent Kinase (CDK) - the 'master regulator' of cell division - operates, researchers at the Francis Crick Institute propose a new explanation for the timing of its action.
Frank Uhlmann of the Crick explains the background to the work: "The cell division cycle is a series of events by which a cell grows and duplicates all its content, before dividing in two. This process is orchestrated by CDK.
"One of the big mysteries is how CDK gets the timing right - first the cellular content, especially chromosomes, must be duplicated and only afterwards must the cell divide."
CDK regulates proteins involved in the cell cycle by chemically adding a phosphate group to them. This process is called phosphorylation and it works like a switch to turn on the protein which is being modified.
Dr Uhlmann adds: "The question is, how does CDK know which proteins to phosphorylate first?"
The researchers used budding yeast cells in their experiments. The principles of cell cycle regulation are the same in yeast and humans, but yeast is much simpler and easier to manipulate. Molly Godfrey in Dr Uhlmann's lab worked with Andrew Jones and Bram Snijders from the Crick's Mass Spectrometry Proteomics Science Technology Platform, who carried out phospho-proteome analysis. This takes stock of all the phosphorylated proteins inside a yeast cell.
The team investigated enzymes called phosphatases that undo phosphorylation by removing the phosphate group. They found one (called PP2ACdc55) that counteracts CDK's phosphorylation on a subset of its targets, thereby helping to explain the timing of CDK action.
Proteins that escape this phosphatase get phosphorylated early in the cell division cycle, while proteins that experience dephosphorylation by the phosphatase have to wait until higher levels of CDK activity accumulate before phosphorylation wins over dephosphorylation.
The researchers also discovered how this phosphatase selects the proteins for which it delays phosphorylation. It was already known that CDK adds phosphates to either serine or threonine amino acids on the surface of proteins. These two amino acids are very similar and until now it was thought that there was no difference between serine and threonine phosphorylation.
But the researchers found that the phosphatase PP2ACdc55 counteracts threonine, but not serine, phosphorylation. Thus threonine-phosphorylated proteins are phosphorylated later.
Dr Uhlmann says: "A multitude of biological processes are regulated by phosphorylation, including, among other things, immunity and growth factor signalling. Our realisation that phosphorylation timing depends on the amino acid that accepts the phosphate group will almost certainly be applicable to the regulation of many other events."
This research complements recent work led by Matthew Swaffer in Sir Paul Nurse's lab at the Crick. Dr Swaffer's paper in Cell provides convincing evidence for the 'activity threshold model' for CDK. This model proposes that CDK controls cell division simply by turning on the proteins needed at different stages of the process based on increases in its own activity level.
Dr Uhlmann says: "These pieces of work show that if we look at protein phosphorylation during the cell cycle, we should always consider both directions: putting the phosphate on (Matthew Swaffer's work) and taking it off again (our paper). Both processes happen at the same time and it's the relative strengths of both reactions that decide on the ultimate outcome of whether a protein is phosphorylated or not."
Dr Uhlmann's paper, PP2ACdc55 phosphatase imposes ordered cell cycle phosphorylation by opposing threonine phosphorylation, is published in Molecular Cell.
