Getting to the heart of cell signalling

EMBL scientists shed light on cellular communication systems involved in cell death and cancer

CELLS rely on a wide range of signalling systems for communication and controlling cell function. Using a structural biology approach, scientists at the European Molecular Biology Laboratory (EMBL) have added important pieces to the puzzle of how cells do this.

Understanding how cells communicate is fundamental to our understanding of biology. Cells rely on a wide range of systems to control, manage and regulate their function and to communicate and interact with neighbouring cells. All processes in the human body come down to signalling activity. Without cell signalling there would be no heartbeat, no nerve impulses and no digestion. Understanding how cells communicate, and how this sometimes goes wrong, is clearly important for furthering our knowledge of human health and disease.

Much research has already been done in the area of cell signalling and much is already known about the systems employed by cells. However, the underlying molecular mechanisms are not yet fully understood. In order to gain a better insight into these systems, EMBL scientists at the Hamburg Unit in Germany have been studying one cell signalling system – the calcium/calmodulin (CaM) dependent protein serine threonine kinases (CaMKs). Kinases – which make up 2% of all proteins from the human genome – are enzymes that catalyse phosphorylation reactions. This is done by transferring a phosphate group from an ATP molecule to the target protein, thus modifying cellular behaviour. The fast and reversible nature of phosphorylation, together with the organisation of kinases into hierarchical pathways or cascades, results in the high sensitivity and strong amplification of the input signals. As a consequence of this, kinases are of great importance in cellular pathways, especially in signalling, and their malfunction has fatal consequences for the organism. With more than 70 predicted members, the calmodulin dependent kinase–related kinases (CaMKs) are one of the largest superfamilies of kinases.

CaMKs rely on intracellular calcium levels inside the cell. This occurs through the modulator protein calmodulin, a universal intracellular calcium receptor, which picks up this signal and binds with the appropriate kinase. This complex can then become an active player in the cell’s machinery. The Hamburg team chose a specific member of the CaMK family called the Death Associated Protein Kinase (DAPK-1) to study how calmodulin binds to these kinases. DAPK-1 is a member of a small subset of CaMKs, the DAP kinase family. DAP kinases have a central role in cell death pathways, including apoptosis, autophagy and necrosis. It has also become a key marker in cancer screens due to its tumour suppressor activity. It suppresses tumour growth by inhibiting cell adhesion and migration and promoting cell death. It is believed to be responsible for destroying premalignant cells, and hence silencing, or absence of DAPK activity can be seen in cancer patients. In addition, mutated versions of DAPK are known to be involved in the development of some cancers. For example, the loss or reduced expression of DAPK has been shown to underlie cases of heritable predisposition to chronic lymphocytic leukaemia (CLL), one of the most common types of adult leukaemia and currently incurable. DAPK silencing occurs in almost all cases of sporadic CLL. Knowing more about this protein and how it functions could bring us a step nearer to new cancer treatments. Since DAPK has physical similarities to other kinases controlled by calmodulin, the Hamburg team hopes that this can also be used as a model for other similar systems.

“Previous research had identified the location of activation and deactivation sites for members of the CaMKs, however the lack of data about the 3D structure of these kinases, limited our ability to precisely understand the underlying molecular and structural mechanisms of what happens when calmodulin binds” explains Matthias Wilmanns, head of the Hamburg group who recently published their work in Science Signalling. “We used a structural approach to the problem to study DAPK and try to fill in the gaps of our knowledge on how everything fits together”.

To examine the 3D structure of DAPK, Wilmanns and his team used X-ray radiation produced by the synchrotrons, or particle accelerators, at the European Synchrotron Radiation Facility (ESRF) in Grenoble and the German Electron Synchrotron (DESY) facility in Hamburg. The X-rays are released when particles are accelerated around the synchrotron. The X-rays are guided through so-called beamlines, experiment tubes which run off at an angle to the synchrotron ring. The scientists placed their samples at the end of this beamline. Just as X-rays are used in medicine to illuminate structures not visible to the eye or even with the aid of a microscope, so can X-rays from synchrotron sources resolve the 3D structures of minute particles such as proteins. The X-rays produced by the synchrotrons are however, much more intense and powerful.

Wilmanns and his group used a technique called X-ray crystallography whereby the protein is first crystallised. This crystal is then put into the X-ray beam. Just as rocks in a fast flowing river will alter the flow of the water, the X-ray beam is bent, or diffracted by the atoms in the crystal. Using complex computer software, the scientists can use the resulting pattern to extrapolate the molecular structure of the atoms making up the protein. In this solid form, the protein can be rotated so as to view the structure for different angles and thus enable the scientist to build up a 3D structure of the protein.

Crystallisation is not an exact science, and each protein has its own optimal conditions (pH, salt or precipitant concentrations) under which it will crystallise. Generally it is harder to get larger proteins, or complexes of proteins to crystallise, however the Hamburg scientists were successful in crystallising the DAPK-calmodulin complex and so were able to see just how the two molecules fit together.

The results showed how calmodulin binds to a particular section of DAPK, and thereby activating it. Additional experiments also showed which amino acids were crucial for calmodulin to bind. The team was also able to show that proper DAPK-CaM complex formation is crucial for DAPK catalytic activity. “This is obviously only the beginning of the story” says Dr. Wilmanns, “these results can help us design drugs which can prevent the calmodulin from binding to DAPK, and stop catalytic activity.”

Since the genetic sequence of the binding position seems to be very similar among members of the CaMK family, the hope is that the findings from the DAPK-calmodulin complex will also be valid for other members of the CaMK family. Calmodulin itself is one of the most widespread regulatory proteins in biological systems, and the results from Hamburg may indeed help us to understand other systems where calmodulin is involved. The team is already planning further experiments with groups from the Weizmann Institute in Israel and other groups from EMBL. 

Pull quote: “Understanding how cells communicate is fundamental to our understanding of biology. Cells rely on a wide range of systems to control, manage and regulate their function and to communicate and interact with neighbouring cells”