Juxtamembrane control of DDR1 kinase activity

The cells in our body are not passive and static building blocks like bricks in a wall; rather, they constantly monitor and react to their environment. They do this by sending and receiving messages in the form of signalling molecules. In order to perceive a particular signal, a cell has to have appropriate sensors. For messages that are received from the cell's environment, the sensor is often a specialised protein molecule called a receptor tyrosine kinase (RTK). One part of the RTK sticks out from the cell and another part is inside the cell. When the outside part interacts with a signalling molecule, the receptor changes its shape. This causes the inside part to become active and carry out a chemical reaction (phosphorylation) that ultimately changes the cell's behaviour. RTKs control many important functions, such as cell division, and their activity must be tightly controlled in order to prevent the development of diseases, such as cancer. Research into how RTKs are controlled is important for understanding normal human physiology, as well as for understanding what goes wrong in disease. RTKs are the targets of many drugs used in cancer therapy, and basic research of RTKs is required for designing more effective drugs.

This project will establish how the activity of an RTK called DDR1 is controlled. DDR1 instructs cells to change their behaviour when collagen is present. We discovered that a part of DDR1, which we named JM4, is needed for DDR1 signalling activity. When JM4 is missing, DDR1 can still bind to collagen, but no phosphorylation reaction results inside the cell. JM4 is also needed when the phosphorylation reaction is done in a test tube rather than in a cell. Therefore, we believe that JM4 is an important control region that regulates the part of DDR1 that carries out the phosphorylation reaction, which is called the kinase.

In this project, we aim to obtain a detailed understanding of how JM4 controls the kinase activity of DDR1. We will determine which part of the phosphorylation reaction is enhanced by JM4 and whether JM4 pushes the kinase into an active shape. In addition to directly affecting the shape of the kinase, JM4 could also affect its activity indirectly by interacting with another cellular component called Src. We already determined that Src increases DDR1 phosphorylation in cells. In this project, we will determine how Src increases DDR1 kinase activity and whether this occurs with the help of JM4.

This research is important because it may provide the basis for designing novel drugs against faulty DDR1 signalling in human disease. Most drugs against RTKs are designed to block the kinase activity by directly blocking the active part of the kinase. Because cells contain hundreds of other kinases with active parts of similar shape, these types of drugs often lead to serious side effects. JM4 is only found in DDR1 (and a similar protein called DDR2). Understanding the precise role of JM4 in controlling DDR1 kinase activity will help in designing drugs that block DDR1 kinase activity, without interfering with the activity of all the other kinases. This is expected to result in drugs with fewer side effects that could be used in diseases with abnormal DDR1 signalling, such as arthritis, fibrosis and cancer.

Receptor tyrosine kinases (RTKs) are an important class of signalling receptors whose dysregulation is associated with disease. The collagen receptor DDR1 is a drug target for kidney disease, fibrosis and many cancers, but in contrast to other RTKs little is known about how the enzymatic activity of DDR1 is regulated. We identified a 26-amino acid segment (JM4) within the DDR1 juxtamembrane region, which positively regulates DDR1 kinase catalytic activity. In this project, we will carry out detailed mechanistic studies in order to define how JM4 controls DDR1 activation.

Using in vitro kinase assays of both isolated cytosolic constructs and full-length DDR1 expressed in cells, we will determine the effect of JM4 on the kinetic parameters of DDR1. Using biophysical techniques, we will investigate the effect of JM4 on DDR1 kinase dimerisation and its affinity for ATP. Using X-ray crystallography, we will determine the structure of the DDR1 kinase with JM4. Furthermore, we will investigate the role of Src in promoting DDR1 kinase activity, characterise the physical interaction of Src with DDR1, and determine whether JM4 provides a binding site for Src. Finally, we will carry out cell biological experiments to define the roles of different Src family kinases in DDR1 activation and downstream signalling.

Collectively, these studies will define the mechanism by which JM4 controls DDR1 kinase catalytic activity. This information will help in the design of more selective drugs against unwanted DDR1 signalling.

The results from this research grant will significantly advance the knowledge base of academic research into receptor tyrosine kinases and could impact academia, industry and clinical practice. Researchers in many fields will profit directly from the fundamental insights and reagents that will be generated by our research (for details, see the section on academic beneficiaries). Although this proposal concerns basic research, it is highly likely that the improved understanding of DDR kinase regulation will result in commercially exploitable opportunities to develop therapies for a number of human diseases including arthritis, lung and kidney disease and metastatic cancer. In particular, pharmaceutical companies aiming to develop anti-DDR compounds will be interested in the results of our studies. We note that a number of pharmaceutical companies have recently started drug development programmes against unwanted DDR signalling and BL has already consulted widely for the pharmaceutical industry (in UK, Germany, Switzerland, France and USA). Although other receptor tyrosine kinases (RTKs) are already the targets of several drugs used in the clinic, our recent research has identified fundamental mechanistic differences between DDRs and canonical RTKs and so a more detailed understanding of the DDR activation mechanism and the control of its kinase activity is likely to be required for any drug development programme. The proposed research could provide thus a framework for future diagnostic and therapeutic applications and the long-term goal (10 years onwards) of this research is to impact the nation's health through the development and subsequent clinical trials of new treatments.

The impact on academic research would be immediate following successful completion of the proposed project; the impact on the pharmaceutical industry would follow with new potential candidates being developed within 5 years and any clinical impact would follow over the next 5-10 years. The research staff associated with the project would benefit from the cross-disciplinary interactions, combining cell culture of bacteria, insect cells and mammalian cells; protein expression and purification strategies; X-ray crystallography; biochemical, cell biological and biophysical techniques; and data management and dissemination. These skills would be generally useful for biomedical research and would be particularly valuable to the pharmaceutical industry. We note that there would also be a positive impact on the training of Masters and PhD students and other research staff working in the applicants' laboratories.