Science for Health
Neuronal dendrites are millimeter-sized elaborate trees in our brains mostly covered with micron sized excitatory postsynaptic sites called dendritic spines. Functioning of the neuronal circuitry depends on the correct development of dendritic arbors and spines. Defects in dendrite and spine development are associated with neurodevelopmental disorders such as autism. Despite the importance of dendrite development, little is known about the intracellular signaling mechanisms that govern this process in mammalian neurons.
Kinases are key regulators of most cellular processes. With more than 500 kinases in the human genome, they are one of the most commonly targeted group of molecules for treatment of diseases such as cancer. Contribution of kinase signaling pathways in neurological diseases are not well understood. A better understanding of such pathways could facilitate the pursuit of targeted treatments for neurological diseases. Despite the ubiquitous roles of kinases in several cellular processes, identification of their downstream signaling pathways has been challenging. We use a novel chemical genetic method for identifying of kinase substrates and phosphorylation sites from complex protein homogenates. We have used this method to identify substrates of NDR1 (Ultanir et. al, 2012) and MST3 (in prep) (Figure1).
Our research aims at identifying kinase signaling pathways that regulate formation and plasticity of dendrite arbors and dendritic spines in the mammalian brain. To address this goal, we use a combination of techniques including imaging, electrophysiology, chemical genetics and biochemistry in rodent hippocampal cultures and mouse models (Figure 1).
Click image to view at full-size
Left: Chemical genetic method for identification of kinase substrates. Endogeneous kinases use ATP to phosphorylate their targets. Analog sensitive (as) NDR1 is generated by a single point mutation at the ATP binding domain. As-NDR1 can use a modified bulky ATP analogs such as (Benzyl-ATP-γ-S) to thiophosphorylate its substrates. Thiophosphate can be covalently captured leading to the identification of substrates and the phosphorylation sites using mass spectrometry. Using this method we identified five novel NDR1 phosphorylation targets, discovering the molecular function of NDR kinase pathway. “P” denotes phosphorylation. Right: Techniques we use to study neuronal development: clockwise imaging of dendritic spines, electrophysiology, in utero electroporations in mice and hippocampal cultures.
Sila Konur Ultanir
Click links to view others working on these themes
© MRC National Institute for Medical Research
The Ridgeway, Mill Hill, London NW7 1AA