Function of the neuronal tyrosine kinase N2-Src in synaptic vesicle exocytosis

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Tyrosine phosphorylation (the addition of phosphate to the amino acid tyrosine) of proteins is performed by enzymes called tyrosine kinases and is used as a signalling mechanism in all cells of the body. Phosphorylation of proteins causes a localised change in the shape and charge of the protein and, depending upon where phosphorylation occurs, can transmit a signal by changing the function of a protein, eg. by altering its affinity for binding to other proteins. The tyrosine kinase C-Src is highly enriched in brain cells (neurons) and is thought to regulate many aspects of brain function, such as development and learning and memory. In neurons, C-Src is concentrated on synaptic vesicles, compartments which store the chemical neurotransmitters that are released by neurons to communicate with each other. An additional type of C-Src is found in the brain, called N2-Src, that differs in a part of the protein which regulates its interactions and can determine which proteins it phosphorylates. Consequently, N2-Src does not bind to the proteins usually bound by C-Src suggesting it has an alternative function. In support of this, we found that N2-Src (but not C- or N1-Src) binds to two proteins, Munc18 and NSF, that are necessary for the release of neurotransmitter by neurons. Furthermore, the tyrosine phosphorylation of Munc18 and NSF has been shown to be important for the release of hormones in other parts of the body. We hypothesise that N2-Src controls the release of neurotransmitter from neurons by binding to and tyrosine phosphorylating Munc18 and NSF. To address the hypothesis, we will first study the interactions between N2-Src and Munc18 or NSF in the test tube to find the specific regions of the proteins which are necessary for binding. We will also discover which tyrosines on Munc18 and NSF are phosphorylated by N2-Src and how phosphorylation affects their function. The information gained from these experiments will allow us to engineer forms of Munc18 and NSF that have lost the ability to bind N2-Src or to be tyrosine phosphorylated. By testing the effects of these engineered proteins upon neurotransmitter release from living neurons we will find out whether N2-Src regulates this process by altering the function of Munc18 or NSF. Since most kinases can phosphorylate tens of proteins, it is unlikely that Munc18 and NSF are its only targets. We will search for other proteins that are phosphorylated in neurons by N2-Src to gain a more complete picture of its function in the brain. Neurotransmitter release is a fundamental process required for normal brain function but is also affected by neurological disease and drugs of abuse. Thus the characterisation of how neurotransmitter release is regulated by the N2-Src tyrosine kinase will aid research into the function of the healthy and diseased brain. More specifically, there is evidence that the levels of neuronal Src kinases are altered following epileptic-like seizures in mice and hence the data from this project will help to better understand the changes in neurotransmission resulting from epilepsy.