Function of the neuronal tyrosine kinase ...

  • Evans, Gareth John Owen (Principal investigator)
  • Dunning, Christopher James Robert (Researcher)
  • Cousin, Michael Alan (Other)

Project: Research project (funded)Research

Project Details


The ubiquitous tyrosine kinase C-Src has two splice variants, N1- and N2-Src, which are exclusively expressed in brain. The uncharacterised N2-Src has a 17 amino acid insert in the so-called N-src loop of its SH3 domain which dramatically affects its binding specificity. We have identified Munc18 and NSF, both necessary for synaptic vesicle exocytosis, as novel interactors of the N2-Src SH3 domain which do not bind to either C- or N1-Src. The hypothesis of the proposal is that N2-Src regulates synaptic vesicle exocytosis by binding and phosphorylating Munc18 and NSF. The planned research will first use a combination of site directed mutagenesis and in vitro binding assays to find the sites of interaction between N2-Src and Munc18 or NSF. The structural features of the N2 N-Src loop that dictate specificity for Munc18 or NSF binding will also be addressed. Since Src tyrosine phosphorylates the proteins it binds, the N2-Src phosphorylation sites on Munc18 and NSF will be determined by MALDI-TOF and confirmed by mutagenesis. The next objective will be to use the binding and phosphorylation mutant proteins generated in the first section to determine how N2-Src affects Munc18 and NSF function. This will involve in vitro biochemical assays and transfection of mutants into cultured neurons and the measurement of synaptic vesicle exocytosis at individual nerve terminals using real time imaging of fluorescent reporters. Finally an innovative method of engineering unnatural inhibitor or nucleotide specificity into the catalytic domain of N2-Src will be used to screen for novel substrates. This will give an indication of how many unique substrates N2-Src phosphorylates and provide new targets for future investigations.

Layman's description

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.

Key findings

This grant has established two novel roles for N2-Src kinase in the brain. First, we have shown that N2-Src processively phosphorylates and binds the N-terminus of Munc18 interacting protein 1 (Mint1) and that this phosphorylation regulates the trafficking of amyloid precursor protein (APP) to synapses. These findings have implications for understanding Alzheimer’s disease pathology, for which aberrant trafficking of APP is thought to be responsible.

Second, we have uncovered a potential role for N2-Src in brain development as a regulator of neuronal differentiation. Neurons, in which levels of N2-Src are disrupted, have defects in the specification of the axon and dendrites. In parallel in vitro experiments, we have explained the behaviour of N2-Src in cells by demonstrating its unique SH3 ligand specificity and high constitutive activity. Two manuscripts describing these data are currently submitted for review.
Effective start/end date21/05/0726/09/10