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Palmitoylation/Phosphorylation interplay affect

Kainate receptors surface expression

Busra P Yucel, 4th year, University of Bristol

BACKGROUND:

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Our brain comprises around 85 billion neurons complicatedly connected to function as a remarkable organic machine. Each neuron has a soma, signal-collecting dendrites, and signal-transmitting axons. Dendrites feature thousands of modulating mushroom-like spines, and synapses form at the connection between axons and dendritic spines, serving as key sites for complex synaptic signalling.

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Kainate receptors (KARs) are one of the ionotropic glutamate receptors localizes on the synapses that are key regulators of neuronal excitability and synaptic transmission. These receptors comprise a combination of five distinct protein subunits, GluK1-5. Dysfunctions in these receptors within the brain can disrupt the delicate balance of neuronal activity and contribute to conditions such as epilepsy, schizophrenia, and autism spectrum disorders.

My research focuses on palmitoylation of GluK2 subunit, which is a postsynaptic modification involving the addition of a palmitic acid to a cysteine residue, rendering the protein hydrophobic and stably anchored to the cell membrane, thus serving as a principal regulator of membrane proteins.

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In this study, to explore the complex interplay between GluK2 palmitoylation and phosphorylation and their influence on KAR surface expression.

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METHODOLOGY:

Throughout my PhD, I learned a wide range of techniques for detecting posttranslational modifications, including the APEGS assay and the Phostag gel technique. I also used confocal imaging and the surface biotinylation. I cultivated skills in cell biology techniques such as PCR, cloning, transduction, truncation, point mutations. Moreover, I conducted a substantial number of Western blot experiments.

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RESULTS:

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In this project, I showed that GluK2 is basally palmitoylated in two cysteine residues C858/C871 and palmitoylation of GluK2 decreased by kainate stimulation (Figure 1A). Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation (Figure 1B) and is insensitive to KA-induced internalisation (Figure 1C-D) (Taken from Yucel et al 2023).

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Figure 1. A: KARs are palmitoylated (upper bands) in cysteine 858 and 871 and KA stimulation causes a significant decrease in double palmitoylation (2). B: Non-palmitoylated mutant is more phosphorylated (upper thick band) by PKC on serine 846 and 868 residues (Orange coloured set induced with PMA (PKC activator)). C, D: Biotinylation and confocal imaging experiments on KAR surface/total numbers after KA stimulation. Surface and total KD-rescue KARs were labelled with GFP or myc antibodies. The non-palmitoylated mutant exhibited lower levels of surface/total expression compared to the wild type (WT), and kainate stimulation (orange coloured in graph C and D) did not have any impact on this mutant (Taken from Yucel et al 2023).

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These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis (Figure 2).

 

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Figure 2. The proposed model. Agonist kainate stimulation leads to depalmitoylation, which subsequently triggers PKC phosphorylation and SUMOylation, resulting in the internalization of kainate receptors (Taken from Yucel et al 2023).

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I recently published this work so more details can be found in Yucel et al 2023.

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FUTURE WORK:

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I am currently in the process of writing my thesis, with plans to submit it in the coming months. In the future, I am hoping to pursue an academic career with a focus on different synaptic protein palmitoylations, compartmentalized palmitoylation and the roles of palmitoylation in synaptic transmission. I want to use more specialized imaging techniques such as the RUSH assay and FRET imaging.

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PS: You will encounter both excitement and challenges throughout your PhD, and there may be times when challenges seem overwhelming. However, it is definitely worth it in the end.

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FUNDED BY:

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CONTACT: 

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Busra P Yucel

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